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Structure-function studies of the gastric inhibitory polypeptide/glucose dependent insulinotropic polypeptide… Gelling, Richard Wayne 1998

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STRUCTURE-FUNCTION STUDIES OF THE GASTRIC INHIBITORY POLYPEPTJDE/GLUCOSE DEPENDENT rNSULINOTROPIC POLYPEPTIDE (GIP) RECEPTOR by RICHARD W A Y N E GELLING B.Sc, The University of Victoria, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Physiology) We accept this thesis as conforming^the required standard UNIVERSITY OF BRITISH C O L U M B I A October 1998 © Richard Wayne Gelling, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of PhuS; olo The University of British Columbia M y Date DE-6 (2/88) A B S T R A C T Incretins are hormones released from the gastrointestinal tract into the circulation during and after a meal that potentiate glucose-stimulated insulin secretion. Glucose dependent insulinotropic polypeptide (GIP) is now accepted as the most important incretin and, along with the glucagon-like peptide 1 (GLP-1 (7-36) amide) has therapeutic potential in Non-Insulin Dependent Diabetes Mellitus (NDDDM). In the present study, a rat islet GIP receptor complementary (c)DNA (GIP-R1) was cloned and characterized. The islet cDNA was identical to that previously identified in a tumor cell line, except for a single nucleotide polymorphism resulting in one amino acid difference (Glu21-»Gln21). When expressed transiently in COS-7 cells or stably in CHO-K1 cells the receptor displayed specific high affinity 1 2 5 I-GIP binding in both saturation (200-300 pM) and competition (IC50 1-8 nM) binding studies, and GIP-dependent increases in cAMP production (EC50 0.069-0.70 nM). Cells expressing GIP-R1 exhibited equivalent signaling in response to porcine and human GIP. In addition, COS-7 cells expressing the GIP-R1 cDNA displayed a biphasic increase in intracellular calcium in response to GIP. Structure-function studies of GIP showed that the peptide could be truncated at its carboxy-terminal at residue 30 (GIP 1-30 amide) without affecting receptor affinity or efficacy. In contrast, amino-terminal truncation of GIP 1-30 resulted in fragments with reduced affinity and lacking receptor activation activity, that antagonized GIP-stimulated cAMP production. Importantly, GIP 6-30amide bound with nearly identical affinity to GIP but was a potent inhibitor of GIP action in vitro, suggesting that this region contains the binding core and that amino-terminal residues are important for receptor activation. ii The latter finding is important given that GTP is metabolized by dipeptidyl-peptidase (DP) IV to biologically inactive GTP 3-42. The analogs Ppa'-GTP 1-30 and D-Ala2-GTP 1-30amide were shown to be resistant to DP IV degradation in vitro, but had slightly reduced affinity and efficacy at the GTP receptor. Such DP IV resistant analogs may be useful in NIDDM treatment. Oligonucleotide-directed mutagenesis was used to examine regions important for ligand binding, receptor activation, and G-protein coupling. Studies of GTP/GLP-1 receptor chimeras indicated that the high affinity GIP binding domain lies within the extracellular amino-terminal of the GIP receptor, while the first transmembrane domain appears critical for GTP-specific receptor activation. A similar region of the GLP-1 receptor may be important for GLP-1 receptor activation. The effect of truncating the carboxy-terminal-tail of the GIP receptor on ligand binding, second messenger coupling, and internalization was examined. Truncation by >37 amino acids greatly decreased expression, and a minimum carboxy-terminal tail length of 13 amino acids appears to be required for receptor expression. In contrast the carboxy-terminal-tail could be truncated by up to 50 amino acids without affecting receptor affinity, and with only small effects on G-protein coupling and receptor internalization. These are the first detailed structure-function studies on GIP and its receptor in a cell system uncomplicated by factors inherent in whole animal preparations or immortalized P-cell lines. Further studies may lead to a better understanding of the apparent reduction of GTP receptors in NIDDM, and the development of GIP analogs that are useful in its treatment. iii T A B L E OF CONTENTS A B S T R A C T ii LIST OF TABLES vii i LIST OF FIGURES: ix ABBREVIATIONS: xii A C K N O W L E D G E M E N T S : xiii CHAPTER 1 INTRODUCTION 14 1.1 OVERVIEW 14 1.2 DISCOVERY OF GIP 16 1.2.1 GIP IS A M E M B E R OF THE VTP/GLUCAGON/SECRETIN SUPERFAMILY 18 1.2.2 GIP GENE STRUCTURE A N D POSTTRANSLATIONAL PROCESSING 19 1.2.3 TISSUE DISTRIBUTION... . 22 1.2.4 GIP R E L E A S E 22 1.2.5 R E G U L A T I O N OF GIP GENE EXPRESSION 25 1.3 BIOLOGICAL EFFECTS 26 1.3.1 ENTEROGASTRONE ACTIVITIES OF GIP : 26 1.3.2 INCRETINS A N D THE ENTEROINSULAR AXIS CONCEPT 27 1.3.3 EVIDENCE FOR GIP AS A N INCRETIN 28 1.3.4 GIP EFFECTS O N OTHER ISLET C E L L TYPES 29 1.3.5 E X T R A P A N C R E A T I C EFFECTS OF GIP 29 1.4 EVIDENCE FOR FURTHER INCRETIN(S) 30 1.4.1 ISOLATION, PROHORMONE PROCESSING, A N D TISSUE DISTRIBUTION OF GLP-1 31 1.4.2 R E G U L A T I O N OF GLP-1 R E L E A S E 34 1.4.3 GLP-1 ACTION O N INSULIN R E L E A S E 34 1.4.4 GLP-1 EFFECTS O N OTHER ISLET C E L L TYPES 35 1.4.5 CONTRIBUTION OF GIP A N D GLP-1 TO THE INCRETIN EFFECT 36 1.5 GIP BINDING SITES 38 1.5.1 ISOLATION OF THE GIP RECEPTOR cDNA A N D G E N E 39 1.5.2 GIP RECEPTOR GENE EXPRESSION A N D TISSUE DISTRIBUTION.... 40 1.6 GLP-1 RECEPTOR BINDING STUDIES 43 1.6.1 ISOLATION OF THE GLP-1 RECEPTOR cDNA A N D G E N E 43 1.6.2 GLP-1 RECEPTOR GENE EXPRESSION A N D TISSUE DISTRIBUTION44 1.7 RECEPTOR SIGNAL-TRANSDUCTION P A T H W A Y S 46 iv 1.7.1 GIP RECEPTOR SIGNAL-TRANSDUCTION MECHANISMS 46 1.7.2 GLP-1 RECEPTOR SIGNAL TRANSDUCTION 48 1.8 GLUCOSE-DEPENDENCE OF GIP AND GLP-1 STIMULATED INSULIN RELEASE 51 1.10 SmUCTURE-FUNCTION STUDIES OF GIP 56 1.10.1 IDENTIFICATION OF A BIOLOGICAL CORE IN GIP 56 1.10.2 GIP IS METABOLIZED TO GIP 3-42 BY DIPEPTIDYL PEPTIDASE IV (EC3.4.14.5) 58 1.11 SmUCTURE-FUNCTION STUDIES OF G-PROTEIN COUPLED RECEPTORS (GPCR) 61 1.11.1 RECEPTOR BINDING DOMAINS 62 1.11.2 RECEPTOR-G PROTEIN COUPLING 64 1.11.3 RECEPTOR ACTIVATION AND CONSTITUTTVELY ACTIVE RECEPTORS 65 1.11.4 RECEPTOR PHOSPHORYLATION AND DESENSLTIZATION 69 1.11.5 RECEPTOR ENDOCYTOSIS 72 1.11.6 DETERMINANTS OF RECEPTOR SEQUESTERATION 75 1.11.7 SEQUESTRATION OF THE SECRETIN/GLUCAGON/ VTP RECEPTOR FAMILY 77 1.12 THESIS STUDIES: HYPOTHESES AND OBJECTIVES 80 CHAPTER 2 METHODS 84 2.1. ISOLATION AND CHARACTERIZATION OF A cDNA ENCODING THE RAT GIP RECEPTOR 84 2.1.1 RNA ISOLATION 84 2.1.2 REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION 86 2.1.3 DNA SEQUENCING A N D ELECTROPHORESIS 88 2.2 CELL CULTURE AND CELL TRANSFECTION 89 2.2.1 MAMALIAN CELL CULTURE 89 2.2.2 TRANSIENT TRANSACTIONS 89 2.2.3 STABLE TRANSFECTIONS 90 2.3 BINDING ANALYSIS 91 2.3.1 125I-GJP PREPARATION 91 2.3.2 TRANSIENT BINDING EXPERIMENTS 93 2.3.3 STABLE CELL-LINE BINDING STUDIES 93 2.3.4 BINDING ANALYSIS 95 2.4 MEASUREMENTS OF cAMP PRODUCTION 99 2.5 CYTOSOLIC C a 2 + MEASUREMENTS 100 2.6. RAT PANCREAS PERFUSIONS 101 2.7 SOURCES OF PEPTIDES 101 2.8 SITE-DIRECTED MUTAGENESIS OF THE RAT ISLET PANCREATIC GIP RECEPTOR 102 2.8.1 OLIGONUCLEOTIDE PHOSPHORYLATION 103 v 2.8.2 CONSTRUCTION A N D EXPRESSION OF H170R 103 2.8.3 CONSTRUCTION A N D EXPRESSION OF CHIMERIC RECEPTORS ... 107 2.8.4 U S E OF ENDOGENOUS RESTRICTION SITES 107 2.8.5 SINGLE STRANDED OLIGONUCLEOTTDE-DTRECTED MUTAGENESIS WITH STRAND SELECTION 108 2.8.6 P O L Y M E R A S E C H A I N REACTION MUTAGENESIS 111 2.8.7 GENERATION OF C A R B O X Y - T E R M I N A L T R U N C A T E D FORMS OF THE R A T GIP RECEPTOR 121 2.9 RECEPTOR DESENSLTIZATION STUDIES 125 C H A P T E R 3 RESULTS 126 3.1 ISOLATION A N D CHARACTERIZATION OF A cDNA ENCODING T H E R A T GIP RECEPTOR 126 3.2 CHARACTERIZATION OF GIP RECEPTOR BINDING 130 3.3 EFFECTS OF R A T ISLET GIP RECEPTOR EXPRESSION O N cAMP F O R M A T I O N 139 3.4 L O C A L I Z A T I O N OF THE CORE GIP BINDING REGION 144 3.5 E X A M I N A T I O N OF RESIDUES IN GIP IMPORTANT FOR RECEPTOR A C T I V A T I O N 156 3.6 GIP A N D GLP-1 RECEPTOR CHIMERAS 166 3.6.1 L I G A N D BINDING OF RECEPTOR CHIMERAS 168 3.6.2 C Y C L I C A M P RESPONSES OF RECEPTOR CHIMERAS 172 3.7 TRUNCATION OF THE C A R B O X Y - T E R M 1 N A L TAIL OF THE GIP RECEPTOR 178 3.7.1 EFFECT OF C A R B O X Y - T E R M T N A L TAIL T R U N C A T I O N O N L I G A N D BINDING 179 3.7.2 EFFECT OF C A R B O X Y - T E R M T N A L TAIL T R U N C A T I O N O N cAMP PRODUCTION 185 3.7.3 EFFECT OF C A R B O X Y - T E R M T N A L TAIL T R U N C A T I O N O N RECEPTOR U P T A K E A N D DESENSITTZATION 188 C H A P T E R 4 DISCUSSION 193 4.1 ISOLATION A N D CHARACTERIZATION OF R A T PANCREATIC ISLET GIP RECEPTOR cDNAs 193 4.1.1 THE R A T ISLET GTP RECEPTOR cDNA 193 4.1.2 CHARACTERIZATION OF GTP RECEPTOR BINDING A N D SIGNALING V I A c A M P 197 4.1.3 EFFECTS OF GTP O N I N T R A C E L L U L A R C A L C I U M 200 4.2 IDENTIFICATION OF THE CORE GTP BINDING REGION 205 vi 4.3 GIP 6-30AMIDE CONTAINS THE HIGH AFFINITY BINDING REGION OF GIP A N D IS A POTENT INHIBITOR OF GIP ACTION IN VITRO 207 4.4 E X A M I N A T I O N OF AMINO-TERMINAL RESIDUES OF GIP IMPORTANT FOR RECEPTOR ACTIVATION 213 4.5 L O C A L I Z A T I O N OF GIP RECEPTOR REGIONS IMPORTANT FOR L I G A N D BINDING 218 4.6 E X A M I N A T I O N OF THE C A R B O X Y - T E R M I N A L TAIL D O M A I N OF T H E GIP RECEPTOR 225 4.7 CONCLUSIONS A N D FUTURE STUDIES 235 REFERENCES: 244 Appendix A 280 vii LIST OF TABLES Table 1. Summary of Preliminary Binding Experiments 133 Table 2. Summary of Competition Binding Studies Comparing Different GIP Preparations 133 Table 3. The Effect of Different GIP Preparations on cAMP Accumulation in wtGIP-Rl Cells and Insulin Release from the Isolated Perfused Rat Pancreas 140 Table 4. Summary of GIP Fragment Binding and cAMP Studies 145 Table 5. Summary of Competitive Binding (IC50) and cAMP Responses (EC50 and % of maximal GIP cAMP production) with GIP analogs 166 Table 6. Binding of 1 2 5 I-GIP (Bmax and IC 5 0 ) (A) and 1 2 5 I-GLP-1 (B) to GIP/GLP-1 Receptor Chimeras in COS-7 and CHO-K1 Cells 172 Table 7. Cyclic A M P Responses to GIP and GLP-in COS-7 and CHO-K1 Cells Expressing Chimeric Receptors 177 Table 8. Summary of 1 2 5 I-GIP binding (Bmax and IC50) and cAMP Responses toGIP Summary of 1 2 5 I-GIP binding (Bmax and IC50) and cAMP Responses with GIP, with wtGIP-Rl and wtGIP-R8 CHO-K1 Cell Lines 178 Table 9. Summary of Binding Experiments with Carboxy-Terminal Tail Truncated Forms of the Rat GIP Receptor 183 Table 10. Summary of cAMP Experiments with Carboxy-Terminal Tail Truncated Forms of the Rat GIP Receptor 188 Table 11. Maximal Receptor Internalization and Slope Values for Receptor Uptake Over the Initial 10 Minutes 191 viii LIST OF FIGURES: Fig. 1. Alignment of GIP Amino Acid Sequences from Several Species 18 Fig. 2. Alignment of the Amino Acid Sequences of GIP with the Related Peptide Hormones Glucagon and Glucagon-Like Peptide-1 (7-36amide) 19 Fig. 3. Organization Of the Human GIP Gene and Duodenal mRNA 21 Fig. 4. Processing of Proglucagon in the Pancreas and Intestine 33 Fig. 5. The Ternary Complex Model (TCM) and Inverse Agonism 67 Fig. 6. Idealized Binding Curves for Saturation (A.) and Competitive (B.) Binding Studies to a Single Binding Site 98 Fig. 7. Double-stranded DNA Mutagenesis 106 Fig. 8. Construction of Chimeric GTP/GLP-1 Receptors Using Endogenous Restriction Sites 109 Fig. 9. Predicted Topography of Chimeric Receptors Constructed Using Endogenous Restriction Sites 110 Fig. 10. Single Stranded Oligonucleotide-directed Mutagenesis with Strand Selection 113 Fig. 11. Predicted Topography of Chimeric Receptors Constructed Using Single Stranded Oligonucleotide-directed Mutagenesis 114 Fig. 12. PCR Based Oligonucleotide-directed Mutagenesis 115 Fig. 13. Predicted Topography of Chimeric Receptors Constructed Using Polymerase Chain-reaction Oligonucleotide-directed Mutagenesis 116 Fig. 14. Primers Used In the Construction of CH-9 117 Fig. 15. PCR Based Strategy for the Construction of CH-9 119 Fig. 16. Predicted Topography of the Chimeric Receptor CH-9 120 Fig. 17. Primer Used In the Construction of Carboxy-Terminal Truncated GIP Receptors 121 Fig. 18. Carboxy-terminal Truncation of the Rat Islet GJP Receptor. 123 Fig. 19. Modified Carboxy-Terminal Tail forms of the Rat Islet GIP Receptor 124 Fig. 20. Amplification of a cDNA Encoding the Rat Islet GIP Receptor Using the Polymerase Chain Reaction 127 Fig. 21. Sequence Alignment Of The Rat Islet Pancreatic GIP Receptor cDNA Isolated Via RT-PCR (Top) to that of the Previously Published Sequence of Usdin et al. (1993) (Bottom) 128 Fig. 22. The Predicted Secondary Structure of the Rat Islet GIP Receptor 129 Fig. 23. Displacement of 125I-spGIP Binding From COS-7 Cells Transiently Expressing GTP-R1 131 Fig. 24. Saturation Binding Curve For Isolated wtGJP-Rl Cell Membranes (A) And Intact Cells(B) 135 Fig. 25. Displacement of 125I-spGIP Binding from wtGJP-Rl Cells 136 Fig. 26. Displacement of 125I-spGIP Binding from wtGJP-Rl Cells by Peptide Hormones of the Secretin/GlucagonATP Family 137 Fig. 27. Displacement of 125I-spGIP Binding from wtGEP-Rl Cells by the GLP-1 Receptor Agonist, Exendin (Ex)-4 and the Truncated Antagonist Form Ex (9-39) 138 ix Fig. 28. Stimulation of cAMP Formation in COS-7 Cells Transiently Expressing pGTP-Rl in Response to Secretin/GlucagonATP Related Peptides 141 Fig. 29. The Effect of Ex-4 and Ex (9-39) on spGIP-stimulated cAMP Formation in the Stable CHO-K1 Clone wtGIP-Rl (A), and GLP-1-stimulated cAMP Formation in Cells Stably Expressing the Rat GLP-1 Receptor (wtGLP-l-Rl) (B) 142 Fig. 30. Cyclic AMP Formation in wtGIP-Rl Cells in Response to 1 uM Secretin/GlucagonATP Related Peptides 143 Fig. 31. Displacement Of 125I-GIP By Different Truncated Forms Of shGIP 147 Fig. 32. Stimulation of cAMP Accumulation in wtGIP-Rl Cells by shGIP and Fragments ofshGIP 148 Fig. 33. Predicted Secondary Structure ofshGIP 149 Fig. 34. Displacement of 125I-GIP Binding to CHO-K1 Cells Expressing the Rat Islet GIP Receptor (wtGIP-Rl Cells) by Truncated Forms of GIP (A), and Stimulation of cAMP Production in wtGIP-Rl Cells by the Same Peptides (B) 152 Fig. 35. Inhibition of 1 nM GIP 1-42 Stimulated cAMP Production in wtGIP-Rl Cells 153 Fig. 36. A. Displacement of 125I-GIP From COS-7 Cells Expressing wtGIP-Rl and H170R cDNAs by shGIP B. 10 nM shGIP-stimulated cAMP Accumulation in COS-7 cells Transiently Expressing wtGIP-Rl and H170R Forms of the GIP Receptor 155 Fig. 37. Displacement of1 T-GIP Binding (A), and Stimulation of cAMP Production (B) by GIP 1-42, N-terminal Truncated (GIP 3-42), and Sequence Modified (Ala^Tyi^-GIP 1-42) Analogs 157 Fig. 38. GIP 3-42 and Ala^Tyi^-GIP Inhibit lnm GIP-stimulated cAMP Production 158 Fig. 39. Displacement of 125I-GIP Binding (A), and Stimulation of cAMP Production (B) by shGIP and D-Ala2-analogs 160 Fig. 40. Displacement of I-GLP-1 Binding (A), and Stimulation of cAMP Production (B) by GLP-1, and the D-Stereoisomer Substituted Analog, D-Ala2-GLP-1 161 Fig. 41. Comparison of spGIP 1-42 and D-Ala2-GIP 1-42 in the Isolated Perfused Rat Pancreas 163 Fig. 42. Comparison of GLP-1 and D-Ala2-GLP-1 in the Isolated Perfused Rat Pancreas 164 Fig. 43. Displacement of 125I-GIP Binding (A), and Stimulation of cAMP Production (B) by shGIP 1-42, and GIP l-30amide Analogs 165 Fig. 44. Predicted Topography of the Chimeric Receptors 167 Fig. 45. Displacement of 125I-GIP Binding by GIP in COS-7 (A) and CHO-K1 (B) Cells Expressing Wild Type or Chimeric Receptors 170 Fig. 46. Displacement of 125I-GLP-1 by GLP-1 in CHO-K1 Cells Expressing the wtGLP-1 Receptor 171 Fig. 47. Stimulation by GEP of cAMP Production in COS-7 (A), and CHO-K1 (B) Cells Expressing the Wild Type GIP Receptor or GIP/GLP-1 Chimeric Receptors 174 Fig. 48. Stimulation by GLP-1 of cAMP Production in COS-7 (A), and CHO-K1 (B) Cells Expressing the Wild Type GLP-1 or GIP/GLP-1 Chimeric Receptors 176 Fig. 49. Carboxy-terminal Truncation mutants of the Rat Islet GIP Receptor 181 Fig. 50. Displacement of 125I-GIP by GIP in CHO-K1 Cell-lines Stably Expressing Carboxy-Terminal truncated and wtGIP Receptors 182 x Fig. 51. Modified Carboxy-Terminal Tail forms of the Rat Islet GIP Receptor 184 Fig. 52. 10 nM GJJP-stimulated cAMP Production with Carboxy-Terminal Tail Truncated Forms of the GIP Receptor 186 Fig. 53. Stimulation by GD? of cAMP Production in Carboxy-Terminal Truncated (A), Deletion, and Alanine Substituted GIP Receptor Mutants (B) Expressed in CHO-K1 Cells 187 Fig. 54. The Effect of Carboxy-Terminal Tail Truncation of the Rat Islet GIP Receptor on Receptor Sequestration in CHO-K1 Cell Lines 190 Fig. 55. Lack of Desensitization of GIP-mediated cAMP Production in wtGIP-Rl Cells 192 Fig. 56. Alignment of the Amino Acid Sequences of the Rat GIP, Human GLP-1, and Human Glucagon Receptors 196 xi ABBREVIATIONS: Amino acid: Three letter Single letter code code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D Cysteine Cys C Glutamine Gin Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine He I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Try W Tyrosine Tyr Y Valine Val V Hormones and receptors*: G-protein-coupled receptors = GPCRs Gastric Inhibitory Polypeptide/Glucose dependent insulinotropic polypeptide = GIP Glucagon-Like peptide-1 = GLP-1 Parathyroid hormone = PTH Parathyroid related peptide = PTHrP Glucagon = GLU Gastrin releasing peptide - GRP Gonadotropin-releasing hormone = GnRH Neurokinin = NK Luteinizing hormone = L H Glicentin-related pancreatic peptide; GRPP Angiotensin II type 1A receptor (AT ] AR) Beta-adrenergic receptor = p-AR Alpha-adrenergic receptor = a-AR Muscarinic acetylcholine receptor = mAchR Growth hormone-releasing hormone = GRH Calcitonin = Cal Miscellaneous: Deoxyribonucleic acid = DNA Ribonucleic acid = RNA Cyclic-adenosine monophosphate = cAMP Phosphotidylinositol 4,5,-bisphosphate = PIP2 Phospholipase = PLC Protein kinase C = PKC cAMP-dependent protein kinase = PKA Guanosine triphosphate (GTP) binding proteins = G-proteins G-protein coupled receptor kinase = GRK P-adrenergic receptor kinase = pARK Non-insulin dependent diabetes = NIDDM Single nucleotide polymorphism = SNP High performance liquid chromatography = HPLC Matrix-assisted laser desorption/ionization-time of flight mass spectrometry - MALDI-TOF MS Carboxy-terminal = CT Amino-terminal = NT 3-phenyl propionic acid/Des-amino-tyrosine = Ppa Synthetic porcine = sp Synthetic human = sh Natural porcine = np Wild type = wt * receptors for hormones are designated by the hormones abbreviation followed byR xii ACKNOWLEDGEMENTS: There are numerous people whom I would like to thank and acknowledge for their contributions, guidance, and support over the last 5 years. I want to point out that I have been exceedingly lucky to carry out my studies with the people in the Department of Physiology at U.B.C. I can think of few places where students are given the scientific and personal encouragement, support, and opportunities that the Department as a whole provides. First of all I would like to thank Chris Mcintosh for all his support and guidance as my mentor. Chris's knowledge and enthusiasm for science have been inspirational and central to my decision to pursue a career in research. I can only hope to one-day learn some of the knowledge Chris possesses as a scientist. In addition to Chris, both Ray Pederson and Mike Wheeler have contributed both as collaborators and advisors in all of the work described in this thesis. More importantly to me, all three have become friends whom I respect and admire, not only as scientists, but also as individuals. I would also like to thank my advisory committee for all their help. Alison Buchan, Ross T. A. MacGillivray, and Ken Baimbridge have all provided valued advice along the way. In addition, Kenny Kwok was always ready to provide insight, cynicism, and help when requested. Tony Pearson was always a source of both entertainment and encouragement, which was greatly valued. The people in R.T.A. MacGillivray's lab deserve particular thanks. Along with Ross, Hung Vu, Bea Tarn and Jeff Hewitt were overly generous with their opinions, advice, and expertise over the last few years. Without them, this would have been a far more time consuming and definitely far less enjoyable experience. I would also like to thank all the support staff within the Department. Marie, John, Joe, Dave, Jack, Zaira, and Nancy perform jobs that all too often go unnoticed. The high degree of professionalism and efficiency was appreciated. Thanks to all the graduate students who have taken the time to socialize and, even in these times of extreme political correctness, have a beer or two. Margaret and Ray Pederson also deserve special mention for all their time and efforts in organizing many of these social functions and of course the infamous "Grad Retreat". Special thanks to Gord and Bryce for their companionship, advice, and total lack of respect. I would also like to thank my family for all their encouragement of my endeavors as a marine biologist. Unfortunately my degree is actually in Physiology. I do not know exactly where this misunderstanding occurred, however their support was omniscient and effective regardless. Special thanks to my parents, Mike and Mauraine, for all their support and encouragement. One could not wish for better parents. I would also like to thank a certain special person, Jeannine Ray, who stood beside me both during the work and writing of this thesis. I will not forget it. To anyone I have forgotten, I give my apologies. Richard Wayne Gelling 13/8/98 xiii 2 4 5 6 8 I 9 10 11 12 13 CHAPTER 1 INTRODUCTION 1.1 OVERVIEW Gastric inhibitory polypeptide/Glucose-dependent insulinotropic polypeptide (GIP) is a 42 amino acid hormone that is produced by the K-cells of the mammalian duodenal and jejunal mucosa, and is released in response to the ingestion of glucose, fat, and some amino acids (reviewed in Brown et al, 1989; Pederson, 1993). GIP was initially identified and isolated on the basis of its ability to inhibit gastric acid secretion (enterogastrone action) (Brown et al, 1970, 1989; Brown, 1971), and later was shown to be a potent stimulant of insulin secretion (incretin) in the presence of hyperglycemia (Dupre et al, 1973). GIP is now widely acknowledged as being one of two established incretins involved in the enteroinsular axis in man (Brown et al, 1989; Pederson, 1993; Fehmann et al, 1995) and other species (Brown et al, 1989; Pederson, 1993), the other being truncated glucagon-like peptide-1 [tGLP-1; GLP-1 (7-3 6amide) and GLP-1 (7-3 7)] (reviewed in Hoist 1994, 1996; Drucker 1998). The latter incretin is now being investigated as a potential therapeutic agent in the treatment of non-insulin dependent diabetes mellitus (NIDDM) (Nauck et al, 1989; Gutniak et al, 1992, 1996; Byrne and Goke, 1996; Todd etal, 1997; Nauck etal, 1997a). Early ligand binding studies identified both high and low affinity GTP binding sites in islet-derived P-cell lines and a gastric tumor cell line (HGT-1) (reviewed in Brown et al 1989; Pederson, 1993; Mcintosh et al, 1996). However, it was not clearly established that GIP receptors were expressed in pancreatic islets, although GIP was shown to increase both intracellular levels of cyclic (c)AMP (Amiranoff et al, 1984, 14 Gallwitz etal., 1993, Lu etal., 1993a) and Ca 2 + ([Ca2+]i) in a tumor cell line (HIT-T15) (Lu etal, 1993a) and isolated islets (Wahl etal, 1992). Usdin and colleagues (1993) recently isolated a complementary (c)DNA encoding a putative seven transmembrane receptor protein with a high degree of structural similarity to receptors in the VIP/secretin receptor family. Expression studies revealed that GIP was the only candidate peptide tested that elicited a high affinity cAMP response 2+ and increased [Ca ]i in reporter cell lines (Usdin et al, 1993). Interestingly, the presence of receptor mRNA transcripts was demonstrated in a number of extrapancreatic tissues by a reverse transcriptase polymerase chain reaction (RT-PCR)-based approach. This included the vasculature and brain, which had not previously been considered as GIP target tissues. Given this unexpected pattern of expression, and that the first partial cDNA was isolated from brain and the subsequent full length cDNA from a tumor P-cell line (RINm5F), it was initially questioned whether the cDNA encoded a pancreatic islet GIP receptor, or a closely related species. In order to answer this question we have used RT-PCR to isolate a GIP receptor cDNA from isolated rat pancreatic islet mRNA. Once isolated the receptor cDNA was functionally expressed in the monkey kidney (COS-7) and Chinese hamster ovarian (CHO-K1) cell lines, and used to study a number of factors concerning receptor binding and activation, and the signal transduction pathways involved. These studies included the following: 1. The receptor binding and adenylyl cyclase stimulating activity of different synthetic preparations of porcine and human GIP were examined. In addition, the mode of 15 action of GIP in increasing [Ca' ]i has been investigated. Furthermore, the stable GIP receptor expressing CHO-K1 cell-line (wtGIP-Rl) was used to investigate the ability of truncated forms of GIP to activate or antagonize receptor activation, allowing the determination of regions of the polypeptide important for receptor binding and/or activation. These studies are important given controversy surrounding the efficacies of different synthetic GIP preparations (Nauck et al, 1993a; Jia et al, 1995), and the potential therapeutic value of the incretins for the treatment of NTDDM. 2. Site directed mutagenesis was used to generate chimeric GIP/GLP-1 receptors to examine which regions of the two highly related receptors were involved in ligand binding, ligand discrimination, and receptor activation. 3. Site directed mutagenesis was also used to truncate the GIP receptor carboxy-terminal (CT) tail, to examine its contribution to receptor expression, ligand binding affinity, G-protein coupling, receptor desensitization, and receptor uptake. These studies identified a five amino acid segment of the GIP receptor CT-tail region that appeared to be critical for functional receptor expression. 1.2 DISCOVERY OF GIP The term enterogastrone was originally proposed by Kosaka and Lim (1930) to describe a blood borne gastric inhibitory chemical messenger released from the small intestine in response to fat. They showed that mucosal extracts, when administered in large doses (mg/Kg), inhibited meal and histamine stimulated acid secretion in dogs. 16 Later, two candidate intestinal hormones, secretin (Bayliss and Starling, 1902) identified on the basis of its ability to stimulate pancreatic secretion, and cholecystokinin (CCK) (Ivy and Oldberg, 1928) on its ability to stimulate gallbladder contraction, initially appeared to meet the requirements of an enterogastrone. Preparations of both secretin and CCK were shown to inhibit acid secretion in the denervated (Heidenhain) canine gastric pouch (Gillespie and Grossman, 1964). However early preparations of CCK were impure, and were demonstrated both to suppress histamine or gastrin stimulated acid secretion (Gillespie and Grossman, 1964; Brown and Magee, 1967) and stimulate acid secretion under fasting conditions (Magee and Nakamura, 1966). Brown and Pederson (1970) found that when 10% pure CCK-pancreozymin (CCK-PZ) was further purified to 40% the gastric inhibitory activity was reduced, while the stimulatory activity in the fasting state was enhanced. They reasoned that the gastric acid inhibitory activity was removed during further purification of CCK-PZ. Using the canine Bickel pouch as a bioassay Brown et al. (1969, 1970) purified the active substance from extracts of hog duodenal and jejunal mucosa and named it gastric inhibitory polypeptide, or GIP (Brown, 1971). 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Human Y A E G T F I S D Y S I A M D K Porcine Bovine Rat Mouse 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Human I H Q Q D F V N W L L A Q K G K Porcine R Bovine R Rat R Mouse R R 33 34 35 36 37 38 39 40 41 42 Human K N D W K H N I T Q Porcine S Bovine S I Rat L Mouse s Fig. 1. Alignment of GIP Amino Acid Sequences from Several Species. Only variations from the human sequence are shown. Sequences were obtained from: human (Moody et al, 1984), pig (Jornvall et al, 1981), cow (Carlquist et al, 1984), rat (Higashimoto et al, 1992), and mouse (Schieldrop etal, 1996). 1.2.1 GIP IS A MEMBER OF THE VrP/GLUCAGON/SECRETIN SUPERFAMILY The complete 42 amino acid sequence of porcine GIP was first reported by Brown and Dryburgh (1971) and later corrected by Jornvall et al (1981). Comparison of GIP sequences from a number of different species indicates a high degree of conservation (>90%) at the amino acid level (Fig. 1). The human sequence differs at two amino acid positions from the porcine and rat sequences, and three positions from the bovine and mouse sequences. The highly conserved nature of different GIP species suggests that GIP has an important regulatory role. The structure of the preproGD? gene (Inagaki et al, 18 1989) indicates that it belongs to the glucagon gene family, and may have arisen from a common ancestral gene (Bell, 1986; Campbell and Scanes, 1992). The peptide products share both sequence similarities and some biological activities, and some of the more closely related peptide members are shown in Fig 2. 1 10 20 30 40 GIP YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ Glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 •••***•**•* ••• ••*••** Fig. 2. Alignment of the Amino Acid Sequences of GEP with the Related Peptide Hormones Glucagon and Glucagon-Like Peptide-1 (7-36amide). Amino acids are presented as their single letter abbreviations. * = completely conserved, • = residues well conserved. Alignment was carried out using the PC Gene software package (IntelliGenetics, 1995). 1.2.2 GIP GENE STRUCTURE AND POSTTRANSLATIONAL PROCESSING The cDNAs encoding both rat (Higashimoto et al, 1992; Tseng et al, 1993) and human (Takeda etal, 1987) GIP, as well as their genes (Higashimoto and Liddle, 1993; Inagaki, et al, 1989) have been isolated. The human GIP gene has been mapped to the long arm of chromosome 17 (Inagaki, et al, 1989). Both human and rat genes consist of 6 exons separated by 5 introns. The human gene exon 1 encodes most of the 5' untranslated (UT) region of the mRNA; exon 2 encodes the distal 5'UT, the signal peptide and a small portion of the amino-terminal cryptic peptide; exon 3 encodes the distal cryptic peptide along with the majority of the mature GIP peptide; exons 4 and 5 encode the remainder of the mature peptide and the carboxy-terminal peptide; and exon 6 19 encodes the 3'-UT region of the mRNA (Fig 3). This organization is conserved in the rat gene and with the genes of other related peptides suggesting a common ancestral gene (Campbell and Scanes, 1992). While the rat gene gives rise to a 144 amino acid prepro-hormone (Higashimoto and Liddle, 1993), the human gene encodes a 153 amino acid precursor (Inagaki, et al, 1989). The differences in preprohormone size are due to an 8 amino acid deletion in the amino-terminal peptide, and a single amino acid deletion within the carboxy-terminal peptide in rat. Mature rat GIP is processed from the large precursor polypeptide by removal of a putative 21 amino acid signal peptide, and further cleavage resulting in the loss of the amino-terminal, and carboxy-terminal cryptic peptides, at single arginine residues 43 and 86, respectively. The human homolog is processed by removal of a 21 amino acid signal peptide, a 30 residue amino-cryptic peptide at position 50, and the carboxy-terminal cryptic peptide at residue 94. The GIP gene and its mRNA are depicted in Fig. 3. 20 signal peptide 5'UT ^ GIP 3'UT Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Fig. 3. Organization of the Human GIP Gene and Duodenal mRNA. Untranslated regions (UT) are represented as clear boxes, the signal peptide region is filled green, sequence encoding the mature peptide is fill red, and regions coding the pro-GIP amino- and carboxy-terminal peptides are blue. Adapted from Inagaki et al., 1989. 21 1.2.3 TISSUE DISTRIBUTION The distribution of GIP was originally shown to be limited to specific endocrine cells (K cells) of the duodenum and jejunum in humans (Polak et al, 1973; Buchan et al, 1978, 1982), but to extend to the ileum in rat and dog (Buchan et al, 1982). Similar distributions of GD? mRNA have been described in human (Inagaki, et al, 1989), and rat (Higashimoto et al, 1992; Tseng et al, 1993). A recent study also identified both GIP-like immunoreactivity and GIP mRNA within the submandibular salivary gland in rats (Tseng et al, 1995). GIP mRNA was localized via in situ hybridization to the ductal cells of the submandibular gland (Tseng et al. 1995). However, the exact identity of submandibular-GD? and any physiological roles it may play remain to be clarified. It is also not known if submandibular -GIP exists in humans. 1.2.4 GIP RELEASE GIP is released from the K-cells in response to nutrient ingestion. It is generally agreed that rR-GIP levels increase 5-6 fold following a mixed meal, however the absolute values measured vary from 12-92 pM during fasting to 35-235 pM after a meal (Alam and Buchanan, 1993). These discrepancies were most likely due to poor cross reactivity of antisera raised against natural porcine GB? for human and rat forms, and/or cross reactivity with as yet unidentified large molecular weight proteins (reviewed in Alam and Buchanan, 1993). The sparse distribution of GIP cells has made it difficult to characterize the factors and molecular pathways involved in regulating GIP secretion. Carbohydrates stimulate 22 GTP release, which is appropriate for its role as an insulinotropic polypeptide. In human (Cataland et al, 1974), dog (Pederson et al, 1975), and rat (Pederson et al, 1982) an oral glucose load increased IR-GIP levels. Ingestion of carbohydrates (glucose, galactose, and sucrose) appears to be necessary as intravenous glucose failed to increase serum IR-GIP levels. While the exact mechanism remains to be determined, the sodium-dependent active transport of monosaccharides is a requirement for the carbohydrate stimulation of GIP secretion (Morgan et al, 1979; Creuzfeldt and Ebert, 1977). Glucose has also been shown to stimulate GIP release in vitro from cultured isolated canine K cells (Kieffer et al, 1994) and from an intestinal cell line (Kieffer et al, 1995a), supporting the hypothesis that glucose affects K cells directly. Ingestion of fat has also been shown to be a potent stimulator of GIP release (Brown et al, 1975; Cleator and Gourlay, 1975; Falko et al, 1982). In man, fat is more potent and results in a more prolonged elevation of circulating IR-GIP levels than glucose (Morgan, 1996). However, as would be expected, the increase in IR-GIP in the absence of elevated glucose levels does not increase insulin secretion (Cleator and Gourlay, 1975; Falko et al, 1982). The chain length of the fatty acids (FA), as well as the degree of saturation, affects the ability of ingested fats to stimulate GIP release. Whereas longer chain (O'Dorisio et al, 1976; Ross and Shaffer, 1981) more highly saturated (Lardinois et al, 1988) FA are potent secretagogues, short and medium chain fatty acids do not stimulate IR-GIP release. The exact nature of the differences in potency is unclear, however it has been suggested that the selective esterification of only the long chain FA and their incorporation into chylomicrons may play a role (Kwasowski et al 1985). 23 It is unclear i f protein or protein-digestion products stimulate GIP release physiologically. While meat or meat extracts were shown not to increase IR-GIP levels (Cleator and Gourlay, 1975), Thomas et al (1976) showed that intraduodenal administration of a mixture of basic amino acids, but not a mixture of aromatic amino acids, resulted in IR-GIP release. Enhancement of sodium-dependent amino acid transport, by increasing N a + K + ATPase activity with corticosteroid or alloxan, increased IR-GIP release (Schulz et al, 1982), suggesting that in common with carbohydrates, active transport of amino acids is coupled to GIP release. GIP release appears to be inhibited by hyperinsulinaemia in both the rat (Bryer-Ash et al, 1994) and human (Takahashi et al, 1991). However some species differences may exist, as inhibition of GIP release was attenuated in the rat, but not in human under hyperinsulinaemic, hyperglycaemic conditions (Bryer-Ash et al, 1994; Takahashi et al, 1991). Regulation of GIP release by the autonomic nervous system is not well understood, with studies indicating that both sympathetic and parasympathetic nerves can stimulate, inhibit or have no effects on GIP release, depending on the experimental conditions (reviewed in Mcintosh, 1991). It has been demonstrated recently that both GIP and GLP-1 are metabolized by circulating dipeptidyl peptidase IV (DP IV) (Mentlein et al, 1993b), both in vivo (Kieffer et al, 1995b; Deacon et al, 1995) and in vitro (Mentlein et al, 1993b; Kieffer et al, 1995b; Pauly et al, 1996) to non-insulinotropic forms of GIP3-42 and GLP-1 (9-36amide) (Brown et al, 1981; Schmidt et al, 1986a; Suzuki et al, 1989; Gefel et al, 1990). Most GIP radioimmunoassays (RIAs) recognize carboxy-terminal epitopes and measure both biologically active and inactive peptide forms, suggesting that previously 24 reported GTP levels are overestimations of the biologically active hormone concentration. The use of RIAs with amino-terminal directed antisera is required to determine the biologically relevant levels of GTP that are reached in the basal and stimulated states. 1.2.5 REGULATION OF GTP GENE EXPRESSION Few studies have examined the regulation of GIP gene expression. Rat GTP mRNA levels in the small intestine and submandibular salivary gland have been shown to increase in response to glucose (Tseng et al, 1994, 1995; Higashimoto et al, 1995) and fat (Tseng et al, 1993; Higashimoto et al, 1995) administration, while only glucose appeared to increase tissue TR-GTP levels (Higashimoto et al, 1995). Increases in mRNA were greatest for long term fat administration (Higashimoto et al, 1995). Schieldrop et al (1996) recently reported that GIP mRNA expression in the intestinal tumor STC&.14 cell line increased 3 fold in response to increasing culture media glucose concentrations from 5 mM to 25 mM. Interestingly, while Higashimoto et al (1995) noted a decrease in both GTP mRNA and tissue TR-GTP levels in response to food deprivation, that was rapidly reversed (within 1 day) by reinstitution of feeding, Sharma et al, (1992) observed a 2 fold increase in GTP mRNA levels in response to food deprivation. It appears, as is the case with secretion, that GIP gene expression is regulated by dietary nutrient content. Given that both the rat and human 5' flanking regions contain a putative cAMP response element, and AP 1 and AP 2 sites (Higashimoto and Liddle, 1994; Inagaki et al, 1989), recognition sites for transcription factors that are regulated by cAMP/protein kinase (PK)A and PKC pathways, it seems likely that other factors are involved in the regulation of GTP gene expression. A very recent study has identified both a distal and proximal 25 promoter, the former containing a G A T A related sequence between positions -193 and -182 that promotes cell-line specific expression of a luciferase reporter construct (Boylan etal, 1997). 1.3 BIOLOGICAL EFFECTS 1.3.1 ENTEROGASTRONE ACTIVITIES OF GBP As stated above, GIP was first isolated on the basis of its gastric inhibitory actions in the denervated stomach (Brown et al, 1969, 1970; Brown, 1971). However, studies in both man (Maxwell et al, 1980) and dog (Soon-Shiong et al, 1979) showed that GIP had only week inhibitory effects on the innervated stomach. Yamagisihi and Debas (1980) reported that, while intra-duodenal administration of oleic acid completely inhibited acid secretion in response to a meal of liver extract, GBP infusion resulted in only 40% inhibition of the acid secretion. In a comparative study, Soon-Shiong et al. (1984) measured the inhibitory effects of GIP on pentagastrin stimulated acid secretion from both the denervated Heidenhain pouch, and the innervated stomach within the same animal. While GIP inhibited acid secretion from the denervated Heidenhain pouch by 80%, only 30% inhibition occurred in the innervated stomach. Further, it was observed that GIP had no effect on acid secretion in the Heidenhain pouch i f it was administered against a background of Bethanechol, suggesting that the enterogastrone effect of GIP might not be direct, but mediated via a second humoral agent under cholinergic control. Mcintosh et al. (1981a) demonstrated that GD? was able to stimulate immunoreactive-somatostatin (IR-SS) release from the isolated perfused rat stomach, and release of IR-SS was inhibited by vagal stimulation, or acetylcholine administration. This suggested that 26 the enterogastrone activity of GIP is mediated by stimulation of the release of neuroendocrine somatostatin from the D-cells of the stomach. Additional studies have indicated a role for the sympathetic nervous system in modulating this pathway (Mcintosh et al, 1981b). While GIP appears to display some of the characteristics of an enterogastrone, it is obviously not the only one, and it likely acts in concert with a number of neuronal and/or humoral agents to achieve the full acid inhibitory action observed in vivo. 1.3.2 INCRETINS A N D THE ENTEROINSULAR AXIS CONCEPT The possible existence of a substance released from the duodenum and capable of modulating carbohydrate disposal was first investigated, without success, by Bayliss and Starling (1902). Moore et al. (1906) proposed that the duodenum produced a "chemical excitant", the absence of which caused diabetes. Their treatment of diabetics with the acid extracts of hog duodenum, although initially promising, was ultimately unsuccessful. Studies using an intestinal extract free of secretin activity, which caused hypoglycemia in dogs (Zunz and L a Barre, 1929; La Barre and Still, 1930), led La Barre (reviewed in Brown et al., 1989) in 1932 to propose the term incretin to describe humoral activity from the gut that enhances glucose deposition. However later studies by Loew et al (1940) failed to demonstrate any effect of several different intestinal extracts on blood glucose levels, and interest in possible insulinotropic intestinal factors declined over the following years. It was the advent of the radioimmunoassay technique, allowing the direct monitoring of insulin levels, which led to renewed interest in the existence of a possible 27 incretin. As oral glucose was far more potent a stimulator of insulin release than intravenous glucose in humans (Elrick et al, 1964; Mclntyre et al, 1964), it was concluded that an additional stimulus for insulin release existed, and that it appeared to originate from the small intestine (Mclntyre et al, 1965). Unger and Eisentraut (1969) proposed the term "enteroinsular axis" to describe the hormonal link between the gut and the pancreatic islets. However, it was known that other factors such as neural connections and nutrients had direct effects on the insulin secreting cells of the islet. Creutzfeldt (1979) therefore suggested that the term "enteroinsular axis" be used to encompass nutrient, neural, and hormonal signals from the gut to all of the islet cells secreting insulin (p-cells), glucagon (a-cells), somatostatin (6-cells), and pancreatic polypeptide (PP-cells). The term 'incretin' should be used to refer more specifically to a hormone of the enteroinsular axis released in response to nutrients, particularly carbohydrates, that stimulates insulin secretion at physiological levels in the presence of glucose (Creuzfeldt, 1979). 1.3.3 EVIDENCE FOR GIP AS AN INCRETIN Evidence of GUP's incretin activity in the enteroinsular axis was first provided by Dupre and Beck (1966) when they demonstrated that impure preparations of CCK exhibited insulinotropic properties. In subsequent studies, Rabinovitch and Dupre (1972) showed that this insulinotropic activity was diminished as the purity of the CCK preparation increased, similar to the loss of gastric inhibitory activity observed by Brown and Pederson (1970). Dupre etal. (1973) demonstrated in man that a purified preparation of GIP, when infused in the presence of elevated glucose, stimulated insulin release. 28 Furthermore, it was shown that GIP had no insulinotropic properties in the euglycemic state, which suggested that GIP was only insulinotropic in the presence of elevated circulating glucose. GIP has since been shown to be insulinotropic in a number of species including human (Elahi et al, 1979) and dog (Pederson et al, 1975), and in vitro with the isolated perfused rat pancreas (Pederson and Brown, 1976, 1978), isolated islets (Siegal and Creutzfeldt, 1985; Shima et al, 1988), and p-cell lines (Kieffer et al, 1993; Lu etal, 1993a,b). 1.3.4 GIP EFFECTS O N OTHER ISLET C E L L TYPES GIP has also been shown to be a stimulator of glucagon release from the islet a-cell. Studies in the perfused rat pancreas (Pederson and Brown, 1978) showed that GIP stimulated glucagon release when glucose levels were below 5.5 mM, but responses were suppressed at higher glucose concentrations. In humans, GIP was unable to reverse either mild or moderate glucose-suppressed glucagon release (Elahi et al, 1979). In contrast GIP was able to reverse glucose-suppressed glucagon release at glucose levels as high as 11 m M in the mouse (Opara and Go, 1991), suggesting species differences may exist. It is unclear i f GIP stimulates somatostatin release from islet 8-cells in a physiologically relevant manner, as only weak stimulation in the perfused rat pancreas has been reported (Schmid etal, 1990). 1.3.5 E X T R A P A N C R E A T I C EFFECTS OF GIP The extrapancreatic effect of GIP on lipid metabolism has received the most attention in recent years (reviewed in Morgan, 1996). Treatment of rat adipocytes with 29 GTP in the nM range, increased glucose uptake both in the presence of insulin (Starich et al, 1985), and when administered alone (Hauner etal, 1988). The demonstration that 1-100 n M GTP also stimulated lipogenesis in adipocytes (Hauner et al, 1988), and rat adipose tissue explants (Oben etal, 1991) supported a positive regulatory role for GTP in de novo triglyceride synthesis. It was also shown that, while GTP alone displayed low lipolytic activity, high concentrations inhibited glucagon- and isoproterenol-stimulated lypolysis (Hauner et al, 1988; Dupre et al, 1976). There is also evidence that GTP can positively regulate lipid deposition from dietary sources. It was demonstrated that increasing dietary fat intake in rats resulted in increased basal GTP and insulin secretion, as well as increasing lipoprotein lipase (LPL) sensitivity to the two hormones (Morgan, 1996). GTP has also been shown to increase L P L activity in fat pad explants (Morgan, 1996) and mouse 3T3-L1 preadipocytes (Eckel et al, 1979), and to increase clearance of plasma trigycerides in the rat (Ebert et al, 1991). Another extrapancreatic effect of GTP that has been examined is the inhibition of glucagon-stimulated hepatic glucose production in rat and man (Hartmann et al, 1986; Elahi et al, 1986). Given that recent studies have failed to identify GTP receptor mRNA in liver, the mechanism by which this occurs is unknown, and it may be an indirect effect (Morgan, 1996). 1.4 E V I D E N C E FOR FURTHER INCRETIN(S) Initial studies suggested that GTP alone could account for the incretin response to glucose ingestion (Elahi et al, 1979). However, infusion of GTP antisera in rats was only shown to reduce, and not ablate, insulin responses to oral (Lauritsen et al, 1981) or 30 intraduodenal (Ebert and Creutzfeldt, 1982) glucose, or to intraduodenal acid accompanied by an intravenous glucose infusion (Ebert et al, 1979b). The degree of suppression (20%-50%) was dependent upon the antibody infusion protocol utilized. These studies suggested the existence of other incretin(s). 1.4.1 ISOLATION, PROHORMONE PROCESSING, A N D TISSUE DISTRIBUTION OF GLP-1 Unlike GIP which was isolated using traditional biochemical techniques, the existence of glucagon like peptides was not determined until the isolation of the proglucagon (PG) cDNA from anglerfish (Lund et al, 1982) and hamster (Bell et al, 1983). Lund and coworkers (1982) identified the glucagon sequence plus one 34 amino acid glucagon-like peptide (GLP) sequence in the carboxy-terminal of PG, while Bell et al (1983) identified two carboxy-terminal glucagon-like sequences in the hamster cDNA, which were referred to as GLP-1 and GLP-2 respectively. Based on data from the hamster cDNA and the human gene (Bell et al, 1983) it was predicted that the preprohormone corresponds to a 20 amino acid signal peptide and a 160 amino acid prohormone (PG1-160). GLP-1, unlike GIP, has a wide tissue distribution, and is found in L-cells of the small intestine (the majority within the ileum), colon and rectum, pancreatic oc-cells, and brain neurons (reviewed in Fehmann et al, 1995). The gene for PG1-160 is expressed in both intestine and pancreas (Novae et al, 1987), but the propeptide is differentially processed in the two tissues (reviewed in 0rskov, 1992 and Fehmann et al, 1995). In the islet a-cell, PG1-30 (glicentin-related pancreatic peptide; GRPP), glucagon (PG33-61), PG64-69 and PG72-158, the major pancreatic proglucagon 31 fragment (MPGF) corresponding to GLP-1, GLP-2 and an intervening peptide II (IP IT), are the main prohormone processing products (Fig. 4). This large unprocessed carboxy-terminal peptide can be further processed to small amounts of PG72-108 [GLP-l(l-37)] in the pig, and PG72-107 [GLP-1 (7-36amide)] in humans (Hoist et al, 1994). Intestinal glucagon cells or L-cells process PG1-160 to glicentin (PG1-69), oxyntomodulin (PG33-69), intervening peptide 2 (IP-2), GLP-1 (1-37) and GLP-2 (Fig. 4). GLP-1 (1-37) is cleaved after position 6 from the amino-terminus yielding GLP-1 (7-37), the majority of which is further processed and amidated to give GLP-1 (7-36amide) 1 (reviewed in 0rskov, 1992 and Fehmann et al, 1995). The latter, mature, peptide form accounts for 80% of the circulating immunoreactive GLP-l/ tGLP-1 in man (0rskov, 1992 and Fehmann et al, 1995). While it has been shown recently that GLP-2 influences hexose transport in intestinal basolateral membranes (Cheeseman and Tsang, 1996), and stimulates intestinal epithelial proliferation (Drucker et al, 1996), the biological relevance of many of the products of proglucagon (MPGF, oxyntomodulin, glicentin) remains unknown and requires further investigation. 1 For the sake of brevity GLP-1 will be used to refer to GLP-1 (7-36amide). Abbreviations for other forms and fragments will be noted in the text. 32 Proglucagon Glucagon GLP-1 GLP-2 16 30 33 61 72 Pancreas GRPP Glucagon MPGF 78 107 GLP-1 158 Intestine 69 78 107 Glicentin 126 158 GLP-1 30 33 69 GRPP Oxyntomodulin IP-2 GLP-2 111 122 Fig. 4. Processing of Proglucagon in the Pancreas and Intestine. Glucagon is the main peptide product with known biological activity resulting from prohormone processing in the islet oc-cell. Processing in the intestine gives rise to GLP-1 and GLP-2. The biological function of the other proglucagon fragments are unknown. Greater than 80% of the circulating GLP-1 levels in humans correspond to GLP-1 (7-37amide). Adapted from Fehmann et al. (1995). 3 3 1.4.2 REGULATION OF GLP-1 RELEASE The pathways involved in the regulation of GLP-1 secretion are unclear. It has been shown that oral ingestion of nutrients stimulates GLP-1 release even though the majority of L-cells exist within the ileum and are therefore unlikely to be directly exposed to high concentrations of nutrients. It has been suggested that GLP-1 secretion may be triggered by neuronal, as well as nutrient, stimuli (Roberge and Brubaker, 1993). Cholinergic stimulation of GLP-1 release has been demonstrated in isolated rat ileum (Reviewed in Fehmann et al, 1995) and, through extrapolation from pharmacological studies in the intestinal cell line STC-1 (Abello et al, 1994), this is thought to be mediated via activation of muscarinic (predominately M3) receptors. A hormonal feed-forward loop, in which nutrients in the duodenum cause release of GIP, which in turn stimulates GLP-1 release, has been postulated to be operational in rats (Roberge and Brubaker, 1993), however studies in humans have failed to show any effect of GIP on GLP-1 levels (Nauck et al, 1993a). Unlike GIP secretion in humans, GLP-1 release appears to be inhibited by hyperglycemia, but not hyperinsulinaemia (Takahashi et al, 1991). 1.4.3 GLP-1 ACTION ON INSULIN RELEASE The main biological action of GLP-1 is thought to be the potentiation of glucose stimulated insulin release. Initial studies found that high concentrations of GLP-1 (1-37), but not GLP-2, stimulated insulin release from perfused rat pancreas (Schmidt et al, 1985). The close sequence homology to GIP and glucagon led some investigators to 34 speculate that truncated GLP-1 would be more insulinotropic than GLP(l-37) (Crueutzfeldt et al, 1993). The potent insulinotropic activity of GLP-1 was first demonstrated in the isolated perfused pancreas from pig (Hoist et al, 1987) and rat (Mojsov et al. 1987). Numerous studies that followed demonstrated that the insulinotropic activity of GLP-1 was greatest in the presence of elevated glucose (reviewed in Creutzfeldt and Ebert, 1993). Of interest was the finding that the amidated and glycine extended forms, GLP-1 and GLP-1 (7-3 7) respectively, did not differ in their biological activity or apparent biological half-lives (Orskov et al, 1993), raising questions as to why GLP-1 (7-3 7) is further processed. The insulinotropic activity of GLP-1 has been confirmed in a number of 0-cell lines (Lu et al, 1993a; Montrose-Rafizadehetal, 1994), isolated islets (D'Alessio etal, 1989; Siegel etal, 1992; Suzuki etal, 1992), and in humans (Kreymann etal, 1987; Nauck etal, 1993a,b). 1.4.4 GLP-1 EFFECTS O N OTHER ISLET C E L L TYPES GLP-1 has been shown to inhibit pancreatic glucagon secretion, and this could enhance the hormone's glucose lowering effects. A potent glucoganostatic action has been demonstrated in vivo in humans (Nauck et al., 1993a; Schirra et al., 1998), and in vitro in the isolated perfused rat (Matsuyama, et al, 1988) and dog (Kawai et al, 1989) pancreas, and isolated human islets (Fehman et al, 1995). GLP-1 has also been shown to be a potent stimulator of somatostatin release from islet 5-cells. In both the perfused rat and dog pancreas GLP-1 is somatostatinotropic (Komatsu, et al, 1989; Kawai et al, 1989) and, additionally, GLP-1 has been demonstrated to stimulate somatostatin release in isolated human islets (Fehmann et al., 1995). Therefore it is unclear i f the inhibitory 35 action of GLP-1 on glucagon secretion is mediated directly on the a-cell, or via the release of islet somatostatin, which then inhibits glucagon release. However recent studies have demonstrated, using autoradiography with 1 2 5 I-GLP-1 (Heller and Aponte, 1995), immunocytochemistry, and single cell RT-PCR, the presence and expression of GLP-1 receptors on a subpopulation (-20%) of dispersed rat a-cells (Heller et al, 1997). This is in contrast to an earlier study, in which Moens et al (1996), failed to detect GLP-1 receptors or receptor mRNA in fluorescence-activated cell sorted (FACS)-sorted rat ct-cells, using Western and Northern analysis. Interestingly, Heller and Aponte (1995) have identified releasable IR-GLP-1 within some a-cells, the secretion of which is inhibited by high glucose levels, suggesting that GLP-1 may play an intra-islet regulatory role when lower glucose levels exist. 1.4.5 CONTRIBUTION OF GIP A N D GLP-1 TO THE INCRETIN EFFECT Although GIP and GLP-1 are widely considered to be the two most important incretins (reviewed in Brown et al, 1989; Pederson, 1993; Hoist, 1994), it is presently unclear as to their relative contribution to the overall incretin response to a meal. As mentioned above, infusion of GIP antisera in rats reduced insulin responses by 20-50%, depending upon the antibody infusion protocol utilized. Subsequently, it was reported that administration of the GLP-1 receptor antagonist exendin (9-39) to rats resulted in an estimated 60% inhibition of insulin responses to intraduodenal glucose (Kolligs et al, 1995) and 48% inhibition of responses to a meal (Wang et al, 1995b). Since immunoneutralization suffers from the drawback of limited accessibility of antibody to 36 the target cells, it is clearly important to develop a OTP antagonist to enable similar in vivo investigations on the role of this peptide. While the investigations described herein were in progress, a OTP fragment, OTP7-30amide, was shown to inhibit GIP-stimulated cAMP production in vitro and, at very high concentrations, to blunt GIP stimulated insulin secretion by approximately 75% in both anaesthetized and conscious rats (Tseng et al., 1996a). Importantly, while GIP7-36amide did not affect GLP-1-stimulated insulin release in the anesthetized rat, there was a 75% decrease in post-prandial insulin levels in antagonist treated rats, although no significant change in glucose handling was observed. Since similar reductions in post-prandial insulin release were observed with the GLP-1 specific antagonist exendin (9-39) in conscious rats (Wang et al., 1995), it seems that the two incretins together can account totally for the potentiation of insulin release resulting from the ingestion of nutrients. Given the conflicting results, it is still unclear which incretin, i f either, is the more important, or if they are redundant systems that co-evolved to ensure blood glucose levels are well maintained. There has been controversy concerning the relative potency of OTP from different animal species. The majority of studies using porcine GIP have shown it to be potently insulinotropic in rats (Pederson and Brown, 1976), humans (Elahi et al., 1979), and other species (reviewed in Brown etal., 1989; Pederson, 1993). However, while earlier studies showed that synthetic human (sh) GIP increased circulating insulin to levels similar to those observed with the natural porcine (np) GIP peptide (Nauck et al., 1989), poor responses to shGIP were subsequently reported in both normal controls and individuals with NTDDM (Nauck et al., 1993a), although these investigators did not comment on this 37 discrepancy. In vitro results suggesting that GLP-1 was insulinotropic at much lower peptide concentrations in rat islets (Siegel et al, 1992) and the perfused rat pancreas (Shima et al, 1988) led Jia et al. (1995) to examine a number of different peptide preparations in the isolated perfused rat pancreas. They observed a greatly reduced insulin response to a synthetic human preparation when compared to np and synthetic porcine (sp) GIP preparations, and suggested that different responses previously reported may in fact have been due to differences in the quality of the synthetic preparations, since the sequence differences between the peptides were relatively minor. Furthermore they found similar glucose and concentration thresholds (-20 pM) for spGTP and GLP-1 (Jia et al, 1995). In a recent study it was found that both GIP and GLP-1 levels correlate with plasma insulin levels throughout the day, strongly supporting their incretin roles in glucose homeostasis (0rskov et al, 1996). However the exact overall and temporal contributions of these two incretins requires further examination. 1.5 GIP BINDING SITES There have been relatively few studies on receptors for GJP, mainly due to the problem of producing biologically active 1 2 5I-GJP peptide and the apparent low expression of such receptors. The presence of GJP receptors was first demonstrated in transplantable hamster insulinoma cells (Maletti et al, 1984) and the insulin secreting hamster 0-cell line, In 111 (Amiranoff et al, 1984, 1985). GIP receptors have since been described on a number of other neoplastic cells, including human insulinomas (Maletti, et al, 1987) and p-TC3 cells (Kieffer et al, 1993). These early studies described high affinity binding sites, ranging from 0.2-7 nM, and most did not detect any eross reactivity 38 in binding with other members of the glucagon/secretin peptide superfamily, although Kieffer etal. (1993) observed that 1 u M glucagon displaced 20% of the 1 2 5I-GIP binding to p-TC3 cells. Several studies identified both high and low affinity binding sites (Amiranoff etal, 1984, 1985; Maletti etal, 1984, 1987) using Scatchard analysis. As a result of chemical (Couvineau et al, 1984), or ultraviolet irradiation (Amiranoff et al, 1986) cross-linking experiments, a 64 kDa 1 2 5I-GIP-labelled protein was identified in membranes from 6-cell tumors, suggesting a molecular weight of 59 kDa for the receptor. Treatment of these membranes, with the reducing agent dithiothreitol, decreased the receptors electrophoretic mobility indicating that the receptor contained intrachain disulfide bond(s). However these linkages did not appear necessary for binding of 1 2 5I-GIP, as pretreatment of membranes did not interfere with labeling of the receptor (Amiranoff et al, 1986). The GTP receptor was also shown to be a glycoprotein containing N-acetylglucosamine, mannose and possibly sialic acid (Amiranoff etal, 1986). 1.5.1 ISOLATION OF THE GIP RECEPTOR cDNA A N D GENE In 1993 Usdin et al. isolated a partial cDNA from rat cerebral cortex, and a full length c D N A from a rat tumor cell line (RTNm5F), encoding a putative seven transmembrane G-protein coupled receptor protein of 455 amino acids, with a high degree of similarity (25-45% identity) to other receptors in the VTP/glucagon/secretin receptor family. Receptors within this sub-family display little sequence identity to other G-protein receptors, such as the P-adrenergic receptors, have large amino-terminal extracellular domains and, as with the VTP/glucagon/secretin superfamily, appear to share 39 a common ancestral gene (Fehmann et al, 1995; Ulrich et al., 1998). Analysis of the predicted amino acid sequence indicated that the receptor shared the greatest identity with the glucagon (44%) and GLP-1 (40%) receptors (Usdin et al, 1993). The region of greatest divergence between these receptors was in the carboxy-terminal tail region, while the amino terminus-extracellular domain was well conserved, with 35% and 39% identity to the GLP-1 and glucagon receptors respectively. The receptor had a predicted molecular weight of 50,063 assuming cleavage of a putative 18 amino acid signal peptide. The presence of three potential glycosylation sites in the amino-terminal region may account for the discrepancy in size observed (~59 kDa) in previous cross-linking experiments (Couvineau et al, 1984; Amiranoff et al, 1986). Expression studies revealed that GIP was the only candidate peptide tested that elicited a high affinity cAMP 2+ response and increased intracellular calcium ([Ca ]i) in reporter cell lines (Usdin et al, 1993). One of the objectives of work described in this Thesis was to isolate and determine if the cDNA isolated from RTN cells (Usdin et al, 1993) was also expressed in primary rat islets, to allow a more complete characterization of the receptor than has been possible with primary tissues and cell lines. 1.5.2 GIP RECEPTOR GENE EXPRESSION A N D TISSUE DISTRIBUTION Studies using Northern blot analysis, RT-PCR, and in situ hybridization in the rat have shown there is widespread tissue distribution of the GIP receptor mRNA, with significant levels in pancreas, stomach, intestine, adipose tissue, adrenal cortex, heart, lung and endothelium of major blood vessels, but not in the spleen or liver (Usdin et al, 40 1993; Yasuda et al., 1994). Several of these regions have not been considered as target 't tissues for GTP suggesting as yet unknown functions exist. Most surprising was the widespread distribution of the GIP receptor mRNA found in the brain (Usdin et al., 1993). In situ analysis with GIP receptor specific oligonucleotides identified mRNA in the telencephalon, diencephalon, brain stem, cerebellum and pituitary (Usdin et al., 1993). Autoradiographic studies using 1 2 5I-GD? for the most part supported the regional localization of GIP mRNA in the brain (Kaplan and Vigna, 1994). For example, binding was observed in several areas of the telencephalon (motor, somatosensory, and auditory), forebrain (anterior olfactory nucleus, lateral septal nucleus, subiculum) and midbrain regions (inferior colliculus). In contrast, although GD? mRNA was detected in the pituitary by RT-PCR (Usdin et al., 1993), and GIP was shown to affect anterior pituitary release of follicle-stimulating hormone (TSIT) and growth hormone (GIT) in ovarectomized rats (Ottlecz et al., 1985), no binding sites were observed in either the pituitary or hypothalamus (Kaplan and Vigna, 1994). The distribution in regions of the brain which are inaccessible to blood borne peptides is especially confusing, in that neither GD? mRNA (Higasimoto et al., 1992; Tseng et al., 1993) nor IR-GD? (Buchan et al., 1982) has been demonstrated in the brain. It is possible that the GIP mRNA is too rare too detect, or a homologous brain peptide exists. Preliminary studies by Usdin et al. (1993) on E12, E17 and E19 day rat embryos did not detect any GD? mRNA in the central nervous system of rats, suggesting that the peptide is not expressed during early development. It may also be that the receptor is expressed in the brain but does not serve a function. 41 Identification of the GIP receptor mRNA in the pancreas, stomach, and adipose tissue supports a direct action of the peptide on these tissues as discussed in section 1.3. The expression of GIP, GLP-1, and glucagon receptors has been examined in FACS purified a- and p-cells, and non-p-cell populations (Moens et al, 1996). Northern blot analysis indicated that all three receptors were expressed at high levels in purified P-cells (>90% pure), whereas only the GIP receptor appeared to be expressed at significant levels in the non-P-cell fraction (>80% a-cells, < 10% p-cells). Comparison of total RNA from a transplantable insulinoma (MSL-G2-IN) and co-derived glucagonoma (MSL-GAN) supported the selective expression of the GIP receptor mRNA on glucagon producing cells (Moens et al, 1996). The lack, or low level expression, of the GLP-1 receptor on the alpha cell was confirmed with Western blots using a GLP-1 receptor specific antibody. These findings support a direct effect of GIP on the a-cell, as was suggested by studies in the perfused rat pancreas (Pederson and Brown, 1978), and strongly support its role in direct regulation of islet cell function. Surprisingly, apart from the localization of the GIP receptor to islet cells of humans (Gremlich et al, 1995), the only other studies to date on receptor expression in other tissues are those on the "ectopic" expression in adrenal cells from a group of patients with Cushing's syndrome (Lacroix et al, 1992; Reznik et al, 1992) and adrenal hyperplasia. Given that Usdin et al. (1993) identified GIP receptor mRNA in the rat adrenal, the food dependent hyper-GIP sensitivity and resulting Cortisol hypersecretion, may be due to overexpression of the GIP receptor or a defect in the pathway normally regulating GIP sensitivity, rather than aberrant expression. 42 1.6 GLP-1 RECEPTOR BINDING STUDIES As with the receptors for GD?, those for GLP-1 were first demonstrated on B-cell lines (Goke and Conlon, 1988) and later, on a number of insulinoma cell lines (reviewed in Fehmann et al, 1995). In agreement with the somatostatinotropic activity of GLP-1, receptors have also been identified on somatostatin secreting cells (Fehmann and Habener, 1991). However, it is controversial as to whether they exist universally on a-cells, or a-cell lines (Matsumara et al, 1992; Fehmann and Habener, 1991; Moens et al, 1996; Heller et al, 1997). Studies with intact cells or cell membranes from insulinoma cell lines displayed a single class of high affinity binding sites, with K d values ranging from 0.2-3.5 n M (reviewed in Fehmann et al, 1995). In similar cross-linking studies to those carried out with GIP, GLP-1 was associated with a single protein of an apparent molecular wt of 63 kDa. Unlike the GD? receptor, binding of 1 2 5 I-GLP-1 was partially displaced by glucagon and the related P G peptide oxyntomodulin. Complete displacement of 1 2 5 I-GLP-1 was obtained with GLP-1 (1-37), but with a significant shift to the right in receptor affinity (Goke and Conlon, 1988). These findings suggest that early observations of insulinotropic effects with high concentrations of GLP-1 (1-37) and oxyntomodulin were due to interaction with the GLP-1 receptor (Thorens and Widmann, 1996). No other related peptides (GIP, secretin, VTP) were shown to compete for GLP-1 binding (Fehmann et al, 1995). 1.6.1 ISOLATION OF THE GLP-1 RECEPTOR cDNA A N D G E N E The GLP-1 receptor cDNA was isolated from a rat islet cDNA library by Thorens (1992) using an expression cloning strategy. Other groups soon followed with the 43 isolation of cDNAs from human islets (Dillon etal, 1993; Thorens et al, 1993), a human gastric tumor cell line (HGT) (Graziano et al, 1993), rat lung (Lankat-Buttgereit et al, 1994), and rat brain (Alvarez et al, 1996). The human GLP-1 receptor gene has been mapped to the long arm of chromosome 6 (Stoffel et al, 1993). Both the rat and human cDNA sequences encode for 463 amino acid receptor proteins that are highly conserved at both the amino acid (-91%) and nucleotide level (87%) (Dillon et al, 1993; Thorens et al, 1993; Graziano etal, 1993). 1.6.2 GLP-1 RECEPTOR GENE EXPRESSION A N D TISSUE DISTRIBUTION The presence of GLP-1 receptor mRNA has been demonstrated in lung, kidney, pancreas, and brain tissues in mouse (Campos et al, 1994), rat (Thorens, 1992; Bullock et al, 1996; Alvarez et al, 1995), and human (Wei and Mojsov, 1995). Furthermore, Wei and Mojsov (1996) have cloned and sequenced GLP-1 receptor cDNA's from brain and heart, and determined that they encode receptors with amino acid sequences identical to the pancreatic receptor. Two groups, using Northern blot analysis and RT-PCR, reported the presence of GLP-1 mRNA in liver from mouse and rat (Wheeler et al, 1993; Campos et al, 1994), and rat skeletal muscle (Wheeler et al, 1993), while Egan and coworkers (1994) identified GLP-1 mRNA via RT-PCR in 3T3-L1 adipocytes. In contrast, two extensive studies using R N A protection assays (Wei and Mojsov, 1995; Bullock et al, 1996), RT-PCR, and in situ hybridization (Bullock et al, 1996) failed to demonstrate GLP-1 mRNA in adipose tissue, liver or skeletal muscle, suggesting that the extrapancreatic actions of GLP-1 in these tissues may be mediated via indirect actions, or another GLP-1 receptor. The latter of these two possibilities seems more likely given that 44 specific I-GLP-1 binding has been demonstrated in membrane preparations from adipose tissue (Valverde et al, 1993) and skeletal muscle (Delgado et al, 1994). Interestingly, functional GLP-1 receptors and GLP-1 receptor mRNA have been demonstrated in several thyroid tumor derived cell lines (Vertongen et al, 1994; Lamari et al, 1996; Beak et al, 1996), suggesting that GLP-1 may play a role either in the regulation of calcitonin secretion from thyroid C-cells, or as a feedback regulator of thyrotropin release from the anterior pituitary. There is also evidence that GLP-1 receptors are involved in the regulation of exocrine secretion. 1 2 5 I-GLP-1 binding and Northern blot studies on highly purified parietal cells (Schmidtler et al, 1994), and in situ hybridization studies (Bullock et al, 1996), have demonstrated that parietal cells express the GLP-1 receptor. In situ hybridization studies also identified GLP-1 receptor mRNA within the crypts of the duodenum, and in large nucleated cells within the aveoli of the lung (Bullock et al, 1996), the latter being consistent with stimulation of mucus secretion from submucosal glands in the trachea (Richter et al, 1993). While it is widely agreed that GLP-1 receptors exist on B-cells (see above references) and 8-cells (Heller and Aponte, 1995 and references therein), it still remains to be determined conclusively whether GLP-1 receptors exist on all islet a-cells or just a subpopulation. Several attempts have been made to study the intra-islet distribution of GLP-1 receptor mRNA. Bullock et al. (1996) observed colocalization of insulin and GLP-1 receptor mRNA, but could not determine cellular specificity. As mentioned in section 1.4.4 studies with FACS isolated islet cells suggested that the GLP-1 receptor was present on 3-cells, but not a-cells (Moens et al, 1996). In more recent studies Heller and 45 coworkers (1997), using double staining of dispersed islet cells with a GLP-1 receptor and islet hormone specific antibodies, identified the GLP-1 receptor on 90% of insulin positive cells, 76% of somatostatin positive cells and 20% of the glucagon positive cells. Using single cell RT-PCR they further demonstrated the presence of GLP-1 receptor mRNA and the P G mRNA within 2 out of 10 P G mRNA positive cells. In contrast, Ding et al. (1997) found that both GIP and GLP-1 stimulated glucagon secretion in FACS sorted a-cells and the majority of cells responded to GLP-1, suggesting that most, i f not all, a-cells have GLP-1 receptors. 1.7 RECEPTOR SIGNAL-TRANSDUCTION P A T H W A Y S 1.7.1 GIP RECEPTOR SIGNAL-TRANSDUCTION MECHANISMS GIP has been shown to stimulate adenylyl cyclase in pancreatic tumor cell lines (Amiranoff et al. 1984, Lu et al, 1993a, Malleti et al, 1987), a gastric cancer cell line (HGT-1) (Gespach etal, 1984), isolated islets (Siegel and Creutzfeldt, 1985), as well as FACS sorted a- and P-cells (Moens et al, 1996), with half-maximal (EC50) values ranging from -200 p M (Moens et al, 1996) to 30 nM (Amiranoff et al, 1984). Although stimulation of adenylyl cyclase has been considered to be the primary mode of action for GIP, it has also been shown to increase influx of extracellular C a 2 + into mouse islets (Wahl et al. 1992), and to increase [Ca 2 +]i levels in HIT-T15 cells (Lu et al, 1993a). Influx of extracellular C a 2 + via voltage-dependent channels (VDCC) appears to be the source of the increased [Ca 2 +]i in HIT T15 cells, as it was blocked by addition of E G T A or the voltage dependent L-type channel antagonist, nimodipine (Lu et al, 1993a). However, there was no evidence that GIP increased inositol-1,4,5-46 trisphosphate (IP3) levels in HIT T15 cells, suggesting that phospholipase (PLC) mobilization of intracellular C a 2 + stores was not involved. In contrast Straub and Sharp (1996) reported that the phosphatidylinositol (D?) 3-kinase inhibitor, wortmannin severely decreased GD? mediated insulin release from HIT T15 cells; an observation they also made with the related peptides VIP and P A C A P (Straub and Sharp, 1996). More recently Ding and Gromada (1997), examined the effect of GIP on voltage clamped individual mouse P-cells, using membrane capacitance as a measure of insulin secretion, whole-cell calcium currents to measure C a 2 + influx, and [Ca 2 +]i via fiira-2 florescence. GIP appeared to stimulate insulin secretion with only a small increase in [Ca 2 +]i (30%), without affecting whole cell calcium currents. The increase in capacitance could be blocked by the protein kinase A (PKA) inhibitor Rp-8-bromo-cAMP, however the authors failed to determine if the [Ca 2 +]i increase from internal stores was blocked in these experiments. It was concluded that GIP increased insulin secretion by the c A M P / P K A pathway at a point distal to the increase in intracellular Ca 2 + . The same authors have also shown that GTP stimulation of a-cells resulted in increased whole cell calcium currents that were at least in part mediated by the cAMP pathway (Ding et al., 1997), suggesting that GD? receptor signaling may differ in different cellular environments. A recent study showed GIP stimulated M A P kinase activity in both a cAMP-dependent and independent manner in CHO-K1 cells stably expressing the human GD? receptor (Kubota et al., 1997). The possible contributions of this and other possible pathways, such as phospolipase A2 (Lardinois et al., 1990), and differences between pathways activated in different target tissues or cell types needs to be further explored. 47 1.7.2 GLP-1 RECEPTOR SIGNAL TRANSDUCTION While some studies on the intracellular signaling of the GJP receptor have been carried-out, there is a much larger body of work examining GLP-1 receptor signal transduction. Given that the receptors share a great deal of identity, and at the physiological level appear to share a similar function, it is likely that they act though similar signaling pathways. However, the receptors differ in their peripheral effects, which may suggest that either different receptor species or isoforms exist, or that the receptors couple differentially in different cell types. It is therefore important not only to study and identify the similarities between GIP and GLP-1 receptor signaling, but also to identify differences that may exist in different cell types. In common with GTP, GLP-1 activation of its endogenous receptor stimulates cAMP production. This has been demonstrated in P-cell lines (Drucker et al, 1987; L u etal, 1993a; Widmann etal, 1994), isolated P-cells (Moens etal, 1996), isolated islets (Ahren et al, 1996) and cell lines transiently (COS-7) or stably expressing (CHL and CHO-K1 cells) expressing GLP-1 receptor cDNA s (Thorens, 1993; Dillon et al, 1993; Wheeler et al, 1993). There was good agreement between the EC50 values obtained in the different cellular systems (-0.5-3 nM) (Fehmann et al., 1995). Interestingly, L u et al (1993 a) noted that both GIP and GLP-1 increased cAMP levels in the presence of 4 m M but not 0.4 m M glucose in HIT T15 cells, suggesting there was some glucose-dependence in incretin mediated cAMP production. However, studies in a number of cell lines (INS-1, RTN 1027-B2 and 1056A) did not demonstrate the same concentration dependence, and a glucose-dependence for incretin-stimulated cAMP generation in islet cells has not been demonstrated (Widmann et al., 1994). 48 Discrepancies exist in the literature concerning the effect of GLP-1 on [Ca ]i in R-cell lines. There have been reports that GLP-1 induces large (Wheeler et al, 1993; Holz et al, 1995) or small (Ahren et al, 1996) increases in [Ca 2 +]i in HIT-T15 cells, small (Ahren et al, 1996) or no increases (Goke et al; 1989) in RJJSTmF5 cells, and large increases in bothPTC-3 (Gromada and Rorsman, 1996) and P-TC6 cell lines (Holz et al, 1995). Widmann and coworkers (1994) observed no increases in [Ca 2 +]i levels in any of the three cell lines (INS-1, RTN 1027-B2 and 1056A) they examined. It is hard to account for all these different observations, often using the same cell lines. However, many of these cell lines are pluripotent, and undergo differentiation or dedifferentiation depending upon the tissue culture conditions (Polak et al, 1993). Additionally, it has been reported that GLP-1 mediated increases in [Ca 2 +]i may be small and relatively insignificant in dispersed islets and FACS sorted P-cells (Fridolf and Ahren, 1993; Ahren et al, 1996; Ding and Gromada, 1997), suggesting that cell-cell interaction in the islet may be important. One consistent observation in the above studies was that P-cell lines responding to GLP-1 with increased [Ca 2 +]i, displayed a glucose-dependence to the response (Wheeler et al, 1993; Holz et al, 1995; Gromada et al, 1996), although the glucose threshold was lower than that seen in P-cells (Cullinan et al, 1994). Most groups have reported significant changes in [Ca 2 +]i in response to GLP-1 in the presence of elevated glucose levels (Yada et al, 1993; Cullinan et al, 1994; Holz et al, 1993, 1995). In contrast, there is little agreement as to the source of the iCa 2 + . Several studies have demonstrated that the effects of GLP-1 on C a 2 + can be mimicked with adenylyl cyclase activators such as forskolin, or membrane permeable cAMP analogs 8-bromo (Br)-cAMP and Sp-49 adenosine-3'5'-monophospothionate triethylamine (sp-cAMP) (Yada et al, 1993; Cullinan et al, 1994; Holz et al, 1995), or they can be blocked by protein kinase A inhibitors such as rp-cAMP or H-89, suggesting that the c A M P / P K A pathway is involved (Gromada et al, 1995; Kato et al, 1996). The removal of extracellular C a 2 + from the medium or addition of V D C C blockers often results in partial attenuation (Gromada et al 1995; Holz et al, 1995), complete blocking (Yada et al, 1993; Cullinan et al, 1994; Fridolf and Ahrens, 1993) or no effect (Wheeler et al, 1993) on GLP-1-dependent C a 2 + responses in the different cellular systems, strongly supporting the involvement of influx from extracellular sources that may or may not be via V D C C . Recent studies have shown that GLP-1 slows calcium channel inactivation in a cAMP-dependent manner manner (Britsch et al, 1995; Kato et al, 1996), while other studies have identified a N a + -dependence of the calcium influx, and a role for voltage-independent non specific cation channels (VTNCCs) (Holz etal, 1993; Fridolf and Ahrens, 1993; Kato etal, 1996). It is not clear i f this is a c A M P / P K A or a G-protein mediated effect on the VINCC itself (Fridolf and Ahrens, 1993; Holz et al, 1993). Ding and Gromada (1997) have recently shown that neither GIP nor GLP-1 increase whole cell calcium currents in FACS sorted cells, suggesting that the small increases observed came from intracellular stores. The same authors have suggested that GLP-1 activation of the c A M P / P K A pathway may result in phosphorylation and sensitization of the ryanodine and/or JP3 sensitive receptors to extracellular calcium mobilized by GLP-1 (Ding and Gromada, 1997; Gromada et al, 1996). Many of the conflicting results are probably due to the different systems • • • 2+ • employed, and a description of the mechanisms by which GLP-1 increases iCa remains 50 to be finalized. It is also likely that both GLP-1 and GIP activate alternative signal transduction pathways in different cell types and in different physiological environments. 1.8 GLUCOSE-DEPENDENCE OF GD? A N D GLP-1 STIMULATED INSULIN R E L E A S E GD 3 and GLP-1 potentiate insulin release only in the presence of elevated glucose levels. Glucose-induced insulin secretion requires glucose uptake, phosphorylation by glucokinase, and cellular metabolism. This is thought to increase the ATP/ADP ratio within the cell, resulting in the closure of ATP-dependent K + channels. Closure of the channels results in depolarization of the B-cell and the opening of VDCCs allowing the influx of Ca 2 + , resulting in the exocytosis of insulin (Reviewed in Holz and Habener, 1992; Fehmann et al., 1995). Exactly how GIP and GLP-1 signal transduction pathways interact with this glucose-signaling pathway has not been determined, and there have been several hypotheses. Increasing cAMP levels, in cells in which the C a 2 + levels are maintained constant, still stimulates insulin secretion suggesting that, as of yet unidentified, proteins of the exocytosis machinery may be activated by P K A phosphorylation (Ammala et al, 1993). As mentioned in section 1.7.2 there is some evidence that GLP-1, and by extrapolation GIP, may slow V D C C inactivation and in this way potentiate insulin secretion (Britsch et al, 1995; Kato et al, 1996) although the extent to which this augments the influx of C a 2 + appears to be minimal (Ammala et al, 1993; Thorens and Widmann, 1996). However, i f calcium induced calcium release (CICR) is actually a characteristic of incretin signal transduction this may be an important priming effect that allows the eventual release of 51 intracellular stores or influx of Ca via membrane V D C C (Gromada and Rorsman, 1996; Gromada et al, 1996). Gromada et al, (1996) suggested that the activation of P K A may lead to the phosphorylation of the IP3 receptor and/or the ryanodine receptor resulting in their sensitization to increases in IP3 or Ca 2 + , respectively. It is also possible that the GLP-1-dependent increases in cAMP levels lead to activation of VTNCC responsible for the Na+-dependent influx of Ca 2 + , augmenting membrane depolarization and activation of V D C C (Holz et al, 1995; Kato et al, 1996; Fridolf and Ahrens, 1993). GLP-1 may act synergistically with glucose metabolism to inactivate the ATP-dependent K + -channels (Holz etal, 1993), however more recent studies have questioned if GLP-1 has effects on K + currents (Britsch et al, 1995; Kato et al, 1996). GLUT2 has recently been shown to be phosphorylated in P-cells in response to GLP-1 (Thorens et al, 1996), resulting in a 40% decrease in the transporter's activity. If and how this phosphorylation acts to potentiate insulin secretion remains to be determined. Both GIP and GLP-1 have also been shown to increase both proinsulin gene expression and biosynthesis in B-cell lines (Fehmann and Goke, 1995; Wang et al, 1995a, 1996; Drucker etal, 1987; Fehmann and Habener, 1992). Interestingly, extended (6-24h) incubations of RIN 1046-38 cells with GIP and GLP-1 both increased the expression of hexokinase I and GLUT1, but not GLUT2 or glucokinase mRNA levels (Wang et al, 1995a, 1996), suggesting that the incretins are able to regulate the glucose sensing elements. It still remains to be determined if GIP and GLP-1 have similar effects in primary P-cells. 52 1.9 GTP A N D THE GTP RECEPTOR IN NON-INSULIN DEPENDENT DIABETES MELLITUS (NTDDM) Fasting GIP levels have been reported to be normal or elevated in individuals with NTDDM when compared to healthy individuals. GTP secretion in response to a meal has also been reported to be increased, normal or blunted in patients with NTDDM, but appears to be normal in those with TDDM (for specific references see reviews by Pederson, 1993; Crueutzfeldt and Ebert, 1993). Crueutzfeldt etal. (1983) determined the integrated IR-GIP response in 141 individuals with NTDDM and found a bimodal distribution with a large group of hypersecretors and a smaller group of hyposecretors when compared to normal controls. Jones et al. (1989a,b) demonstrated that GTP infusion under basal glycemic conditions resulted in stimulation of insulin release in individuals with NTDDM. This suggests that the P-cells in GIP hypersecretors may be constantly stimulated throughout the day. Studies on obese patients found similar discrepancies in both fasting and food stimulated GTP release, however the test meal used and the rate of gastric emptying may have played a role in the elevated responses observed (Creutzfeldt and Ebert, 1993). When obese subjects were further subdivided on the basis of having normal or impaired oral glucose tolerance (OGT), obese individuals with impaired OGT always displayed exaggerated GIP responses, while obese or lean individuals with normal OGT displayed normal (Creutzfeldt et al, 1978), or elevated GTP levels (Salera et al, 1982). In contrast, in a recent study of post-menopausal women with impaired OGT, GTP levels were found to be decreased while GLP-1 levels were unchanged (Ahren et al, 1997), suggesting that a decrease in GIP secretion contributes to the impaired OGT in this group. 53 It is clear that individuals with N I D D M have blunted or ablated incretin responses (Nauck et al, 1986, 1996; Hoist et al, 1997), but obese individuals are still GIP responsive (Amland et al, 1985; Elahi et al, 1984), and therefore may have inappropriate GIP-mediated insulin release if they are glucose-intolerant. Furthermore, it has been proposed that the hyperinsulinaemia that accompanies impaired OGT in obesity may lead to desensitization of the K-cell to normal feedback inhibition by insulin and result in hypersecretion of GIP (Creutzfeldt et al, 1978). Interestingly, the hyperGIPemic response could be reversed by starvation or dietary restriction in the obese individuals with impaired OGT (Willms et al, 1978; Ebert et al, 1979a; Deschamps et al, 1980). It has not been determined if mutations within the genes for GJP or GLP-1 are linked to NIDDM. The glucose-dependence of both GIP and GLP-1 stimulated insulin secretion makes them prime candidates for the treatment of NIDDM. Unlike the sulfonylureas currently used to treat NIDDM, the incretins only stimulate insulin secretion in the presence of elevated glucose, and therefore never induce hypoglycemia. As mentioned above, responses to GIP infusion in individuals with N I D D M have been shown to be severely blunted or absent (Nauck et al, 1993a; Jones et al, 1989; Krarup et al, 1987; Elahi et al, 1994). However, some studies have shown that, while GD? may have little effect on insulin release in individuals with NIDDM, the incretin response is maintained for GLP-1 at supraphysiological levels (Nauck et al, 1993a,b, 1996; Elahi et al, 1994; Hoist et al, 1997). Furthermore, GLP-1 has been demonstrated to normalize fasting (Nauck et al, 1993b, 1996; Nathan et al, 1992), and reduce postprandial (Gutniak et al, 1992, 1994) glycaemia. However, the latter finding may have been in part due to 54 inhibition of gastric emptying by GLP-1 resulting in slower nutrient uptake (Wettergren et al, 1993; Willms et al, 1996). Inhibition of gastric emptying probably also accounted for the observed decrease in postprandial glycaemic excursions in GLP-1 treated insulin-dependent diabetics (Dupre et al, 1995) and may compromise its use in the treatment of NTDDM (Nauck et al, 1997b). The R-cell insensitivity to physiological levels of both GTP and GLP-1 suggests that there may be a defect at the receptor level responsible for loss of the incretin effect. The fact that GTP responsiveness is more severely affected than GLP-1 suggests that the GIP receptor signaling pathway may be impaired to a greater extent than that of the GLP-1 receptor, and that GTP plays a more important role in glucose homeostasis than GLP-1. While initial genetic studies have failed to link the GLP-1 receptor to NTDDM (Tanizawa et al, 1994; Zhang et al, 1994), more recent studies have identified two missense mutations in the human GTP receptor gene, Glyl98-»Cys (Glyl98Cys) and Glu354-»Gln (Glu354Gln), within the predicted second extracellular loop and sixth transmembrane domains of the receptor, respectively (Kubota et al, 1996). When expressed in CHO cells the Glu354Gln mutant displayed similar function to the wild type (wt) receptor, whereas Glyl98Cys displayed a shift to the right in its half-maximal stimulation value (EC50). However linkage studies failed to show any linkage between NTDDM in Japanese subjects and either of the two mutations. Further studies are needed to confirm or exclude a role for the GTP receptor in NTDDM. Given that NTDDM is a polygenic disease, even i f mutations exist in either incretin receptor, they may only be a minor risk factor or a major risk factor in a small number of individuals (Tanizawa et al, 1994). This is exemplified by studies on transgenic mice homozygous for a targeted null 55 mutation in the GLP-1 receptor gene, which displayed mild fasting hyperglycaemia, and reduced glucose tolerance (Scrocchi et al, 1996). Pederson (personal communication) found the same mice to have normal fasting glucose, and mild hyperglycaemia in OGTT, suggesting unidentified environmental factors, such as food source, are involved in the development of impaired glucose tolerance in this model. It also suggests that GIP is in part able to compensate for the loss of the GLP-1 stimulatory activity at the P-cell. Further examination of the contribution of GIP/GLP-1 to N I D D M and their receptors is required to determine their exact role, and possible exploitation as therapeutics, in NIDDM 1.10 SmUCTTJRE-FUNCTION STUDIES OF GIP 1.10.1 IDENTIFICATION OF A BIOLOGICAL CORE IN GIP Studies from a number of laboratories have demonstrated that limited truncation of GIP at the carboxy-terminus has relatively minor effects on its insulinotropic activity. Thus porcine GIP 1-38 (Moroder etal, 1978) and bovine GIP 1-39 (Sandberg etal, 1986) exhibited similar insulinotropic activities to GIP 1-42 in the perfused rat pancreas. Further truncation produced two fragments, GIPl-31 and GIP1-30, with very interesting characteristics. The former peptide was shown by Malleti et al. (1987) to have a receptor binding affinity an order of magnitude lower than GIP1-42. shGIP l-30amide was shown to be equally insulinotropic to npGIPl-42 in the perfused rat pancreas (Wheeler et al., 1995; Morrow etal, 1996), but to have only 30% of the somatostatin stimulatory activity in the perfused rat stomach. Rossowski et al. (1992) showed that spGIPl-30amide and npGD?l-42 had similar inhibitory activity on bombesin-stimulated amylase secretion, but 56 spGD?l-30amide was far less potent at inhibiting pentagastrin stimulated acid release than npGTPl-42. These studies suggest that different regions of the GD? molecule are involved in different biological actions; i.e. the somatostatinotropic domain lies in the carboxy-terminal region, while the insulinotropic region exists in the amino-terminus. Studies involving the truncation of the amino terminus of GD? produced a confusing picture. Jornvall et al. (1981) identified a contaminant in a npGD? preparation that corresponded to npGIP3-42, and which was found to be biologically inactive (Brown etal, 1981; Schmidt etal, 1986a). The enzymatically produced bovine (b) GDM-42 was shown to have similar affinity for the GIP receptor on hamster insulinoma cell membranes, and still retained 10% of the insulinotropic activity in the perfused rat pancreas when compared to the intact peptide (Maletti et al. 1986). These results were in apparent conflict with studies that showed that npGIP 15-42, produced by cyanogen bromide cleavage (Pederson and Brown, 1976), and bGIP 17-42, produced by enterokinase cleavage (Carlquist et al, 1984; Maletti et al, 1986), displayed significant insulinotropic activity. Maletti et al. (1986) also found that the fragments bGIP 1-16 and bGDM9-30 failed to compete for 1 2 5I-GIP binding, and concluded that the insulinotropic domain lay between residues 17-38. However, this was in contrast to the observations that GIP 17-42 did not stimulate cAMP production in RINm5F cells (Gallwitz et al, 1993), and GD? 19-42 failed to stimulate insulin release from isolated islets (Schmidt et al, 1986b), whereas GIP1-42 was active in both systems. From the above observations, Morrow et al. (1996) predicted that the insulinotropic activity of the peptide must reside in residues 15-30, and then used the perfused rat pancreas to examine a number of fragments within this region for 57 insulinotropic activity. It was found that while spGTP 15-30 was devoid of any significant activity, cleavage to pGIP17-30 (with enterokinase) and to pGIP19-30 (with trypsin) restored -30% of the insulinotropic activity seen with the intact peptide. A synthetic preparation of spGIP17-30 was also found to be insulinotropic, supporting the results with the enzymatically-produced fragment. A confusing observation, made later by the same group was that these peptides failed to displace 1 2 5I-GD? binding to (3-TC3 cells, suggesting the peptide's site of action may be at a receptor other than the GIP receptor (Morrow, Pederson and Mcintosh, personal communication). The related hormone glucagon has recently been shown to be metabolized at target tissues to glucagon9-29 or "mini-glucagon", which has antagonistic effects on glucagon's positive ionotropic effects, and insulinotropic activity (Blache et al, 1994). Ohneda and Ohneda (1994) reported that further truncation of glucagon to fragments 23-29, 21-29, 17-29 restored some insulinotropic activity to the peptides. Given that this region is well conserved between glucagon, GIP and GLP-1 it may be that it is a structurally similar region containing sequences required for receptor interaction and activation. Identification of such a common sequence should assist in the design of analogs with antagonist or improved agonist activity. 1.10.2 GIP IS METABOLIZED TO GD? 3-42 B Y DIPEPTDDYL PEPTIDASE IV (EC3.4.14.5) Dipeptidyl peptidase IV (DP IV) is a selective serine protease which preferentially hydrolyses peptides after a penultimate N-terminal proline (Xaa-Pro) or alanine residue (Xaa-ala) (Heins et al, 1988), requires that amino acids in positions PI and P2 be L -58 isomers in the trans conformation (Fisher et al, 1983), and the N-terminus be protonated (Demuth and Heins, 1995). DP TV has a wide tissue distribution and has been shown to degrade a number of regulatory peptides such as substance P, human a-relaxin, human pancreatic polypeptide, human chorionic gonadotropin, prolactin, neuropeptide Y , peptide Y Y , and P-casomorphin (Mentlein, 1988; Nausch et al., 1990; Wang et al, 1991; Mentlein et al, 1993a). Once cleaved these peptides become susceptible to cleavage by other exopeptidases (Mentlein et al, 1988). It was of particular interest that the 44 amino acid growth hormone-releasing hormone (GRIT), a member of the VTP/glucagon/secretin family, was rapidly degraded by DP IV to biologically inactive GRH3-44 both in vitro and in vivo (Frohman et al, 1986). Amino-terminal substituted analogs of GRH: des-amino (Nrl^-Tyr 1-, D-Tyr 1-, and D-Ala 2 -GRH were all found to be resistant to DP IV cleavage (Frohman et al, 1989). Earlier studies with D-Ala 2-GRHl-29amide and D-Tyr 1-GRHl-29amide, and to a lesser extent des-amino-Tyr1-GRHl-29 truncated G R H analogs, showed they demonstrated an increased potency compared to GRHl-29amide in stimulating growth hormone (GH) release both in vivo (Lance et al, 1984) and in vitro (Heiman et al, 1985). It is possible that this was due to an increased biological half-life due to their resistance to DP IV degradation, however the authors thought it was more likely that the analogs had higher affinity for the G R H receptor (Lance et al, 1984). Many of the peptides of the glucagon/VTP/secretin family share a considerable sequence similarity at their amino-terminus beginning with either a Tyr-Ala, His-Ala, or His-Ser (Rosselin, 1986). It therefore seemed likely that a possible source of the contaminating GTP 3-42 in early porcine GTP preparations (Jornvall et al, 1981) was due to DP TV degradation. Mentlein et al (1993b) demonstrated that GTP and GLP-1 were 59 substrates of circulating DP IV and cleavage renders these non-insulinotropic (Brown et al, 1981; Schmidt etal, 1986a; Suzuki etal, 1989; Gefel et al, 1990). The kinetics of DP IV degradation of GIP 1-42 and GLP-1 by purified placental DP IV was rapid and suggested that this may be an important pathway for the degradation of these two peptides. This proposal was supported by Kieffer et al (1995b) who demonstrated, by high performance liquid chromatography (HPLC), degradation of physiological levels of 1 2 5I-GJP and 1 2 5 I-GLP-1 to 1 2 5I-GIP3-42 and 1 2 5I-GLP-l(9-36), following injection into rats. The degradation was very rapid, with over 50% of the injected label being degraded within the first 2 minutes, supporting a possible physiological role in the regulation of incretin actions. Recent studies by Pauly et al. (1997) using the highly sensitive and accurate technique of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) have further confirmed that GIP 3-42 and GLP-1 (9-36) are the major degradation products resulting from incubation with human serum. This degradation was blocked both in vitro and in vivo by the inclusion or administration of the DP IV specific inhibitor Ile-thiazolidide, resulting in an early peak in plasma insulin and better glucose handling in the anesthetized rat (Pauly, 1996). Experiments in patients with N I D D M have indicated that GLP-1 is rapidly degraded to the appropriate DP IV hydrolysis product (Deacon et al, 1995), suggesting that a defect in DP TV, resulting in prolonged exposure to biologically active GIP and GLP-1, does not underlie the reduced responsiveness of these individuals to incretins. It is therefore of interest to determine i f DP IV inhibitors, or alternatively DP IV resistant analogs of GIP or GLP-1 can be used in the treatment of NIDDM. 60 1.11 STRUCTURE-FUNCTION STUDIES OF G-PROTEIN COUPLED RECEPTORS (GPCR) Since the isolation and cloning of the fj-adrenergic receptor (Dixon et al, 1986), the development of molecular biological techniques has allowed the cloning of receptors without protein purification, and resulted in an explosion in the number of GPCRs isolated and identified (reviewed in Houslay, 1992; Simonsen and Lodish, 1994). Additionally, the use of site-directed mutagenesis has facilitated studies into the structure and function of GPCRs by allowing the manipulation of different regions of the receptor to determine their specific functions. In general these studies have looked at regions important to receptor binding, receptor activation, G-protein coupling, receptor desensitization, sequestration and/or downregulation. The 02-adrenergic receptor (AR) has been the most thoroughly characterized of these receptors (for recent reviews see, Hein and Kobilka, 1995; Lefkowitz et al., 1993). While many of the findings with the P2-AR can be extrapolated to other G-protein coupled receptors, many studies with individual receptors continue to identify different or additional regions involved in receptor structure and function. Given that GD? or its receptor may play a role in NTDDM, and that GIP or GIP-analogs may be useful in the treatment of NTDDM, an understanding of GTP receptor physiology and structure-function will greatly aid in identifying possible defects in diseased states, and in the design of possible useful peptidomimics. 61 1.11.1 RECEPTOR BINDING DOMAINS The first step in receptor activation requires that the ligand associate with high affinity and in a specific manner with its appropriate receptor. Among the GPCRs there is diversity in the structural determinants of ligand binding (See Strader et al, 1994, and Ulrich et al, 1998 for reviews). The membrane-spanning regions constitute the binding domain for the cationic amine receptors (Strader et al, 1994), whereas both the T M spanning segments and extracellular loops contribute to receptor binding of opioid peptides (Fukuda et al, 1995; Varga et al, 1996). In contrast, the binding of glycoprotein hormones is thought to be primarily to a long glycosylated extracellular domain (reviewed in Combarnous, 1992). The N-terminal (NT) extracellular domain and extracellular (EC) loops are also considered to determine binding of the neurokinins (Strader etal, 1994). Less is known regarding the binding domains of the secretin-VIP sub-class of receptors. The use of molecular biology techniques to synthesize cDNA's encoding chimeric proteins is a common technique used to examine the function of a small specific region or domains of a protein in the absence of the rest of the endogenous sequence, but allowing its expression in an appropriate manner or environment. This methodology has been used successfully in the examination of the secretin-VTP and other subclasses of receptors. Chimeric Secretin/VD? receptor studies demonstrated that the NT-domains of these proteins were involved in specific ligand binding (Holtmann et al, 1995; Gourlet et al, 1996). However, the NT region of the VD? receptor appeared to play a greater role in VTP binding than the secretin receptors, and EC loops one and two, but not three have been shown to also contribute to secretin binding (Holtmann et al, 1995a,b, 1996). This 62 conclusion was supported by findings using chimeric proteins consisting of the secretin and, the more distantly related, PTH receptors (Turner et al, 1996a). Receptors consisting of the PTH receptor NT and secretin receptor body bound 125I-secretin and were responsive to secretin, while the inverse protein (secretin receptor NT and PTH receptor body) bound and responded to PTH. Similarly, studies on calcitonin/glucagon receptor chimeras indicated the NT-domain of the calcitonin receptor is involved in binding, but that the body of the receptor is required for signaling (Stroop et al, 1995; Bergwitz et al, 1996). Interestingly, neither chimera examined in that study (glucagon receptor NT/calcitonin body or calcitonin NT /glucagon body) bound or responded to glucagon, suggesting that its binding required regions present in both NT and body regions of the receptor. Other evidence has been presented suggesting that the NT domain of the glucagon receptor is involved in ligand binding (Buggy et al, 1995; Carruthers et al, 1994; Unson et al, 1995). While some GLP-1/glucagon receptor chimera (Buggy et al, 1995) and antibody (Unson et al, 1996; Buggy et al, 1996) studies suggested that the membrane proximal region of the glucagon receptor was important for ligand binding, other site directed mutagenesis and receptor chimera studies suggested a more distal region (residues 29-32) was involved in ligand selectivity and binding (Graziano et al, 1996). Findings by both Buggy and colleagues (1995) and Unson et al (1996) suggested that the first EC domain may also be important for glucagon receptor binding. Interestingly, the extracellular domain of the closely related GLP-1 receptor, when expressed alone, has intrinsic GLP-1 binding activity (Wilmen et al, 1996). By analogy it seems likely that the N-terminal region of the GIP receptor is involved in its ligand binding, however this 63 remains to be confirmed. It is also of interest to determine exactly how the closely related GIP, GLP-1, and glucagon receptors discriminate between their highly conserved ligands. 1.11.2 RECEPTOR-G PROTEIN COUPLING Upon binding their ligand, GPCRs transduce the signal from the extracellular surface to the intracellular side by coupling to a hetertrimeric GTP binding (G)-protein(s), which can then initiate a variety of intracellular biochemical signals. Extensive mutagenesis and chimeric receptor studies on rhodopsin, and the p-adrenergic and cholinergic receptors, have indicated that all of the intracellular loops of the seven transmembrane class of receptors are involved in G-protein binding, although the N- and C-termini of the third intracellular loop are considered to be of primary importance for both G-protein binding and conferring specificity of action (O'Dowd et al, 1988; Wong et al, 1990; Liggett et al, 1991; Hedin et al, 1993; Burstein et al, 1998). While no studies of these regions have been carried out with the GD? receptor, residues within the proximal region of the third and first intracellular loops of the closely related GLP-1 receptor (Takarh et al, 1996; Mathi et al, 1997; Heller et al. 1996), and the second and third intracellular loops of the glucagon receptor (Chicchi et al, 1997) were shown to be involved in G-protein coupling. In contrast, less is known regarding the function of the C-terminal (CT)-tail of G protein-coupled receptors. This region has been implicated in receptor desensitization and endocytosis (Reneke et al, 1988; Hausdorf et al, 1990b; Huang et al, 1995a), and the membrane proximal region of the P2-adrenergic receptor CT-tail has been shown to 64 be important for G protein coupling (ODowd et al, 1988). Additionally, the CT-tail has also been suggested to play a role in directing transport of the receptor to the plasma membrane (Parker and Ross, 1991) and restricting lateral membrane movement (ODowd et al., 1988; Parker and Ross, 1991). Studies on CT-tail truncated members of the secretin-VTP-glucagon family of receptors, suggested that reduction in the length of the C-terminal tail increased agonist binding of the PTH/PTH-RP (Iida-Klein et al., 1995), calcitonin (Findlay et al, 1994), and glucagon (Unson et al, 1995) receptors, whereas effects of truncation on adenylyl cyclase responses varied from inhibition (Findlay et al, 1994) to stimulation (Iida-Klein et al, 1995). In more recent studies it was shown that approximately half the GLP-1 receptor's CT (Widmann et al, 1997), and the majority of the glucagon receptor's CT-region (Buggy et al, 1997) could be truncated with no effect on receptor affinity or expression. 1.11.3 RECEPTOR ACTIVATION AND CONSTITUTTVELY ACTIVE RECEPTORS The binding of a receptor and its ligand, results in a conformational change that allows the receptor to associate with G-proteins, causing the G-protein to exchange bound GDP for GTP resulting in the G-protein activation. As high affinity binding only occurs when the receptor-ligand complex is also associated with a G-protein, a model accounting for the formation of such a "ternary complex" was initially proposed by DeLean and co workers (1980) and is shown in Fig. 5A. However, this model failed to explain results from studies of the cti- (Cotecchia et al, 1990; Kjelsberg et al, 1992), ot2- (Ren et al, 1993), and pV (Samama et al, 1993) adrenergic receptors (AR), that identified mutations resulting in receptors that were active even in the absence of bound ligand. Nor could it 65 account for the observations that increasing pVadrenergic receptor density appeared in some cases to increase basal activity (Samama et al, 1993; Adie and Milligan, 1994), and that ectopic expression of a receptor could result in high basal activity (Costa and Herz, 1989; Costa et al, 1990; Tiberi and Caron, 1994; Jakubik et al, 1995). These data suggested that GPCR's could exist in an equilibrium between both an inactive (R) and active (R*) state (Samama etal, 1993), the latter of which can associate in the absence of ligand with G-protein and account for "basal" receptor activity. This "allosteric" ternary complex model (Samama et al, 1993) (Fig. 5B) illustrates the possible conformations and associations of receptor, ligand, and G-protein. In this model receptors do not have to bind their ligand to be activated. Agonists act to stabilize receptors in the R* state shifting the equilibrium to the active state resulting in an increase in receptor signaling, while inverse, or reverse, agonists act to stabilize the receptor in the inactive state (Fig. 5C) (Samama etal, 1993; see Milligan etal, 1995, and Scheer and Cotecchia, 1997 for recent reviews). While in vitro evidence for inverse agonism has existed for a long time (Milligan et al, 1995; Scheer and Cotecchia, 1997), more direct evidence has been presented using transgenic mice overexpressing the P2-adenergic receptor in a heart specific manner (Bond et al, 1995). These mice displayed both increased basal cAMP levels and increased cardiac contractility, and the pVadenergic ligand ICI-188551 acted as an inverse agonist, decreasing ventricular systolic pressure both in vivo and in vitro. 66 B. H + R + G « *" H R + G H + R + G « * H R + G H + R G « * H R G H + R* + G « » HR* + G H + R * G — H R * G C. H = Agonist: HR 15= HR* H = Inverse Agonist: H R M = ^ HR* Fig. 5. The Ternary Complex Model (TCM) and Inverse Agonism. Representative diagrams of possible receptor (R)/hormone (H) and G-protein interactions. A . The T C M as originally proposed by Delean et al. (1980). B . The T C M as later modified by Samama and coworkers (1993) where it is possible for R to undergo an allosteric transition to the R* intermediate which can then interact with H and G. C. While agonists act to stabilize the R* form of the receptor, shifting the equilibrium towards the "active" form (R*), inverse agonists stabilize the majority of receptor in the inactive (R) state. Adapted from Samama et al. (1993). 67 Studies have also identified naturally occurring mutations in GPCRs resulting in a number of pathological states such as autosomal dominant retinitis pigmentosa and night blindness (rhodopsin receptor), familial male precocious puberty (luteinizing hormone receptor), hyperfunctioning thyroid adenomas (thyroid stimulating hormone receptor) and inherited hyperpigmentation of a mouse strain (melanocyte-stimulating hormone receptor) (reviewed in Milligan et al, 1995 and Scheer and Cotecchia, 1997). Recently, the first example of such constitutively activating mutations in the glucagon/secretin/VIP receptor family were identified (Schipani et al, 1995, 1996). Substitution of a histidine residue at the junction of the first intracellular loop (IC) with arginine (H170R), or a threonine in the sixth transmembrane (TM) domain with proline (T410P), results in constitutively active forms of the PTH/PTHrP receptor associated with a rare form of dwarfism, Jansen-type metaphyseal chondysplasia (Schipani et al, 1995, 1996). The histidine residue at the base of the second TM is conserved in all members of the glucagon/secretin/VIP receptor family suggesting that it may play a universal role in regulating spontaneous receptor activation (Schipani etal, 1995). The availability of constitutively active receptors has assisted in the identification of inverse agonists (Samama et al, 1993; Jakubik et al, 1995; Gardella et al, 1996). While it remains to be shown that inverse agonists have any benefit over neutral antagonists in clinical applications, it can be imagined that they may be of great use in the treatment of pathologies due to constitutively active receptors (Bond et al, 1995; Milligan etal, 1995). 68 1.11.4 RECEPTOR PHOSPHORYLATION A N D DESENSITIZATION The exposure of a receptor to high and/or continual exposure of an agonist can lead to a reduced cellular response. The attenuation of a ligand's signal can occur at many levels, including ligand removal, uptake or degradation, receptor phosphorylation, receptor sequestration or internalization and, finally, downregulation of both receptor protein and gene expression (for recent reviews see Hausdorff et al, 1990a; Chuang et al, 1996; Ferguson^al, 1996a; Bohmetal, 1997). The term desensitization, in the case of GPCRs, usually refers to the rapid plateau or return to near basal levels seen within a few minutes of agonist stimulation. This attenuation of the cellular signaling occurs in seconds or minutes and involves receptor phosphorylation, resulting in the uncoupling of receptor from G-proteins (Hausdorff et al, 1990a; Chuang et al, 1996; Ferguson et al, 1996a; Bohm et al, 1997). The rapid phosphorylation associated with receptor desensitization is mediated by two classes of serine/threonine protein kinases: the second messenger activated protein kinases, P K A and P K C , and the G-protein coupled receptor kinases (GRKs) that preferentially phosphorylate agonist-activated GPCRs. Receptor desensitization can be functionally differentiated into agonist-dependent, or homologous desensitization, and agonist-independent or heterologous desensitization. Homologous desensitization of a receptor refers to the blunting or reduced sensitivity of a response to specific ligands of that receptor. In contrast, heterologous desensitization of a receptor is a result of the activation of a separate, different type of receptor and subsequent second messenger-dependent protein kinase phosphorylation (Hausdorff et al, 1990; Ferguson et al, 1996a; Chuang etal, 1996; Bohm etal, 1997). 69 While in general terms it can be said that heterologous desensitization is mediated mostly by second messenger-dependent protein kinases, and homologous desensitization is mediated mostly by GRKs, the relative contribution of the two kinase types to homologous desensitization depends on the cell type, receptor type, or even the concentration of agonist being examined (Ferguson et al, 1996a; Chuang et al, 1996). For example, P K A phosphorylation appears to be important for homologous desensitization of the P2-adenergic receptor at lower agonist concentrations (Hausdorff et al, 1989; Lohse et al, 1990a), while at higher concentrations G R K mediated phosphorylation plays a larger role in this receptor desensitization (Hausdorff et al, 1989; Lohse et al, 1990a; Roth et al, 1991). As mentioned in section 1.11.2 the CT-tail of many G-protein coupled receptors have been implicated in receptor desensitization and sequestration. For the prototypic 02-adenergic, and many other receptors, residues within the CT-tail region have been implicated in receptor phosphorylation, desensitization, and sequestration (Hausdorff et al, 1990; Chuang et al, 1996; Ferguson et al, 1996a; Bohm et al, 1997). With other receptors that either have short CT-tails, and/or few Ser/Thr residues in the CT-tail region such as the ct2-adenergic and m2 muscarinic (m2-AchR) receptors, the large third intracellular loops appear to be the site of G R K phosphorylation (Liggett et al, 1992; Eason et al, 1995; Ferguson et al, 1996a). As mentioned above, G R K phosphorylation of receptors is most efficient when the receptor is activated. In addition, the residues phosphorylated differ from those modified in heterologous desensitization in both position and the fact that no identifiable consensus sequences, such as those for P K A and PKC, have been identified for the six GRK' s identified to date (Ferguson et al, 1996a; 70 Chuang et al, 1996; Bohm et al, 1997; Palczewski, 1997). It therefore appears that the tertiary structure of the receptor induced by receptor activation rather than exposure of a linear recognition sequence is the critical determinant for phosphorylation (Hausdorff et al, 1990; Ferguson et al, 1996a; Chuang et al, 1996; Bohm et al, 1997; Palczewski, 1997). In studies with the rhodopsin and 02-adenergic receptors GRK-mediated phosphorylation was not found to be sufficient for full inactivation, suggesting that some other "arresting agent" was required for full receptor desensitization. Wilden et al, (1986) identified a 48-kDa protein, now called arrestin, that bound to phosphorylated rhodopsin. Benovic and co-workers (1987) noticed that a crude preparation of PARK1 (GRK-2) could fully desensitize the P2-AR while purified PARK1 could not, suggesting that a similar arrestin like protein may exist in this system. This led to the isolation of P-arrestin 1, that was able to reestablish the ability of purified PARK-1 to desensitize P -AR in reconstitution assays (Lohse et al, 1990b, 1992). To date six distinct arrestins have been identified. While it is widely accepted that phosphorylation of GPCRs by GRK-1 , 2 and 3 results in the binding of arrestins, thus further uncoupling the receptor from G-proteins (Lohse et al, 1990b, 1992; Pippig et al, 1993), it is unclear i f GRKs 4-6 target arrestins to receptors (Ferguson et al, 1996; Chuang et al, 1996; Bohm et al, 1997; Palczewski, 1997). Less is known about the heterologous and homologous desensitization of the secretin/glucagon/VrP receptor family. Similar to the P2-adenergic and other GPCRs, the CT-region appears to be the major site of phosphorylation in response to agonist stimulation of the receptors for secretin (Ozcelebi et al, 1995; Holtmann et al, 1996b), 71 PTH/PTHrP (Blind et al, 1995, 1996), glucagon (Heurich et al, 1990; Savage et al, 1995; Buggy et al, 1997) and GLP-1 (Widmann et al, 1996a,b, 1997; Thorens and Widmann, 1996). While P K C and/or P K A phosphorylation have been implicated in the phosphorylation and desensitization of the PTH/PTHrP, glucagon and GLP-1 receptors (Blind et al, 1995, 1996; Savage et al, 1995; Thorens and Widmann et al, 1996; Widmann et al, 1996a,b), in all cases antagonism of P K C and P K A attenuated heterologous desensitization but failed to fully block the receptor desensitization due to homologous desensitization. This suggests that GRKs play a role in the desensitization of GPCRs of the secretin/glucagon/VTP receptor family (Blind et al, 1995, 1996; Savage et al, 1995; Thorens and Widmann, 1996; Widmann et al, 1996a,b). However, to date the only direct evidence of G R K mediated phosphorylation within this subfamily of receptors is the observation that P A R K 1 phosphorylates the recombinantly expressed PTH/PTHrP receptor CT-tail in vitro (Blind et al, 1996). It remains to be demonstrated whether GRKs are involved in the in vivo regulation of secretin/glucagon/VTP receptor phosphorylation and desensitization. In addition, it has not been determined if arrestins play a role in the attenuation of the signals mediated by these receptors. 1.11.5 RECEPTOR ENDOCYTOSIS Receptor internalization appears to play a role in the desensitization of receptors in certain cellular environments, for example the u-opioid (Pak et al, 1996) and secretin receptors (Holtmann et al, 1996) when expressed in CHO-K1 cells. However, it is now widely accepted that, for the majority of receptors, the kinetics of internalization are too slow to account for the majority of desensitization responses. Further, results from 72 studies on chemical inhibition of internalization with sucrose or concanavalin A , and internalization deficient receptor mutants, indicate that desensitization can occur independently of receptor uptake (reviewed in Hausdorff et al, 1990; Ferguson et al, 1996; Chuang et al, 1996; Bohm et al, 1997). It now seems more likely that receptor uptake is important to the resensitization of a cell to a ligand, and may also contribute to receptor downregulation (Hausdorff et al, 1990; Ferguson et al, 1996; Chuang et al, 1996; Bohm et al, 1997). Once internalized, one or more of the following may occur to the receptor-ligand complex (reviewed in Shepherd, 1989): 1. Class I. The ligand is dissociated from the receptor due to vesicle acidification and sorted to lysosomes for degradation, while the receptor is recycled to the membrane. 2. Class II. Both receptor and ligand are recycled to the membrane. 3. Class III. Both receptor and ligand are targeted to lysosomes. 4. Class IV. Both the receptor and ligand are translocated to the opposite side of a polarized cell. Most of the GPCRs examined to date appear to belong to the Class I group, though it is possible that a small number of receptors in each cycle are targeted to the lysosome and can, over the long term, account for the decreased receptor number observed in receptor downregulation (Hausdorff et al, 1990; Ferguson et al, 1996; Bohm et al, 1997). It now appears likely that receptor internalization is more often involved in receptor recycling, dephosphorylation, and resensitization of the target cell to the ligand post stimulation. Evidence supporting this proposal includes: the observation that sequestered pools of the 73 P 2 - A R receptor are less phosphorylated than membrane associated receptors; these vesicles are associated with high receptor phosphatase activity; receptor resensitization can be inhibited by using phosphatase inhibitors; and finally the fact that inhibition of receptor sequestration prevents both receptor dephosphorylation and resensitization (reviewed in Furguson etal, 1996; Bohm et al, 1997). Recently, a membrane-associated G-protein-coupled receptor phosphatase (GPCRP) that is inactive at neutral pH but active at lower pH in vitro has been implicated in receptor dephosphorylation in the acidified endosome (Garland et al, 1996; Krueger et al, 1997). Given that GPCRP and the phosphorylation sites of GPCRs exist in the neutral intracellular environment, the acidification of endocytotic vesicles may act to both dissociate ligands and cause receptor-mediated conformational activation of GPCRP (Garland et al, 1996; Krueger et al, 1997). There is a great deal of evidence that activated receptors uncoupled from their G-proteins are internalized into early endosomes either via clathrin coated pits, or caveolae mediated pathways. The exact pathway responsible for a given receptor, like the kinases responsible for desensitization, probably depends both on receptor specific determinants and the cellular environment it exists in (Ferguson et al, 1996). For example, dominant-negative mutants of P-arrestin and dynamin, a GTPase required for clathrin coated vesicle formation, both inhibited wt p 2 -AR sequestration, but not that of the angiotensin II type 1A receptor (ATIAR ) , suggesting that both arrestins and dynamin are essential in p 2 -adenergic clathrin-coated vesicle mediated sequestration (Ferguson et al, 1996b, Zhang et al, 1996). However, while A T I A R internalization was independent of P-arrestin and dynamin function, overexpression of P-arrestin increased the number of A T i A R s 74 internalized via clathrin coated pits, suggesting that some plasticity exists by which pathway a receptor can be internalized (Zhang et al, 1996). 1.11.6 DETERMINANTS OF RECEPTOR SEQUESTERATION To date no clear universal determinants of GPCR sequestration have been identified. It appears that multiple domains or motifs, as well as receptor phosphorylation state and receptor conformation, may all play a part individually, or combined, to induce receptor internalization (reviewed in Ferguson et al, 1996a; Bohm et al, 1997). Early studies suggested that regions required for G-protein coupling were also required for sequestration. In support of this was the observation that receptor coupling efficiency often correlated to a receptor's ability to be sequestered. (Strader et al, 1987: Ferguson et al, 1996a; Bohm et al, 1997). However, further studies identified receptors that were uncoupled yet sequestered normally (Mahan et al, 1985; Cheung et al, 1990; Hausdorff et al, 1990; Petrou et al, 1997). There is also a large body of evidence indicating that the two activities (second messenger coupling and sequestration) are functionally distinguishable (reviewed in Ferguson et al, 1996a; Bohm et al, 1997). It was initially suggested that receptor phosphorylation acts as an internalization signal. Subsequently it was found that removing (by CT-truncation) or mutating all P K A and GRKs phosphorylation sites, or inhibition of kinase activity failed to inhibit 0 2 - A R sequestration. This has led to the belief that phosphorylation is not essential for receptor sequestration (Lohse et al, 1990a; Hausdorff et al, 1989; Ferguson et al, 1995). It has been demonstrated recently that overexpression of P-arrestin 1 or 2 rescued sequestration deficient mutants of the P2-adenergic receptor (P2 - A R Y326A), an effect that was 75 complemented by the overexpression of GRK-2 (Ferguson et al, 1996b). However, dominant-negative mutants of P-arrestin inhibited sequestration of both wt receptor and P2-AR Y326A, even when receptor phosphorylation was restored by the over-expression of GRK-2 (Ferguson et al, 1996b). These observations strongly suggest that, at least in the case of the P2-AR receptor, phosphorylation acts to stabilize the conformation required for sequestration and/or promotes the association with other cellular elements required for sequestration (Hausdorff et al, 1989). However, it is possible that phosphorylation is important for many other receptors, as removal of the Ser/Thr rich CT-tails of the GRP, alb-adrenergic, PTH/PTHrP, calcitonin, A T I A R , neurotensin, and GLP-1 receptors (Benya et al, 1993; Lattion et al, 1994; Findlay et al, 1994; Huang et al, 1995b; Thomas et al, 1995; Hermans et al, 1996; Widmann et al, 1996), or the middle region of the third intracellular loop of mAChRs (Lameh et al, 1992: Moro et al, 1993) impairs their ability to be sequestrated. Attempts have also been made to identify endocytic signals or motifs similar to the tyrosine residues containing sequences identified for a number of single transmembrane (STM) receptors. Studies of a NP(X)2,3Y motif found near the cytoplasmic face of many GPCRs, that resembles the N P X Y sequestration signal identified for the low density lipoprotein (LDL) and insulin receptors (Ferguson et al, 1996; Bohm et al, 1997), have demonstrated that mutation of the conserved tyrosine residue in both the P2-AR (Barak et al, 1994) and the Neurokinin 1 receptor (NK1-R) (Bohm et al, 1997b) impaired receptor internalization. However similar mutations in the GRP and A T i A R did not impair sequestration (Slice et al, 1994; Hunyady et al, 1995), suggesting that this was is not an universal GPCR internalization sequence. Further 76 investigation indicated that the N P X Y motif may be a critical domain in receptor isomerization from low to high affinity (R->R*) (Barak et al, 1995). This suggests that interaction of kinases not only requires interaction with multiple domains, but that the receptor must be in an appropriate (active like) conformation to be efficiently sequestered. A conserved " D R Y " motif ( D R Y X X V / I X X P L ) , found within the second intracellular domain of many GPCRs (but not members of the secretin/glucagonATP receptor family), is important for internalization of the ml mAChR and the GnRH receptors (Moro et al, 1994: Arora et al, 1995). Interestingly, Tyr residues in the CT-tail of the neurokinin receptor (Bohm et al, 1997b), and A T I A R (Thomas et al, 1995), and possibly the PTH/PTHrP receptor (Huang et al, 1995b) may act in a similar manner as those found in S T M receptors. Additionally, negative regulatory sequences have been identified in the membrane proximal region of the thyrotropin-releasing hormone receptor (TRHR) and PTH/PTHrP receptors (Nussenzveig et al, 1993; Petrou et al, 1997; Huang et al, 1995b). However, as stated above, none of these signals appear to be universal to GPCR's and the regions important to each receptor, or more closely related receptors, may have to be determined individually. 1.11.7 SEQUESTRATION OF THE SECRETIN/GLUCAGON/ VIP RECEPTOR F A M I L Y Few studies have looked at receptor internalization of members of the secretin/glucagonATP receptor family, and most of these have focused on the contribution of the CT-tail region to receptor internalization. CT-truncation of the 77 secretin receptor had little effect on receptor sequestration in CHO-K1 cells (Holtmann et al, 1996b), while internalization of the glucagon (Buggy et al, 1997) and GLP-1 receptors was inhibited (Widmann et al, 1995, 1997). Both glucagon and GLP-1 receptor internalization was dependent on the phosphorylation of Ser residues within the CT-tail regions (Buggy et al, 1997; Widmann et al, 1995, 1997). For the GLP-1 receptor, the degree of receptor internalization was dependent on the number of phosphorylation sites mutated, with the degree of inhibition being correlated to the number of sites removed. However not all sites appeared to contribute equally to receptor internalization, with the most distal of three Ser doublets in the GLP-1 receptor CT-tail being less critical than the two more internal Ser doublets for receptor internalization (Widmann et al, 1996a). These findings are consistent with those found for the glucagon (Buggy et al, 1997), and P T H receptors (Huang et al, 1995a,b) in which the more distal regions of these two receptors could be deleted with little or no effect on sequestration. The regulation of the more distantly related calcitonin and PTH/PTHrP receptors' internalization appears to be more complicated than that seen for the GLP-1 and glucagon receptors. Both positive and negative regulatory signals have been identified in the membrane proximal region of the PTH/PTHrP receptor (Huang et al, 1995b). While the majority of the calcitonin receptor CT-tail could be deleted without impairing receptor uptake, intermediate truncations resulted in receptors that were either sequestration deficient or resulted in wild-type (wt) like or even slightly improved internalization (Findlay et al, 1994). PTH/PTHrP receptor sequestration may involve phosphorylation as its CT-tail is Ser/Thr rich and is phosphorylated in response to receptor activation 78 (Blind et al, 1995, 1996). However given the few Ser/Thr residues in the CT-tail of the calcitonin receptor, and the lack of correlation with loss of internalization with loss of these residues, it is unlikely that phosphorylation of the calcitonin CT-tail is required for sequestration (Findlay et al., 1994). Given the great divergence in the CT-tail regions between members of the secretin/glucagonATP receptor family, it is not surprising that multiple receptor specific CT-tail regions are involved in receptor internalization. Both the GLP-1 and PTH/PTHrP receptor appear to be internalized by via clathrin coated pits, as internalization of both could be blocked by treating cells with hypertonic sucrose (to disrupt clathrin lattices) (Widmann et al., 1995; Huang et al., 1995b). However, both of these observations were made in fibroblast or COS 7 cell lines, and it remains to be determined if the same endocytic pathway is important in vivo. No studies to date have examined the downregulation (actual depletion of receptor number) of receptors within the secretin/glucagonATP family. In fact little is actually known concerning the downregulation of most GPCRs, although enhanced degradation and reduced synthesis are likely candidates (Bohm et al, 1996). Downregulation of many GPCR's mRNA levels has been associated with decreased stability of the transcript following long term agonist treatment (reviewed in Bohm et al, 1996). A 35 kD f3-adrenergic receptor mRNA binding protein (P-ARB) has been identified that preferentially binds to one or more A U U U A pentamers found within the 3 'UTR of the P2-adrenergic receptor resulting in increased agonist induced destabilization (Port et al, 1992; Tholanikunnel et al, 1995). The protein was also found to destabilize thrombin receptor mRNA, and it may be responsible for the post-transcriptional regulation of many other receptor mRNA's containing A U U U A pentamers within their 3'UTRs 79 (Tholanikunnel et al, 1995). The existence of such regulation of the GIP receptor or other members of the secretin/glucagon/VTP family remains to be determined. 1.12 THESIS STUDIES: HYPOTHESES A N D OBJECTIVES Studies on numerous G-protein coupled receptors have identified regions with common functions. However, it has become apparent that in most cases it is difficult to identify distinct structural motifs, and structure-function studies have to be performed for each individual receptor. As discussed previously, it is now thought that GTP is the most important incretin in healthy individuals (Hoist et al., 1997; Nauck et al., 1997a), and a defect in GTP-mediated insulin secretion may play a central role in the aetiology of NTDDM (Hoist et al, 1997). It is therefore important to identify structural features of both GD? and its receptor that are important for normal physiological regulation of glucose homeostasis. Studies undertaken in this thesis were designed to examine a number of questions concerning the identity, function, and structure of the rat islet GD? receptor and GIP. The rationale, specific hypotheses and objectives are listed below: As mentioned in section 1.5.1 Usdin et al. (1993) originally isolated a partial cDNA clone from rat cerebral cortex, and used this sequence to isolate a full length GTP receptor cDNA clone from the rat tumor J3-cell line, RTNm5F. Surprisingly, the RTNm5F cDNA was shown to be expressed in a wide variety of tissues not normally thought to be targets of GTP action (Usdin et al, 1993). As neither GIP immunoreactivity (Buchan et al, 1982), nor mRNA (Higasimoto et al, 1992; Tseng et al, 1993) have been detected in the brain, this suggests that the receptor isolated by Usdin et al. (1993) may encode a receptor for a highly related ligand present in the brain, and it may not be the GTP 80 receptor present in primary islet cells. The receptor for GIP would also be expected to display high specificity and affinity for its ligand, and activate signal transduction pathways associated with GIP-mediated insulin secretion (see section 1.7.1). To determine if the GIP mRNA isolated by Usdin et al. (1993) was also expressed in primary rat islet cells, and to more thoroughly examine its signal transduction, the following studies were undertaken. Hypothesis 1. The pancreatic islet GIP receptor is the product of an identical or alternatively spliced form of the cDNA identified in the tumor cell line, RIN5mf, and may display major or minor sequence differences. Objectivel. To prepare a pancreatic GIP receptor cDNA from isolated rat islet mRNA and compare its sequence with that of a cDNA previously isolated from a (3-cell tumor cell line, RIN5mf. Hypothesis 2. The islet GIP receptor exhibits strong specificity with respect to ligand binding and initiation of signal-transduction pathways. Objective 2. To compare and contrast ligand binding and activation of adenylyl cyclase by members of the secretin-glucagon superfamily in COS-7 and CHO-K1 cells expressing the pancreatic islet GIP receptor. A great deal of controversy exists in the literature concerning the potency of synthetic human GIP in both humans (Elahi et al., 1979; Nauck et al., 1989, 1993a) and rat (Jia et al., 1995). Once isolated, the rat islet GIP receptor mRNA, when expressed in COS-7 and CHO-K1 cells allowed the examination of the potencies of different species of GJP. Hypothesis 3. Different species of GIP display different affinity and efficacy at the rat islet GIP receptor. Objective 3. To compare the affinity and efficacy of different commercial preparations of synthetic GIP on COS-7 and CHO-K1 cells expressing the rat 81 pancreatic islet GD? receptor, and compare these to the in vivo activity in the isolated perfused rat pancreas. Studies to date (see section 1.10) have suggested that the biological core of GTP exists within residues 1-30, while smaller fragments may possess reduced activity (Maletti et al., 1986; Carlquist et al., 1984; Morrow et al., 1996). However, many of these peptide preparations were generated by enzymatic cleavage and may not have been pure. Furthermore, all were tested with either insulinoma cell lines or the perfused rat pancreas, both systems that contain the highly related GLP-1 and glucagon receptors, with which the fragments may crossreact, further confusing interpretation of the results. The stable rat islet GIP receptor expressing CHO-K1 cell line provided an isolated system in which to examine the ability of different GD? fragments to bind and/or activate its receptor. Hypothesis 4. The biological core of GD? exists within the region of amino acids 1-30, and further truncation will result in fragments exhibiting high affinity and biological activity. Objective 4. To utilize stable CHO-K1 cell lines expressing the rat islet GIP receptor to examine the effect of truncation of the biologically active GD? 1-30 fragment, to identify regions and residues important for receptor binding and activation. While many studies concerning the structure-function of the P-AR, and the more distally related oc-AR and mAchR have been carried out, far fewer have examined receptors of the secretin/glucagon/VTP family. The isolation of the rat islet GD? receptor mRNA allowed us to examine regions of the GIP receptor important to its ligand specificity, binding, and signal transduction. Based on findings presented in section 1.11 the following hypothesis were proposed: 82 Hypothesis 5. The amino-terminal extracellular domain of the GD? and GLP-1 receptor contains the majority of the selective ligand-binding region, while regions important for activation require multiple receptor regions. Objective 5. To construct rat GIP/human GLP-1 receptor chimeras to define regions important for high affinity ligand binding, ligand selectivity, and receptor activation. Hypothesis 6. The carboxy-terminal intracellular tail of the GD 3 receptor contains regions important for effective coupling to second messenger activation, desensitization, and sequestration. Objective 6. To use site directed mutagenesis to construct increasing truncation of the rat islet GIP receptors carboxy-terminal tail, and compare and contrast the ability of these truncated receptors to generate cAMP, undergo desensitization, and be sequestered in response to ligand stimulation. 83 CHAPTER 2 METHODS 2.1. ISOLATION A N D CHARACTERIZATION OF A cDNA ENCODING THE R A T GIP RECEPTOR Isolation of the rat GIP receptor cDNA was performed in collaboration with Dr. Michael B . Wheeler. Standard molecular biology techniques used in the studies were based on those described in Molecular Cloning, A Laboratory Manual by Sambrook et al., 1989, and further details are provided in this section when modifications of the original procedure were made. Chemicals and enzymes were all of molecular biology grade unless otherwise stated; commercial sources are listed in the text. A l l primers were synthesized on an Applied Biosystems 380A D N A synthesizer in the laboratory of Dr. Ross T.A. McGillivray. They were eluted from the column with 2 ml 14.8 M NH4OH, incubated at 55°C for 12-16h, and dried in a Speed-Vac. 2.1.1 R N A ISOLATION Normal precautions for work with R N A were taken: all solutions used were treated with 0.1% diethyl pyrocarbonate (DEPC) for 12 h, and then autoclaved to inactivate the DEPC, or made from fresh chemical stocks with DEPC treated distilled (d)H20; all glassware, gel boxes, tip holders, etc. were treated with Absolve® (Dupont, Wilmington), and rinsed with DEPC treated dH 2 0; and all plasticware used was RNase free grade. 84 Rat pancreatic Islets were isolated by Robert Pauly or Charlene Fell, as described by Van der Vliet et al., 1988. Total R N A was isolated from the rat islets using the acid guanidinium thiocyanate-phenol-chloroform extraction method of Chomczynski and Sacchi (1987). Islets were removed from the isolation medium by centrifugation, and lysed in approximately 0.5 ml of solution D (4 M guanidinium isothiocyanate, 25 m M sodium citrate, 0.5 % sarcosyl, and 0.1 M 2-P-mercaptoethanol). Sequentially, 0.1 volume of 2 M sodium acetate (pH. 4) and an equal volume of buffer saturated phenol, and 0.2 volumes of chloroform/iso-amyl alcohol (49:1), were added per volume of solution D used in the initial homogenization, and the mixture vortex mixed briefly. Samples were then incubated on ice for 15-20 min., and centrifuged at 10-12,000 X g for 20 min at 4°C. The R N A containing aqueous phase (top) was transferred to a fresh RNase free tube, and mixed with an equal volume of isopropanol. After at least l h at -20°C the precipitated R N A was sedimented at 10-12,000g for 20 min at 4°C, and the resulting pellet resuspended in 300 pi of solution D. The pellet was transferred to a fresh 1.5 ml Eppendorf tube, and precipitated with isopropanol as before. The R N A was then washed with 70% ethanol, dissolved in DEPC treated dH 2 0 (50-100 pi), and the concentration determined using sample absorbance at A26o and A 26o/A 2 8o ratios. Integrity was assessed from appearance of the R N A when run on a formaldehyde-agarose (1.1%) gel. The total R N A used for reverse transcription had an A 26o/A 28o ratio of at least 1.7. Total R N A was stored either as an isopropanol precipitate or in DEPC treated H 2 0 with 1 pi (10TJ) of RNase inhibitor (RNAguard, Pharmacia), until use. 85 2.1.2 R E V E R S E TRANSCRIPTION-POLYMERASE C H A I N R E A C T I O N Total R N A (5 ug) from rat pancreatic islets was primed with 0.5 ug oligo deoxythymidine (dT) and 0.5 ug random hexamers (Pharmacia) by incubating at 95°C for 10 min, and then chilled on ice for 5 min. Reverse transcription (RT) was then carried out with 400U Superscript II™ reverse transcriptase (Gibco B R L , Grand Island, NJ) in I X RT buffer (50 mM Tris-HCl (pH 8.3), 75 mM KC1, 3mM M g C l 2 , and 10 m M dithiothreitol (DTT)) containing 10 U of RNase inhibitor (Pharmacia), and 1 m M dNTPs (Pharmacia), at 42°C for l h in a total volume of 20 uL. The reaction was terminated by heating the reaction tube to 85°C for 15 min. 80 uL of CIH2O were added to the mixture to bring the total volume to 100 uL. To amplify the coding region of the rat islet GD? receptor, cDNA oligonucleotide primers were designed based on the published sequence of Usdin et al. (1993) for a GIP receptor cDNA cloned from a rat insulinoma cell line (RTNm5F), corresponding to nucleotides 163-184 (S '-AGGATGCCCCTGCGGCTGTTGC-S') and 1537-1515 (5'-G T C C T A G C A G T A A C T T T C C A A G A - 3 ' ) . Amplification of 1-10 uL of the cDNA was carried out with 100 pmol of each primer, 200 u M dNTPs and 5U of Taq polymerase (Perkin Elmer Cetus, Norwalk, CT) in 1 X PCR buffer E (67 m M Tris-HCl, pH 9.0, 1.5 m M M g S 0 4 , 166 m M N I L S O ^ and 10 mM 2-fj-mercaptoethanol) in a total volume of 50 uL. The following PCR conditions were used: denaturation at 95°C for 3 min, annealing at 62°C for 1 min, and an extension time of 1 min at 72°C for one cycle, followed by 35 cycles with 1 min denaturation, annealing and extension steps. A single product of the appropriate size (-1.4 kb) was obtained, and three individual PCR reaction products (GIP-R1, Gn*-R2, and GD»-R3) were isolated, gel purified, and ligated into the T A 86 cloning vector pCRII (Invitrogen Co, San Diego, CA) as per the manufacturer's instructions. The pCRII vector takes advantage of the one base pair 3' overhang, usually an adenosine (A), that Taq polymerase adds to the end of an amplified product. The linearized vector is prepared with one base pair thymidine (T) overhang at its 3' ends, at a site within the multiple cloning site (MCS), making small "sticky ends" for the A overhangs present on the PCR product. This results in higher efficiency subcloning than other methods commonly used to subclone PCR generated products. Three individual clones (GTP-R1, GTP-R2, and GIP-R3) were mapped by restriction analysis and partial dideoxy-sequencing (T7-Sequencing Kit, Pharmacia Biotech, Sweden). The cDNAs pGTP-Rl and pGIP-R2 were subcloned into the Hind JWXba I site of the expression vector pcDNA 3 (Invitrogen), and the complete sequence of the pGTP-Rl coding strand was determined (See Fig 21). Alignment with the published sequence identified only one nucleotide difference in pGIP-Rl, resulting in a single amino acid difference (Glu21-»Gln21). This single nucleotide change was confirmed in the two other individual clones by partial sequencing, suggesting that this may be due to a polymorphism in the rat gene. The rat and human GLP-1 receptor cDNAs were the kind gift of Dr. M . B. Wheeler and their isolation and characterization are described elsewhere (Wheeler et al., 1993; Dillon et al. 1993). Both cDNAs were sub-cloned into pcDNA 3 for use in the described experiments. 87 2.1.3 D N A SEQUENCING A N D ELECTROPHORESIS Purified plasmid D N A (1-2 pig) sequence was determined using a T7-Sequencing Kit (Pharmacia) as per the manufacturer's instructions, with the following modifications. Up to 7 uL of the plasmid D N A , 10 pmol of sequencing primer (2 uL), and 2 uL of dimethylsulfoxide (DMSO) were added to a 1.5 mL tube, heat denatured for 3-5 min at 95°C, then snap frozen in a dry ice-ethanol bath. The sample was then thawed and 2 uL of annealing buffer were added. This mixture was centrifuged rapidly (<10s), and incubated at room temperature (RT) for 10 min. Six microlitres of labeling mix (3 uL labeling mixture, 1 uL a 3 5 S-dATP, and 2 uL of T7 D N A polymerase) were added, mixed gently, and incubated at room temperature for 5-10 min. A microtitre plate was prepared with 2.5 uL of termination mixtures for the four nucleotides G, A, T and C, and preheated on a heating block set to 42-50°C. Sequencing mixture (4.5 uL) was added to each of the four termination mixtures, and incubated for a further 10 minutes. The reaction was stopped by the addition of loading buffer, and the samples were stored at -20 °C until separated by electrophoresis on a sequencing gel. Samples were subjected to electrophoresis on a 6% acrylamide, 8 M urea, l X t r i s -borate- disodium ethylenediaminetetraacetate (EDTA) (TBE) buffered gel. The top chamber of the Model S2 sequencing apparatus (GTBCO) was filled with Vi X T B E and the bottom chamber with 200 ml of 1 X TBE. The gel was pre-warmed by running at 50-60 W for 30 min. Samples were then loaded, and subjected to electrophoresis at 60 W for 30 min. at which time 200 mL of 3 M sodium acetate was added to the bottom chamber. Electrophoresis was continued for a further 3-4 h, until the cyanol bromide (second migrating dye in loading buffer) was 3/4 the way down the gel. The gel plates were then 88 separated, the gel absorbed onto 3 M M Whatman filter paper, and dried on a gel dryer. Once dried the gel was exposed to an autoradiograph film for 24-48h, and the sequence was read manually. 2.2 C E L L C U L T U R E A N D C E L L TRANSFECTION Generation of all stable expressing cell lines was carried out by the author. Transient expression experiments were carried out in collaboration with the laboratory of Dr. M . B. Wheeler. 2.2.1 M A M A L I A N C E L L C U L T U R E Green Monkey Kidney (COS-7) and Chinese Hamster Ovary (CHO-K1) cells were cultured in D M E M supplemented with 10% fetal calf serum (FCS), and DMEM/Ham's F12 supplemented with 10% newborn calf serum (NBS), respectively. A l l culture media contained 50 units/mL penicillin G, and 50 ug/mL streptomycin (Culture media and antibiotics from Gibco). Cells were grown in 75 cm 2 flasks until 80-90% confluency, and split 1:10-1:20 for maintenance of exponentially growing cultures. 2.2.2 TRANSIENT TRANSFECTIONS A l l plasmid D N A used in transient or stable transfections was purified using Qiagen Maxi Plasmid Kits (Chatsworth, CA) as per the manufacturer's instructions. The plasmid D N A isolated had A26o/A2go ratios of 1.7-1.8. For transient expression, COS-7 cells (3.0 x 106 cells/dish) were seeded in 10 cm dishes (Becton Dickinson, Lincoln Park, NJ) and cultured in D M E M supplemented with 10% fetal bovine serum (Gibco, Grand 89 Island, NJ). Cells were transfected 48h. later with the appropriate cDNA at concentrations of either 5 ug (for cAMP and C a + + imaging) or 10 ug (for binding experiments), using the DEAE-dextran method (Sambrook et al., 1989). Briefly, 1 mL of Chloroquine in phosphate buffered saline (PBS) (13 mg/mL) was added to 9 mL of a stock solution consisting of 11 mg/mL DEAE-dextran in PBS. To prepare the transfection medium, this solution was filter sterilized (protecting from light) just prior to transfection, and 1.2 mL added to 21.5 mL D M E M plus 2.5 mL Nu-serum (Collaborative Biomedical Products, Boston, MA) . The cDNA was added to 5 mL of transfection media, mixed by inversion, added directly to the cells, and incubated for 3-4 h. The transfection medium was removed by aspiration, and the cells shocked with 2-3 mL of a solution of 10% D M S O in PBS for 1.5 min, then allowed to recover overnight in 5-7 mL. of culture medium. 12-16 h following transfection, the cells were passed into 6, 12, or 24 well plates (for cAMP or C a 2 + imaging studies), or 10 cm dishes (for binding or C a 2 + imaging studies), and cultured for an additional 48 h before experiments were conducted. 2.2.3 S T A B L E T R A N S A C T I O N S Production of permanent (stable) CHO-K1 cell lines expressing the GD? receptor was carried out using the CaP0 4 co-precipitation method of Sambrook et al.(1989), with minor modifications. Cells were grown on 10 cm dishes until approximately 90% confluent. Fresh culture medium (5-7 mL) was added to the cells at least 3 h before the transfection. Ten micrograms of the cDNA was added to 62.5 uL of 2.5 M CaCfe (filter sterilized) and the total volume was brought up to 500 pi with sterile dH-20. To this 90 solution, 500uL of 2 X N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-Buffered Saline (HBS) (280 mM NaCI, 10 mM KCI, 1.5 mM N a 2 H P 0 4 , 12 m M Dextrose, and 50 mM HEPES, pH 6.95) were added. The mixture was vortex mixed briefly, and incubated for 30 min at room temperature to allow a precipitate to form. The D N A / H B S / C a 2 + mixture (1 mL/plate) was added directly to plates containing 5-7 mL of media. After a 3.5-4h incubation, cells were then shocked for 1.5 min in 15% glycerol/1 X HBS, and allowed to recover for 12-16h. The cells were then split onto two dishes and those expressing pGIP-Rl, or other constructs, were isolated by G418 selection (800 ug/mL) (Gibco), changing the media every 2-3 days as needed. After 10-12 days individual clones were isolated and further selected for high level expression by screening for GIP stimulated cAMP production and 1 2 5I-GIP binding as described below. One clone, designated wtGTP-Rl, was selected for further characterization. It was determined later that a pooled pGIP-Rl expressing cell line, isolated and maintained under high stringency selection (800 pg/ml), resulted in high level expression and maximal cAMP similar to the levels seen in the original wtGIP-Rl clone. In later studies pooled clones were obtained to reduce the selection time. 2.3 BINDING ANALYSIS 2.3.1 1 2 5I-GTP PREPARATION 1 2 5I-GJP used in receptor studies was prepared by the chloramine T method of Kuzio et al (1974), and purified by gel filtration and High Performance Liquid Chromatography (HPLC) as described by Verchere (1991), with minor modifications. Porcine GIP, 5 pg in a silconized test tube, was dissolved in 100 uL of 0.4 M phosphate 91 buffer (pH 7.5), and 10 uL (lmCi) of N a 1 2 5 L and 10 uL of chloramine T (4 mg/mL in 0.4 M phosphate buffer, pH 7.5) added. The reaction was stopped 15s later by the addition of 20uL sodium metabisulphite (14.8 mg/ml in 0.4 M phosphate buffer, pH 7.5). The mixture was then applied to a column (0.5 X -10 cm) of Sephadex® G-15 (Pharmacia), prepared in a 10 ml disposable pipette and equilibrated for 4-6h in 0.2 M acetic acid containing 2% RIA grade bovine serum albumin (BSA). On the day of the iodination 2% aprotinin (Trasylol™, Sigma) was added to the column buffer, and equilibrated for at least l h before the iodination mixture was applied. H P L C purification of the peak 1 2 5I-GTP fractions from the gel filtration chromatography was performed on uBondapak C-18 column (Walters Associates Inc., Milford, M A ) using 2 Beckman HOB solvent delivery module pumps with a programmable Beckman model 421A controller, and a model 170 Radioisotope Detector (Beckman Instruments Inc., San Ramond, CA) to monitor radioactivity eluting from the column. Separation of the different 1 2 5I-GTP species was accomplished with a gradient of CH3CN in water containing 0.1% trifluoroacetic acid run over 35 min. Fractions were injected using a needle syringe (Hamilton CO., Reno, NV) , washed on to the column in 31% CH3CN. This concentration was maintained for 10 min, following which a linear gradient to 38% CH3CN was run over 10 min. The column was then washed by increasing the C H 3 C N concentration to 70% over 5 min, where it was held for 5 min, and then returned to 31% over 5 min. The second major peak eluting at approximately 18 min post-injection was collected from multiple runs in a siliconized tube with 0.5 ml of 2.5% RIA grade B S A and 50% apoprotin. The amount of radioactive label purified was determined in a y-counter, and aliquots of 3-6 X 106 cpm were lyophilized and stored at -92 20°C until use. The specific activity of the label was determined to be approximately 250-350 uCi/ug using a homologous displacement assay as previously described (Verchere, 1991). 2.3.2 TRANSIENT BINDING EXPERIMENTS Seventy two hours post transfection COS-7 cells were detached from dishes using phosphate buffered saline (PBS) containing 0.1 mM disodium ethylenediaminetetra-acetate (EDTA), washed twice in binding buffer (DMEM, supplemented with 20 m M HEPES, 0.1% bovine serum albumin (BSA), 0.05 mg/ml bacitracin, pH 7.4) (BB1) and preincubated for 30 min at 37°C. Cells (5 X 105/well) were then incubated for 30 min at 37°C with 50,000 cpm of radiolabeled peptide (approximately 90-130 pM), in the presence or absence of unlabeled peptide, in a final volume of 200 pi. After incubation, cell suspensions were centrifuged at 12,000 x g, washed once in ice cold binding assay buffer, and the cell associated radioactivity in the pellet measured in a y-ray counter. 2.3.3 S T A B L E C E L L - L I N E BINDING STUDIES While some initial studies were carried out as above, most whole cell studies with CHO-K1 cells stably expressing pGTP-Rl, or other receptor constructs, were carried out with the cells attached in 24 well culture plates. Cells were washed 2X with binding buffer (BB1) with the same composition as BB1, apart from DMEM/Ham's F12 replacing D M E M . In initial studies, involving comparison of synthetic GIP preparations, cells were incubated for 1 h at either room temperature (RT) or 37°C, and saturation binding experiments with both whole cells and membranes were incubated for 4-6h at 93 4°C, with 50,000 cpm I 2 5I-GTP in the presence or absence of cold competing peptide. In subsequent studies, cells were incubated at 4°C for 12-16h to ensure that steady state conditions for all levels of receptor expression were achieved. At the end of the incubation period cells were washed 2X with ice cold buffer, solubilized with 0.1M NaOH (0.5 mL), and transferred to test tubes for counting of cell associated radioactivity (Wheeler et al., 1995). Non- specific binding was defined in all experiments as the cell associated activity observed in the presence of 1 u M GD?. Cell numbers were determined by counting cells trypsinized from control wells, in a haemocytometer, at the end of the experiments. Cell membranes were isolated as described by Samama et al (1993). wtGD?-Rl cells in two 1500 cm 2 tissue culture roller bottles (Becton Dickinson) were grown until confluent. Cells were then washed twice in PBS, detached in 0.1 m M EDTA/PBS, and sedimented by centrifugation at 1200 R P M at RT. Cells were then resuspended in 10 mL of buffer A (5mM Tris-HCl, pH 7.5, 5mM EDTA) and re-sedimented by centrifugation at 40,000 X g. The pellet was resuspended in 3 ml of buffer A, homogenized in a Teflon-glass homogenizer, and centrifuged a second time. The pellet was resuspended in 3mL of the same buffer and the protein concentration determined using the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL). Approximately 26 mg total protein were obtained, diluted in buffer A to a concentration of approximately 2 mg/mL, and stored in aliquots at -80°C until use. Saturation binding studies with membranes or cells used a similar protocol and differed only in the buffers used. Whole cell experiments were carried out in the binding buffer described above, while membrane experiments were done in 50 m M Tris-HCl, pH7.2, 5 mM M g C l 2 , 1 m M EDTA, 0.1 % RIA grade BSA, 94 and 0.1 mg/ml bacitracin (BB2). Transfected CHO-K1 cells (3-8 X 105 cells/well), or 100 ug protein/tube were incubated with increasing amounts of radiolabeled peptide (-700-1.4 x lO 6 cpm) added, and allowed to incubate at 4°C for 4-6h. Cells and membranes were then centrifuged at 12000 X g and the pellet washed 2X with ice cold BB1 or BB2, respectively, and the pellet-associated binding determined. Total binding and the nonspecific binding at each concentration tested were determined as described above for cellular binding. 2.3.4 BINDING ANALYSIS In all experiments specific binding was analyzed using the nonlinear regression analysis program Prism (GraphPad, San Diego, CA). Saturation binding analysis compared data fitted to both one and two site models using the following equations: One site model: Y= Bmax*X/(Kd+X) Two site model: Y=Bmaxl*X/(Kdl+X) + Bmax2*X/(Kd2+X) Both equations describe the binding of a ligand to a receptor that follows the law of mass action where X is the concentration of free ligand, Y is the specific binding measured, Bmax is the maximal binding, and Kd is the concentration of ligand required to reach half-maximal binding (in either cpm or molar concentration) (Fig. 6). In the two-site model K d l and Kd2 are the concentration of ligand required to reach half-maximal binding for two different receptor populations with different affinities for the ligand. In 95 both models it is assumed that the reaction has reached steady-state; only a small fraction (less than 10% in practice) of the labeled ligand is bound and therefore the free concentration is essentially the same as the original concentration added to the reaction; and that there is no cooperativity in the binding, ie. the binding of ligand to one site does not effect the affinity at another site. Data from competitive binding experiments were fitted to both single and two site models using the following equations: Competitive one site Model: Y= Bottom + (Top-Bottom)/(l+10(X-LogIC5o)), Competitive two site model: SPAN=Bottom+Top Partl=Span*Fractionl/(l+10(X-LogIC5ol)) Part2=Span*(l-Fractionl)/(l+10(X-LogIC502)) Y=Bottom + Parti +Part2 Where Y is the bound label and X is the log value of the concentration of unlabeled competitor. Fraction one is the fraction of all sites that display affinity 1, and fraction two is the population of receptors with affinity 2. Span refers to the difference between the Bmax (Top) (maximal binding in the absence of competitor), and the bottom plateau (Bottom), or NSB (see Fig. 6B.). As the specific activity of 1 2 5 I-GIP was not determined for every labeling, IC50 values (inhibitory concentration displacing half the Bmax) are presented. The same assumptions made for saturation binding analysis are made in 96 competitive binding analysis. Idealized binding curves for saturation and competitive binding experiments are shown in Fig. 6. In both saturation and competitive binding experiments, the program compared the fit of the results to both the appropriate one and two site models. The program used an F-test to compare the goodness of fit of the two equations, and the simpler equation was assumed to be correct i f an F ratio corresponding to a P value of 0.05 or greater was obtained. 97 A . c Bmax-c m o 0-1 i r 0 Kd Concentration of Free Ligand B. D) bottom-1 i 1 0 IC50 log [Concentration of Competitor] Fig. 6. Idealized Binding Curves for Saturation (A.) And Competitive (B.) Binding Studies to a Single Binding Site. The K d is the concentration of labeled ligand required to give half-maximal binding, while the I C 5 0 is the concentration of cold ligand required to inhibit half of the maximal binding of labeled ligand. See text for details. 98 2.4 M E A S U R E M E N T S OF cAMP PRODUCTION COS-7 cells (24 hours after transfection) and CHO-K1 clones expressing GJP-R1 were passed into multiwell plates and cultured for an additional 48 h. Cells were then washed in assay buffer ( D M E M for COS-7 and DMEMZHam's F12), containing 0.1% B S A and 0.05% bacitracin, and preincubated for 30-60 min, followed by a 30 min stimulation period with test agents at appropriate concentrations. In all transient experiments with COS-7 cells the stimulation media contained 1 m M isobutylmethyl-xanthine (IBMX) to prevent cAMP breakdown by phosphodiesterases and allow measurement of total cAMP production. In initial studies (as noted in the text) with CHO-K1 cells, I B M X was not included in the stimulation buffer as it was originally thought that the availability of a homogeneous population of cells expressing high levels of the GIP receptor would produce very high and consistent levels of cAMP. However, while initial studies demonstrated that GIP stimulated cAMP production in a concentration dependent manner, the variation in maximal cAMP levels obtained and estimated EC50 values varied greatly day to day. In later studies it was found that the addition of 1 m M I B M X in the stimulation media and normalization of cAMP levels to fmol/1000 cells gave highly consistent EC50 values. Maximal cAMP levels while more consistent in the presence of I B M X did vary over time, and this did not appear to correlate with passage number (both increasing and decreasing at later passages). For this reason experiments were conducted over a short a period of time as possible (30-60 days), and maximum levels were normalized to % of wtGIP-Rl maximal levels. 99 Following the stimulation period, cells were extracted in 0.5-lml of 70% ethanol, transferred to Eppendorf tubes, centrifuged at 7000 rpm at 4°C to remove the cell debris, and the supernatant transferred to a second tube. Samples were then dried in a Speed-vac for a time of 6-18h and stored at -20C until they were assayed. On the day of the assay, samples were reconstituted in 0.5 ml 0.5 M sodium acetate buffer and cAMP levels were determined using a cAMP radioimmunoassay (RIA) kit (Biomedical Technologies Inc., Stoughton, M A ) as per the manufacturer's instructions. 2.5 CYTOSOLIC C a 2 + M E A S U R E M E N T S Cytosolic calcium measurements in the initial characterization of the rat islet GD? receptor signaling were carried out by Dr. M . B . Wheeler and Dr. J. Georgiou in the Departments of Medicine and Physiology, at the University of Toronto. Later studies on characterization of the GIP receptor C a 2 + signaling in CHO-K1 cells and the point mutant H170R (see section 3.4, Appendix A) in COS-7 cells were carried out at the University of British Columbia by Dr. P. Squires. These results were important for establishing GD? receptor linked C a 2 + signaling and, subsequently, in identifying the effect of cellular environment and receptor mutation on this signaling pathway. As these results were important for discussion of the results presented in this thesis, but the author did not perform the C a 2 + measurements himself, the methods and results are included in Appendix A. 100 2.6. RAT PANCREAS PERFUSIONS The rat pancreas perfusion assays were performed with the assistance of Dr. Raymond Pederson as previously described (Pederson et al., 1982). Briefly, overnight fasted rats were anaesthetized (60mg/kg pentobarbital) and the pancreas and associated duodenum isolated. Perfusate consisted of a modified Krebs-Ringer buffer containing 3% dextran (Sigma) and 0.2 % BSA, gassed with 95% 0 2/ 5% C 0 2 to achieve a pH of 7.4. The peptide preparations were delivered as a linear gradient of 0-1 nM over a 45-50 minute period in the presence of 16.7 mM glucose. Immunoreactive insulin was determined using an established RIA as previously described (Pederson et al., 1982). Results are expressed as the mean integrated insulin response in mU over the 50 min. perfusion time. 2.7 SOURCES OF PEPTIDES Synthetic peptides were obtained from the following commercial or private sources. Peptides used for comparing the biological activity of different GIP preparations and determining receptor specificity were obtained from: Peninsula Laboratories (Belmont, CA): synthetic human (sh) GIP Lot #9408164, synthetic porcine (sp)GIP Lot #033785, GLP-1 (7-37) Lot #019860, shGLP-2 Lot #008674, vasoactive intestinal peptide (VIP) Lot #015174; Novo Biolabs (Bagsvaerd, Denmark): porcine glucagon Lot #G4211963; Bachem California (Torrance, CA): shGIP Lot #ZK887, spGIP Lot #758C, exendin-4 (Ex-4) Lot #ZL765, exendin (9-39) (Ex-9-39) Lot #ZL777. Natural porcine (np) GIP was purified as described elsewhere (Brown, 1971). ShGIP 1-30 free acid (OH) and spGIP 17-30 were the kind gifts of Dr. N. Yanaihara (Shizuoka, Japan) and Dr. S. St. 101 Pierre (ENTRS Sante, Montreal, Canada) respectively. SpGIP 19-30 was prepared by tryptic digestion of spGIP 17-30, and purification of the major product by reverse phase (RP-) HPLC. The identities of the peptides were confirmed by sequence analysis. The peptides shGIP 18-21, shGD? 21-28, GLP-1 (7-36) 21-28 and glucagon 21-28 were synthesized by solid-phase techniques by the Nucleic Acid-Protein Service Unit, U B C , and purified by RP- HPLC. As it was determined that both sh and spGIP were equipotent and had identical affinity for the rat islet GD? receptor, all further peptides for investigations were based on the human sequence, and shGD? preparations were obtained from Bachem (Torrence, CA.) or Hukabel (Montreal, QUE). Dr. T. O'Dorisio, S. Cataland and O. Succek (University of Ohio, Columbus) kindly provided GIP 10-30. GD? 7-30, D-Ala2-Gn», D-Ala 2 -GLP-1, and GIP 3-42 were obtained from Hukabel (Montreal, QUE). GIP 1-30amide, GD? 6-30amide, D-Ala 2-GIP l-30amide, D-Tyr^GD? l-30amide, Desamino-Tyr^GD? l-30amide, D-Glu3-Gn> l-30amide, and D-Ala 4-GD? l-30amide were synthesized by Dr. D.H. Coy (Peptide Research, Department of Medicine, Tulane School of Medicine, New Orleans, L A , USA). A l l peptides were shown to be homogeneous on H P L C and of the correct molecular weight by MALDI-TOF Mass Spectrometry. 2.8 SITE-DIRECTED MUTAGENESIS OF THE R A T ISLET PANCREATIC GIP RECEPTOR The experimental design of mutant forms of the receptors was carried out in collaboration with Dr. Michael B. Wheeler. Several different methodologies were utilized, and specific constructs and the methodologies used for their production are described below. 102 2.8.1 OLIGONUCLEOTIDE PHOSPHORYLATION Non-PCR based oligonucleotide-directed mutagenesis protocols require that the 5' end of the mutant oligonucleotide is phosphorylated to allow the ligation of the 5' termini of the synthetic oligonucleotide in the extension and ligation reaction step (see below). In all protocols utilized below the same basic protocol was used to phosphorylate the primers: 1-2.5 pg of the mutagenic oligonucleotide in 1 X kinase buffer (70 m M Tris-HC1, pH 7.6, lOmM MgCl 2 ) plus 1 mM rATP was incubated with 10-25 U of T 4 polynucleotide kinase at 37°C for lh. Incubating the mixture at 65°C for 10 min. terminated the reaction. 2.8.2 CONSTRUCTION A N D EXPRESSION OF H170R Double stranded oligonucleotide-directed mutagenesis was utilized to generate a point mutation in the GIP receptor resulting in the substitution of an arginine at position 170 in place of the endogenous histidine residue. This mutation in the related PTH/PTHrP receptor had been shown previously to result in a receptor with constitutive signaling activity (Schipani et al., 1995, 1996). A commercial kit (Morph™ Site-Specific Plasmid D N A Mutagenesis Kit, 5-prime 3-prime, Boulder, CO) utilized two steps allowing the use of double stranded template, while obtaining an acceptable rate of mutagenesis. The mutagenic primer, corresponding to coding nucleotides 501-520 (5'-A T T A C A T T C G C A T G A A C C T G - 3 ' ) , was designed with a single base change (underlined) at position 510 (A->G) resulting in the amino acid substitution of an 103 arginine for histidine at amino acid residue 170. The protocol is described briefly below. A l l materials, except primers and template, were from 5-prime 3-prime (Boulder, CO). 1. Double stranded template (-30 fmol; pBKS-/GTP-Rl) and 100 ng of the 5'-phosphorylated mutagenic primer, in a total volume of 20 ul 1 X MORPH™ Annealing buffer, were denatured at 100°C for 5 min, cooled in an ice water bath for 5 min, and incubated at RT for 30 min. 2. A replacement strand was synthesized in vitro resulting in a mixture of hemi-methylated half-mutant plasmid and, fully methylated wild type (wt) target plasmid (Fig.7). The reaction conditions were as follows: 8 uL of 3.75 X MORPH™ Synthesis buffer (buffered 4 dNTP's and rATP solution) along with 3U of T 4 D N A polymerase and 4U of T 4 D N A ligase were added to the primer template mixture and incubated at 37°C for 2h. Heating the reaction to 85°C for 15 min. stopped the reaction. 3. The next step involved the addition of the restriction enzyme Dpn I, and digestion of the mixture for 30 min. The preference of Dpn I for the fully methylated target plasmid D N A resulted in the preferential degradation of the wild type target D N A into linear D N A of low transformation efficiency, while the hemi-methylated heteroduplex mutant D N A was left in the circular form, and "transformation competent". This eliminated much of the non-mutant dsDNA that would increase 104 the number of wild type progeny in the subsequent transformation into an E. coli host. 4. Immediately after the 30 min. digestion the entire reaction volume was transformed into the MORPH mutS" cells. The use of a strain of E. coli (mutS") deficient in methyl-dependent DNA repair system prevented the normal tendency to identify the methylated strand as correct and a higher loss of mutant than non-mutant plasmid in the propagation step. In these studies approximately 40-60% colonies isolated and screened carried mutant plasmids. Six clones containing the mutation were identified by dideoxy-sequencing, two of which were subcloned into the Hind IWXba I site of pcDNA 3 for expression. 105 Double-stranded Target plasmid DNA Non-methylated and phosphorylated oligonucleotide Denature target plasmid Anneal to mutageneic oligonucleotide Non- mutagenized target plasmid T4 DNA polymerase T4 DNA Bgase dNTP's -X-. . . Mutagenized pkmid DNA (Hemi-methyhted) Dpn I Digestion for 30 min. ] Hemi-methylated mutated plasmid is resistant to Dpn I digestion. Transform E. coli mulS strain and plate. Screen Colonies for mutants Fig. 7. Double-stranded D N A Mutagenesis. Generation of the GIP H170R receptor mutant was carried out using the commercially available mutagenesis kit (Morph™ Site-Specific Plasmid D N A Mutagenesis Kit, 5-prime 3-prime). See text for detail. 106 2.8.3 CONSTRUCTION A N D EXPRESSION OF CHIMERIC RECEPTORS The rGTP-R and hGLP- lR wild type (wt) receptor cDNAs were cloned into the expression vector pBKS' (Stratagene, San Diego, CA) for modification. Receptor cDNAs were endonuclease-digested utilizing common endogenous sites or sites introduced into homologous regions (see below for specific constructions) to facilitate chimeric construction. Recombinant receptors were generated by ligating the N-terminal fragment of one receptor to the C-terminal fragment of the other into the Hind IWXba I site of pcDNA 3 for expression (Invitrogen, San Diego, CA). Introduction of mutations and the generation of common restriction sites were performed using the strand selection technique developed by Kunkel (1985), and two different PCR based methodologies. The chimeric forms generated and the methodologies used are described below: 2.8.4 U S E OF ENDOGENOUS RESTRICTION SITES Several chimeric receptors did not require the introduction of non-endogenous occurring restriction sites. CH-1: generated using the Kpn I site at coding nucleotide 264 in both receptors, resulting in a receptor encoding the first 88 amino acids of the GIP receptor and residues 99-463 of the human GLP-1 receptor. CH-4 and CH-6: N-terminal regions of the receptors, consisting of the extracellular NT domain, first E C loop, T M domains 1 and 2, and part of 3, and the first intracellular loop, were exchanged using the common Sea I restriction sites at nucleotide 664 in rGIP-R and nucleotide 703 in hGLP-1R (Fig. 8). The general methodology is outlined in Fig. 8, and the cartoons of the individual receptors are presented in Fig. 9. 107 2.8.5 SINGLE STRANDED OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS WITH STRAND SELECTION This methodology of ssDNA oligonucleotide mutagenesis originally described by Kunkel (1985) utilizes E. coli strains that are dUTPase (dut-) and uracil N-glycosylase (ung-) deficient, and is outlined in Fig. 10. These strains have increased levels dUTP due to the lack of functional dUTPase, resulting in the incorporation of dUTP rather than dTTP into the replicating D N A . Due to the lack of uracil N-glycosylase, the spuriously incorporated dUTP molecules are not removed by normal D N A repair mechanisms. M l 3 derived plasmids with target sequences can be transformed into a duflung' strain (such as CJ236) to generate dUTP containing ssDNA template. This template can then be used to synthesize a mutant heteroduplex molecule in the presence of dTTP, which is then transformed into an ung+ strain. The uracil containing parental strand is selected against, while the mutant strand is preferentially replicated, increasing the number of mutant progeny to as high as 80%. The following chimeric receptor cDNAs were produced utilizing this methodology. 1. CH-2 and CH-5: The putative NT domain of h G L P - l R was replaced by all but the two distal amino acids of the rGIP-R NT, and vice versa, by introducing a Xho I restriction site at nucleotide 392 in GIP-R and nucleotide 424 in GLP-1R (Fig. 11). This modification resulted in no amino acid changes in the 108 rGIP-R hGLP-1 H i n d HI Xba k S e a l S e a l Digest with H i n d H U S c a l H i n d m I k S e a I H i n d m H i n d l R Digest with X b a U S c a I 1 Xbal S e a I pcDNA3 or pBKS cut with Hindni/Xbal X b a l Ligation S e a I Fig. 8. Construction of Chimeric GIP/GLP-1 Receptors Using Endogenous Restriction Sites. When possible restriction sites common to the GD? and GLP-1 receptors were utilized to construct cDNA's encoding chimeric receptors. Generation of the construct CH-4 was accomplished by utilizing common Sea I sites in the two receptor cDNAs. 109 wtGIP-Rl w t G L P - l - R l 223-455 Fig. 9. Predicted Topography of Chimeric Receptors Constructed Using Endogenous Restriction Sites. Regions corresponding to G I P - R are in blue, those o f the GLP-1 -R are in red. See text for details 110 wtGIP-Rl, and a Phe to Glu substitution at amino acid 143 in the wtGLP-1 receptor. The latter modification to the GLP-1 receptor was shown to generate a receptor that was functionally similar to the wtGLP-lR. 2. CH-3: constructed by introducing a Nhe I site at nucleotide 480 of the hGLP-1 cDNA (Leu to He substitution at amino acid 161), allowing ligation of the N -terminal Hind III/Nhe I fragment of rGJP-Rl to the carboxy-terminus of hGLP-Rl (Fig. 11). 2.8.6 P O L Y M E R A S E C H A I N REACTION MUTAGENESIS The method of Vallette et al. (1988) was used to introduce silent Bss H H sites at coding nucleotides 539 and 567 by introducing a single nucleotide change of A—»C at codons 180 and 190, of GIP-R1 and GLP-R1 respectively. Full-length clones, with the introduced Bss HIT restriction site, were obtained as the result of three rounds of the polymerase chain reaction (see Fig. 12). The Expand High Fidelity P C R system (Boehringer Mannheim, Laval, Quebec), a commercial mixture of two thermal D N A polymerases, Taq and Pwo, was used to reduce the occurrence of PCR mutations 1. Round 1: 100 pmol of an appropriate mutagenic primer (A) and the vector specific flanking primer (B; 5'-G G A G T A C T A G T A A C C C T G G C C C C A G T C A C G A C G T T G T A A - 3 ' ) (Fig. 12), ~1 fmol of pBKS-/GIP-Rl or pBKS7hGLP-l R template, 200 p M dNTPs, and 2.6 U of Expand High Fidelity PCR system were added together in a total volume of 50 pi, in I X Expand Buffer, supplemented with 2 m M MgCU (Boehringer 111 Mannheim) and cycled 25 cycles: 30s denaturation at 95°C, 30s anneal at 58°C, and a 1.5 min extension at 72°C. Buffer conditions did not change in any of the following steps. 2. Round 2: -600 fmol (200 ng) of gel purified Round 1 PCR product used to linear amplify 1 fmol of the same template from which it was derived, under similar conditions: 2 min denaturation at 95°C, 30s anneal at 55°C, and 2 min extensions at 72°C. 3. Round 3: 100 pmol of primers C (5 ' - C G A G A A A C A G C T A T G A C C AT-3') and D (5 ' -GAAGTACTAGTAACCCTGGC-3 ' ) (Fig. 12) were added to the Round 2 reaction mixture and cycled 25 times as follows: 30s denaturation at 95°C, 30s anneal at 55°C, and 2 min extensions at 72°C. The PCR products of three individual reactions were gel purified, and restriction digested with Hind YWXba I and subcloned into pcDNA 3 for expression, and further manipulation. Clones were transiently expressed, and binding studies were performed as a quick screen for extraneous PCR errors prior to use in generating chimeric receptors. cDNAs used to construct both CH-7 and CH-8 were sequenced on one strand to confirm no other PCR errors occurred. The N-terminal Hind m/BssH. II fragment of the GIP-R1 cDNA was ligated to the GLP-R1 BssH IVXba I fragment (CH-7) in the Hind Ill/Xba I sites of pcDNA 3 for expression and sequencing (Fig. 13). The inverse of this construct was also generated in a similar way and designated CH-8 (Fig. 13). 112 SS DNA with uracil incorporated M13 replicative form Transform dut-/ung-strain of E. coli Cloned gene of interest Add oligonucleotide Synthesize second strand Transform ung+ E. coli Most wt uracil containing DNA is selected against. Mutated form is not degraded. Isolate and screen plasmids for mutation. Fig. 10. Single Stranded Oligonucleotide-directed Mutagenesis with Strand Selection. The method originally described by Kunkel (1985) was used to generate chimeric receptors CH-2, CH-5 and CH-3. See text for details. 113 wtGIP-Rl w t G L P - l - R l 162-463 Fig . 11. Predicted Topography of Chimeric Receptors Constructed Using Single Stranded Oligonucleotide-directed Mutagenesis. Regions corresponding to GIP-R are in blue, those of the G L P - l - R are i n red. See text for details 114 mutagenic primer (A) — Target cDNA 25 cycles of PCR. ^ -M- -^primer B vector ^ sequence unique primer sequence Round 1 PCR product. Gel purify use to linearly amplify target cDNA. 1-3 rounds: Denature, 95°C for 2 min anneal, 55PC for 30s, and extend at 72°C for 2 min. \ \ Add vector specific primer C and unique sequence specific primer D to round 2 reaction. Primer C cycle 25 more times; 30s denaturation at 95°C, 30s anneal at 55°C, 2 min extensions at 72°C Primer D _A_ Digested with Hind III/Xba I and subcloned into pcDNA 3 (Invitrogen) for expression and sequencing to confirm mutation and cDNA integrity. Fig. 12. PCR Based Oligonucleotide-directed Mutagenesis. A PCR based strategy was used to introduce BssH II restriction sites into both the GIP and GLP-1 receptor cDNAs. This modification was used to construct the chimeric receptors C H - 7 and CH-8 and are shown in Fig. 13. 115 wtGIP -Rl w t G L P - l - R l 191-463 181-455 Fig. 13. Predicted Topography of Chimeric Receptors Constructed Using Polymerase Chain-reaction Oligonucleotide-directed Mutagenesis. Regions corresponding to GIP-R are in blue, those of the GLP-1-R are in red. See text for details 116 The second methodology used PCR to introduce a type II restriction enzyme recognition sequence (Earn 1104 I) at the 5' and 3' end of PCR fragments. The fact that this Type II restriction enzyme cleaves at regions outside its consensus sequence allows the production of sticky D N A fragments with sticky ends unrelated to the recognition sequence. This allowed the two cDNAs of interest to be ligated together at structurally homologous regions (i.e. transmembrane domains), requiring only one conserved amino acid at the point of ligation (see Fig. 14). A mutagenesis kit (Seamless™ Cloning Kit, Stratagene) was used as described below. 5'P-Gal 5' - AT ACTCJTTC A C C A T G A T T A C G C A A G C G C - 3 ' 3'P-Gal 5' - A T ACTCTTC A T G G T C A T AGCTGTTTCCTG-3 ' GIP Phel59 5' - A T A C T C T T C T G A A C A A A C T T A A A A T G A G T - 3 ' GLP Phel69 5' - A T A C T C T T C C T T C A G A C A C C T G C ACCTGC-3 ' Fig. 14. Primers Used In the Construction of CH-9. Primers were designed with three random nucleotides, Earn 1104 I sequences (underlined), followed by receptor specific sequences one nucleotide prior to the codon (bold) used for ligation (Fig. 14). Two other primers, designed with vector specific sequences (5' and 3'P-Gal), were also used in conjunction with one of the receptor specific primers as shown in Fig. 15. A recipient (GLP-R1 carboxy terminal amino acids 169-463 plus vector) and insert (GIP-R1 NH3+-terminal to amino acids 1-159) PCR products were obtained as follows: 10-15 fmol of target vector (pBKS/GIP-Rl or GLP-R1) in 1 X Pfit Buffer, 200 p M of each dNTP, 20 p M of each of the appropriate primers (see Fig. 14 and 15), and 2.5 117 \] Pfu polymerase were cycled as follows: one cycle 95°C for 3 min, 58°C for lmin, and 72° for 6 min (GLP-R1 reaction) or 1 min (GIP-R1 reaction), followed by 12 cycles of 95°C for 45s, 58°C for 45s, and 72° for 6 min (GLP-R1 reaction) or 1 min (GIP-R1 reaction). Pfu polymerase was used as it is a "proof reading" polymerase (having 3"-5' exonuclease activity) with a lower error rate than Taq. This was followed by the addition of 50 ul of one times Pfu buffer containing 200 u M dATP, dGTP, dTTP, and 1 m M 5-methyl (m 5)dCTP, and the reactions were cycled 5 more times as above. PCR products where then extracted once with phenol-chloroform (1:1), and precipitated with 0.1 vol of 3 M sodium acetate and 2.5 vol of ice cold ethanol (98%). The pellet was resuspended in 50 ul of TE (10 m M tris-HCl, pH 7.5, ImM EDTA) and run on agarose gels to estimate PCR product concentration. 10 ul of each PCR product in I X Universal buffer (Stratagene), were then digested with 24U of Earn 1104 I in a total volume of 50 ul, at 37°C for 1 hour. The presence of the m 5 d C T P in the PCR products renders endogenous Earn 1104 I sites that exist within the vector or target sequences resistant to digestion, while the non-methylated sites within the primers can be digested. Five-fifteen ul of this digestion mixture were then ligated in 1 X ligase buffer, with ImM rATP, 0.25 U T 4 Ligase (Stratagene), and 4U of Earn 1104 I in a total volume of 20 ul, at 37°C for 30 min. 2-5 ul of the ligation reation were transformed into Epicurian Coli X L 1-Blue M R F ' (Statagene) supercompetent cells. Only one clone corresponding to the expected ligation product was obtained. It was cloned into pcDNA 3 (Invitrogen) for expression and sequencing, and was designated CH-9 (Fig. 16). 118 GIP-R1(1-159) GLP-1 RI (170-463) Fig. 15. PCR Based Strategy for the Construction of CH-9. Primers containing Earn 1104 I sites allowed the generation of 3 base pair overhangs 4 nucleotides from the recognition sequence. This allowed the two cDNAs to be joined at a single conserved amino acid. 119 w t G I P - R l w t G L P - l - R l CH-9 1-159 170-463 Fig. 16. Predicted Topography of the Chimeric Receptor CH-9. CH-9 was constructed using a polymerase chain-reaction oligonucleotide-directed mutagenesis methodology. Regions corresponding to GIP-R are in blue, those of the GLP-1 -R are in red. See text for details 120 2.8.7 GENERATION OF C A R B O X Y - T E R M I N A L T R U N C A T E D FORMS OF THE R A T GIP RECEPTOR Preparation of cDNAs encoding truncated forms of the GJP-R1 utilized primers directed to specific regions of the CT tail of the receptor designed to introduce a stop codon at the site of interest (Fig. 17). These primers were used with a common 5' primer specific for the beginning of the coding region (5 ' -AGGATGCCCCTGCGGCTGTTGC-3') to generate cDNA encoding truncated forms of the receptor. 1255-1236, 5 ' -CTACTGCCCCAGGTGCGGACGTG-3 ' (418) 1216-1196, 5 ' - C T A G A G A C G C A G A C G G C G G A T C T C - 3 ' (405) 1198-1175, 5 ' -CTAGATCTCCGACTGTACCTCTTTGTTGAT-3 ' (400) 400ala5, 5 ' -CTATGCTGCTGCTGCTGCGATCTCCGACTGTACCTCTTTGTT-3 ' 386ala9, 5' CT A T G C T G C T G C T G C T G C T G C T G C T G C T G C T A C C T C T T T G T T G A T G A A G C A G -3' Fig. 17. Primers Used In the Construction of Carboxy-Terminal Truncated GIP Receptors. A l l PCR products were initially cloned into pCR U (Invitrogen), before subcloning into the Hind DT/ Xba I, or Not II Nsi I sites of pcDNA 3 (Invitrogen) for sequencing and expression. One other truncated receptor was generated by digesting the wt rGrP-R in the vector Bluescript-KS with Sad at nucleotide number 1278 and religating with the vector's Sac I site prior to insertion into the expression vector. The resulting construct 121 encoded the first 427 amino acids of the GIP receptor plus 6 unrelated amino acids (QRVGCI) encoded for by the vector sequence and was designated GIP-R-427+. A construct with the membrane proximal residues 397-400 deleted (GIP-R-AQSEI) was generated using the ssDNA oligonucleotide mutagenesis strategy described in section 2.7.5. Figures 18 and 19 illustrate the CT-tail truncation and modifications resulting from the mutagenesis. 122 393 TM7-NKEVQSEIRRLRLSLQEQCPRPHLGQAPRAVPLSSAPQEAAIRNALPSGMLHVPGDEVLESYC GIP-R-400-QSEI GIP-R-405-QSEIRRLRL GIP-R-418-QSEIRRLRLSLQEQCPRPHLGQ GIP-R-427+-QSEIRRLRLSLQEQCPRPHLGQAPRAVPLSSQRVGC1 \ Fig. 18. Carboxy-terminal Truncation of the Rat Islet GIP Receptor. The single letter code for amino acids is used. Four cDNAs were constructed encoding forms of the rat GIP receptor carboxy-terminal truncated at the indicated residues. Serine residues, which are potential phosphorylation sites, are in red text. The six residues resulting from vector sequence in construct GIP-R-427+ are underlined. See Text for details. 123 TM7-NKEVQSEIRRLRLSLQEQCPRPHLGQAPRAVPLSSAPQEAAIRNALPSGMLHVPGDEVLESYC GIP-R-396A9 = TM7-N KE VAAAAA AAAA GIP-R-400A5 = TM7-NKEVQSEIAAAAA GIP-R-AQSEI = TM7-NKEVRRLRLSLQEQCPRPHLGQAPRAVPLSSAPQEA AIRNALPSGMLHVPGDEVLESYC Fig. 19. Modified Carboxy-Terminal Tail forms of the Rat Islet GIP Receptor. The single letter code for amino acids is used. Serine residues, which are potential phosphorylation sites, are in red text. TM7 = transmembrane region 7. See Text for details. 124 2.9 RECEPTOR DESENSITIZATION STUDIES. In desensitization studies, the CHO-K1 cell line wtGIP-Rl was preincubated in the presence or absence of various concentrations of GD? for 0-120 min. in cAMP assay buffer as described in section 2.4. After this initial preincubation, cells were washed three times with assay buffer, and then stimulated for 15-20 min. with various concentrations of GD? in the presence or absence of D3MX. Cells were then extracted in 70% ethanol, and assayed for cAMP production as described in section 2. 125 CHAPTER 3 RESULTS 3.1 ISOLATION A N D CHARACTERIZATION OF A cDNA ENCODING THE R A T GIP RECEPTOR Total R N A was isolated from purified rat pancreatic islets and first strand cDNA prepared using the polymerase chain reaction with oligonucleotide primers designed to amplify the coding region of the rat islet GD? receptor cDNA, based on the published sequence of the RTN5mF cell receptor cDNA (Usdin et al., 1993). A single product of appropriate size (~1.4Kb) was amplified, as determined by agarose gel electrophoresis (Fig. 20). Three individual PCR reaction products (GIP-R1, GIP-R2, and GEP-R3) were subcloned into the T A cloning vector pCRTI (Invitrogen). Subsequently, a Hind IWXba I fragment of the cDNA pGIP-Rl was subcloned into the Hind IWXba I site of the expression vector pcDNA 3, and the complete sequence of the coding strand was determined (Fig. 21). Alignment with the published sequence identified only one nucleotide difference in pGD?-Rl, resulting in a single amino acid difference (Glu21-»Gln21). This single nucleotide change was confirmed in the two other independently generated clones by partial sequencing, suggesting that it may be due to a single nucleotide polymorphism (SNP) in the rat gene. The predicted secondary structure of the seven-transmembrane receptor is presented in Fig. 22. 126 1 2 3 -1.4 Kb Fig. 20. Amplification of a cDNA Encoding the Rat Islet GIP Receptor Using the Polymerase Chain Reaction. Lane 1, 1 Kb ladder; Lane 2, Rat Islet 1st strand cDNA as template; Lane 3, d H 2 0 negative control. A single product of approximately 1400 bp was obtained following amplification of rat islet cDNA. Clones from three individual PCR reaction were subcloned for further sequence analysis. 127 AGCaTGCCOCTQCGGCTCTTGCTT^^ I I I I!MI I I I I I I I I I I!I I I I I I I I I I I!I I I I I!I! I I I ! M I I I I I I I I I I I I I I I I 11 I 11 I I I 111 I I I I I I I I I I I I I 11 I I I I I I I I I 11 AGGATQOOXTGCGGCTGTTGCITCTQC^ /^AGCGCTGSaCKGrrACGOT III i n n i n n nun i n n i n n i i i i i i i i i i i i i i i i i i i i IIIIII i n n i n n i n n i n n i n n i n n i i i n i i n n n n ACCAGCGCTGGGAGCGTTAOSGCTGGGMTGC^^ CTGCreGAACTACACGQCTGCCMCAOCACTGCCCGGCT 300 TGTGGTAGTGATGGCCAATGGGGATCTrcGAGAGACCACACTC I I I II I I I I I I I I I I I II I I I I II I II II I II I I II I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I III I I I I I I I I I I M l I I I I TGTGCTAGTGATGGCCAATGGGCMCITGGAGAGAC^ QOCTGCAGGTCGTCTATACAGTCGa^ACTO^ III l l l l l IMMMI I I I l l l l l M i l l M i l l M i l l I I I I I I IMI IIIIII M i l l M i l l M i l l M i l l I M M I I M I IIIIII M i l l MM GOCTGCAGGTCGTGTATACAGraSGCTACTC^^ TAATTACATTCACATGAACCTGTTCACGTCTTTCATOT 600 I I I I I I I I I I I I I I I I I I I I II II II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I III I I I I I I I I III I TAATTACATTOCATGAACCTCTTCACCT^ AACCAGACCCCTAOCCTGTGGAACCAGG^ I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I II I II I I I I I I I I I I I I I I I I I I I I IIII I I I I II I II I I AACCAGACOXTACCCICTGGAACCAG(XOT^ TGGAGGGTGTCTATCTGCACCATCTGCTGGTOT II I I I I I I I I II I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I II III I I I I I I I I IMII I II IIII TGGAGGCTGTGTATCTGCACCATCTGCTGGTOT TTTOm»TOlXCTGGGTGATCGTCAGGTAOCTGTAC^ 900 I I I I I I I I I I I H I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I II I M l I I I III I I I III I I I II TTTCGTCATOXCTGGGreATCGTCAGCTAarre ATCrTAATAAOCATCTTGATCAATTTCC^^ I I I I I I I I I I I I I I I I I I I II II I I II I I I I II I II II I IIII I I I I I I I I I I I I I I I I I I II I I I II I I I I I I I I I I I I II I I I III I I I II I II I II I ATCCTAATAACO^TCTTGATCAATTTCCTCATCTTT^ GACTAAGGCTGGCTCGCTCCACGCTGACACTGATGO^ 1100 Ml l l l l l l l l l l IIIIII I M i l l l l l l l l l l l M i l l l l l l l l l l l l IIIIII l l l l l l l l l l l l l l l l l l l l l l l l l M i l l IIIIII l l l l l MM GACTAAGGCTGQCTCGCTCCACGCT^ QCGCTTTGCCAMCreGCCTTTGAMTCTTOT III l l l l l MM I IIIIII l l l l l l l l l l l l l l l l l l l l l l l l l III II III III M i l l M i l l l l l l l M i l l III II l l l l l IIIIII l l l l l MM GCGCTTTGCCAMCTGG03TTTGAMTCTTCTTM ATCCGCCGTCTGCGTCTCAGCCT^ 1300 I I I I I I I I I I I I I I I I I I I I I I II I I I I I I I II I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I IIII I I I I I I II III I I I I I I II I ATCCGXGTCTQCGTCTCAGanTCAGGA CCATCCGCAATGCCTTGOCCTCTGGGATGCTGCATGTQCCTGGAGA 1374 I I I I I I I I I I I I I I II II I I I I I I I II I I I I I I I IIII I II I I I I I I II II III I II II I II I IIIIII I II II CCATOCQCMTGCCTTGCCCTCTGGGA^ Fig. 21. Sequence Alignment Of The Rat Islet Pancreatic GIP Receptor cDNA Isolated Via RT-PCR (Top) to that of the Previously Published Sequence of Usdin et al. (1993) (Bottom). Sequence of the primers used in PCR reactions are underlined and the one nucleotide difference found in three individual clones isolated is noted by 128 Fig. 22. The Predicted Secondary Structure of the Rat Islet GIP Receptor. 129 3.2 CHARACTERIZATION OF GIP RECEPTOR BINDING Initial studies on GIP action used peptide purified from pig intestines (pGIP), and it was shown to be strongly insulinotropic in rats (Pederson and Brown, 1976), humans (Andersen et al, 1978), and other species (reviewed in Brown et al, 1989; Pederson, 1994). However, it was subsequently reported that synthetic preparations of human GIP exhibited greatly reduced insulinotropic activity compared to both the native porcine hormone and synthetic preparations of pGIP (Nauck et al, 1989, 1993a). In comparative studies, using the isolated perfused rat pancreas, Jia and co-workers (1995) showed that commercially available preparations of shGIP exhibited much lower insulinotropic activity than synthetic pGIP preparation. It was suggested that the sequence differences between the human and porcine peptides could account for the difference in potency, but that problems in chemical synthesis were the most likely source. To establish that the PCR-amplified cDNA isolated from rat islet tissue encoded a GIP-specific receptor, and to compare receptor binding affinity of spGIP and shGIP preparations, binding analyses were initially performed on COS-7 cells transiently expressing GIP-R1. Both peptide preparations inhibited the specific binding of 125I-spGIP to COS-7 cells in a concentration-dependent manner and with similar potencies (Fig. 23). The IC50 values for displacement were 7.6 ± 1.2 nM and 8.9 ± 1.8 nM (n = 3) for spGJP and shGIP, respectively (Fig. 23). Similar displacement results were obtained with COS-7 cells transfected with a second GIP receptor cDNA, GJP-R2 (spGJP IC50 7.2 nM; shGIP IC50 8.0nM, n =2). There was a complete absence of specific binding to control non-transfected COS-7 cells, to cells transfected with the expression vector pcDNA 3, or to the rat GLP-1 receptor. 130 I 1 1 1 1 1 1 1 0 -11 -10 -9 -8 -7 -6 -5 L o g 1 0 [GIP] M Fig. 23. Displacement of 1 2 5I-spGIP Binding From COS-7 Cells Transiently Expressing GTP-R1. Both spGIP and shGIP displayed similar affinity for the rat islet GIP receptor when expressed in COS-7 cells (see Text for details). Curves are representative of 3 individual experiments. 131 Since stable CH0-K1 cell lines expressing GTP-R1 provided a more practical approach to assess ligand binding, the high level expressing stable CHO-K1 clone, referred to as wtGIP-Rl, was used to characterize GIP receptor binding further. Saturation isotherms obtained with both intact cells and membranes gave monophasic binding curves with K d values of 204 ± 17 p M and 334 + 94 p M (n = 3-6), respectively (Fig 24). wtGIP-Rl was determined to express approximately 12-15 X 104 receptors/cell or 11.8 ± 0.10 fmol of receptors/mg of cell membrane. In competitive binding studies, data were found to fit most consistently to a one site model at all temperatures examined (4°C, RT, and 37°C). IC50 values were lower at 4°C (1.2 -1.7 nM) than at room temperature (3.1-3.7 nM) or 37°C (1.2-8.9 nM) for spGIP (Table 1). Interestingly, Bmax values obtained at 37°C were consistently decreased in comparison to those obtained at either 4°C, after a 4 hour incubation, or RT after a 1 hour incubation (Table 1). As experiments performed at room temperature gave more reproducible IC50 values then those at 37°C, with a convenient investigation time, all initial experiments were performed at RT. Given that Jia et al. (1995) had seen a marked difference in the insulinotropic potencies of spGJP and shGIP, it was surprising that no differences between the affinities of these peptides was observed in COS-7 cells. To determine i f differences in the quality of different synthetic preparations could account for the lack of biological activity, the wtGIP-Rl cell line was used to examine the affinity of two different spGD? and shGIP preparations in competitive binding experiments. Again, there were no differences between mean IC50 values for different preparations of either porcine or human GJP [shGJP-1 (Bachem) 2.6 ± 0.8 nM, spGJP-1 (Bachem) 3.7 ± 1.5 nM, shGJP-2 (Peninsula) 3.1 ± 0.9nM, spGTP-2 (Peninsula), 3.6 ± 0.4 nM], or 132 between the preparations from the two different commercial suppliers (P > 0.05, n = 3; Fig. 25, Table 2). Natural porcine GIP (enterogastrone UI; EGIIT) also had a similar IC50 value (~3nM, Table 2), further validating the use of more readily available synthetic preparations for GIP studies. Non-transfected CHO-K1 cells or cells transfected with vector alone did not display specific I 2 5 I-GJP binding. Competitive binding Conditions IC 5 0 (nM)n = 2 Bmax (% of 4°C Bmax) n = 2 4°C, 4h 1.2-1.7 100% RT, l h 3.1-3.7 89% 37°C, l h 1.2-8.9 54% Table 1. Summary of Preliminary Binding Experiments. Experiments were carried out with wtGIP-Rl cells to optimize conditions for later experiments. GIP preparation spGIP-1 spGIP-2 shGIP-1 shGJP-2 E G H I IC 5o(nM) 3.7 ±1.5 3.6 ±0.4 2.6 ±0.8 3.1 ±0 .9 3.0* Table 2. Summary of Competition Binding Studies Comparing Different GIP Preparations. E G HI (Enterogastrone UI) = porcine GIP purified from intestine. n=3 for all data except * where n = 2. The binding specificity of the receptor was examined further in wtGIP-Rl cells, using several structurally related mammalian peptides, including GLP-l(7-36) (tGLP-1), GLP-2, glucagon, and VIP (Fig. 26). In addition, given that the GLP-1 agonist, exendin (Ex)-4, and antagonist, Ex (9-39), venom peptides isolated from Heloderma suspectum, 133 have been demonstrated to stimulate and antagonize, respectively, the incretin response in the rat (Kolligs et al, 1995; Wang et al, 1995), it was of interest to determine whether these peptides interact with the rat islet GD? receptor. Interestingly, while none of the structurally related mammalian hormones tested inhibited 125I-spGD? binding, both Ex (9-39) and Ex-4 demonstrated significant low affinity binding to the GD5 receptor; with -39% and 21% displacement of 125I-GIP binding, respectively, at a concentration of lpM (Fig. 27). Although these results demonstrated the high specificity of the GIP receptor for its native ligand, they also suggest that Ex (9-39), an antagonist of the GLP-1 receptor, may have a similar action at the GJP receptor when used in the micromolar or higher concentration range. 134 A. 4000-, u-f 1 1 1 1 0 2 . 5 X 1 0 0 5 5 . 0 X 1 0 0 5 7 .5X10 0 5 L O x l O 0 6 Free (DPM) B . 60000-, 0 4 , , 1 0 5 . 0 X 1 0 0 5 LOx-IO 0 6 1 .5X10 0 6 Free (DPM) Fig. 24. Saturation binding curve for isolated wtGIP-Rl cell membranes (A) and intact cells (B). Data were analyzed using the curve-fitting program PRISM (GraphPad). The curve was monophasic for both membranes and cells expressing the rat islet GIP receptor, and three individual experiments yielded mean Kd values of 334 ± 94 pM (membranes) and 204 ± 17 pM and a binding capacity of 59 ± 0.5 pM/mg of membrane protein (124,560 ± 14,800 sites/cell). 135 I 1 1 1 1 1 1 1 0 -11 -10 -9 -8 -7 -6 -5 Log 1 0 [GIP] M Fig. 25. Displacement of 1 2 5I-spGIP Binding from wtGIP-Rl Cells. A CHO-K l cell-line stably expressing pGTP-R was used to compare the affinity of two shGIP and spGIP preparations. No significant differences between the I C 5 0 values obtained were observed (see Text and Table 2). Curves are representative of 3 individual experiments. 136 Fig. 26. Displacement of 1 2 5I-spGIP Binding from wtGIP-Rl Cells by Peptide Hormones of the Glucagon/VTP/Secretin Family. None of the related hormones tested displaced specifically bound 1 2 5 I -spGJP. Curves are representative of 3 individual experiments. 137 Fig. 27. Displacement of 1 2 5I-spGJP Binding from wtGTP-Rl Cells by the GLP-1 Receptor Agonist, Exendin (Ex)-4 and the Truncated Antagonist Form Ex (9-39). Ex-4 and Ex (9-39) inhibited binding by 21% and 39%, respectively, at 1 pM. Curves are representative of 3 individual experiments. 138 3.3 EFFECTS OF RAT ISLET GD? RECEPTOR EXPRESSION ON cAMP FORMATION To correlate GD? receptor binding to activation of the adenylyl cyclase (AC) system, cAMP accumulation was determined by radioimmunoassay in COS-7 cells expressing pGD?-Rl. In the presence of the phosphodiesterase inhibitor, D3MX (1 mM), synthetic porcine GD? evoked a concentration-dependent increase in cAMP accumulation (EC50 = 870 ± 1 5 0 pM). This effect was not significantly different from that observed with shGD? (810 ± 160 pM) (Figure 28), indicating that the human and porcine species of GD? also share similar biological activities. No significant increases in cAMP accumulation were observed with any of the structurally related peptides tested previously in binding experiments, with the exception of Ex-4, which produced a small response (2.6 + 0.3 fold over basal) at the highest concentration tested (luM). In control experiments, with cells expressing the vector, or the GLP-1 receptor (GLP-1R), GIP (100 nM) was unable to evoke a cAMP response, further demonstrating the specificity of responses in wtGIP-R transfected COS-7 cells. The wtGIP-Rl cell line was subsequently used to examine the effects of different GIP preparations (Summarized in Table 3), related hormones, and fragments on GD?-stimulated activation of cAMP production. Due to the large numbers of cells expressing GEP-R1 in each well, it was anticipated that the addition of D3MX would not be required for cAMP assays in these studies. Comparisons of cAMP responses to different synthetic GIP preparations did not reveal any significant differences at any GD? concentration examined (Table 3). The preparation of synthetic human GD? used in the present study also produced an identical insulin response to spGD? in the isolated perfused rat pancreas 139 (integrated 50 min insulin responses: shGDM 172.7 ±17.5 mU, spGD? 176.2 ± 32.7 mU, shGIP-2 175.0 ± 26.8 mU, spGIP-2, 180.8 ± 17.5 mU, Table 3) in contrast to the commercial preparations of shGIP assayed by an identical procedure in earlier studies (Jia etal, 1995). Treatment shGIP-1 spGfP-l shGIP-2 spGIP-2 GD? (nM) Cyclic A M P 0.1 5.8 ±0.3 6.0 ±0 .1 5.7 ±0.6 6.5 ±0.3 1.0 7.6 ± 1.0 7.8 ±0.3 6.6 ±0.5 8.2 ± 0.4 10 9.6 ± 1.2 9.4 ± 1.2 8.8 ±0.3 10.0 ±2.5 Integrated insulin 1.0 172.7 ±17.5 176.2 ±32.7 175.0 ±26.8 180.8 ±17.5 Table 3. The Effect of Different GIP Preparations on cAMP Accumulation in wtGD?-Rl Cells and Insulin Release from the Isolated Perfused Rat Pancreas. cAMP levels are expressed as fold-increase over basal, and insulin levels as integrated insulin responses in mU of insulin over 50 min. Data = mean ± S.E.M. of > 3 individual experiments. Since the GLP-1 receptor antagonist Ex (9-39) and agonist Ex-4, displayed binding to the rat pancreatic islet GIP receptor, the ability of these peptides to alter cAMP accumulation in the absence and presence of GIP was examined. In wtGIP-Rl cells, and in contrast to experiments with transiently transfected cells, Ex-4 did not increase cAMP measurably at concentrations as high as l u M , nor did it antagonize GIP-stimulated cAMP increases (Fig. 29A). Importantly, Ex (9-39) did not have any effect on basal cAMP accumulation, or on responses to 10 nM spGD? (Fig. 29A). In agreement with studies reported by Fehmann et al. (1994), both Ex-4 and Ex (9-39) exerted their expected agonist and antagonist effects, respectively, in cells expressing the GLP-1 receptor (Figure 29B). As binding data would predict, none of the structurally related mammalian hormones (GLP-1, GLP-2, glucagon or VIP) had any effect on cAMP accumulation in wtGIP-Rl cells at concentrations as high as 1 u M (Fig. 30). 140 1 2 5 - , 100-7 5 H ° Q. 50H 2 5 H 0 J 0 V A O o spGIP-1 shGIP-2 tGLP-1 Ex-4 Ex (9-39) -10 -9 - 8 Log 1 0 [Peptide] M Fig . 28. Stimulation of c A M P Formation in COS-7 Cells Transiently Expressing p G I P - R l in Response to Secretin/Glucagon Related Peptides. E C 5 0 values for shGIP-1 and shGIP-2 were not significantly different (P > 0.05). Ex-4 caused a 2.6 fold increase over basal at a concentration of 1 u M . 141 125-1 Ji = 100 3-2 a. a O a. Q.S </> < B. 75-50H 25H «7 *u1 Q. (9 £ S c o ST 0-2 a. 1<3 —5 uJ s x _ c EC H - T -r- + 125 •o 9) C ffl O 100 11 V 2 T a-a. o. _i s o § 75-50-25-ra in ra m ** • o s a> 3 T O Figure 29. The Effect of Ex-4 and Ex (9-39) on spGTP-stimulated cAMP Formation in the Stable CHO-K1 Clone wtGIP-Rl (A), and on GLP-1-stimulated c A M P Formation in Cells Stably Expressing the Rat GLP-1 Receptor (wtGLP-l-Rl) (B). Neither form of Exendins influenced GTP-stimulated cAMP production in wtGTP-R l cells (A), whereas Ex-4 increased cAMP levels and Ex(9-39) inhibited 10 n M GLP-1-stimulated cAMP production in wtGLP-l-Rlcells (B). 142 11 < o ° E 15-, p 10 a o o O Fig. 30. Cyclic-AMP Formation in wtGIP-Rl Cells in Response to 1 p M Secretin/Glucagon Related Peptides. spGD? was the only peptide tested that elicited a significant increase in cAMP accumulation in a CHO-K1 cell line expressing the rat islet GIP receptor. The data are representative of two individual experiments carried out in triplicate. 143 3.4 L O C A L I Z A T I O N OF THE CORE GD? BINDING REGION In studies aimed at localizing the region of GD* that is required for receptor binding, the ability of several GD? fragments (shGD? l-30amide, spGD? 17-30 and spGIP 19-30) to displace 125I-spGD? binding from wtGD?-Rl cells was first examined. In addition, based on the recent identification of the region 21-29 as a biologically active fragment of glucagon (Ohneda and Ohneda, 1994), several fragments with sequences based on the homologous region 21-26 in GIP, GLP-1 and glucagon were synthesized and tested. These included: GDP 21-26, GIP 18-28, GLP-1 21-26, and Glucagon 21-26 (Glu 21-26). To increase the sensitivity of the system, and allow true steady state conditions to be obtained all of these, and later, competition binding experiments were carried out at 4°C for 12-16 h. A synthetic preparation of GD 3 1-30-free acid (OH), prepared by Dr. N . Yanaihara (Shizuoka, Japan) was found to exhibit significantly reduced affinity (IC50 = 39 ± 17 nM) when compared to shGD? (Table 4). In contrast, GIP l-30amide from an alternative source (Dr. D.H. Coy, Tulane School of Medicine, New Orleans, L A ) displayed nearly identical affinity for the rat islet GIP receptor to shGD5 (IC 5 0 : shGIP 1.2 + 0.5 nM; n = 7 vs. shGD* l-30amide 3.1 ± 0.9 nM, n = 6) (Fig. 31). MALDI-TOF analysis of the two GD 3 1-30 preparations indicated that GIP 1-30-OH contained contaminating additional peptides of lower molecular mass, while the GD? 1-30amide preparation was of high purity (Dr. H - U . Demuth, personal communication). An under-estimation of the GD?-1-30-OH peptide mass in the stock solutions therefore probably accounted for the decreased apparent affinity observed. In contrast to the high affinity binding of GIPl-30amide, neither the fragments sharing homology within the 144 region of residues GIP 21-26 nor the peptides spGIP 17-30 or spGIP 19-30, displaced specific 1 2 5 I-GIP binding (Fig. 31, Table 4). cAMP Studies Binding Studies Peptide I C 5 0 (nM) E C 5 0 (pM) Inhibition of 1 n M GIP stimulated cAMP production shGIP #1 1.21 ±0.46 112±25 shGIP 1-30-OH 39 ± 17* N D N D shGIP l-30amide 3.01 ±0.69* 120 ± 45 N D GIP 17-30 - - -GIP 19-30 - - -GIP 21-26 - - -GIP 18-28 - - -GLP-1 21-26 - - -Glu 21-26 - - -shGD? #2 2.39 ± 1.15 310 ± 2 6 GTP 15-42 1980 ±420* - + (40.9 ±3.6%) GTP 10-30 562 ± 3 7 * - + (50.8 ±2.3%) GIP 7-30 177 ± 2 5 * +/- + (73.0 ±0.7%) GIP 6-30amide 3.08 ±0.57 +/- + (94.0 ± 2.2%) Table 4. Summary of GIP Fragment Binding and cAMP Studies. Control shGD? values (#1 and #2) precede the data for the peptides tested. Maximal cAMP inhibition observed is in parenthesis. (-) no displacement, cAMP accumulation, or inhibition of cAMP accumulation observed; (+) strong or (+/-) weak stimulation of c A M P accumulation or inhibition. Data are representative of n > 3 experiments carried out in triplicate. * = significantly different from responses to shGIP 1-42 (p < 0.05 or less). Results of initial cAMP studies with wtGIP-Rl cells proved to be highly variable from day to day, making estimates of EC50 values difficult to calculate. The addition of 1 m M D3MX to inhibit phosphodiesterase activity and stabilize cAMP for the duration of the stimulation period alleviated this problem, without affecting basal cAMP levels (-1-3 fmol/1000 cells). Interestingly while EC50 values were very consistent, maximal cAMP levels shifted over time (compare maximal levels observed in Fig. 32 with Fig. 39B). This necessitated that a group of experiments be completed within a limited time range. 145 The addition of 1 m M I B M X to the incubation buffer was performed in all further experiments. GD? l-30amide was the only truncated form of GD? tested that stimulated cAMP production. Despite the small shift in affinity observed in binding experiments, cAMP responses to GD* l-30amide did not differ from GD?2 in either maximal levels achieved (GD? 351 ± 6 3 fmol/1000 cells vs. GIP l-30amide 347 ± 24 fmol/1000 cells), nor in efficacy ( E C 3 0 values: GIP 112 ± 25 p M vs. GIP l-30amide 120 ± 45 pM, n =4-5) (Fig. 32). None of the fragments tested potentiated or antagonized lOnM shGIP-stimulated cAMP accumulation (Table 4). Examination of the predicted secondary structure of GIP using P C G E N E indicated that GIP should share a "coil-helix-coil " structure exhibited by members of the glucagon superfamily of peptides (Bodanszky, 1974; Sasaki et al, 1975) (Fig. 33). It was predicated that the high affinity binding region of the peptide may be localized to, and require, the secondary structure found within residues 10-30 (comprising the putative large central helical stretch). To test this theory, a number of synthetic fragments (based on the human sequence) were synthesized corresponding to residues GD? 10-30 and amino-terminally extended forms, GD? 7-30 and GD? 6-30amide. In addition a carboxy-terminal fragment, GIP 15-42 corresponding to the predicted "turn-helix" region (Bodanszky, 1974) of GD?, allowed examination of the contribution of this region to binding and activation of the receptor. 2 For brevity, GD 5 will be used to refer to the full-length molecule GIP 1-42. 146 \ Fig. 31. Displacement of 1 2 5I-GTP by different truncated forms of shGIP. Of the fragments tested only GIPl-30amide displaced 1 2 5 I-GIP binding from wtGIP-Rl cells. There was a small but significant shift to right in the I C 5 0 value obtained for GIP l-30amide in comparison to the full length peptide (GIPl-30amide: 3.01± 0.69 nM, n = 6, vs. shGIP 1-42: 1.21 ± 0.46 nM, n = 7: P < 0.05). 147 I 1 1 1 1 1 0 -13 -11 -9 -7 -5 L o g 1 0 [Peptide] Fig. 32. Stimulation of cAMP Accumulation in wtGIP-Rl Cells by shGD? and Fragments of shGIP. GIP l-30amide was the only peptide tested that increased cAMP levels in a concentration dependent manner. There were no differences in efficacy or maximal cAMP levels between GD? l-30amide and shGD? 1-42 (See Table 4). 148 Fig. 33. Predicted Secondary Structure of shGD?. The method of GGBSM was used to predict the secondary structure using the sequence analysis package PCGENE (IntelliGenetics, 1995). 149 Competitive binding experiments on wtGIP-Rl cells with 12iI-GD? revealed no significant difference between the receptor binding affinities for GTP and GTP 6-30amide, with mean IC50 values of 2.39 ± 1.15 nM and 3.08 ± 0.57 nM (n = 3), respectively (Fig. 34A, Table 4). Both GTP 7-30 and GTP 10-30 also displaced 125I-GTP in a concentration-dependent manner, but the mean IC50 values, 177.15 ± 25.09 nM and 562.36 ± 37.47 nM (n = 3) respectively (Fig. 34A), were approximately 74 (GTP 7-30) and 235 (GTP 10-30) -fold greater than that for GTP. GTP 15-42 displayed the lowest affinity of the fragments (an approximate 641-fold decrease in affinity) with a mean IC50 value of 1.98 + 0.42 uM (n = 3). Neither GTP 10-30 nor GTP 15-42 fully displaced 123I-GTP to non-specific binding levels obtained with 1 uM GIP. The maximal displacements obtained with GIP 10-30 and GIP 15-42 were 93.9 ± 1.8% and 74.4 ± 2.0% (n = 3), respectively, at 10 uM peptide (Fig. 34A). Synthetic human GIP, stimulated cAMP production in wtGTP-Rl cells in a concentration-dependent manner to a maximum of 138 ± 3 1 fmol/1000 cells (n = 3) at a concentration of 1 uM, with an EC50 of 310 ± 26 pM (Fig. 34B). While GTP 10-30 and GTP 15-42 produced no change in cAMP levels at concentrations as high as 10 uM, both GTP 6-30amide and GIP 7-30 consistently caused small increases in mean cAMP levels, of 1.58 ± 0.54 and 3.49 ± 2.96 fmol/1000 cells (n = 3) respectively, at a concentration of 10 uM (Fig. 34B, Table 4). However, these increases were less than 1.14 and 2.53% respectively of those obtained with GTP at a concentration of 1 uM. As the fragments exhibited extremely weak, or absent, agonist activity, their ability to antagonize 1 nM GTP-stimulated cAMP production was examined. In 150 agreement with the competitive binding studies, the order of potency of the fragments as inhibitors of GJP-stimulated cAMP production was GIP 6-30amide > GIP 7-30> GIP 10-30> GIP 15-42 (Fig. 35, Table 4). GIP 10-30 and GIP 15-42 caused significant inhibition only at a concentration of 10 p M (50.8 ± 2.3% and 40.9 ± 3.6%, respectively, n = 3). In contrast, GIP 7-30 inhibited GIP-induced responses by 34.2 ± 3.3% and 73.0 ± 0.7% at 1.0 and 10 p M respectively (n = 3). GTP 6-30amide was the most potent antagonist, significantly inhibiting cAMP production by 58.0 ± 2.5%, 87.0 ± 0.4%, and 94.0 ± 2.2% (n = 3), at concentrations of 0.1, 1.0 and 10 p M respectively (Fig. 35, Table 4). In some experiments it was observed that the peptide antagonists actually decreased basal cAMP, levels suggesting they may be acting as inverse agonists (Milligan et al., 1995; Scheer and Cotecchia, 1997), stabilizing receptors in the low affinity, G-protein uncoupled state as discussed in section 1.11.3. To examine the nature of the antagonism of these peptides in more detail, site directed mutagenesis was used in an attempt to generate a constitutively active form of the GIP receptor. Based on several reports of constitutively active receptors within the Secretin/VIP family, both naturally occurring (Schipani et al, 1995, 1996) and generated by mutagenesis (Schipani et al, 1995), GTP receptor mutants were prepared containing arginine substituted for histidine at position 170 at the intracellular face of the predicted second transmembrane domain. The resulting receptor construct was referred to as H170R. 151 B. 125-100-o £Q 75 S3 50 25-0-200 Jfi "5150 u Cu o S o < °100 o £ 50 r~ 0 •11 -9 -7 Log! o [Peptide] shGIP 1-42 GIP 10-30 GIP 7-30 GIP 15-42 GIP 6-30amide 0-1 0 = ° O shGIP 1-42 A GD? 6-30amide V GIP 7-30 • GIP 10-30 • GIP 15-42 B — B — B = B = 8 — B — 0 -13 -11 -9 -7 L o g 1 0 [peptide] Fig. 34. Displacement of 1 2 5I-GD? Binding to CHO-K1 Cells Expressing the Rat Islet GD? Receptor (wtGIP-Rl Cells) by Truncated Forms of GIP (A), and Stimulation of cAMP Production in wtGIP-Rl Cells by the Same Peptides (B). Data are representative of three individual experiments, each carried out in triplicate. See text and Table 6 for details. 152 Fig. 35. Inhibition of 1 n M GIP 1-42 Stimulated cAMP Production in wtGTP-Rl Cells. Cells were preincubated with GIP fragments for 15 min. prior to stimulation with 1 n M GIP 1-42 for 30 min. Significant inhibition of cAMP production occurred at concentrations of 100 n M GIP 6-30amide, 1 p M GIP 7-30, 10 u M GIP 10-30 and GIP 15-42. * = significance at p<0.05 and ** = significance at p<0.01. 153 In transient experiments, COS-7 cells expressing H170R, displayed similar affinity to shGIP (IC 5 0: H170R 4.18 + 0.4 nM vs., GIP-R1 5.65 ± 3.96 nM. n =3) in competitive binding studies (Fig. 36A), and similar expression levels (Bmax, 78.3 ± 2.9% of wt, n=3) when compared to cells expressing the wild type receptor (Fig. 36A). In cyclic A M P studies with cells transiently expressing the H170R or wtGIP-Rl cDNAs, no significant differences in basal or 10 nM-stimulated cAMP production (Fig. 36B) were evident. In addition, C a 2 + imaging experiments revealed no differences between the [Ca 2 +]i responses of COS-7 cells expressing wt and H170R cDNAs (See Appendix A, Fig. A3). It was therefore not possible to examine whether the GIP fragments are capable of demonstrating reverse agonism. 154 A. Fig. 36. A . Displacement of 1 2 5IGTP From COS-7 Cells Expressing wtGIP-Rl and H170R cDNAs by shGIP. While H170R expression levels (Bmax) were 78.3 ± 2.9% of wtGIP-Rl, there was no significant difference between the point mutant and the wtGIP-Rl receptors in their affinity for ligand. B. 10 n M shGEP-stimulated cAMP Accumulation in COS-7 Cells Transiently Expressing wtGIP-Rland H170R Forms of the GD? Receptor. No changes in basal or 10 n M GIP-stimulated cAMP levels were observed. 155 3.5 EXAMINATION OF RESIDUES IN GD? IMPORTANT FOR RECEPTOR ACTIVATION It has been shown previously that the first two residues at the amino-terminus of GD5 (Tyr^Ala2) and GLP-1 (FfiV-Ala2) are required for biological activity (Brown et al, 1981; Schmidt et al, 1986a; Suzuki et al, 1989; Gefel et al, 1990), and that GIP and GLP-1 are metabolized by DP IV to GJP 3-42 and GLP-1 9-36amide (Mentlein et al, 1993b; Kieffer et al, 1995b; Pauly et al, 1997), respectively. The effect of removing residues 1 and 2 from the N-terminus of GIP and reversing the order of the first two amino acids on receptor binding and receptor activation was examined. Both GD> 3-42 and Ala^Tyi^-GIP displayed reduced affinity for the receptor in competition binding studies (GIP 3-42 IC5o = 58.42 ± 18.76 nM, n = 5; Ala'-Tyi^-GIP IC 5 0 = 67.04 ± 20.26 nM, n = 3 vs. GIP 3.56 ± 0.81 nM, n = 6.) (Fig. 37A, Table 5). Both peptides were found to be devoid of the ability to stimulate cAMP production in wtGIP-Rl cells at concentrations as high as 1 pM (Fig. 37B). However, both peptides antagonized GD»-stimulated cAMP accumulation by 45.59 ± 5.23 % and 91.04 ± 0.95 % (GJP 3-42), and 40.54 ± 4.62 % and 90.91 ± 0.42 % (Ala1-Tyr2-GD>), at 1 pM and 10 pM (n = 3-7) respectively (Fig. 38). The observation that removing or modifying the first two residues of GIP reduced the affinity of the peptides below that for GH* 6-30amide, suggested that the sequence and/or conformation of the N-terminal putative "coiled region" was important for both high affinity binding and receptor activation. 156 A. • GIP 1-42 V GIP 3-42 i i i i i 1 0 -13 -11 -9 -7 -5 Log10 [Peptide] Fig. 37. Displacement of 1 2 5I-GTP Binding (A), and Stimulation of cAMP Production (B) by GIP 1-42, N-terminal Truncated (GD? 3-42), and Sequence Modified (Ala^Ty^-GD? 1-42) Analogs. Mean I C 5 0 and E C 5 0 values are summarized in Table 5. 157 Fig. 38. GD? 3-42 and Ala^Tyr'-GIP Inhibit InM GLP-stimulated cAMP Production. wtGfP-RT cells were preincubated with the peptides at the given concentrations, then stimulated with InM GD 3 in the presence of the GIP fragment or modified form at the appropriate concentration. Data shown are the mean ± S.E.M. of 3-7 individual measurements. ** = significantly different from responses to shGIP 1-42 (p < 0.01 or less). 158 Additional amino terminally modified forms of GD?, GLP-1 and GIPl-30amide were synthesized to examine the contribution of specific N-terminal residues for receptor affinity and activation. As mentioned earlier, dipeptidyl peptidase IV attenuates the biological activity of both GD* and GLP-1 (Mentlein et al, 1993b; Kieffer et al, 1995b; Pauly et al, 1997). Requirements for enzymatic activity of this enzyme are that amino acids in the PI and P2 positions are L-isomers in the trans conformation (Fisher et al, 1983) and that the N-terminus is protonated. An Desamino-Tyrosine amino-terminal deprotonated form (3-phenyl propionic acid (Ppax)-GIP l-30amide) and a number of D-isomer substituted forms (see Table 5) of truncated GIP were synthesized for us, by Drs. H. U . Demuth and D. Coy to allow examinination of the contribution of the N-terminal residues to receptor affinity and activation. Additionally, these studies incorporated attempts at synthesizing DP TV resistant forms of GIP. D-Ala2-GIP and D-Ala2-GIP l-30amide were found to exhibit a slight shift to the right in their IC5o (11.52 ± 1.08 nM and 10.26 ±2.76 nM, n = 5, respectively) and EC5o (1.78 ± 0.86 nM, n = 4 and 681 ± 210 pM, respectively, n = 5) values, when compared to GIP binding (IC50 = 3.56 ± 0.8, n=6) and cAMP production (EC 5 0 = 248 ± 68 nM, n=7) (Fig.39, Table 5). D-Ala2-GLP-1 displayed a similar small decrease in potency in cAMP production (EC50 = 692 ±177 pM, n = 3) and a small shift to the right in displacement of 125I-GLP-1 (IC50 = 5.85 ± 3.52 nM), in comparison to GLP-1 (EC 5 0 = 255 ± 36 pM, and IC50 = 117 ± 0.31, n=3) when tested on the wtGLP-Rl cell line (Fig. 40). These small differences, while statistically different for the GIP analogs, were not different for the single GLP-1 analog tested. 159 A. 125 100 1 75 m w 50 25 0 n — D • shGD? 1-42 O D-Ala2-GD? 1-42 • D-Ala2-GIP l-30amide -11 -9 -7 Log10 [Peptide] B. o w i s 160 120 80 40 0 L B - ^ -13 -11 -9 Log10 [Peptide] • shGD? 1-42 O D-Ala2-Gn> 1-42 • D-Ala^GIP 1-30amide Fig. 39. Displacement of 1 2 5I-GIP Binding (A), and Stimulation of cAMP Production (B) by shGIP and D-Ala2-analogs. Mean I C 5 0 and E C 5 0 values are summarized in Table 5. 160 A. 6OO-1 =! 8 5 o 400H 200-O GLP-1 • D-Ala2-GLP-1 -11 -9 -7 Log10 [Peptide] -5 Fig. 40. Displacement of 1 2 5 I-GLP-1 Binding (A), and Stimulation of cAMP Production (B) by GLP-1, and the D-Stereoisomer Substituted Analog, D-Ala 2 -GLP-1. Mean I C 5 0 values were GLP-1: 1.17 ± 0.31 n M and D-Ala 2 -GLP-1: 5.39 ± 3.52 n M (A). Mean E C 5 0 values were GLP-1: 255 ± 36 pM, and D-Ala 2 -GLP-1: 692 ± 177pM(B). 161 Results consistent with the similar cAMP responses were obtained in the perfused rat pancreas, with D-Ala2-GTP having a marked but significantly smaller integrated insulin response in comparison to the endogenous hormone (GIP =188.3 ±6 .1 mU, n = 5, vs. D-Ala2-GJP = 154.5 ± 4.9 mU) (Fig. 41). D-Ala2-GLP-1 did not differ significantly in its stimulation of insulin secretion when compared to GLP-1 (160.1 ± 3.240 mU, n = 4 vs. 174.7 ± 15.4 mU n = 6, respectively) (Fig. 42). Other analogs were tested only in binding and cAMP studies. In binding analysis of the analogs (Fig. 43A, Table 5), displacement curves for both D-Ala4-GIP l-30amide and D-Tyr^GIP l-30amide were shifted to the right in comparison to GIP [IC50S = 30.67 ± 6.62 nM and 29.28 ± 6.83 nM, respectively, vs. GIP IC5o = 3.56 ± 0.81. P < 0.05, (n = 4-6)]. Ppa^GJP l-30amide and D-glu3-GIP l-30amide did not differ significantly from GJP in their affinity for the rat islet GIP receptor (Fig. 43A, Table. 5). As would be predicted from the binding studies, in cAMP experiments D-Ala4-GTP l-30amide and D-Tyr^GJP l-30amide displayed displacement curves significantly shifted to the right, with E C 5 0 values of 158.4 ± 85.3 nM and 13.6 ± 0.7 nM, respectively (n=4), compared to 248 ± 68 pM (n = 7) for GJP. While D-Glu3-GJP l-30amide displayed a small shift in efficacy (EC5o = 469 ± 126 pM) compared to GIP, Ppa^GJP l-30amide, despite its high affinity, had a greater reduction in receptor activation (EC50 = 930 ± 143 pM). 162 A. 300mg% Glucose + 0 • I n M peptide Time (min.) Fig. 41. Comparison of spGIP 1-42 and D-Ala 2-GTP 1-42 in the Isolated Perfused Rat Pancreas. A . A linear gradient of 0 to 1 n M of the indicated peptide was perfused in the presence of 300 mg% glucose. B. Integrated insulin release (area under the curve) was slightly decreased for D-Ala 2-GTP 1-42 (154.5 ± 4.9 mU) in comparison to spGIP 1-42 (188.3 ±6 .1) . (p<0 .05 ,n = 4-5). 163 A. 300mg% glucose + 0 •1nM peptide Fig. 42. Comparison of GLP-1 and D-Ala 2 -GLP-1 in the Isolated Perfused Rat Pancreas. A . A linear gradient of 0 to 1 n M of the indicated peptide was perfused in the presence of 300 mg% glucose. B. Integrated insulin release (area under the curve) was similar for both D-Ala 2 -GLP-1 and GLP-1 (n = 4-6). 164 A. ^ ^ O shGIP 1-42 A D-Tyi^ -GIP l-30amide • Ppa^ GIP l-30amide f V D-Glu -^GIP l-30amide i i i i i 0 -11 -9 -7 -5 Log10 [Peptide] B. 160r c . . 0 JO 1 "§ 120r TJ O 80 r | o <r.i. 40r 0L •13 -11 -9 Log 1 0 [Peptide] -7 -5 O shGIP 1^2 A D-Tyr1-GIP l-30amide • Ppa^GIP l-30amide V D-Qu3-GIP l-30amide • D-Ala4-GIP l-30amide Fig. 43. Displacement of 1 2 5I-GTP Binding (A) and Stimulation of cAMP Production (B) by shGIP 1-42, and GIP l-30amide Analogs. Mean I C 5 0 and E C 5 0 values are summarized in Table 5. 165 Analog ICJO (nM) E C 5 0 (nM) % of maximal GD? cAMP production (%) hGTP 3.56 ±0.81 0.248 ± 0.068 100% D-Ala 2-GTP 11.52 ±1.08* 1.780 ±0.864 102.2 ± 3.03 D-Ala2-GD» l-30amide 10.26 ±2.76* 0.681 ±0.210 94.5 ±3 .7 D-Tyr^-GTP l-30amide 29.28 ±6.83* 13.6 ±0 .7* 113.6 ±4.79 Ppa 1- GIP l-30amide 4.85 ± 1.33 0.930 ±0.143 107.4 ±7.71 D-Glu 3-GIP l-30amide 3.84 ±0.55 0.469 ±0.126 103.5 ±7.08 D-Ala 4-GIP l-30amide 30.67 ± 6.62* 158.4 ±85.3* 50.9 ±7 .6* GIP 3-42 58.42 ± 18.76* - -Ala'-Tyr^-GIP 67.04 ±20.26* - -Table 5. Summary of Competitive Binding (IC50) and cAMP Responses (ECj 0 and % of maximal GD? cAMP production) with GD* analogs. IC50 and EC50 values were determined by nonlinear regression analysis (n=3-7). (-) = non-detectable. (*) = differ from GIP by at least P < 0.05 as determined by one way A N O V A . 3.6 GIP A N D GLP-1 RECEPTOR CHIMERAS Studies with structurally related receptors of the glucagon superfamily, have suggested that the amino-terminal (NT) region of the secretin, VIP (Holtmann et al., 1995a,b; Gourlet et al., 1996), and glucagon (Buggy et al., 1995; Carruthers et al., 1994; Unson et al., 1995, 1996; Garziano et al., 1996) receptors play a role in ligand binding. Therefore, in an attempt at identifying regions of the GD? and GLP-1 receptors required for ligand binding and receptor activation, GIP/GLP-1 receptor chimeric cDNAs encoding differing portions of one receptor's NT-domain ligated to the C-terminal coding regions of the other were constructed as described in Sections 2.7.3-2.7.6, and depicted in Fig. 44. 166 CH-1 1-88 CH-2 CH-3 1-151 Kpn Xho I 89-463 Nhe I 143-463 162-463 CH-5 1-142 Xho I 133-455 CH-4 1-222 CH-6 1-235 Sea I Sea I 236-46." 223-455 CH-7 1-180 CH-8 1-190 CH-9 1-159 BssH II BssHll Earn 1104 191-463 181-455 70-463 Fig. 44. Predicted Topography of the Chimeric Receptors. Regions corresponding to GIP-R are in blue, those of the GLP-1-R are in red. 167 3.6.1 LIGAND BINDING OF RECEPTOR CHIMERAS Receptor binding and displacement studies were performed to determine the effect of domain exchange on ligand-receptor binding. Most receptor constructs were analyzed in both transient and stable expression systems. For initial localization of binding specificity, constructs were generated encompassing the putative NT domain, intracellular domain (IC)-l, extracellular domain (EC)-l, and part of the TM-3 of each receptor, displayed on the corresponding C-termini (CH-4, CH-6, Fig. 44). Interestingly, CH-4 displayed near normal affinity for GIP when expressed transiently or stably when compared to the wtGIP-R: IC 5 0 values (nM) in COS-7 cells CH-4 2.74 ± 0.87 vs. wt 6.42 ± 1.22; CHO-K1 cells CH-4 8.33 ± 0.14 vs. wt 1.33 ± 0.19, respectively (Fig. 45; Table 6). The similar affinity was observed despite the fact that this and subsequent constructs were not expressed with the same efficiency as the wt receptors (Table 6). CH-4 did not bind I 2 5I-GLP-1, and therefore lacked the necessary regions for high affinity GLP-1 binding (Fig. 46, Table 6). Neither CH-5 nor CH-6 displayed detectable binding of 1 2 5I-spGTP or 125I-GLP-1 (Table 6). To date receptor antibodies for the GD? or GLP-1 receptors are not available, and it was therefore not possible to establish whether the lack of binding was due to absent cell surface expression of the chimeric receptors, or loss of the ability of the modified receptors to bind ligand. To localize GD> binding specificity further, smaller portions of the GIP-R NT were used: CH-3, encompassing the first NT domain and a portion of TM-1 of the GD? receptor, and CH-2, in which only the first 132 amino acids of the GIP NT domain was displayed on the GLP-1 R (Fig. 44), both bound GTP, however there was a 5 to 10 fold shift in affinity compared to wtGIP-R (Fig. 45, Table 6). CH-1, consisting of the first 88 168 amino acids of the GD? receptor and C-terminal residues 89 to 463 of the GLP-1 receptor (Fig. 44), bound neither 125I-GIP (Fig. 45; Table 6) nor 125I-GLP-1 in either COS-7 or CHO-K1 cells. Thus, despite an apparently minor shift in binding affinity, it is quite clear that the majority of GIP-R specificity for its ligand is localized to the first 132 amino acids of the GD? NT domain. In an attempt at restoration of binding affinity to that seen with CH-4, constructs encoding chimeric receptors with further NT extensions of the GD? receptor into the first transmembrane domain (CH-9) and into the second transmembrane (CH-7) were constructed. However, all these receptors failed to bind 125I-GD? (Fig. 45; Table 6). While both COS-7 and CHO-K1 cells expressing the hGLP-1 receptor bound 125I-GLP-1 with high affinity (IC5o 7.5 ± 1.2 nM and 1.47 ± 0.42 nM, respectively) none of the chimeric receptors tested were capable of binding 125I-GLP-1 (Fig. 46, Table 6). Therefore it appears that GLP-1 receptor requires multiple receptor regions in order to bind GLP-1. 169 I 1 1 1 1 0 -11 -9 -7 -5 Log 1 0[GIP] Fig. 45. Displacement of 1 2 5I-GTP Binding by GTP in COS-7 (A) and CHO-K1 (B) Cells Expressing Wild Type or Chimeric Receptors. Data are the means ± S.E.M. of 4-7 individual experiments. Expression level and I C 5 0 values are summarized in Table 6. 170 I 1 1 1 1 0 -11 -9 -7 -5 L o g 1 0 [GIP] Fig. 46. Displacement of 1 2 5 I-GLP-1 by GLP-1 in CHO-K1 Cells Expressing the wtGLP-1 Receptor. Curves are representative of at least three individual experiments. Expression levels (Bmax) and I C 5 0 values are summarized in Table 6. 171 A. B . Receptor COS-7 CHO-K1 COS-7 CHO-K1 Bmax (% of wt) Bmax (% of wt) I C 5 0 (nM) ICso (nM) wtGTP-Rl 100 100 6.42 ± 1.22 1.33 ±0.19 w t G L P - l - R l - - - -CH-1 - - - -CH-2 15.5 ± 1.5* 6.4 ± 1.9* 31.7±8.18* 27.8 ± 11.9* CH-3 27.9 ± 8.7* 4.8 ±1.7* 23.6 ± 10.6 9.04 ± 1.07* CH-4 15.2 ±1 .5* 13.3 ± 3 . 1 * 2.74 ±0.87 8.33 ±0.14* CH-5 - - - -CH-6 - - - -CH-7 - N D - N D CH-8 - N D - N D CH-9 - - - -Receptor COS-7 CHO-K1 COS-7 CHO-K1 Bmax (% of wt) Bmax (% of wt) I C 5 0 (nM) I C 5 0 (nM) wtGIP-Rl - - - -w t G L P - l - R l 100 100 7.5 ±1.2 1.47 ±0.42 CH-1 - - - -CH-2 - - - -CH-3 - - - -CH-4 - - - -CH-5 - - - -CH-6 - - - -CH-7 - N D - N D CH-8 - N D - N D CH-9 - - - -Table 6. Binding of 1 2T-GD? (Bmax and IC 5 0 ) (A) and 1 2 T-GLP-1 (B) to GIP/GLP-1 Receptor Chimeras in COS-7 and CHO-K1 Cells. IC50 and EC50 values were calculated by nonlinear regression analysis (n = 3-7). N D = Not determined; - = Non-detectable. 3.6.2 C Y C L I C A M P RESPONSES OF RECEPTOR CHIMERAS In COS-7 cells expressing the rat wild type GD? receptor, treatment with 10 n M GIP resulted in stimulation of cAMP production to a maximum value of 1323 ± 1 4 1 pmol/well (n = 9), compared with 25.5 ± 17.2 pmol/well (n = 12) in controls wells (Fig 172 47A). In COS-7 cells expressing CH-3 and CH-4 increases in cAMP production in response to 10 nM GIP were 37.6 ± 3.3% and 20.5 ± 3.8% (n=3) of that seen with the wtGD? receptor (Fig. 47A, Table 7). In agreement with the binding data, none of the chimeras consisting of the NT-region of the hGLP-Rl responded to 100 nM GLP-1 stimulation (Table 7). Unexpectedly, however, COS-7 cells expressing chimeras CH-2 and CH-3 responded to stimulation with 100 nM GLP-1 with 84.8 ± 9.6% and 102.5 ± 23.0%, respectively (n=3), of the cyclic AMP response of cells expressing hGLP-Rl (Fig. 48A). The failure to detect significant binding of 125I-GLP-1 in these cells is probably a result of the presence of sufficient receptors to stimulate measurable cyclic AMP responses, but of either insufficient number or too low an affinity to allow the detection of 125I-GLP-1 binding. Stably transfected CHO-K1 cells were used to examine the cAMP responsiveness of the different receptor forms more fully. The maximal increases in cAMP levels, at concentrations up to 1 pM GIP (Fig. 47B), were: CH-3 29.3 ± 10.2% and CH-4 42.4 ± 11.2% of wt GJP-R1 cell line responses (180.1 ± 21.2 fmol/1000 cells, n=7). In agreement with COS-7 experiments, cells expressing CH-2 showed no significant cAMP responses to GIP up to 1 pM. The mean EC5o value for CH-4 (Fig. 47B, Table 7) was not significantly different from that of the wtGJP-Rl, however there was a significant shift to the right in the concentration-response curve for CH-3 (p<0.05) (Fig. 47B, Table 7). It is unlikely that this and other shifts in EC50 values are due to reduced receptor expression, since a low level expressing clone, wtGJP-R8, exhibited an EC50 value similar to that seen with the high level expressing wtGJP-Rl cell line (summarized in Table 8). 173 A. i 1 1 • 1 1 1 0 -12 -9 -6 Log 1 0 [GIP] Fig. 47. Stimulation by GIP of cAMP Production in COS-7 (A), and CHO-K1 (B) Cells Expressing the Wild Type GIP Receptor or GIP/GLP-1 Chimeric Receptors. Data are the mean ± S.E.M. of 4-7 individual experiments. Maximal cAMP increases and EC™ values are summarized in Table 7. 174 As seen in the transient expression experiments, GLP-1 stimulated cAMP production in a concentration-dependent manner in CH-2 and CH-3 expressing cell lines (Figure 48B). The levels of cAMP production in CH-2 and CH-3 cells were 30.5 ± 2.0% and 13.2 ± 1.9%, respectively, of the maximal levels seen in CHO-K1 cells stably expressing the GLP-1 receptor (wtGLP-Rl). The E C 5 0 values for CH-2 (81.4 ± 19.6 nM, n=3) and CH-3 (5.99 ± 0.68 nM, n=3) were shifted to the right compared to wt GLP-R1 (103 ± 1 1 pM, n=3). In agreement with COS-7 cell line studies none of the other chimeras responded with increased cAMP production in the presence of GLP-1 (Fig. 48, Table 7). 175 A. 750"i ® 500 H 2 ~ 2 5 0 H B. 500n =\ 4004. o u § 300H o 5 200-1 100-0-0 1 1 Basal • • 100 nM GLP-1 S S 5 -12 -9 Log 1 0 [GLP-1] i -6 • v*GLP- l -R l A CH-1 V CH-2 • CH-3 O CH-4 Fig. 48. Stimulation by GLP-1 of cAMP Production in COS-7 (A), and CHO-K1 (B) Cells Expressing the Wild Type GLP-1 or GJTVGLP-1 Chimeric Receptors. Data are the mean ± S.E.M. of 3-7 individual experiments. Maximal cAMP increases and EC« n values are summarized in Table 7. 176 Receptor GIP-Stimulated cAMP accumulation GLP-1-Stimulated cAMP accumulation COS-7 % of wt max CHO % of wt max CHO E C 5 0 (nM) COS-7 % of wt max CHO % of wt max CHO E C 5 0 (nM) wtGTP-Rl 100 100 0.49 ± 0.21 - - -wtGLP-Rl - - - 100 100 0.103 ± 0.011 CH-1 - - - - - -CH-2 - - N D 84.8 ±9.6 30.5 ± 2.0* 81.4± 19.6 * CH-3 37.6 ± 3.3* 29.3 ± 10.2* 17.L+-3.5* 102.5 ± 23.0 13.2 ± 1.9* 5.99 ± 0.68 * CH-4 20.5 ± 3.8* 42.4 ± 11.2* 0.47 ± 0.17 - - -CH-5 - - - - - -CH-6 - - - - - -CH-7 - N D N D - N D N D CH-8 - N D N D - N D N D CH-9 - - - - - -Table 7. Cyclic A M P Responses to GIP and GLP-in COS-7 and CHO-K1 Cells Expressing Chimeric Receptors. Data are expressed as % of maximal wild type levels and EC50 values, calculated by nonlinear regression analysis (n=3-7). N D = Not determined; -= Non-detectable.* = significantly different from wt (p < 0.05, n = 3-7). 177 Cell line Binding cAMP production. Bmax (% of wt) IC 5 0 (nM) % of wt Max. E C 5 0 (pM) wtGIP-Rl 100% 4.87 100% 490 + 210 wtGIP-R8 4.9% 1.37 9.59 + 0.29 70.3 ± 43 Table 8. Summary of I-GIP binding (Bmax and ICso, n =2) and cAMP Responses to GIP, with wtGIP-Rl and wtGIP-R8 CHO-K1 Cell Lines. Data expressed as % of maximal wild type levels (n = 3) for the low-level, stable GD? receptor expressing CHO-K l cell line, wtGIP-R8. 3.7 TRUNCATION OF THE C A R B O X Y - T E R M I N A L TAIL OF THE GIP RECEPTOR As discussed in sections 1.11.2, 1.11.4, and 1.11.5 the carboxy-terminal (CT) tail of G-protein linked receptors has been identified as a region that is important for G-protein interaction, receptor desensitization, and sequestration. A number of carboxy-terminal truncated forms of the receptor were constructed to examine the effect on receptor binding and activation of adenylyl cyclase, desensitization, and receptor internalization. Receptor truncations were designed to examine the effects of loss of a given CT-tail region on receptor binding and signaling, and the effect of loss of possible serine (Ser) phosphorylation sites on receptor desensitization and internalization. One receptor construct was derived using a convenient CT restriction site (Sac I), which resulted in removal of the most distal 30 amino acids of the receptor (GD?-R-427+), including two Ser residues (440 and 453). Since an additional six amino acids encoding vector sequence (QRVGCI) were included, the receptor's final length was 433 residues (Fig. 49). The other constructs prepared were: 178 GIP-R-418-further truncation to 418 residues, resulting in the deletion of an additional two Ser residues (426 and 427). GIP-R-405-truncation to 405 residues resulted in the loss of a putative casein kinase JJ phosphorylation site (Ser406). GIP-R-400-truncated by a further 5 residues resulted in the deletion of a highly basic sequence (RRLRL) within a region shown to be important to G-protein coupling in the 02-adenergic receptor (O'Dowd et al., 1988; Liggett et al., 1991). Fig. 49 depicts the different constructs generated. 3.7.1 EFFECT OF CARBOXY-TERMINAL TAIL TRUNCATION ON LIGAND BINDING Receptors truncated at residues 405, 418 and 427+ exhibited high affinity binding in competition binding studies similar to that seen in cells expressing the 455 amino acid wild type receptor (wtGIP-Rl), when expressed both transiently in COS-7 cells (Table 11), and stable CHO-K1 cell lines (Fig. 50A, Table 9). Receptor expression level as determined from Bmax values indicated that GIP-R-427+ was expressed at least as efficiently (125 ± 33%, CHO-K1) as the wt receptor in both cell systems (Table 9). However, in cells expressing the 418 and 405 amino acid forms of the receptor, maximal binding was 74 ± 16% and 29 ± 3% of that seen with the wtGIP-Rl cell line, respectively, suggesting these receptors were not as efficiently expressed at the membrane level as the longer forms of the protein. In contrast, when the receptor was truncated at amino acid 400 no detectable 125I-GD? binding was observed (Fig. 50A). The 179 other three constructs bound GD? with affinity similar to that of the full length GTP receptor in both COS-7 (Table 9) and CHO-K1 cells (Fig 50A, Table 11). In order to determine whether GIP-R-400 is expressed it would be preferable to determine its localization (membrane or intracellular) by immunocytochemistry. However, this was not possible due to the lack of a GTP receptor antibody. As the CT-tail length appeared to be important for efficient expression at the cell surface, an alternative approach to examine the importance of residues 401-405 was taken. A sixth construct with five alanine (400As) residues added on to GIP-R-400 was constructed to give a similar length to that of 405 (Fig. 51). Both COS-7 (Table 9) and CHO-K1 cells, stably expressing the 400As receptor, specifically bound 125I-GIP with a small but significant shift to the right in the displacement curve compared to the wtGIP-Rl cell-line (Fig. 50B, Table 9). Two other constructs were then generated to examine the region just distal to the seventh transmembrane domain, one with residues 397-405 replaced with 9 Ala residues (Gn>-R-396A9), and the other with the residues 397-400 deleted (GJP-R-AQSEI). Neither of the constructs displayed specific 125I-GD? binding in competition binding experiments (Fig. 50B, Table 11). 180 393 ' 4 ^ 5 TM7- • NKEVQSEIRRLRLSLQEQCPRPHLGQAPRAVPLSSAPQEAAIRNALPSGMLHVPGDEVLESYC \ GIP-R-400-QSEI GIP-R-405-QSEIRRLRL GIP-R-418-QSEIRRLRLSLQEQCPRPHLGQ GIP-R-427+-QSEIRRLRLSLQEQCPRPHLGQAPRAVPLSSQR G I Fig. 49. Carboxy-terminal Truncation of the Rat Islet GIP Receptor. 181 B. 125-j 100-o m 75-m 50-25-0-125-i 100-o m 75-m 50-25-0-r -0 -1— -11 Log10[GIP] i — •11 O wtGIP-Rl • GIP-R-400 A GTP-R-405 O GIP-R-418 • GIP-R-427+ O wtGIP-Rl • GIP-R-4OOA5 A GIP-R-396A9 • GIP-R-AQSIE -5 Log10[GIP] Fig. 50. Displacement of 1 2 5I-GIP by GIP in CHO-K1 Cell-lines Stably Expressing Carboxy-Terminal truncated and wtGIP Receptors. Curves are representative of at least 4 individual experiments. Expression levels (Bmax) and I C 5 0 values are summarized in Table 9. 182 COS-7 CHO-K1 Construct IC50 (nM) Bmax % of wt I C 5 0 (nM) Bmax % of wt wtGIP-Rl 1.79 100.0 2.24 ± 0.35 100 GIP427+ 1.73 100.8 2.73 ± 0.23 125.5 ±33.8 GIP418 0.42 70.4 2.28 ± 0.27 74.0 ± 16.1 GIP405 1.47 29.8 2.16 + 0.12 29.3 ± 2.94* GIP400 - - - -GrP400ala5 0.42 ND 3.79 ±0.21 * 12.0 ± 1.1* GIP396ala - - - -GIP-QSIE - - - -Table 9. Summary of Binding Experiments with Carboxy-Terminal Tail Truncated Forms of the Rat GD? Receptor. (-) = not detectable, ND = not done. (*) = significance difference from wt, p < 0.05, n =6. 183 TM7-NKEVQSEIRRLRLSLQEQCPRPHLGQAPRAVPLSSAPQEAAIRNALPSGMLHVPGDEVLESYC GIP-R-396Ag = TM7-NKEVAAAAAAAAA GIP-R^OOAg = TM7-NKEVQSEIAAAAA GIP-R-AQSEI = TM7-NKEVRRLRLSLQEQCPRPHLGQAPRAVPLSSAPQEA Al RNALP SGM LHVPGDEVLE SYC Fig. 51. Modified Carboxy-Terminal Tail forms of the Rat Islet GIP Receptor. 184 3.7.2 EFFECT OF C A R B O X Y - T E R M I N A L TAIL T R U N C A T I O N O N cAMP PRODUCTION To assess the effect of truncation on signal transduction, cAMP responses to GD? were determined with cells expressing the truncated receptors both transiently in COS-7 cells and stably in CHO-K1 cells. In COS-7 cells, maximal GD? (10 nM)-stimulated cAMP levels observed for receptors GIP-R-427+ (100.3 ± 6.5 pmolAvell) and GIP-R-418 (93.4 ± 14.6 pmolAvell) were not significantly different (p < 0.05, n = 3) from those with the wtGDP-R (104 ± 17 pmolAvell) (p > 0.05; n=3). However, GIP-R-405 (60.3 ± 12.7 pmolAvell) and GD»-R-400 (5.3 ± 0.7 pmolAvell) displayed significant decreases in maximal cAMP production (57.7 ± 12.1% and 5.7 ± 0.7% of wtGIP-R maximum, respectively) (n=3, p<0.05)) (Fig. 52A, Table 10). Of particular interest was the fact that extension of the receptor tail length to 405 amino acids, in the GD>-R-400A5 construct, restored the levels of cAMP production to 67.7 ± 6.9% of GrP-R-405 (Fig. 52B). However, neither GrP-R-386A9 nor GTP-R-AQSffi responded to 10 n M GTP stimulation (Fig. 52B, Table 10), suggesting that either these receptors were not expressed, or that regions important to G-protein coupling were changed or deleted. When expressed in CHO-K1 cells, maximal cAMP production with all of the truncated receptors was decreased when compared to that obtained in the wtGD > -Rl cell line (Fig. 53, Table 10). Surprisingly, even though there was only a small decrease in the affinity of GIP-R-405A5 for GD?, there was a large increase in the EC50 value (1.16 + 0.32 uM) when compared to the wt receptor (69 + 27 pM) (Fig. 53, Table 10). None of the other truncated forms of the receptor differed significantly from the wt receptor in their EC50 values (Fig. 53, Table 10). In agreement with results in COS-7 experiments, 185 150 u = Q. g 9- Q. I O O H B. 3 0 0 n I I Basal H 10 n M GIP Fig. 52. 10 n M GJP-stimulated cAMP Production in Carboxy-Terminal Tail Truncated Forms of the GIP Receptor. A : Of all the receptor constructs initially examined, only 400 did not respond to 10 n M GD? stimulation, while receptors with truncations up to amino acid 405 responded. B : When the 400 construct was extended to a length of 405 amino acids by the addition of 5 alanine residues 400A 5, cAMP responsiveness was restored to 67.7 ± 6.9% of that seen with the GD?-R-405. A third construct with residues 397-400 deleted (AQSEI) was also found to not respond to 10 n M GIP stimulation. 186 A. 400 J2 •S 300 8 S _ 200-i 100-0-- i — -13 -11 -9 Log1 0 [GIP] o A V O wtGIP-Rl GIP-R-400 GIP-R-405 GIP-R-418 GIP-R-427+ B. 125-i 100-s M < o 75-o ^ 50-25-0-V 0 -13 - 1 — -11 —r~ -7 Log10[GIP] V GIP-R-405 • GIP-R-400A5 O GTP-R-400A9 • GIP-R-AQSEI Fig. 53. Stimulation by GIP of cAMP Production with Carboxy-Terminal Truncated (A), Deletion, and Alanine Substituted GD? Receptor Mutants (B) Expressed in CHO-K1 Cells. Data are the mean ± S.E.M. of 3-6 individual experiments. Maximal cAMP increases and E C 5 0 values are summarized in Table 10. 187 neither GTP-R-396A9 nor GTP-R-AQSEI were responsive to GIP stimulation at concentrations as high as luM (Fig. 53, Table 10). Receptor COS-7 CHO-K1 % of wt max % of wt max E C 5 0 (pM) wtGIP-Rl (455) 100 100 69 ± 2 7 427+ 95.9 ±6.2 55.1 ± 12.5 * 47 ±20 418 89.3 ± 14.0 23.3 ±4.9* 15 ±0 .9 405 57.7 ± 12.1 8.1 ± 1.9* 11 ±0 .7 400 5.0 ±0.7* - -400ala5 - 31.3 ±6.8* 1163 ±320 * 396ala9 - - -AQSEI - - -Table 10. Summary of cAMP Experiments with Carboxy-Terminal Tail Truncated Forms of the Rat GIP Receptor. (-) = not detectable. (*) =significance difference from wt, p < 0.05, n =3-6. 3.7.3 EFFECT OF CARBOXY-TERMINAL TAIL TRUNCATION ON RECEPTOR UPTAKE AND DESENSITIZATION As serine and threonine residues are often phosphorylated in response to receptor activation, and can lead to both homologous receptor desensitization and receptor sequestration, the effect of truncation of the CT-tail on receptor uptake and homologous desensitization was examined. All the constructs were internalized over time as assessed by the increase in acid resistant pool in receptor uptake studies (Fig. 54A). The wtGIP-Rl receptor clone 188 displayed a rapid increase in acid resistant binding over time, reaching maximal levels (64.9 ± 2.7% of the total bound) within 120 minutes (Fig. 54A, Table 11). Maximal internalization of the truncated receptors did not differ significantly from that seen for the full length GIP-R.-455 cell line at time points from 60-120 min (Fig. 54A, Table 11). Further incubation times of up to 4 hours failed to reveal a difference in maximal uptake of the different receptor constructs in CHO-K1 cells. Closer examination of the initial uptake suggested that it was linear in nature, and the rate of initial uptake was examined for the different constructs (Fig. 54B). Surprisingly, truncation of the tail by 28 residues (GIP-R-427+) and 37 residues (GIP-R-418) caused significant decreases in the rate of internalization over the first 10 min compared to that seen for the wt cell line (Fig. 54B, Table 11). However, further truncation of the C-terminal tail by 50 amino acids (GIP-R-405) partially restored the rate of receptor uptake and, with the GIP-R-400A5 construct, the rate of uptake was not significantly different from that of the wt receptor (Fig. 54B, Table 11). These data suggest that truncation of the more distal region of the receptor interfered with receptor sequestration, possibly by stabilizing a conformation unable to interact as efficiently with other proteins involved in the receptor uptake machinery. Further truncation of the receptor to 405 amino acids (GIP-R-405 and GTP-R-400A5) appeared to restore efficient receptor sequestration activity to the GD? receptor, possibly by relieving conformational inhibition. More detailed mutational analysis is required to determine i f this is due to removal of a negative sequestration motif or exposure of a positive sequestration signal, as has been suggested for other receptors (Huang et al., 1995b; Findlay et al., 1994). 189 A. i 1 1 1 1 0 30 60 90 120 Time (min) B. i 1 1 1 1 0 3 6 9 12 Time (min) Fig. 54. The Effect of Carboxy-Terminal Tail Truncation of the Rat Islet GIP Receptor on Receptor Sequestration in CHO-K1 Cell Lines. A : No significant difference in maximal receptor sequestration was observed between the wtGIP receptor and four truncated receptors examined. However, uptake of wtGIR-Rlwas more rapid and was significantly greater than that seen for some CT truncated receptors at time points 5-40 min. B : Initial rate of receptor uptake was greater for the wtGIP-Rl receptor than that seen for GIP-R-418, GJP-R-427+, and GIP-R-405, while no significant difference between GnM00A5 and the wt GJP-R1 uptake rates was observed. Maximal sequestration (% of total binding), and rate of internalization (Slope) values are summarized in Table 11. 190 Construct Maximal Internalization (% of Total Bound) Slope (%/min) GIP-R-455 64.9 ± 2.7 2.94 ± 0.26 GTP-R-400A5 61.8 + 2.9 2.53 ± 0.24 GIP-R-405 60.3 ±3.3 2.02 ±0.16 * GIP-R-418 73.8 ±2.4 1.64 ±0.16 * GIP-R-427 69.8 ± 1.0 1.60 ±0.08 * Table 11. Maximal Receptor Internalization and Initial Slope Values for Receptor Uptake Over the Initial 10 Minutes. Values are the mean ± S.E.M. of at least 3-4 individual determinations. * = Significant difference from wtGIP-Rl, p<0.05. Attempts at examining receptor desensitization with the wtGIP-Rl cell line were undertaken, but were without success (Fig. 55). wtGIP-Rl cells pre-incubated with GD? (1 nM-luM) , for 15 min-2 hours did not differ in their subsequent responses to 1 or 10 nM GIP, in comparison to untreated wtGIP-Rl cells. Nor did their E C 5 0 values differ significantly i f dose-response curves were generated after pre-treatment. This suggests that the GIP receptor does not undergo homologous desensitization, or at least not in CHO-K1 cells. 191 Fig. 55. Lack of Desensitization of GIP-mediated cAMP Production in wtGIP-Rl Cells. Cells were pre-incubated with 10 n M GIP for 20 min., washed three times with assay buffer, then challenged again with 10 n M GD?. cAMP levels were normalized to % of untreated cells . 192 CHAPTER 4 DISCUSSION 4.1 ISOLATION A N D CHARACTERIZATION OF R A T PANCREATIC ISLET GIP RECEPTOR cDNAs The pancreatic islet GD? receptor has proven difficult to study due to its apparent low level of expression, and difficulties in obtaining islets in sufficient quantities for detailed investigations. In light of these limitations much of the information regarding GJP receptor expression has been obtained from membranes of multipotential islet-derived tumor cells that express a variety of neuroendocrine peptide receptors (reviewed in Pederson, 1993; Mcintosh et al., 1996; Fehmann et al., 1995). The first objective of the studies undertaken in the current thesis was to isolate cDNAs encoding the rat islet GD 3 receptor to enable study of its ligand binding and intracellular signaling properties. 4.1.1 THE R A T ISLET GIP RECEPTOR cDNA Islet receptor cDNAs (GIP-R1, GTP-R2 and Gff-R3) were isolated, and sequencing demonstrated that they were homologous to the cDNA obtained by PCR from a rat tumor cell line (RTNm5F) (Usdin et al, 1993). However all three receptor cDNAs differed from the sequence reported by Usdin et al. (1993) at one nucleotide (Fig. 21) resulting in a single amino acid difference (Glu21—>Gln21) (Fig. 22), suggesting that this is a single nucleotide polymorphism (SNP) in the rat gene, rather than a PCR based sequencing error. Nevertheless, it is evident that the GIP receptor expressed endogenously in rat islets 193 (Wheeler etal., 1995) is the same as that expressed in RINm5F cells and the brain (Usdin etal, 1993). Similar GTP receptor cDNAs to that described in this Thesis have also been isolated from the hamster cell line FTTT-T15 (Yasuda et al. 1994), human islets (Gremlich et al, 1995), and a human insulinoma cDNA library (Volz et al, 1995). The human 466 and hamster 462 amino acid GD? receptors displayed a high degree of identity with the rat, 79%, and 86% respectively (Volz et al, 1995; Yasuda et al, 1994), with the greatest divergence residing in the signal peptide and carboxy-terminal tail regions (Gremlich et al, 1995). Both groups that isolated human receptor cDNAs reported partial cDNAs. One of these displayed a small deletion in the second extracellular loop (Gremlich et al, 1995), and the second was an alternatively spliced, truncated form of the receptor due to the deletion of a 62 base pair exon, which resulted in a frame shift within the fourth transmembrane domain (Volz et al, 1995). The receptors were non-functional when expressed in C H L cells. Gremlich et al. (1995) also isolated both a short and long form of the human receptor; the latter most likely arising from a partially spliced pre-mRNA resulting in the insertion of an additional 27 amino acids at the juxtamembrane region of the cytoplasmic tail. Surprisingly, this long form was shown to be expressed and to function in a similar fashion to the fully spliced cDNA, encoding a receptor of 466 amino acids (Gremlich etal, 1995). The GTP receptor gene has been mapped to human chromosome 19 (Gremlich et al, 1995; Stoffel et al, 1995). Cloning and sequencing (Yamada et al, 1995) revealed that the -13.8 kb gene contains 14 exons. The exon-intron organization is conserved among members of the VTP/glucagon/secretin receptor family (Yamada et al, 1995), 194 being similar to that seen with the genes for both the mouse parathyroid hormone receptor (McCuaig et al, 1994), and the human and mouse glucagon receptors (Lok et al, 1994; Burcelin etal, 1995). A l l GPCRs share the same basic secondary structure consisting of an extracellular amino-terminal "head", seven transmembrane domains, with three extracellular (EC) and three intracellular (IC) loops, and an intracellular carboxy-terminus (CT) tail (see Fig. 22). The rat pancreatic GD 3 receptor is a member of the Secretin/Glucagon/VTP receptor sub-family (Usdin et al, 1993; Donnelly, 1997; Ulrich et al, 1998) of the GPCR superfamily (Probst et al, 1992). Members of the Secretin/Glucagon/VTP receptor family lack any significant sequence identity to other GPCRs, such as the P-adrenergic and rhodopsin receptors, but share substantial identity with each other (Segre and Goldring, 1993; Donnelly, 1997; Ulrich etal, 1998). Members of this family bind moderately large peptides, and include the receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) (Spengler et al, 1993), calcitonin (Lin et al, 1991), calcitonin gene-related peptide (Aiyar et al, 1996), parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) (Juppner etal, 1991), growth hormone-releasing factor (GHRH) (Godfrey et al, 1993), corticotropin-releasing factor (CRF) (Chang et al, 1993), glucagon (Jelinek et al, 1993), GLP-1 (Thorens, 1992) and insect diuretic hormone (Reagan, 1994). As mentioned in section 1.5.1 the rat islet GTP receptor shares the greatest identity with the glucagon and GLP-1 receptors (-40%) (Fig. 56). 195 Signal peptide rGIP-R MP LRLLLLLLWLWGLSLQRAETDSEGQTTGEL—YQRWERYGWEC 43 hGLP-lR MAGAPGPLRLALLLLGM VGRAGPRPQGATVSLWETVQKWREYRRQC 46 hGlu-R MP-PCQPQRPLLLLLLL LACQPQVPSAQVMDFL—FEKWKLYGDQC 43 rGIP-R QNTLEATEPP-SGLACNGSFEMYACWNYTAANTTARVSCPWYLPWYRQVA 92 hGLP-lR QRSLTEDPPPATDLFCNRTFDEYACWPDGEPGSEVNVSCPWYLPWASSVP 96 hGlu-R HHNLSLLPPP-TELVCNRTFDKYSCWPDTPANTTANISCPWYLPWHHKVQ 92 ** * * * ******** rGIP-R AGFVFRQCGSDGQWG SWRDHTQCENP—EKNGAPQDQKLIL-E 132 hGLP-lR QGHVYRFCTAEGLWLQKDNSSLPWRDLSECEESKRGERSSPEEQLLFLY- 145 hGlu-R HRFVFKRCGPDGQWV-RGPRGQPWRDASQCQMD—GEEIEVQKEVAKMYS 139 rGIP-R RLQWYTVGYSLSLATLLLALLILSLFRRLHCTRNYIHMNLFTSFMLRAG 182 hGLP-lR IIYTVGYALSFSALVIASAILLGFRHLHCTRNYIHLNLFASFILRAL 192 hGlu-R SFQVMYTVGYSLSLGALLLALAILGGLSKLHCTRNAIHANLFASFVLKAS 189 * * * * * * * * * * * * * * * * * * * * * * rGIP-R AILTRDQLLPP-LGPYTGNQTPT LWNQA LAACRT AQILT Q YC VGAN Y 228 hGLP-lR SVFIKDAALKWMYST-AAQQHQWDGLLSYQDSLSCRLVFLLMQYCVAANY 241 hGlu-R SVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQYGIVANY 239 rGIP-R TWLLVEGVYLHHLLVWRRSEKGHFRCYLLLGWGAPALFVIPWVIVRYLY 278 hGLP-lR YWLLVEGVYLYTLLAFSVLSEQWIFRLYVSIGWGVPLLFWPWGIVKYLY 2 91 hGlu-R CWLLVEGLYLHNLLGLATLPERSFFSLYLGIGWGAPMLFVVPWAVVKCLF 289 * * * * * * * * * * * * * * * * * * * * * * * * rGIP-R hGLP-lR hGlu-R rGIP-R hGLP-lR hGlu-R ENTQCWERNEVKAIWWIIRTPILITILINFLIFIRILGILVSKLRTR^R E DEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVI CI WSKLKANLMC ENVQCWTSNDNMGFWWILRFPVFLAILINFFIFVRIVQLLVAKLRARQMH * * * * CPDYRLRLARSTLTLMPLLGVHEWFAPVTEEQAEGSLRFAKLAFEIFLS KTDIKCRLAKSTLTLIPLLGTHEVIFAFVMDEHARGTLRFIKLFTELSFT HTDYKFRLAKSTLTLIPLLGVHEWFAFVTDEHAQGTLRSAKLFFDLFLS 328 341 339 378 289 378 * * * * * * * * * * * * * * * * * * rGIP-R SFQGFLVSVLYCFINKEVQSEIRR LRL SLQEQCPRP 414 hGLP-lR SFQGLMVAILYCFVNNEVQLEFRKSWERWRLEHL HIQRDSSMK 434 hGlu-R SFQGLLVAVLYCFLNKEVQSELRRRWHRWRLGKVLWEERNTSNHRASSSP 439 rGIP-R HLGQAPRAVPL — SSAPQEAAIRNALPSGMLHVPGDEVLESYC 4 55 h GLP-1 R PLKCPTSSLSSGATAGS SMYTATCQASCS 4 63 hGlu-R GHGPPSKELQFGRGGGSQDSSAETPLAGGLPRLAESPF 477 Fig. 56. Alignment of the Amino Acid Sequences of the Rat GTP, Human GLP-1, and Human Glucagon Receptors. Amino acids are presented as their single letter abbreviations. * = residues completely conserved, . = residues well conserved. Putative transmembrane domains are overlined. Alignment was carried out using the PC Gene software package (IntelliGenetics, 1995). 196 The receptor has a large amino-terminal head (-135 amino acids) with three putative N -linked glycosylation sites (asparagines 59, 69, and 74) and 6 conserved cysteine residues, as well as two other highly conserved cysteine residues in the first and second E C loops that are thought to form disulfide bonds that may be important in the maintenance of receptor structure. 4.1.2 CHARACTERIZATION OF GIP RECEPTOR BINDING A N D SIGNALING V I A cAMP In initial binding studies, cells expressing GD?-R1 transiently, or stably, demonstrated a single class of high affinity binding sites (IC50 = 1.2-7.9 nM) in competitive binding studies (Fig. 23, Table 1), values within the range of those reported in tumor cell lines (reviewed in Pederson, 1993;McIntosh et al, 1996), and with expressed receptor cDNAs from the hamster B-cell line, HIT-T15 (Yasuda et al. 1994), human islets (Gremlich et al, 1995), and a human insulinoma c D N A library (Volz et al, 1995). Intact wtGIP-Rl cells, and membranes purified from these cells, both bound 1 2 5 I -spGIP in a concentration dependent manner in saturation binding studies (Fig. 24). Non-linear regression analysis indicated that the isotherms were monophasic, with K d values of 200-300 pM, and an expression level of 12-15 X 103 receptors per cell, or 59.2 ± 5.2 pmol of receptors/mg of protein. In addition, synthetic porcine GD? yielded concentration-dependent increases in cAMP in wtGIP-Rl cells with an EC50 of 8.7 xlO" 10 M (Fig. 28), a value which is in agreement with those previously reported in HIT cells (Lu et al, 1993a) and the originally isolated RTN cell cDNA (Usdin et al, 1993). More recently, others have demonstrated that the cells expressing hamster (Yasuda et al, 1994) 197 and human (Gremlich et al, 1995; Volz et al, 1995) GD? receptors responded to GD 5 with increases in cAMP levels, with EC50 values ranging from 300 p M (Usdin et al, 1995) to 15 n M (Yasuda et al, 1994). Volz et al (1995) reported a technically inaccurate value of 0.12 fM, since these authors extrapolated to below tested peptide concentrations. The considerable range of reported EC50 values probably results from technical differences in the determination of cAMP levels, and in the cell lines used in the experiments. Importantly, none of the related peptide hormones tested in the current study (GLP-1, GLP-2, glucagon, or VTP) displaced 1 2 5I-spGIP (Fig. 26) or stimulated cAMP production (Fig. 30) in the n M range, demonstrating that GIP-R1 encoded a rat islet GrP-specific receptor. This lack of cross reactivity has been confirmed by others for the rat (Usdin et al, 1993), hamster (Yasuda et al, 1994), and human receptor (Gremlich et al, 1995; Volz et al, 1995) indicating the absolute specificity of the GD? receptor from different animal species for its ligand. Recently, the use of the GLP-1 receptor antagonist Ex (9-39) has provided important information supporting a role for GLP-1 in the enteroinsular axis (Kolligs et al., 1995; Wang et al., 1995; D'Alessio et al., 1996; Schirra et al., 1998). In the current studies, exendin-4 and exendin (9-39) were the only non-GD? peptides that displaced 1 2 5 I -spGTP binding (Fig. 27), a finding that was later confirmed with the human GIP receptor (Gremlich et al, 1995), suggesting that at high concentrations these peptides may antagonize or potentiate GD 5 action. While COS-7 cells expressing G f f - R l displayed a small increase in cAMP levels at the highest concentration of Ex-4 tested (1 pM), neither Ex-4 nor Ex (9-39) increased cAMP levels in the equivalent CHO-K1 cell line (wtGIP-R l ) (Fig. 28 and 29B). In addition, neither Ex (9-39) nor Ex-4 were able to inhibit or 198 augment GIP stimulated cAMP production in wtGIP-Rl cells (Fig. 29A), while in parallel studies with cells expressing the rat GLP-1 receptor, Ex (9-39) inhibited GLP-1 -stimulated cAMP production by approximately 65% (Fig. 29B). These findings suggest that interaction of the venom peptides with the GIP receptor is very weak, and support recent studies in the rat (Kolligs et al, 1995; Wang et al, 1995) and humans (Schirra et al., 1998) indicating that Ex (9-39) does not alter the contribution of GIP to the enteroinsular axis in vivo, or decrease its insulinotropic action in RTN5 AH cells (Wang et al, 1995) at the concentrations used. Although GTP has potent insulinotropic properties and facilitates glucose disposal, controversy exists in the literature as to the relative effectiveness of GTP and GLP-1 in stimulating insulin release. Some investigators have found GTP to be equipotent to GLP-1 (Schmid et al, 1990; Suzuki et al, 1990) while others have found that GLP-1 has markedly greater insulinotropic activity (Siegel et al, 1992; Shima et al, 1988). In addition, GTP was shown to be strongly insulinotropic in humans in one study (Nauck et al, 1989) while a subsequent report by the same authors demonstrated that GIP was a poor stimulant of insulin secretion (Nauck et al, 1993a). Some of these differences may be explained by variability in the relative potencies among several commercially available GTP preparations. Having a single species of the rat GIP receptor expressed in COS-7 and CHO-K1 cells provides an ideal model with which to address this issue. The present studies revealed no significant differences between porcine and human GTP formulations tested with respect to binding properties or ability to stimulate cAMP accumulation. This suggests that the rat GIP receptor does not have preferential affinity for either peptide, and that the Hisl8->Argl8 and Ser34-»Asn34 amino acid differences 199 between porcine and human GTPs do not influence ligand-receptor interaction. These studies also revealed no significant difference in the affinities or potencies of synthetic GD? preparations obtained from two different suppliers (Fig. 26, Table 2, and Table 3). Unlike earlier peptide preparations (Jia et al., 1995), the current batches of spGD? and shGIP were also equipotent as insulinotropic agents in the isolated perfused rat pancreas (Table 3). Since porcine and human GIP bind and activate the rat islet GIP receptor equally, such a heterologous system can be used for structure-activity studies on these peptides. Also, while the present studies cannot conclusively confirm that the discrepancies seen in the literature concerning the insulinotropic potency of shGD? are due to variations in the synthetic preparations, they do indicate that GDP-R1 expressing cell lines represent an ideal model to assess such preparations in the future. 4.1.3 EFFECTS OF GIP O N I N T R A C E L L U L A R C A L C I U M Usdin et al. (1993) examined the RINm5F GD 3 receptor signaling pathway utilizing a cell line consisting of HEK293 cells expressing apo-aequorin which, when reconstituted with the chromophore coelenterazine, emits light on the binding of calcium. Cells transfected with the RINm5F cell GIP receptor displayed increases in [Ca 2 +]i in response to GD 3 , however the mechanism underlying the increase was not determined. As discussed in section 1.7.1 and 1.7.2 there has been considerable discrepancy in the literature concerning the effects of both GIP and GLP-1 on changes in intracellular calcium. However, characteristics of Ca2+-signaling in response to GLP-1 have been more extensively examined, and provide clues as to how GIP and its receptor may signal through this system. Real time spectrofluorimetry studies on GIP-dependent Ca 200 responses were performed in Toronto using cells transfected with the wild type cDNA described in the current Thesis (see Appendix A). The data are discussed here since both the design and interpretation of experiments were performed collaboratively. COS-7 cells expressing the GD? receptor displayed a biphasic [Ca 2 +]i response in the presence of GD? (50 nM) with an acute transient phase, termed PI , which then decreased to a sustained level over basal, termed P2 (Fig. A l ) . Phase P2 was abolished under conditions in which extracellular calcium was removed from the medium, suggesting that this response was dependent on C a 2 + influx across the plasma membrane, while PI persisted under C a 2 + free conditions, strongly suggesting that it represents C a 2 + release from intracellular stores (Fig A l , Table A l ) . To examine the nature of PI further, cells were treated with the endoplasmic reticulum Ca 2 +-ATPase inhibitor thapsigargin. As thapsigargin is known to deplete the intracellular inositol triphosphate-sensitive C a 2 + stores (Lytton et al, 1991), loss of the peak transient phase after this treatment supports the possibility that PI represents a GEP-induced mobilization of C a 2 + from intracellular sources. Although the ability of the GIP receptor to couple to intracellular mobilization of C a 2 + has not previously been described in pancreatic islets, the related incretin GLP-1 in part increases [Ca 2 +]i through the release of intracellular C a 2 + stores in mouse (Cullinan et al., 1994) and human (Gromada et al, 1998a,b) P-cells. Furthermore, L-type Ca2+-channel blockers do not block the 2+ [Ca ]i response in mouse and human B-cells once the initial GLP-1 mediated increase has been initiated (Cullinan et al., 1994; Gromada et al, 1998a), suggesting that entrance of C a 2 + from extracellular sources acts as a feed forward stimulus of C a 2 + release (calcium-induced calcium release; CICR). A similar response to GD? does not appear to 201 be present in COS-7 cells, as the transient (PI) response was unchanged in the absence of extracellular Ca 2 +. It is possible that the overexpression of the GD? receptor results in inappropriate coupling to G-proteins and mobilization of Ca 2 + from intracellular sources, alternatively it may be that GJP receptor is acting via an alternative Ca2+-signaling pathway in non-beta cells such as COS-7 cells. Given the contradictions in the literature concerning GIP and GLP-1 mediated increases of [Ca2+]i, it is likely that the cellular environment in which the response is observed is very important. For example both ourselves, with CHO-K1 cells expressing the rat islet GIP receptor (Appendix A), and others (Gremlich et al, 1995; Volz et al, 1995) with the GIP receptor expressed in Chinese hamster lymphoblast (CUL) cells, have observed that GIP stimulation of the receptor in these cellular environments resulted in no increase in [Ca2+]i levels. In addition, studies on several tumor cell lines (Lu et al, 1993a; Widmann et al, 1994) and on CHO-GR1 cells stably transfected with the hamster GIP receptor revealed no GTP-dependent increases in D>3 levels (Yasuda et al, 1994). An D°3-mediated increase in [Ca2+]i is therefore unlikely. Similar contradictions exist concerning the GLP-1 receptor. Interaction of GLP-1 with its receptor results in the activation of a number of other signal transduction pathways that, as with GD? receptor signal transduction, appear to depend on the cell type used for expression studies. For example, expression of the receptor transiently in COS-7 cells was reported by one group to increase levels of IP3 and [Ca2+]i (Wheeler et al, 1993), while a second group did not see consistent increases in TP3 levels, and no effect on [Ca2+]i when expressed in the same cell line (Widmann et al, 1994). Dillon and coworkers (1993) reported that GLP-1 increased [Ca2+]i levels in COS-7 cells expressing the human GLP-1 receptor, as with the 202 GD? receptor, but no GLP-1-dependent increases in [Ca + ] i were observed in C H L cells expressing the human GLP-1 receptor (Thorens et al, 1993). This was unlikely to be due to species differences, as the rat GLP-1 receptor also failed to increase [Ca 2 +]i in C H L cells (Widmann et al, 1994). In contrast, GLP-1 was found to stimulate large increases in [Ca 2 +]i in the human embryonic kidney cell line (HEK293) stably expressing the human GLP-1 receptor (Gromada et al, 1995). Receptors for several other members of the Secretin/VD? receptor family, including those for PTH/PTHrP (Abou-Samra et al, 1992), glucagon (Jelinek et al, 1993), calcitonin (Cal) (Force et al, 1992; Houssami et al, 1994), and the P A C A P receptor family (Spengler et al, 1993), are capable of mobilizing intracellular C a 2 + stores. Given the apparently wide distribution of the GIP and the GLP-1 receptor in extrapancreatic tissues, it is quite possible that, as with glucagon and PTH/PTHrP receptors, the GIP receptor couples to different species of G-protein and signal transduction pathways in different tissues (Spengler etal, 1993; Wakelam etal, 1986). How are the effects of GD? and GLP-1 on cAMP production and intracellular C a 2 + levels linked to insulin secretion? Lu etal. (1993a) using the glucose responsive HIT-T15 cell line as a model, demonstrated that insulin secretion was associated with both GD?-induced increases in cAMP levels and C a 2 + influx through VDCCs. This correlates with findings that GLP-1 stimulated increases in [Ca 2 +]i can be blocked with L-type channel blockers, both in isolated p"-cells (Fridolf and Ahren, 1993; Holz etal, 1995; Yada etal, 1993) and cell lines (Gromada et al, 1995; Lu et al., 1993a). The overall conclusion from such studies is that GLP-1, and probably GIP, target either the ATP-sensitive K + channel and/or V D C C to increase [Ca 2 +]i through cAMP-dependent protein kinases. The 203 current findings, that GD? evoked a concentration-dependent increase in cAMP accumulation, support this model. However, since COS-7 cells do not bind dihydropyridines, and do not exhibit [Ca2+]i responses to KCI or forskolin (M.B. Wheeler, personal communication), it is unlikely that the GD?-induced increases in Ca 2 + influx were mediated by cAMP-dependent effects on VDCCs. This possibility is supported by the observation that increases in [Ca2+]i during the sustained phase (P2) were not sensitive to the L-type calcium channel antagonist nifedipine. Since P2 was dependent on extracellular Ca 2 + , GJP may activate a voltage-independent Ca2+/cation channel. Recent studies using the mouse insulin-secreting tumor cell line BTC6 (Holtz et al., 1995) and rat islets demonstrated that a component of the tGLP-1 induced increase in [Ca2+]i is not sensitive to nifedipine or membrane depolarization (Holtz et al., 1995, Kato et al., 1996). Holz and coworkers (1985) suggested that, in addition to its actions on ATP-sensitive K + channels, GLP-1 modulates B-cell Ca 2 + influx through voltage-independent Ca 2 + channels or non-specific cation channels. A similar conductance, that is activated by maitotoxin, has been identified in mouse B-cells (Worley et al., 1994), and a P-cell line (Leech and Habener, 1997). In addition Soergel and co-workers (1990) have demonstrated that this maitotoxin-activated conductance is associated with stimulation of insulin secretion from the HIT B-cell line. Whether the voltage-independent Ca influx evoked by GD5 in COS-7 cells and GLP-1 in BTC6 cells represent the same receptor-mediated Ca 2 + entry pathway remains to be determined. Also undetermined is the significance of the heterogeneity in the Ca 2 + flux patterns observed in individual cells (Fig. A2). It is possible that the magnitude of the Ca 2 + response is dependent on the level of receptor expression, representing another potential mode of agonist regulation. Such a 204 relationship has been documented for the PTH/PTHrP (Guo et al, 1994) and the calcitonin receptor isoforms Cla and Clb, where an increase in the Ca 2 + sensitivity to CT was positively correlated with receptor number (Houssami et al, 1994). In summary, it is likely that GIP, like GLP-1, stimulates insulin release via cAMP-dependent pathways at sites both proximal and distal to increases in [Ca2+]i. Such pathways probably involve phosphorylation of ATP-sensitive K + channels, VDCCs, and/or VINCCs, resulting in potentiation of glucose-dependent cellular depolarization, increased intracellular calcium levels, and increased recruitment of secretory granules from the reserve pool to the readily releasable pool (Ding et al, 1997; Gromada et al, 1998a, b). However in other cell types the modes of action may differ, and it will important to examine the effects of GIP and GLP-1 in such cells to determine whether there are cell-specific signal-transduction pathways. 4.2 IDENTIFICATION OF THE CORE GIP BINDING REGION In humans, GIP has been shown to be a potent incretin (Brown et al, 1989; Pederson et al, 1993; Fehmann et al, 1995) and, since incretins and their analogs have promising therapeutic potential for the treatment of NTDDM (Nauck et al., 1989; Gutniak et al, 1992; Byrne and Goke, 1996; Todd et al, 1997; Nauck et al., 1997a), it is important to develop an understanding of the regional sequence requirements for receptor interaction. As discussed in section 1.10, structure-activity studies on GIP showed that limited truncation at the C-terminus had relatively minor effects on its insulinotropic activity. Thus, GIP 1-38 (Moroder et al, 1978) and GTP 1-39 (Sandberg et al, 1986) were found to exhibit similar activity to GTP 1-42 in the perfused rat pancreas. GIP 1-31 205 was shown subsequently by Maletti et al. (1987) to exhibit 10-fold weaker receptor binding affinity than GIP 1-42, but to have similar adenylyl cyclase stimulating activity. A further analog, GD? l-30amide, was also shown to stimulate insulin secretion (Pederson etal., 1990) and inhibit bombesin-stimulated amylase secretion (Rossowski et al., 1992). Further studies on N-terminally truncated forms of GTP showed that GH» 17-42 possessed significant insulinotropic activity (Maletti et al., 1986; Carlquist et al., 1984). Morrow and co-workers (1996) examined the effect of shorter GD? fragments in the perfused rat pancreas, based on the premise that amino acids 15-30 appeared to be crucial for activity, and showed that GJP 17-30 and GIP 19-30 exhibited low, but significant, insulinotropic activity. Overall these studies suggested that the region of GIP important for insulinotropic activity lies between residues 17 and 30, but that the N -terminus of the molecule is also important for receptor binding. However, many of the fragments used in these studies were produced enzymatically, and tested in bioassays complicated by the presence of GLP-1 and glucagon receptors, with which the fragments could conceivably cross-react. The cloning of the rat islet GD? receptor and its expression in CHO-K1 cells provided an ideal system to reexamine, in isolation, the interaction of some of these fragments with the GIP receptor, as well as better define which residues were involved in both receptor binding and receptor activation. None of the fragments generated based on the conserved region of amino acids 21-28 of GIP, GLP-1 and glucagon (GIP 18-28, GIP 21-26, GLP-1 21-26, and Glucagon 21-26), affected 125I-GD> binding or cAMP production. Interestingly, GIP 17-30 and GD> 19-30 neither displaced 125I-Gn» binding, nor stimulated cAMP accumulation in wtGD*-R l cells (Fig 31,32, and Table 4), despite having been demonstrated to exert some 206 insulinotropic activity in the perfused pancreas (Morrow et al., 1996). This suggests that they may be acting either via interaction with another related receptor or an unidentified isoform of the GD? receptor. However, preliminary studies indicate that neither of these fragments interact with the rat GLP-1 receptor (P. Dan and C.H.S. Mcintosh, personal communication). In contrast, GIP l-30amide displayed high affinity GD? receptor binding (3.01 ± 0.69 nM) and, despite a small shift to the left in its displacement curve compared to GIP (1.21 ± 0.46 nM) (Fig. 31, Table 4), exhibited equivalent activity in cAMP experiments both in its EC50 and maximal cAMP levels obtained (Fig.32, Table 4). 4.3 GIP 6-30AMIDE CONTAINS THE HIGH AFFINITY BINDING REGION OF GD? AND IS A POTENT INHIBITOR OF GIP ACTION IN VITRO While these observations confirmed that GTP l-30amide interacted with the GIP receptor, and that the fragment contained the majority of the determinants for high affinity binding, the exact N-terminal requirements of receptor binding were still unclear. It was reasoned that limited truncation of GIP 1-30 at the N-terminus should retain the region which is important for receptor binding and could result in peptides with either agonist or antagonist activity. Examination of the predicted secondary structure (PC Gene) (see Fig. 33), and comparison to the crystal (Sasaki et al., 1976) and lipid/aqueous interface NMR (Braun etal., 1983) structures of glucagon, suggested that residues 10-30 contain a putative extended alpha helical region when GD? is associated with its receptor (Sasaki etal., 1976). This is a conformation that has been suggested to play an important role in receptor binding for members of the glucagon superfamily (Bodanszky, 1974). 207 Interaction with the rat GIP receptor with this predicted a-helical stretch (GD? 10-30), and two other fragments with amino-terminal extensions from the a-helix boundary predicted to begin at Tyr 1 0 (GIP 7-30 and GIP 6-30amide), were therefore studied. Importantly, the results showed that the core region responsible for high affinity binding lies within residues 6-30, since GIP 6-30amide displays a receptor affinity equivalent to that of the intact peptide (IC50s = 2.39 ± 1.15 nM and 3.08 ± 0.57 nM, respectively). Further truncation of the N-terminus, by one and four amino acids, resulted in peptides with 74-fold (GIP 7-30) and 235-fold (GIP 10-30) lower affinity, as determined from competitive binding assays. Truncation of the full length GD? polypeptide by 15 amino acids (GIP 15-42) resulted in a 641-fold decrease in the peptide affinity when compared to that observed for the endogenous peptide (Fig. 34A). Despite the ability of all four peptides to bind to the GIP receptor, they demonstrated either extremely weak or absent stimulant activity on adenylyl cyclase (Fig.34B), suggesting that they could act as antagonists. This possibility was confirmed, with GD? 15-42, GD3 10-30, and GD? 7-30, inhibiting GD? (1 nM)-stimulated cAMP production in the uM range, and GIP 6-30amide inhibiting by 58 ± 2.5% at a concentration of 100 nM (Fig 35). The current results regarding the antagonist activity of GD? 7-30 are in agreement with a recent report that C-terminally amidated rat GD? 7-30 inhibited GTP stimulated cAMP production in cells transfected with GIP receptor cDNA, and in 0-TC3 cells, and displaced 125I-GD? binding to 0-TC3 cells (Tseng et al., 1996a). However, although in vitro GD? 6-30amide exhibits much higher receptor binding affinity and is a more potent inhibitor of GIP stimulated adenylyl cyclase activity than GTP 7-30, it has not so far proven possible to show inhibition of insulin release with either GD? 6-30amide or 7-30 208 during oral glucose tolerance tests in the rat (Pederson and Mcintosh, personal communication). One possible explanation for this discrepancy is that Tseng et al. (1996a) assessed the inhibition by GIP 7-30amide of responses to a GIP infusion in the absence of stimulating glucose levels in the anesthetized rat, and of insulin release following a 30 min feeding period, after an overnight fast. Both protocols resulted in low level insulin secretion (Tseng et al, 1996a). It therefore appears that while GD? 7-30 and GIP 6-30amide may be useful for antagonizing GD* action in cell culture and other in vitro applications, they are of limited use in examining the in vivo actions of GD?, probably due to increased degradation and short half-life in the whole animal. The observation that N-terminally truncated fragments of GD* could still bind to the receptor, but did not result in activation, suggests that while the binding core is located between residues 6-30, other residues in the N-terminus are critical for receptor activation. This appears to be a common theme for ligands of the secretin/glucagonATP receptor family, as the carboxy-terminal region of glucagon (Hruby, 1982), secretin, VIP (Gourlet et al, 1996; Robberecht et al, 1986; Turner et al., 1986), calcitonin, and PTH (Bergwitz et al, 1996; Stroop et al, 1996) have been shown to contain the core sequences critical for binding, while the amino-termini of the ligands are required for receptor activation. Additionally, several N-terminally truncated VD? (Turner et al, 1986), secretin (Konig et al, 1984; Kofod et al, 1991), and PTH/PTHrP (Gardella et al, 1996) fragments have been demonstrated to antagonize the action of these hormones in vitro. However, in a similar fashion to GIP 7-30 and GIP 6-30amide, these fragments were of limited use due to their lack of potency (Ulrich et al, 1998). 209 Recently, the PTH/PTHrP antagonists [Leu 1 1, D-Trp12]hPTHrP-(7-34)amide and [D-Trp 1 2, Tyr34]bPTH-(7-34)amide were demonstrated, using constitutively active mutants of the human PTH/PTHrP receptor (Schipani et al, 1995, 1996), to be the first examples of inverse agonism within the secretin/glucagon/vTP receptor family (Gardella etal, 1996). As discussed in section 1.11.3, GPCRs can exist in either the inactive R or active R* conformation, with receptors in the basal state existing at an equilibrium that favours the inactive R form (Samama et al, 1992; Milligan et al, 1995; Scheer and Cotecchia, 1997). As inverse agonists stabilize the receptor in the inactive R form, they may be of use in the treatment of some disease states resulting from constitutively active receptors, or increased basal receptor sensitivity due to ectopic production and/or overexpression of a receptor. Given that some forms of food-dependent Cushing's syndrome are characterized by the presence of either ectopic production or overexpression of the GD? receptor in the adrenal (Lacroix etal., 1992; Reznik etal, 1992), a GD? receptor inverse agonist may be useful in treatment of this disease. However, the GD? receptor point mutant generated in these studies, H170R, did not differ from the wt receptor in its affinity for GD?, in basal or stimulated cAMP production (Fig. 36), or in its ability to invoke increases in [Ca 2 +]i (Appendix B , Fig. A3). It was therefore not possible to determine i f any of the peptides found to antagonize GIP-stimulated cAMP production could act as inverse agonists. Tseng and Lin (1997) recently confirmed that rat receptors containing the H170R mutation, when expressed in human embryonic kidney cells, did not differ from the wild type in affinity or in stimulation of cAMP production. However, mutations of the homologous His residue in the glucagon receptor did result in increased basal cAMP 210 levels, with a blunted glucagon-stimulated response (Hjorth et al, 1998), while point mutants of both the glucagon and GD? receptors at conserved Thr352 and Thr340 residues, at the base of the 6th transmembrane domain, resulted in receptors with increased basal cAMP production but unaltered ligand-induced cAMP production. This mutation is similar to a second naturally occurring mutation identified originally in the PTH/PTHrP receptor (Schipani et al, 1995, 1996), suggesting that it is located in a region involved in tethering the receptor in an inactive R state, and/or transducing the ligand binding signal within this family of GPCRs. However, it is important to note that while a T340P mutation resulted in constitutively active GD* and PTH/PTHrP receptors, a T352A but not a T352P mutation resulted in constitutive activation of the glucagon receptor, indicating that differences exist in the sequence requirements for this activity in different receptors. It has been demonstrated that the N-terminal residues His1, Gly4, Phe6, He7, Asp9, and the C-terminal residues Phe22 and He23 of GLP-1 are critical for binding (Gallwitz et al, 1994; Adelhorst et al, 1994), while His1 (Unson et al, 1991), Phe6 (Unson et al, 1993; Azizeh etal, 1997), Asp15, and Tyr 1 0 (Azizeh et al, 1996) of glucagon have been demonstrated to be important for receptor binding and activation. Similarly, in the current study, it was found that addition of the hydrophobic amino acids He7 and Phe6, dramatically increased the binding affinity of GD* fragments 7-30 and 6-30amide for the GIP receptor, when compared to the GIP fragment 10-30 (Fig. 34, Table 4). This region is thought to form a small hydrophobic patch in the three related peptide hormones, and it appears to be required for receptor binding by glucagon (Azizeh et al, 1997), GLP-1 (Gallwitz et al, 1994; Adelhorst et al, 1994) and GD5. Studies directed at examining 211 GLP-1 and glucagon structure/activity using chimeric peptides indicated that the divergent carboxy-terminal residues of GLP-1 are important for the ligand's ability to discriminate between the GLP-1 and glucagon receptors, while the N-terminal residues of glucagon are important for binding to its receptor (Hjorth et al, 1994). In addition, a chimeric peptide consisting of the N-terminal 14 amino acids of glucagon and the carboxy-terminal 15 residues of GLP-1 bound to both receptors with high affinity (Hjorth et al, 1994). In contrast, substitution of GD? residues at non-conserved positions in the N-terminal 22 amino acids of GLP-1 disrupted binding to the GLP-1 receptor, especially Tyr 1 3 ->Ala 1 3 and Glu 1 5 -»Asp 1 5 , while none of the chimeric peptides bound effectively to the GIP receptor, suggesting that GD? required both N - and C-terminal residues for efficient binding (Gallwitz etal, 1996). Comparison of the sequences of the three peptides (see Fig. 2) indicates that the central region of GD? (-residues 12-20) has the least identity, further supporting a role for this region in GIP receptor specific interactions. These observations, taken together with data presented in the current Thesis, support the conclusion that N-terminal residues of the peptides in the glucagon/secretin/VD? superfamily are important for receptor recognition and activation. However, for the closely related peptides GIP, glucagon, and GLP-1 divergence of amino acid sequence in different regions of the peptides allows nearly absolute discrimination by the ligands between the three known receptors, but differences in ligand binding domains must also play a part in ligand selectivity. 212 4.4 E X A M I N A T I O N OF AMINO-TERMINAL RESIDUES OF GIP IMPORTANT FOR RECEPTOR ACTIVATION. The importance of the N-terminal residues His 1 or Tyr 1 has been demonstrated for a number of members of the secretin/glucagonATP superfamily. Substitution, truncation, or modification of L-His 1 in analogs of glucagon (Lin et al, 1975; Sueiras-Diaz et al, 1984; McKee etal, 1986; Unson etal, 1991) and GLP-1 (Gefel etal, 1990; Gallwitz et al, 1996) resulted in peptides with some reduction in affinity, but with a much greater reduction in their ability to activate their appropriate receptors (Adelhorst et al, 1994; Unson et al, 1987). Interestingly, substitution of D-Tyr 1 and D-Ala 2 for the endogenous L-isomers in truncated G R H (GRH 1-29) resulted in peptides with increased in vivo potency (Lance et al, 1984; Heiman et al, 1984). Important studies by Frohmann et al. (1986) indicated that G R H was metabolized to biologically inactive G R H 3-44 both in vivo and in vitro by the enzyme DP IV (Frohman et al, 1989), and that des-amino-Tyr1-, D-Tyr 1-, and D-Ala 2 -GRH were resistant to cleavage by this enzyme, suggesting that increased biological half-life may play a role in the increased potency previously observed. It has now been well established that DP IV is involved in the degradation of GIP and GLP-1 to GIP 3-42 and GLP-1 9-36amide (Mentlein et al, 1993b; Kieffer et al, 1995b; Pauly et al, 1997), rendering these fragments non-insulinotropic (Brown et al, 1981; Schmidt etal, 1986a; Suzuki etal, 1989; Gefel etal, 1990). The current studies have shown that more extensive degradation results in peptides, such as GIP 6-30, GD? 7-30 and GD 5 15-42, that interact with the GD 5 receptor but do not activate GJP-receptor mediated cAMP production. However, since these fragments also inhibit GIP-mediated 213 stimulation of cAMP production, it suggested that the major product of GD? metabolism in vivo, GD? 3-42, might also inhibit the action of the intact peptide in vivo. Both GD? 3-42 and an analog with the first two amino acids inverted (Ala^Tyi^-GD?) were found to display reduced affinity in competition binding studies and to be devoid of the ability to generate cAMP (Fig. 37) at concentrations as high as 1 uM. As predicted, given the previous N-terminal fragment data, both analogs were capable of inhibiting GIP (lnM)-stimulated cAMP production in the u M range (Fig. 38). The reduced affinity (-16-18 fold) of this fragment compared to that of GIP 6-30amide, suggests that extension of the GD? fragment from Phe6 to Glu 3 reduced the ability of the fragment to bind to the receptor. Given that Tyr 1 appears to be a critical residue for both binding and receptor activation, the presence of an aromatic hydrophobic residue such as Phe6 rather than the more hydrophilic Glu 3 may account for the difference in binding especially if, as has been suggested for a number of other member of the glucagon superfamily, the N-terminus associates with regions within the transmembrane regions of the receptor (Turner et al., 1996a, b). It has recently been suggested that GLP-1 9-36amide may act as an antagonist of the GLP-1 receptor (Grandt et al, 1996; Knudsen and Pridal, 1996). However it is clear from the current studies and that of Knudsen and Pridal (1996) that the reduced affinity of the metabolites, and their relatively low circulating concentrations, would preclude significant inhibition of hormone-receptor interaction in vivo. In order to define the contribution of the N-terminal amino acids for receptor activation further, additional N-terminally modified analogs of GIP were examined. In parallel with these studies, attempts were initiated at developing DPIV-resistant forms of 214 GIP, that could form the basis for future development of long-acting analogs with equal or greater insulinotropic activity. This involved studying the effect on receptor binding and ability to stimulate cAMP production of changing individually the structure (Ppa1-GJP l-30amide), chirality (D-Tyr 1-GIP l-30amide, D-Ala 2-GJP, D-Ala 2-GJP 1-30, D-Glu 3-GIP l-30amide), or identity (D-Ala 4-GJP l-30amide) of the first four amino acids of the GIP molecule. As mentioned in the Results section, all of these peptides, including AW - T y ^ - G J P and GIP 3-42, but excepting D-Glu 3 - and D-Ala 4-GIP l-30amide, were resistant to DP IV degradation in vitro (D.-H. Demuth, personal communication). D-Ala 2-GIP and the C-terminally truncated D-Ala 2-GIP l-30amide displayed similar shifts in their affinity (11.52 ± 1.08 nM and 10.26 ± 2.76 nM, respectively) and efficacy (EC5os; 1.78 + 0.86 nM and 0.68 ± 0.21 nM, respectively) compared to those for GTP (IC 5 0: 3.56 ± 0.81 nM; E C 5 0 : 0.25 ± 0.07 nM) in studies with wtGTP-Rl cells (Fig. 3 9, Table 5). A similar small, but significant, decrease was seen in the integrated insulin response to D-Ala 2-GTP compared to the endogenous hormone (154.5 ± 4.9 mU vs. 188.3 +6.1 mU, respectively) in the isolated perfused rat pancreas (Fig. 41), suggesting that modifications to the shorter fragment resulting in changes in affinity or potency should accurately reflect changes made to the full length hormone. In contrast, D-Ala 2 -GLP-1 had nearly identical affinity and efficacy to GLP-1, when tested on CHO-K l cells stably expressing the GLP-1 receptor (Fig. 40) and in the isolated perfused rat pancreas (Fig. 42), suggesting that the chirality of A l a 2 is less important for receptor recognition and activation than with GTP. Similar to studies on the importance of His 1 in glucagon (Sueiras-Diaz et al, 1984; McKee et al, 1986), or Tyr 1 in G R H (Lance et al, 1984; Heiman et al, 1984), 215 modification of Tyr1 in GIP resulted in variable changes in binding and signalling, depending on the modification made. The substitution of des-aminoTyr (Ppa1) in GIP 1-30amide led to no change in receptor affinity but resulted in a 3- to 4-fold shift in efficacy (Fig. 43, Table 5), while D-Tyr'-GD? l-30amide displayed an approximate 8-fold decrease in receptor affinity which was associated with a surprising 55-fold reduction in its EC50 value in comparison to GIP (Fig. 43; Table 5). The smaller shift in efficacy seen for Ppa^GD? l-30amide, compared to that seen for D-Tyr^GD? l-30amide, suggests that the chirality of the Tyr1 is more important than the protonated amide group for receptor activation. The substitution of L-Ala 3 with D-Glu3 or L-Gly4 with D-Ala4 in GIP l-30amide did not result in analogs that were resistant to DP IV hydrolysis (D-H Demuth, personal communication). D-Glu3-GIP l-30amide did not differ significantly from GIP in affinity (IC50S: 3.84 ± 0.55 nM vs. 3.56 ± 0.81 nM, respectively) or efficacy (EC50s: 0.248 ± 0.068 nM vs. 0.469 ± 0.126 nM, respectively) (Fig. 43, Table 5). The glycine at position 4 is completely conserved in glucagon, GLP-1, and most other members of the glucagon superfamily, and introduction of D-Ala4 into GD? l-30amide resulted in an 8-fold decrease in affinity, similar to that seen for D-Tyr^GD? l-30amide, and a 639- fold decrease in EC50 value compared to those of GD? (Fig. 43: Table 5). D-Ala4-GIP 1-30amide was also the only peptide tested that differed from GD? in maximal cAMP levels achieved (50.9 ± 7.6% of GIP max.) in concentration-response studies (Fig. 43, Table 5), suggesting that addition of even the small space filling methyl group/and or altering the chirality of alanine resulted in some steric hindrance to binding, and greatly disrupted structures required for receptor activation. Given that substitution of L-Ala at the 216 conserved position in GLP-1 resulted in a similar decrease in affinity and potency it seems likely that a conformational change rather than the chirality is responsible (Gallwitz etal, 1994; Adelhorst etal, 1994). These studies indicate that DP IV resistant forms of GIP can be synthesized that are of high (Ppa^GIP l-30amide) or slightly decreased (D-Ala 2-GJP, D-Ala 2-GJP 1-30amide) affinity and efficacy. Recent studies have indicated that similar modifications of A la 2 of GLP-1 with Ser, Gly, Thr or alpha-aminoisobutyric acid, resulted in DP TV-resistant GLP-1 analogs that displayed similar shifts in receptor affinity and stimulatory activity in the perfused porcine pancreas when compared to GLP-1 (Deacon et al, 1998a). Initial studies by Pederson (personal communication) have failed to demonstrate that D-Ala 2-GIP, D-Ala 2-GJP l-30amide, and D-Ala 2 -GLP-1 display any increased insulinotropic ability in vivo when compared to the endogenous peptides. This may be due to the fact that DP IV resistant analogs of GIP, like those of GLP-1 (Deacon et al, 1998a), have increased N-terminal stability but are still cleared by other mechanisms such as renal metabolism (Deacon et al, 1996) or degradation by other proteases to fragments non-detectable by RIA (Deacon et al, 1998a). Interestingly, the first pharmaceutical reports of combined DP IV-resistant and fatty-acylated forms of GLP-1 have just appeared (Clodfelter et al, 1998). Acylation was reported to decrease the plasma clearance of the peptide, and the DP IV stability extends its activity, resulting in increased biological activity in vivo (Clodfelter et al, 1998). Whether similar modifications of GIP can result in increased biological activity remains to be determined. 217 4.5 LOCALIZATION OF GIP RECEPTOR REGIONS IMPORTANT FOR LIGAND BINDING A chimeric receptor approach has been shown to be useful for delineating regions important for ligand binding and signal-transduction (Strader et al., 1994; Ulrich et al., 1998). As discussed in section 1.11.1, from previous studies on GPCRs it has been concluded that ligand binding involves the extracellular NT region of glycoprotein receptors (reviewed in Combarnous, 1992) and TM domains for cationic amines and small neuropeptides (Strader et al, 1994). Peptides of the secretin-glucagon family are probably too large to be completely accommodated by a transmembrane pocket since their structures are predicted to fold into a relatively large one or two-helical conformation. In recent years significant evidence has appeared implicating the large cysteine rich, and therefore presumably highly folded, extracellular NT domain as a likely ligand binding target for members of the secretin-glucagon family of receptors (Holtmann et al, 1995; Gourlet et al, 1996; Stroop et al, 1995; Bergwitz et al, 1996; Buggy etal. 1995, 1996; Carruthers etal, 1994; Unson etal, 1995, 1996; Wilman etal, 1996). Most of this information has been concerned with the receptors for secretin, VIP, CT, and PTH/PTHrP. Much less is known concerning the binding of GIP, GLP-1, and glucagon to their receptors, and the current studies were designed to define more closely regions important for binding and signaling of GIP and GLP-1. Chimeras, consisting of portions of the GIP and GLP-1 receptors, which share approximately 40% overall sequence identity (-35% in the NT) (Usdin et al, 1993), and appear to signal via identical intracellular mechanisms (Usdin et al, 1993; Wheeler et al, 1995; Thorens et al, 1992; Lu et al, 1993a; Gromada et al, 1998b), were utilized to 218 define regions involved in ligand binding. One important finding of the present study is that expression of the first 132 N-terminal residues of the GD? receptor on the body of the GLP-1 receptor (CH-2) bound 125I-GD» with slightly reduced affinity in COS-7 cells (IC50s: 31.7 ± 8.18 nM (CH-2) vs. 6.42 + 1.22 nM (wtGIP-Rl)) and CHO-K1 cells (IC50s: 27.8 ± 11.9 nM (CH-2) vs. 1.33 ± 0.19 nM (wtGDMU)). Extension of the GE» receptor to include the first 222 amino acids, consisting of the NT, EC-1, TM-1 and -2, and part of TM-3 (CH-4) was sufficient for full ligand binding when expressed in COS-7 cells, or with a slight decrease in affinity seen in the more sensitive CHO-K1 cell line (Fig. 47, Table 6). In contrast, in cAMP studies, GIP-stimulated cAMP production was only observed in cells expressing CH-4, suggesting that while the N-terminus of the GD? receptor was sufficient, when expressed on the body of the GLP-1 receptor, to bind GIP, additional receptor regions, critical to receptor activation, existed within the region of TM-1 to TM-3. To address this question, a number of other chimeric receptors, consisting of further C-terminal extensions, were constructed (CH-3, CH-9, and CH-7; See Fig. 44), however only CH-3, which extended the N-terminus an additional 19 amino acids into the first TM-domain, displayed I25I-GIP specific binding with an affinity equal to that seen for CH-4 in CHO-K1 cells (IC50s: 9.04 ± 1.07 nM (CH-3) vs. 8.33 ± 0.14 nM (CH-4)) (Fig. 45; Table 6). Addition of these 17 amino acids, putatively of TM-1, also restored cAMP responsiveness to the chimeric receptor, indicating that the first transmembrane region of the GIP receptor is important both for GIP binding and receptor activation. All the chimeras that displayed Gff-specific binding and cAMP production, exhibited decreased expression levels (Table 6) and, predictably, lower maximal cAMP 219 production (Fig. 47, Table 7). Indeed the extension of the CH-3 by an additional 8 amino acids (CH-9) or into TM-2 (CH-7) resulted in receptors that did not appear to be expressed. However without GIP or GLP-1 receptor specific antibodies it was not possible to determine i f the receptors were inactive, or just not expressed at the cell membrane. It is difficult to ascertain why these hybrids were expressed less efficiently than the wt receptor, however it is suggested that the most likely explanation is less efficient processing, since receptor binding affinities and, in the case of CH-4, its EC50 value were not dramatically altered. While all chimeric receptors did not display detectable 1 2 5 I-GLP-1 (Fig. 46, Table 6), CH-2 and CH-3 both responded with increases in cAMP levels when stimulated with GLP-1 (Fig. 47, Table 7). Surprisingly, maximal cAMP production in COS-7 cells was the same as that seen for cells expressing the wtGLP-1 receptor. In contrast, GLP-1 stimulation in CHO-K1 cell lines expressing CH-2 and CH-3 resulted in maximal cAMP levels that were 30.5 ± 2.0 % and 13.2 + 1.9 %, respectively, of those seen in the wtGLP-1 cell line. In addition, there is no obvious explanation for the apparent higher efficacy of GLP-1 with CH-2 (81.4 ± 19.6 nM) than CH-3 receptors, which are expressed at a higher level and consist of less of the GLP-1 receptor body. However, despite these discrepancies it is apparent that the body of the receptor is capable of interacting with GLP-1 and stimulating cAMP accumulation. The observation that none of the chimeras consisting of GLP-1 N-terminal regions expressed on the C-terminal GIP receptor (CH-5, CH-6, and CH-8) bound or signaled in response to GIP or GLP-1 may also have been due to inefficient processing. Alternatively, binding of GLP-1 and glucagon may be more complex than that for GIP 220 and have absolute requirements for multiple binding regions. Observations that glucagon/Calcitonin (Cal) (Stroop et al, 1995, 1996), and GLP-1 (NT)/glucagon (Buggy etal, 1995) receptor chimeras have been generated that are expressed at the cell surface, but have disrupted ligand binding, suggest that it is possible that GLP-1 binding was disrupted in all constructs examined in the current studies. Again, without receptor antibodies this remains to be determined. Importantly, the studies described here show that the GD? receptor N T demonstrates high affinity binding of GIP in the presence of a heterologous first extracellular loop, and that the distal part of NT and TM-1 appear to be important for cAMP activation of both the GIP and GLP-1 receptors. The reduced binding affinity seen for GIP with CH-2 compared to CH-4 suggests that the first E C loop is involved in ligand binding and that TM-domains 1-3 may also be involved. Additionally, the reduction in affinity observed in CHO-K1 cells expressing CH-4, compared to the wt receptor, suggest that regions in the CT of the GIP receptor, most likely EC-loop 3, and/or TM-domains 4, 5, 6 and 7 may contribute to ligand binding. The importance of the NT and first extracellular loop for binding is similar to the glucagon receptor, as deduced from studies on deletion/truncation mutants (Carruthers et al, 1994; Unson et al, 1995), and GLP-1/glucagon receptor chimeras (Buggy et al, 1995). However, in addition to the NT-domain and first extracellular loop, the third EC-loop has also been implicated in ligand binding of the PTH/PTHrP (Lee et al, 1994) and PTH2 (Clark et al, 1998), and glucagon (Unson et al, 1995; Buggy et al, 1995) receptors. There are both similarities and major differences between the NT requirements for the GDVGLP- l receptor sub-group and other members of the secretin-glucagon family. 221 Holtmann et al. (1995) showed that secretin-VIP receptor chimeras, consisting of the complete NT of the secretin receptor and the C-terminus of the VIP receptor, exhibited low affinity responses to secretin, whereas this region of the GIP receptor expressed on the GLP-1R C-terminus was capable of ligand binding with an affinity similar to that of the wt receptor, albeit with a significantly reduced level of binding. The requirement of the GD? receptor for the NT, EC-1, and associated T M domains, for maximal signaling is similar to the secretin receptor, whereas only the extracellular NT was required for high affinity binding and signal-transduction in the VIP receptor. Similar studies with Cal/Glucagon (GR) receptors revealed that expression of the N -terminal region of the Cal receptor on the GR receptor body bound Cal with a slight reduction in affinity (12 n M vs 0.3 nM) compared to the wt Cal receptor, but did not signal in response to glucagon or Cal (Stroop et al, 1995), similar to the chimera CH-2 described in this Thesis. In contrast, chimeras consisting of the GR N-terminus and Cal receptor body did not bind detectable amounts of 125I-glucagon or 1 2 5 I-Cal, but did respond with increased cAMP levels when stimulated with Cal, in a manner similar to CH-2 when stimulated with GLP-1 (Stroop etal, 1995). The observation that the GR N-terminus lacked binding ability when expressed on the Cal receptor body, and the fact that others have demonstrated that the glucagon receptor can be expressed with its N-terminus deleted, suggests that glucagon (Unson et al, 1995; Hjorth et al, 1998) requires multiple regions, both N-terminal and more distal, to efficiently bind and signal. However the GLP-1 receptor body, like that of the Cal receptor, appears to act as a low affinity binding site that can be activated by its ligand. The inability to determine i f CH-5 (GLP-1 NT/GIP receptor body) was expressed or not 222 prevents conclusions regarding the similarity of the involvement of the GD? receptor body in binding and signaling to the Cal and GLP-1 receptors or to that of the glucagon receptor. The complexity of the phenotypes seen with the different chimeric receptors argues against a single model of receptor binding and activation. However the most interesting observation here was that extension of the GD» extracellular N-terminus by 19 amino acids into the TM-1 (CH-3) restored GIP-dependent cAMP responsiveness, but did not disrupt GLP-1 signaling. In addition, extension of the GD» receptor sequence to include TM-2, EC-1, and part of TM-3, restored the absolute GD» specificity of the chimera CH-4. This suggests that the first 2/3 of TM-1 of the GLP-1 receptor, or the two amino acids predicted to exist at the membrane border, in some way act as a selective filter, discriminating between GIP and GLP, while the GIP specific filter exists somewhere in the bottom of TM-1, TM-2, EC-1, and possibly TM-3. Recently, the second transmembrane domain of the PTH receptor has been shown to contain a single Ile234 residue that, when changed to the corresponding Asnl92 residue in the secretin receptor results, in a receptor that responds to both ligands. The reciprocal substitution in the secretin receptor (Asnl92Ile) results in a secretin receptor that can signal in response to both PTH and secretin. In addition, point mutations of charged residues in the TM-domains of the PTH and secretin receptor suggest they are important for receptor activation and ligand binding (Turner et al, 1996a), supporting an argument for TM-domains playing a role in ligand binding and receptor activation by peptide hormones, somewhat like that seen for cationic amines and small neuropeptides 223 (Strader et al, 1994). Further studies are required to determine if, and exactly what, residue(s) are involved in the proposed GD? and GLP-1 fdters. While no studies reported in the current Thesis examined which specific regions of GD? associate with the different regions of the receptor, studies with hybrid PTH(1-34)/Cal ligands and N-terminal PTH/Cal receptor chimeras indicated that the C-terminal regions of the peptides bind to the N-terminus of their respective receptors, and that the N-terminus of the ligand associates with the body of the receptor (Bergwitz et al, 1996). In addition, Stroop and co-workers (1995) demonstrated, using GR/Cal receptor chimeras, high affinity binding associated with the N-terminus of the receptor and a lower affinity association with the body of the Cal receptor, which helped decrease the off rate and allow activation by Cal. Additional studies using a number of analogs of Cal, showed that the ability of an analog to form an a-helical stretch was positively correlated with the analog's affinity for the NT-Cal/GR chimera (Stroop et al, 1996). While the ability to activate the NT-GR/Cal receptor chimeras was also dependent on the analogs helical nature, conservation of the N-terminal sequence residues 1-6 was also essential for receptor activation (Stroop etal, 1996). Both Stroop and Co-workers (1996) and Hjorth and Schwartz (1996) have proposed similar models for the calcitonin, GLP-1, glucagon, and other members of the secretin/glucagon/VTP receptor family. In these models the disulfide bonds formed by six conserved Cys residues in the N-terminus form a globular domain that initially binds or captures the ligand with lower affinity. Once tethered, the peptide is brought into close proximity to the external face of the receptor, resulting in high affinity binding and receptor activation. In light of the results from the GIP/GLP-1 receptor chimera and 224 structure-activity studies examining residues important for GD? receptor activation, it is tempting to speculate that the C-terminal 6-30 amino acids of the GIP molecule interact with the N-terminal region and specific external loops of the receptor, resulting in high affinity binding similar to that of GD? l-30amide or GDP. The N-terminal 6 residues essential for activation of the receptor could do so by interaction with regions within a c binding pocket including TM-1 and, possibly, TM-2 and E C loop 1. A similar model for the binding of GLP-1 to its receptor can also be visualized. While more detailed receptor mutagenesis, perhaps coupled with hybrid ligand studies, are required to explore this proposed model, the GIP chimeras described in the current Thesis should provide a basis on which to plan future experiments. NT-GR/GIP receptor chimeras may be expressed more efficiently then the NT-GLP-1/GD? receptor chimeras, and thus aid in defining regions of the GBP receptor body important for receptor activation and ligand selectivity. 4.6 E X A M I N A T I O N OF THE C A R B O X Y - T E R M I N A L TAIL D O M A I N OF T H E GD? RECEPTOR As discussed in section 1.11.2 the intracellular loops of the heptahelical receptors have been implicated in G-protein recognition, coupling and activation (O'Dowd et al, 1988; Wong et al., 1990; Liggett et al, 1991; Hedin et al, 1993; Burstein et al, 1998; Takarh et al, 1996; Huang et al, 1996; Mathi et al, 1997; Heller et al, 1997; Chicchi et al, 1997), but there is no consensus as to the importance of the C-terminal tail with regard to these functions. Indeed, there appears to be considerable variability in the importance of this region among the different G-protein coupled receptor types. For 225 example, O'Dowd et al. (1988) showed that the N-terminus of the C-terminal tail of the human B2-adrenergic receptor was critical for coupling to G protein activation of adenylyl cyclase, whereas shortening of the C-terminus of the avian 3-adrenergic receptor resulted in increased basal and agonist-stimulated cyclic A M P production, and reductions in agonist EC50 values (Parker and Ross, 1991). There have been few studies on the importance of the C-terminal tail of the secretin- VTP receptor family, and the only relatively consistent finding has been an increase in affinity for agonists with CT-truncated mutants, as reported for the PTH/PTH-RP (Iida-Klein et al, 1995), calcitonin (Findlay et al, 1994), and glucagon (Unson et al, 1995) receptors. In the case of the GD? receptor, removal of up to 50 amino acids from the C-terminal tail had no significant effect on receptor binding affinity (IC 5 0s: 2.73 ± 0.35 n M (wtGD»-Rl) vs. 2.16 ± 0.12 n M (GD°-R-405)). This is similar to the human glucagon receptor, for which 62 of the amino acids in the CT were shown not to be required for binding (Buggy et al, 1997). It is evident from the GFP-induced cyclic A M P responses of the truncation mutants that the majority of the C-terminus of the receptor is not essential for coupling to adenylyl cyclase, since a mutant consisting of as few as thirteen of the sixty-three amino acids was capable of stimulating adenylyl cyclase. The ability to remove a substantial portion of the C-terminal tail while retaining G protein coupling is in agreement with studies on other heptahelical receptors. In similar mutational analysis experiments to those described here, progressive truncations of the C-terminal tail of the opossum (Huang et al, 1995a) and human (Schneider et al, 1994) PTH/PTH-RP receptors resulted in no significant alterations in cyclic A M P production. Similarly, removal of the distal two-thirds of the TSH (Chazenbalk et al, 1990) and luteinizing hormone/chorionic gonadotropin 226 receptors (Rodriguez et al, 1992) also resulted in no change in ligand activation of adenylyl cyclase. In contrast, C-terminally truncated forms of both the rat PTH/PTHrP (Iida-Klein et al, 1995) and avian P-adrenergic (Parker and Ross, 1991) receptors were found to signal adenylyl cyclase with much higher efficacy than the wt receptor, and evidence was presented suggesting that the C-terminal tail decreases PTH/PTHrP receptor affinity for Gs (Iida-Klein et al, 1995). The situation with the PTH/PTHrP is probably complicated, however, since pertussis toxin-sensitive inhibitory effects of PTH on adenylyl cyclase were observed only in wt receptors, and it was proposed that the CT plays a crucial role in interactions between receptors and inhibitory G-proteins. In contrast to the PHT/PTHrP receptor, a reduction, rather than an increase, in maximal cAMP production was observed in the current studies with truncated GIP receptors. This can probably be partially explained by reduced plasma membrane expression levels, as discussed further below. Nevertheless, significantly lower EC50 values (4-6-fold) were obtained for GIP-R-418 and GIP-R-405 (Table 10). One possible interpretation of this result is that CT shortening removes specific amino acids that induce less efficient receptor-induced Gs coupling to adenylyl cyclase. The truncated region contains both serine and threonine residues, and phosphorylation could result in such reduced coupling. Although it is not known whether the C-terminus of the GD? receptor is phosphorylated following agonist binding, it seems probable since phosphorylation of the closely related GLP-1 receptor by both protein kinase C and other receptor kinases has been established (Widmann et al, 1997). Another possible explanation, which has been proposed for the GLP-1 receptor, is that over-expression 227 leads to desensitization of the receptor, a decrease in EC50 (Fehmann et al, 1997), and reduced maximal rates of cAMP production. If expression level plays a role with the GD? receptor, then the increase in efficacy observed with Gn»-R-418 and GD?-R-405 may have been due to the lower expression levels (Table 9, 10). Indeed in earlier studies using the GD?-R8 clone that expressed the GD? receptor at ~5 % of that seen for wtGD?-Rl, EC50 values were decreased 7-fold, while maximal cAMP levels were -10% of that seen for the wt receptor (Table 8). All receptor truncations, except GIP-R-427+, resulted in some decrease in maximal binding levels. Truncation probably decreases the efficiency of receptor insertion in the plasma membrane, and the decrease in maximal cyclase stimulation with GIP-R-405 reflects this reduced membrane expression. Cells transfected with the truncated mutant GIP-R-400 exhibited neither binding nor an ability to stimulate adenylyl cyclase. Although this could be due to either lack of receptor expression in the plasma membrane, or dramatically reduced agonist binding to an expressed receptor, the former is more likely. There is probably a minimum length for efficient folding of a heptahelical receptor and its translocation from the endoplasmic reticulum and insertion into the plasma membrane. Such a lack of expression explains the inability to detect biological responses with these severely truncated mutants. Similar suggestions were made to explain the lack of detectable binding with extensively truncated PTH-PTH-RP receptors (Huang et al, 1995a) and, more recently, with the human glucagon receptor (Buggy et al, 1997). In the latter study, using similar CT mutation and alanine substitution techniques to those used here, it was shown conclusively that, as with the GD? receptor, the majority of the CT of the glucagon 228 receptor could be deleted without compromising membrane insertion. However a glucagon receptor mutant (RT410) equivalent, apart from one less amino acid, to GIP-R-400 was shown by immunostaining not to be expressed in the plasma membrane, whereas a further mutant, RT415, almost equivalent to GIP-R-405 was expressed. Unfortunately, the absence of a GIP receptor antibody precluded us from performing similar immunostaining studies to those of Buggy etal. (1997). More recently it has been shown that extension of the rat gonadotropin-releasing hormone receptor (GnRHR) CT, which normally consists of only 2 amino-acids, with an additional 51 amino acids from the catfish (ct) GnRHR CT tail had no effect on binding affinity, but increased the number of the receptor binding sites ~5-fold, and augmented the D?3 response (Lin et al., 1998). In addition, truncation of the catfish receptor tail-region of these chimeras resulted in decreased expression compared to the full-length chimera. Taken together, the three studies provide convincing evidence that a C-terminal peptide length of between 10 and 15 amino acids, in the case of the GD? and glucagon receptors, is necessary for membrane expression of this receptor sub-family. In contrast, for some receptors, such as the mammalian GnRHR, as few as ~2 CT amino acids are sufficient for expression. However this may have required changes in mammalian GnRHR expression to correct for the apparent decreases in efficient membrane expression and decreased coupling to some second messenger pathways (Lin et al, 1998). Interestingly, COS-7 cells expressing the truncated mutants had similar expression levels, but higher maximal cAMP levels when compared to maximal levels seen with the 229 truncated CH0-K1 cell lines (Table 9, 10). The reason for this is unclear, however it may be that the receptor expression in the COS-7 system is so high that enough spare receptors exist to activate near wt-maximal Gs-mediated adenylyl cyclase activity, even in the case of disrupted coupling. In the case of the CHO-K1 system receptor expression is lower, and therefore more sensitive to both alterations in receptor coupling and changes in receptor levels. For example, GIP-R-427+ was expressed at similar levels to those seen as the wt receptor in both systems. However, while its EC50 value did not differ from that seen for the wtGIP-Rl cell line (wtGIP-Rl: 69 ± 27 p M vs.GB?-R-427+: 47 ± 20 pM), maximal cAMP levels were significantly decreased in the CHO-K1 cell system. If the above suggestion is correct, given the relative expression levels (Table 9) of the truncated receptor in the CHO-K1 system, there was a definite decrease in the ability of the receptor to stimulate maximal cAMP levels (Table 10). This suggests that some of the distal 50 amino acids act to couple the GD? receptor in a positive manner to Gs-mediated adenylyl cyclase activity. In the case of GIP-R-427+, the additional six vector-encoding amino acids do not appear to be responsible for the loss of coupling as recent studies with the GD? receptor truncated at 427 using site directed mutagenesis produced a receptor with very similar characteristics to those seen with GIP-R-427+ (M.B. Wheeler and C.H.S. Mcintosh, personal communication.). Further, GDP-R-405 was expressed at ~ 30%, and maximal cAMP values were only 8.1 ± 1.9%, of those obtained with the wtGD?-Rl cell line. In contrast, as discussed above, the wtGD?-R8 clone expression levels, and maximal cAMP accumulation, were 4.9% and 9.59 ± 0.29% respectively of wtGIP-Rl levels. These results threfore suggest that GD°-R-427+, GIP-R-418, and GIP-R-405 coupled ~2-, 3-, and 3.5-fold less efficiently to cAMP production, respectively, 230 assuming that a receptor with a membrane expression level of 10% would be expected to display maximal cAMP levels of at least 10% that of the wt receptor clone. Two possibilities were considered regarding the structure of the CT necessary for membrane expression. Either the specific sequence R-R-L-R-L, at positions 401-405, was required (O'Dowd et al, 1988; Liggett et al, 1991), or the chain length itself was the determining factor, and the specific amino acids were immaterial. A mutant receptor was therefore prepared which extended the C-terminal chain with five alanines to produce a 405 amino acid protein (GTP-R-4OOA5). The level of receptor binding and the maximal level of cAMP production with this receptor mutant were similar to those produced with GIP-R-405. However, there was a marked increase in the EC50 value for cAMP production (1163 ± 320 pM) compared to the full-length receptor (69 ± 27 pM) indicating that specific amino acids within the 400-405 region influence the efficacy of G protein coupling. Attempts were made to define the importance of the membrane proximal region further, but were unsuccessful due to the fact that neither GIP-R-396A9 nor the deletion mutant GIP-R-AQSEI bound 125I-GD?, and therefore their expression or lack thereof could not be determined. However, despite the decrease in EC50 observed with GJP-R-4 O O A 5 , what is important is that there must be considerable redundancy in the specific amino acids in this region of the CT which can allow G-protein coupling, and the length of the tail appears to be the critical factor. Since the proximal CT of the glucagon receptor (403-415 NKEVQSELRRRW) is almost identical to that of the GIP receptor (393-405 NKEVQSEIRRLRL) it is likely that a similar level of redundancy exists for this receptor. Specificity of G-protein binding in 231 the secretin-VIP receptor family probably resides elsewhere within the intracellular domains, and evidence has been presented recently indicating a critical role for a single amino acid (K334) in the N-terminal portion of the IC3 loop of the GLP-1 receptor for efficient coupling to adenylyl cyclase (Takhar et al, 1996). This region is highly conserved among the secretin-VD? family, and will probably prove equally important for GIP receptor activation. The C-terminal tails of a number of G-protein coupled receptors, including those for yeast a-mating factor (Reneke et al, 1988), calcitonin (Nygard et al, 1997) and GLP-1 (Widmann et al, 1997) have been shown to be phosphorylated on serine and threonine residues, and to be involved in both receptor desensitization and internalization, although the majority of the CT of the fi-adrenergic receptor was not required for receptor sequestration (Strader et al, 1987). Studies on desensitization and internalization of the GIP receptor failed to produce unambiguous evidence for a major role for the CT in these events. In fact we were unable to demonstrate receptor desensitization in either the high level expressing wtGIP-Rl clone (Fig. 55) or the low-level expressing clone wtGIP-R8. We failed to find any major effect of receptor truncation on short-term desensitization of cAMP signaling in response to GIP. A similar observation was also made by Buggy et al. (1997) with the homologous glucagon receptor, although homologous and heterologous glucagon receptor desensitization has been demonstrated in hepatocytes (Savage et al, 1995). Additionally, both the human and rat GLP-1 receptor have been shown previously to undergo homologous desensitization in response to receptor phosphorylation (Widmann et al, 1995, 1997). The protocol used by us was similar to that described by Widmann et al 232 (1997) for the GLP-1 receptor, and the CT of the GIP receptor contains serines that could potentially be involved in phosphorylation and subsequent desensitization. However, there is only one putative casein kinase II site, and no consensus sequences for P K A or P K C phosphorylation in the C-terminus (PCGENE). Other workers have shown that reduced responsiveness to chronic infusion of 10 n M GIP occurs in rats after approximately 4 hours (Tseng et al, 1996b). In expression studies using a P-galactosidase reporter cell line, desensitization of the cAMP response was observed after 16 hours (Tseng et al, 1996b). Incubation periods greater than 2-4 hours were not examined in our studies as the rapid desensitization of the receptor was of initial interest. It is possible that the GD? receptor does not undergo rapid desensitization, either in vivo or in vitro, and long-term down regulation resulting in loss of membrane localized receptor is the mechanism by which GBP responsiveness is attenuated, in a manner analogous to that seen for the p-opioid receptor (Pak et al, 1996). Alternatively, it is possible that expression of the GIP receptor in cells in which it is not normally found, such as CHO-K1 cells, may ablate its ability to desensitize homologously, possibly due to lack of the appropriate GRK. Expression of the wt GD? receptor in islet cell lines, as well as determining the GRKs expressed in the specific islet cell types, should help clarify i f the GD? receptor, and truncated mutants described here, are susceptible to desensitization, and the cellular components required. It has been suggested that the maintenance of an insulin response to pharmacological GLP-1 treatment and the loss of GIP responses in N I D D M patients (Hoist et al, 1997), may be due to a defect in GIP receptor function or expression (Hoist et al, 1997). The delineation of potential pathways of desensitization and down-233 regulation of the GD? receptor is therefore important since chronic elevation of GD? levels has been reported in some NDDDM patients (Ebert and Creutzfeldt, 1980; Elahi et al, 1994). Such a condition may lead to long term desensitization (see below), resulting in a decreased GIP responsiveness. One possible scenario is that the GD? receptor does not undergo rapid desensitization but that it is internalized (see below) and cycled in response to ligand activation. Over time this could lead to GD? receptor down-regulation at the protein level due to increased receptor uptake, degradation, and/or decreased receptor expression. In contrast, GLP-1 receptors, which are rapidly desensitized, may have reduced overall receptor cycling and degradation. Since both receptors signal via identical second messenger pathways it is conceivable that elevated GIP levels may also lead to heterologous down-regulation of GLP-1 receptors, and be partially responsible for the apparent defect in GLP-1 responsiveness in NDDDM patients. A l l CT truncated GIP receptor forms were internalized to a similar maximal level (-60-70%) over time, in response to ligand exposure (-80-100 p M GD?) (Fig. 54, Table 11). However the wt receptor internalization was more rapid at early time points suggesting that truncation may have decreased the rate at which the receptor was internalized. Examination of initial uptake rates from time points 1-10 min revealed small changes in the initial rate of uptake (% Total binding /min), although it is unclear as to whether these would have a major impact on overall responsiveness to GD? (Fig. 54). However it does suggest that either a positive internalization signal exists in the distal 27 amino acids, and/or truncation allows exposure of a negative sequestration signal even when the receptor is unoccupied. 234 Interestingly, GIP-R-400A5 in which residues 401-405 were replaced with 5 Ala residues restored the internalization rate to that seen for the wt receptor (Table 11), suggesting that the membrane proximal basic L-R-R-L-R sequence may be acting as a negative internalization signal similar to those described for the PTH/PTHrP and thyrotropin-releasing hormone receptors (Huang et al., 1995b; Petrou et al., 1997). However more detailed analysis of this region is required to confirm i f such a sequence exists. These findings are in contrast to the closely related GLP-1 and glucagon receptors, in which internalization was shown to be very sensitive to removal of CT serine residues (Widmann et al., 1997; Buggy et al., 1997). It is possible that experimental differences explain these divergent results, but it is alternatively possible that other regions of the internal domains of the GD? receptor are involved in the processes leading to internalization and desensitization. Further studies are required to determine the importance of the more membrane proximal residues to desensitization and internalization. 4.7 CONCLUSIONS A N D FUTURE STUDIES The cloning of the cDNA and gene for the GTP receptor has expanded the horizons of incretin research. The involvement of GDVand or its receptor in NTDDM has long been speculated upon (Pederson et al., 1993; Crueutzfeldt and Ebert, 1993; Hoist et al., 1997), and the studies described here are the first to examine the structure-function relationships of both GJP and its cloned receptor in isolation from other complicating factors present in cell lines and whole animal bioassays. These findings are important i f 235 the intelligent design of therapeutic analogs of GD? for the treatment of NDDDM and other pathophysiological states, such as food-dependent Cushings disease (Lacroix et al, 1992; Reznik etal., 1992) are to be undertaken. Importantly these studies have shown: • A cDNA isolated from pancreatic islets was shown to encode a cDNA identical to that initially isolated by Usdin and co-workers (1993) from the CNS and a pluripotent p-cell line cDNA library, establishing that GD> receptors encoded by these different cell types are products of the same gene. A fact that has been further confirmed by the cloning of the human homologue from a human islet cDNA library (Gremlich et al., 1995). • Heterologous expression of the GH»-R1 cDNA in COS-7 and CHO-K1 cells resulted in both 1 2 5I-GrP specific binding and GD?-stimulated cAMP production with IC50 and EC50 values comparable to those observed by others in cell lines and in vivo. Surprisingly, GD? stimulated an increase in [Ca 2 +]i in COS-7 cells and not in CHO-K l cells, suggesting the cellular environment in which the receptor is expressed may effect the signal transduction pathways activated. GIP-stimulated changes in [Ca 2 +]i in COS-7 cells were biphasic with a rapid thapsigargin sensitive peak, resulting from release from intracellular stores, followed by a lower level extended phase that was due to influx of extracellular C a 2 + that was not via VDCCs. The exact nature of this influx remains to be determined, however it may be that a nonspecific cation channel similar to that described for rat P-cells and some P-cell lines (Holz et al, 1995; Kato et al, 1996) is involved. 236 Synthetic preparations of porcine and human GD? displayed similar affinity and efficacy with cloned rat islet GO? receptors. In addition, all peptide preparations displayed similar potency in the isolated perfused rat pancreas, unlike a previous shGD? preparation (Jia et al, 1995). While these data cannot confirm that much of the controversy concerning the potency of GIP in different systems is due to the heterogeneity of GIP preparations, the wtGD?-Rl cell line provides a quick and easy bioassay to examine preparations of GIP. Indeed it was this assay that allowed us to determine that one batch of shGIP was devoid of biological activity. It was later determined via N-terminal sequencing and MALDI-TOF spectrometry that this preparation had the first two amino acids reversed (Ala^Tyr 2-GIP). It was demonstrated using the high level GD? receptor expressing CHO-K1 cell line, wtGn»-Rl, that the majority of the residues of GIP required for high affinity binding and receptor activation reside within the region 1-30. In addition it was demonstrated that N-terminal truncation of this fragment resulted in peptides with reduced binding affinity, and antagonist activity. One fragment, GD 3 6-30amide, displayed equal affinity to GD?l-42 for the GD? receptor, but did not activate the GD? receptor at concentrations as high as 10 uM. While GL° 6-30amide proved to be potent antagonist of GD? in vitro, it did not display any antagonist activity in vivo (R.A. Pederson, personal communication). 237 Modification of the N-terminal residues of GIP, to render the peptide DP IV resistant, had varying effects on both receptor affinity and ability to activate adenylyl cyclase. Importantly, the modification of Tyr1 and Ala2 to Ppa1 and D-Ala2, respectively, demonstrate that it is possible to design GD? analogs resistant to DP IV degradation with only small effects on affinity and efficacy. Furthermore, modifying the stereochemistry of Tyr1 (D-Tyr^GD? l-30amide) and the sequence of Gly4 (D-Ala4-GIP l-30amide) had the greatest effect on affinity and efficacy at the GD? receptor. This suggests that Tyr1 of GIP is involved in a stereospecific interaction with the receptor that is important to receptor activation, while substitution of the highly conserved Gly4 must also disrupt the ability of the N-terminus of the GD? molecule to assume an active conformation. These observations, along with the N-terminal truncation data indicate that the central, putatively a-helical region of GD?, is important for receptor binding while the N-terminal 4-6 amino acids are important for both binding and receptor activation. This appears to be part of a conserved structural architecture of members of the glucagon superfamily (Bergwitz et al, 1996; Stroop et al, 1996; Hjorth and Schwartz, 1996; Ulrich etal, 1998). Similar to other members of the secretin/glucagonATP receptor family (Holtmann et al, 1995; Gourlet et al, 1996; Turner et al, 1996a; Stroop et al, 1995; Bergwitz et al, 1996; Wilman et al, 1996), the N-terminus of the GD? receptor appears to contain the majority of the structural requirements for high affinity GD? binding. Interestingly while GTP did not stimulate cAMP production of one chimeric receptor consisting of the N-terminus of the GD? receptor when expressed on the body of the GLP-1 238 receptor, GLP-1, which did not appear to bind, was able to stimulate cAMP production. Extension of the E C N-terminus of the GIP receptor by 19 amino acids into the first T M domain partially restored GIP responsiveness and did not affect GLP-1 activation. Further extension of the GIP receptor protein to TM-3 further restored cAMP production in response to GD? and ablated any responsiveness to GLP-1. This suggests that regions important to GD* receptor activation exist in the first 2/3 of the TM-domain. Additionally this region of the GLP-1 receptor appears to contain a ligand-specific "filter" sequence, as has been describe for the PTH/PTHrP and secretin receptors (Turner et al, 1996b). Additional regions of the GD? receptor, which may include the remainder of TM-1, TM-2, EC loop 1, and possibly TM-3, appear to be required to maintain the Gff-specific selectivity of the receptor. It was found that the majority of the CT-tail of the GD 3 receptor can be truncated with no effect on receptor affinity, and only minor effects on receptor coupling to cAMP generation and receptor internalization. However, truncation of the CT tail by greater than 27 specific amino acids resulted in reduced expression levels at the plasma membrane as determined from Bmax levels obtained in competition binding studies. This observation is similar to that seen for the glucagon (Buggy et al, 1997), P T H (Huang et al, 1995a) and CT (Findlay et al, 1994) receptors, although different effects were observed on receptor affinity and G-protein coupling. Interestingly it was not possible to demonstrate homologous desensitization of the wt GD? receptor in the CHO-K1 cell system, suggesting that the GD» receptor does not undergo rapid homologous desensitization or at least not in the CHO-K1 cell system employed. 239 The described studies provide a basis on which to develop further research on important aspects of the physiology of GD? and its receptor. Areas in which they are likely to have an important impact and further developments that are likely to arise are outlined below. The transfected cell lines can clarify the confusion in the literature over human responsiveness to synthetic human GD? preparations. It had been noted previously that some shGD? preparations exhibited peptide heterogeneity and low biological activity (Jia et al, 1995), and this may explain some of the variability in responsiveness of N I D D M patients reported. It is therefore essential that clinical studies using synthetic preparations of GTP are repeated, both in healthy individuals and individuals with NDDDM, with well standardized, biologically active peptide, to establish whether GD° or GD? analogs are potential therapeutic agents in the treatment of some patients with N I D D M . Since both shGIP and spGD? were equipotent in binding and stimulating the heterologously expressed rat islet GD» receptor this system is appropriate for screening different GIP preparations. Knowledge of the minimal structural requirements of high affinity GIP binding of the GIP antagonist, GD? 6-30amide and N-terminal sequence requirements provides some clues as to structural motifs that theraputic analogs will have to maintain to conserve binding affinity and/or receptor activation. Interestingly, although GD? 6-30amide was found to be a potent antagonist, and the analogs D-Ala 2-GIP and D-Ala2-Gn» l-30amide were DP IV-resistant, in vitro, neither antagonism of GIP action nor prolonged biological activity, respectively, were observed in vivo. This suggests that other considerations such 240 as systemic half-life due to renal clearance (Deacon et al, 1996) will have to be considered. The recent report that a DP TV-resistant analog of GLP-1, with the addition of a fatty acyl-chain, had improved biological activity (Clodfelter et al, 1998), suggests that similar modification of the DP TV resistant GIP analogs and the high affinity antagonist GEP 6-30amide may improve activities of these peptides in vivo by increasing their half life. Another exciting possible therapeutic strategy is the use of DP IV antagonists to prolong the biological half-life of endogenously released GD? and GLP-1, a strategy that has been shown to inhibit GD> and GLP-1 degradation (Kieffer et al, 1995b; Pauly et al, 1996; Deacon et al, 1998b) and improve glucose tolerance in the rats (Pauly et al, 1998). The major drawback of using peptide based analogs to treat any pathology is that they require some form of invasive application, although there are reports of using a GLP-1 buccal tablet (Gutnaik et al, 1996, 1997), and other investigators have suggested that microencapsulation may prove to be suitable for administration of GLP-1 analogs (Nauck et al, 1997a). In the case of NIDDM, an analog, most likely non-peptide in nature, with a long biological half life that could be taken orally and that acts via the GD* or GLP-1 receptors would be preferred over the more complicated and expensive peptide formulation. It is therefore important to understand the structural requirements of the peptide critical for receptor activation since this region, or even specific residues within this region, could be targeted. The studies presented here suggest that such analogs should interact with the N-terminus for binding and at least the first T M domain in order to activate the GTP receptor. However, it appears that regions of TM-2, similar to the 241 PTH and secretin receptors (Turner et al, 1996a, b), may also be involved. Additional screening of individual residues in this region may allow the design of small inorganic GTP-mimics that specifically interact with the activation region of the GD? receptor, and may or may not require interactions with other such regions of the receptor. Interestingly, the majority of the GD? receptor CT tail could be removed with only small effects on coupling with adenylyl cyclase and receptor internalization, and no effect on receptor affinity. However the membrane proximal region of the tail appears to be important for receptor expression, and may contain residues or sequences that are involved in the inhibition of receptor sequestration. Alanine scanning of this region should help to identify which, if any, of the residues are involved. However it appears that other regions of the GD? receptor are involved in G-protein coupling, similar to those described for the GLP-1 receptor (Takhar et al, 1996; Mathi et al, 1997). Desensitization of the GIP response was not observed in CHO-K1 cells expressing the wt GIP receptor cDNA. It would be surprising if the GD? receptor was dramatically different from the glucagon and GLP-1 receptors (Widmann et al, 1995, 1997; Savage et al, 1995). Indeed the fact that Buggy and co-workers (1997) did not observe homologous desensitization in response to agonist stimulation of the glucagon receptor expressed in CHO-K1 cells, while its rapid desensitization has been demonstrated in isolated hepatocytes, suggests that both the GD? and glucagon receptors require cellular elements to undergo homologous desensitization that are not present in CHO-K1 cells. However 4 hours of GD? infusion were required to elicit desensitization with in vivo studies in rats (Tseng et al, 1996b). This slow time frame of desensitization suggests that in vivo desensitization is at least in part due to downregulation of the 242 receptor at the protein level. Such a response may have important implications in some N I D D M individuals that display elevated GD? levels, and could account for the lack of a GD? response and reduced responsiveness to GLP-1. However such a possibility is just speculative at this point and examination of desensitization in islets and B-cell lines should help to clarify the role of desensitization in GBP receptor physiology. While the exact role of GBP in N I D D M remains to be determined, available evidence indicates that GD? is the most important incretin, at least in healthy individuals (Hoist et al, 1997; Nauck et al, 1997a). It is therefore essential to develop a greater understanding of GBP receptor function, both in healthy and diseased states, and to elucidate further the structure-function requirements of GBP and its receptor, in order to develop GD? analogs that may prove useful in the treatment of N I D D M . It is hoped that the work described in this Thesis will help direct future research into these areas. 243 REFERENCES: Abello, J., Ye, R, Bosshard, A., Bernard, C , Cuber, J. C , and Chayvialle, J. A. 1994, Stimulation of glucagon-like peptide-1 secretion by muscarinic agonist in a murine intestinal endocrine cell line: Endocrinology, 134, p. 2011-2017. Abou-Samra, A. B., Jiippner, H., Force, T., Freeman, M . W., Kong, X . F., Schipani, E., Urena, P., Richards, J., Bonventre, J. V., Potts, J. T.,Jr., Kronenberg, H. M. , and Segre, G. 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E., Myhre, J., and Christiansen, J. ,Holst JJ. 1993, Truncated GLP-1 (proglucagon 78-107-amide) inhibits gastric and pancreatic functions in man: Digestive Diseases & Sciences, 38, p. 665-673. Wheeler, M . B., Lu, M. , Dillon, J. S., Leng, X . H., Chen, C , Boyd, A. E. 3rd. 1993, Functional expression of the rat glucagon-like peptide-I receptor, evidence for coupling to both adenylyl cyclase and phospholipase-C: Endocrinology, 133, p. 57-62. Wheeler, M . B., Gelling, R. W., Mcintosh, C. H. S., Georgiou, J., Brown, J. C , and Pederson, R. A. 1995, Functional expression of the rat pancreatic islet glucose-dependent insulinotropic polypeptide receptor: ligand binding and intracellular signaling properties: Endocrinology, 136, p. 4629-4639. Widmann, C , Burki, E., Dolci, W., and Thorens, B. 1994, Signal transduction by the cloned glucagon-like peptide-1 receptor: comparison with signaling by the endogenous receptors of p cell lines: Molecular Pharmacology, 45, p. 1029-1035. Widmann, C , Dolci, W., and Thorens, B. 1995, Agonist-induced internalization and recycling of the glucagon-like peptide-1 receptor in transfected fibroblasts and in insulinomas: Biochemical Journal, 310, p. 203-214. Widmann, C , Dolci, W., and Thorens, B. 1996a, Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate: Molecular Endocrinology, 10, p. 62-75. Widmann, C , Dolci, W., and Thorens, B. 1996b, Heterologous desensitization of the glucagon-like peptide-1 receptor by phorbol esters requires phosphorylation of the cytoplasmic tail at four different sites: Journal of Biological Chemistry, 271, p. 19957-19963. Widmann, C , Dolci, W., and Thorens, B. 1997, Internalization and homologous desensitization of the GLP-1 receptor depend on phosphorylation of the receptor carboxyl tail at the same three sites: Molecular Endocrinology, 11, p. 1094-1102. Wilden, U., Hall, S. W., and Kuhn, H. 1986, Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments: Proceedings of the National Academy of Sciences of the United States of America, 83, p. 1174-1178. Willms, B., Werner, J., Hoist, J. J., Orskov, C , Creutzfeldt, W., and Nauck, M . A. 1996, Gastric emptying, glucose responses, and insulin secretion after a liquid test meal: effects of exogenous glucagon-like peptide-1 (GLP-l)-(7-36) amide in type 2 (noninsulin-dependent) diabetic patients: Journal of Clinical Endocrinology & Metabolism, 81, p. 327-332. Willms, B., Ebert, R., and Creutzfeldt, W. 1978, Gastric inhibitory polypeptide (GIP) and insulin in obesity: II. Reversal of increased response to stimulation by starvation of food restriction: Diabetologia, 14, p. 379-387. Wilmen, A., Goke, B., and Goke, R. 1996, The isolated N-terminal extracellular domain of the glucagon-like peptide-1 (GLP)-l receptor has intrinsic binding activity: FEBS Letters, 398, p. 43-47. 278 Wong, S. K., Parker, E. M , and Ross, E. M. 1990, Chimeric muscarinic cholinergic: beta-adrenergic receptors that activate Gs in response to muscarinic agonists: Journal of Biological Chemistry, 265, p. 6219-6224. Worley, J. F.,3rd, Mclntyre, M . S., Spencer, B., Mertz, R. J., Roe, M . W.,and Dukes, I. D. 1994, Endoplasmic reticulum calcium store regulates membrane potential in mouse islet P-cells: Journal of Biological Chemistry, 269, p. 14359-14362. Yada, T., Itoh, K., and Nakata, M . 1993, Glucagon-like peptide-1 -(7-36)amide and a rise in cyclic adenosine 3',5'-monophosphate increase cytosolic free Ca 2 + in rat pancreatic p-cells by enhancing Ca2+channel activity: Endocrinology, 133, p. 1685-1692. Yamada, Y. , Hayami, T., Nakamura, K., Kaisaki, P. J., Someya, Y. , Wang, C. Z., Seino, S., and Seino, Y. 1995, Human gastric inhibitory polypeptide receptor: cloning of the gene (GIPR) and cDNA: Genomics, 29, p. 773-776. Yamagishi, T. and Debas, H. T. 1980, Gastric inhibitory polypeptide (GIP) is not the primary mediator of the enterogastrone action of fat in the dog: Gastroenterology, 78, p. 931-936. Yasuda, K., Inagaki, N . , Yamada, Y. , Kubota, A., Seino, S., and Seino, Y. 1994, Hamster gastric inhibitory polypeptide receptor expressed in pancreatic islets and clonal insulin-secreting cells: its structure and functional properties: Biochemical & Biophysical Research Communications, 205, p. 1556-1562. Zhang, J., Ferguson, S. S., Barak, L. S., Menard, L., and Caron, M . G. 1996, Dynamin and • -arrestin reveal distinct mechanisms for G protein-coupled receptor internalization: Journal of Biological Chemistry, 271, p. 18302-18305. Zhang, Y. , Cook, J. T., Hattersley, A. T., Firth, R., Saker, P. J., Warren-Perry, M . ,Stoffel M , and Turner, R. C. 1994, Non-linkage of the glucagon-like peptide 1 receptor gene with maturity onset diabetes of the young: Diabetologia, 37, p. 721-724. Zunz, E. and LaBarre, J. 1929. Contributions a l'etude des variations physiologiques de la secretions interne du pancreas: relations entre les secretions externe et interne du pancreas. Archives Internationales de Physiologie et de Biochimie, 31, p. 20-44. 279 Appendix A. CYTOSOLIC C a 2 + M E A S U R E M E N T S C U V E T T E FLUORJMETRY: Free cytosolic calcium concentrations [Ca 2 +]i were determined using a Hitachi F2000 spectrophotometer as described by Nasmith and Grinstein (1997). Briefly, COS-7 cells were loaded in D M E M with 2 p M fura-2 A M (Molecular Probes, Eugene, OR) for 20 min at 37°C. Aliquots of these cell suspensions were washed by sedimentation, and approximately 2.5 x 105 cells resuspended in K R P D buffer (140 m M NaCI, 4 m M KCI, 10 m M glucose, 10 m M HEPES, 1 mM MgC12, with or without 1 m M CaCl 2 , pH 7.4) and placed into a cuvette (#. 67.775, Sarstedt, Germany) with magnetic stirring in a thermostatically controlled (37°C) chamber. Fluorescence was measured at 37°C with excitation at 335 nm and emission at 510 nm. [Ca 2 +]i calibration was performed using ionomycin and M n 2 + with a K d of 224 n M and a ratio Fmax/Fmin of 3 as previously determined (Nasmith and Grinstein, 1997). SINGLE-CELL MICROFLUOROMETRY A confocal laser scanning microscope (CLSM, Biorad-600) was used to analyze GIP-evoked C a 2 + fluxes in individual COS-7 cells transfected with pGIP-Rl. Briefly, COS-7 cells were loaded with lOpM INDO-3AM (Molecular probes), for lhr in K P R D / D M E M V / V , 0.5% DMSO, and 0.01% pluronic acid. Subsequently, cells were washed twice in D M E M and changes in [Ca 2 +]i analyzed in the presence of 50nM GIP. Fluo-3 A M was excited using the 488 nm line of the argon laser, and emitted fluorescence 280 detected through a low pass fdter with cutoff at 515 nm. Images were collected digitally and a false color scale generated for quantitative purposes, where blue corresponds to lower and red to higher [Ca 2 +] levels. The changes in fluorescence were measured using CONRAD, a program for PC analysis and the preparation of confocal images, written by T.A. Goldthorpe, Department of Physiology, University of Toronto. Studies carried out by Dr. Paul E. Squires were essentially carried out as described above for the cuvette studies. Briefly, CHO-K1 or COS-7 cells were loaded with 5 u M fura-2-AM in D M E M at 37°C for 20 min. Individual cells were imaged with an Attofluor™ digital fluorescence microscopy system (Atto Instruments, Rockville, MD). A l l records were corrected for background fluorescence. In contrast to the cuvette studies, only the relative changes in [Ca 2 +]i are presented. RESULTS EFFECTS OF GD? O N [Ca 2 +]i Usdin et al (1993) demonstrated that the RTNm5F cell GIP receptor when expressed in a calcium reporter cell line (HEK293 expressing apo-aequorin), yielded an increase in [Ca 2 +]i in the presence of 100 nM GD 3 . The present series of experiments were designed to examine the linkage between GTP-R1 and [Ca 2 +]i. In COS-7 cells expressing GIP-R1 and loaded with the intracellular C a 2 + indicator fura 2-AM, 50 n M GIP increased [Ca 2 +]i with an acute transient phase, followed by a sustained elevation of [Ca 2 +]i (Fig. A l . A ) . The net increases in the transient (PI: A[Ca 2 +]i) and sustained phases (P2: A[Ca 2 +]i) were 114±18.1 and 36± 6.1 n M respectively (n > 3). A[Ca 2 +]i PI was further shown to be concentration-dependent with net increases of 49 ± 3.8 and 11 ± 4.5 at 5 n M and 0.5 n M 281 spGD? respectively (n = 3). To determine whether the source of the increased [Ca ]i elicited by GD? was from an intracellular or extracellular source, the above experiment was repeated first in a nominally C a 2 + free environment (Table A l ) , and then in the presence of 4 m M E G T A (Fig. A2.B). Under both conditions the transient first phase response was reduced but not eliminated (70±7.6 and 40±7.6 n M respectively vs. 114±8.1 n M in controls, p < 0.05). In contrast, the second phase responses were completely eliminated, in fact spGD? appeared to induce C a 2 + efflux from the cell (-10 ± 2.4 and -17 ± 2.4 nM). These data are consistent with the transient increase of [Ca 2 +]i originating primarily from an intracellular C a 2 + pool, and the sustained phase of [Ca 2 +]i increase resulting from an extracellular source(s). Treatment COS-7 A [Ca 2 +]i COS-7 A [Ca 2 +]i PI (nM) P2(nM) 50nMGIP 114 ± 8.1 36 ±6 .1 50 n M GTP + 10 u M nifedipine 110 + 5.3 42 ± 2.9 50 n M GTP (Ca2+-free media) 70 ± 7.6 -10 ±2 .4 50 n M GD* + 4 mM E G T A 40 ± 7.6 -17 ±2 .4 Table A l . Effects of spGIP on [Ca 2 ]i in COS-7 cells expressing GIP-R1. Data presented are the mean ± S E M of at least three individual experiments. P I , peak transient phase; P2, plateau phase. To characterize further the first phase [Ca 2 +]i response, cells were exposed to the sarcoplasmic/ endoplasmic reticulum C a 2 + ATPase inhibitor thapsigargin in nominally C a 2 + free medium (Fig. A2.D). Thapsigargin (50 nM) initially caused an increase in [Ca 2 +]i followed by a plateau phase suggesting depletion of intracellular C a 2 + stores. Subsequent addition of GD* (50 nM) did not elicit an increase in [Ca 2 +]i strongly suggesting that the PI response was primarily due to the mobilization of C a 2 + from intracellular stores. To characterize the C a 2 + entry pathway, COS-7 cells expressing GD»-282 R I were pretreated with the L-type V D C C blocker nifedipine (10 uM) (Fig A l . C ) . Nifedipine had no effect on either the sustained increase in [Ca 2 +]i (42±2.9 vs. 36±6.1 nM, p>0.05), or the immediate acute rise in [Ca 2 +]i (110±5.3 vs. 114± 8.1, p>0.05) (Table A l ) . This result is in contrast to previous findings in insulin-secreting HIT cells (Lu et al., 1993) and more recently in the 3TC6-F7 cell line (unpublished results) where E G T A (4 mM) pretreatment, or L-type C a 2 + channel blockers prevented spGD? induced increases in [Ca 2 +]i. The activation of voltage-sensitive C a 2 + channels by spGD? in COS cells was further discounted since KC1, used at a concentration that should depolarize the cell (50 mM), was unable to stimulate C a 2 + entry. The P2 response was also not elicited by forskolin (10 uM) or D3MX (data not shown) indicating that spGIP-induced increases in [Ca 2 +]i were unlikely to be mediated by a protein kinase A (RKA)-mediated pathway. Confocal microscopy was used to determine the relative number of COS-7 cells responding to spGD? and to examine C a 2 + fluxes in individual cells. In cells loaded with fluo 3 - A M the majority were observed to have similar resting C a 2 + fluorescence levels (Fig. A2.B). In response to the addition of 50 nM GD?, approximately 10-60% of cells in any given field showed an increase in C a 2 + fluorescence intensity (Fig. A2.C). This percentage is similar to that observed in control transfection experiments using p C M V b-gal (Invitrogen) as a reporter system to assess transfection efficiency. The changes in fluorescence for each cell (indicated by numbers in Fig. A2.B) were normalized to respective control values (DF/F) and plotted against time (Fig. A2.D). The pattern of fluorescence, although similar among cells, varied greatly in overall intensity. When averaged however, the calcium response pattern was remarkably similar to that observed by fluorimetry (Fig. A L A ) . That is, a rapid initial phase followed by a sustained second 283 phase. This is in direct contrast to the observations in CHO-K1 cells expressing GTP-R1. No GIP-stimulated changes in [Ca 2 +]i were observed in the wtGTP-Rl cell line, suggesting that the cellular environment the receptor is expressed in may determine the signal transduction pathways activated by a receptor. Alternatively extremely high expression levels obtained with the transient COS-7 cell line may be required to activate the Ca2 +-signaling pathway. 284 c. 4 0 0 -1 3 0 0 -CM 2 0 0 ti O 1 o o -4 0 0 -GIP 2 S 3 0 0 ™ 2 0 0 " (0: O 1 0 0 -GIP Nif ^ i r - 1 — r 2 0 0 1 B. 3 0 0 2 0 0 1 0 0 Thaps GIP R |p 1 0 0 2 0 0 3 0 0 Time (sec) 1 1—I 1 1 l 1 0 0 2 0 0 3 0 0 Time (sec) Figure A l . Effects of spGIP on [Ca2+]; in suspensions of COS-7 cells. (A) The effect of spGIP on [Ca2+]; was measured in COS-7 cell suspensions 72 h post-transfection with pGIP-Rl. the cells were loaded with fura-2 and then spGIP (50nM) was added at time point indicated by the arrow. In control experiments GD? was unable to evoke a [Ca2+]; response in cells expressing the GLP-1 receptor under identical conditions (not shown). (B) to determine the source of the spGIP-induced increase in [Ca2+]j, GIP-R1 transfected COS-7 cells were pre incubated in 4mM EGTA and stimulated with 50mM spGIP. (C) Alternatively, the cells were pretreated with 10pM nifedipine or (D) the Ca2+-ATPase inhibitor, thapsigargin (50nM). tracings are representative of at least three independent experiments. 2 8 5 Figure A 2 . Effect of s p G I P on [Ca 2 + ]j in individual C O S -7 cells. Confocal microscopy was employed to determine the pattern of changes in [Ca2"*"];. (A) Nonconfocal image acquired using the Bio-Rad transmitted light attachment showing COS-7 cells transfected with GIP-R1. Scale bar, 50 urn. (B) Confocal image of the same cells as in (A) loaded with fluo-3 A M , showing resting C a 2 + levels. Relative fluorescence appears in colour scale, with blue representing lower and white representing higher C a 2 + fluorescence. (C) In response to 50nM spGIP, fluorescence was followed in five identified responding cells (1-5). (D) Changes in C a 2 + fluorescence, normalized to resting fluorescence (%AF/F) for each cell, were analyzed over time from the identified cells (1-5). GIP was added at time zero. (E) The C a 2 + signals for cells 1-5 were averaged to show the C a 2 + signal in a population. 286 THE EFFECTS OF THE H170R POINT M U T A T I O N O N GTP-MEDIATED [Ca 2 +]i SIGNALING. Responses of COS-7 cells expressing the GIP-R1 cDNA to 10 nM GD? were similar to those originally obtained using single cell fluorimetry except the onset of the response was slightly delayed and the secondary phase appeared to have an decreased duration (Fig. A3.A). COS-7 cells expressing the point GIP receptor point mutant H170R did not appear to differ in their Ca2+-responsiveness compared to cell expressing the wt GIP receptor cDNA (Fig. A3.B). It was concluded that the substitution of His 17s0 with Arg did not affect the receptor's ability to couple to changes in [Ca 2 +]i. 287 A. l O n M G I P 2.0-. i i i i 1 0 200 400 600 800 B. Time (s) 0 .0 J i 1 i i i 0 200 400 600 800 Time (s) Fig. A3. Changes in [Ca 2 +]i in Response to 10 n M GIP Treatment in COS-7 Cells Expressing GIP-Rl(A) or H170R (B). 10 n M GIP stimulated rapid [Ca 2 +]i transients followed by slower secondary decay in [Ca 2 +]i. Experiments are representative of 3 individual transfections. The number of cells responding on a given coverslip varied from 5-60%. 288 

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