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Molecular cloning of a human putative 7-transmembrane segment (7TMS) receptor Federsppiel, Bartolome S. S. 1992

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MOLECULAR CLONING OF A HUMAN PUTATIVE 7-TRANSMEMBRANESEGMENT (7TMS) RECEPTORbyBARTOLOME S. S. FEDERSPPIELB.Sc., The University of Antwerp (Belgium), 1987A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Biochemistry)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAOctober 1992Bartolome S.S. Federsppiel, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)BiochemistryDepartment ofThe University of British ColumbiaVancouver, CanadaOctober 5th, 1992DateDE-6 (2/88)I. Abstract:Seven transmembrane-segment (7TMS) receptors are highlyhomologous proteins that mediate a variety of cell functions throughthe interaction with G-proteins. They are important mediators in theresponse to neurotransmitters, hormones and cytokines. Moreover,two of these receptors have been found to be proto-oncogenes or topossess transforming activity. During the immune response they areinvolved in inflammation, producing and modulating this processthrough the interaction with ligands that are primarily chemotacticand activating factors. A family of such ligands has already beencharacterized and their receptors, when known, were all of the 7TMStype. To further investigate this category of proteins in cellsinvolved in immunity a group of oligonucleotides was patterned onpredicted conserved areas of chemotactic 7TMS receptors, and alsoon divergent non-homologous sequences. To facilitate subcloningprocedures restriction sites where engineered at the 5'-end of theprimers. Human monocyte mRNA was converted to single strand cDNAby antisense priming and reverse transcription. This cDNA was usedas a template for the in vitro enzymatic amplification by thepolymerase chain reaction (PCR) with upstream and downstreamprimers. The amplified cDNA (388 bp) was cloned in plasmidBluescript and sequenced by the chain termination method. Homologysearches for the sequence revealed that the fragment belonged tothe 7TMS superfamily. The cDNA was radiolabeled and used to screena human fetal spleen library constructed in the x-phage system. Aclone was isolated that when sequenced revealed a complete openreading frame for a 352 residue protein. This polypeptide was foundto be a human homologue of a neuropeptide receptor: neuropeptidetyrosine (NPY) subtype 3. Apart from having the typical topology ofthe 7TMS superfamily it also displayed a high degree of homology tochemotactic factor receptors. Northern blot analysis demonstratedthat this protein is expressed in locations outside the nervoussystem like spleen and thymus.iiiII. Table of contentsI. Abstract	  iiII. Table of contents 	  i vIII. List of tables	  v iiIV. List of figures 	  viiiV. List of abbreviations	  xVI. Acknowledgements	  xiiiVII. Introduction 	  11. Inflammation 	  12. Seven-transmembrane segment receptors	  53. Receptors for intercrines	  74. Neuropeptides 	  1 05. Neuropeptide tyrosine and its receptor 	  1 36. Project purpose	  1 5VIII. Materials and Methods	 171.Materials 	 1 72. Plasmids 	 1 83. Bacteria 	 1 94. The human fetal spleen cDNA library	 1 95. Synthesis of oligonucleotides	 2 16. Purification of rabbit or human leukocytes	227. Purification of monocytes	 228. Extraction of total cell RNA 	 239. Extraction of RNA from tissues	 2510. Reverse transcription	 2511. Polymerase chain reaction (PCR) amplification 	 2612. Electrophoretic analysis of nucleic acids	2713. Purification of DNA from agarose gels	 2714. Restriction enzyme digestion of plasmid DNA andamplified cDNA 	 2815. Ligation 	 2916. Preparing competent cells 	 3017. Transformation 	 3018. Minipreps for plasmid DNA	 3119. Mapping of recombinant plasmids	 3220. Plasmid DNA purification for sequencing insert	 3221. Sequencing reactions	 3421.1. Alkaline denaturation of double stranded DNA 3621.2. Annealing reaction	 3621.3. Labeling reaction 	 3721.4. Termination reactions	 3721.5. Casting of polyacrylamide gels for sequencing 38iv21.6. Denaturing gel electrophoresis	  3922. Large scale preparation of plasmid DNA	  3923. Purification of insert cDNA from recombinantplasmid	 4024. Screening of the cDNA library	  4024.1. Preparation of host cells	  4124.2. Titration procedure	  4124.3. Screening procedure	  4224.4. DNA labeling reaction	  4224.5. Hybridization 	  4324.6. Rescue protocol	  4424.7. Mapping of phagemids	  4625. Northern blot analysis of tissue and cell RNAs	 4626. Chromosomal localization 	  47IX. Results	  491. Experimental strategy	  492. Primer oligonucleotides   503. The polymerase chain reaction	  534. Purification of DNA from agarose gels	  555. Insertion of cDNA into plasmid Bluescript	  566. Results from total white-blood cells cDNA	  586.1.Primers specificity 	  587. Results from monocytes cDNA	 597.1. Amplification of cDNA and genomic DNA	  597.2. Library screening	  607.3. In vivo excision of the recombinant s9a	  607.4. Sequence information of the clone s9a	  637.5. ORF for the protein humnyr3	  668. Chromosomal localization	  689. Northern blots	  68X. Discussion 	  711. Structural and functional determinants of the 7TMSsuperfamily 	  712. Virally encoded 7TMS proteins 	  823. 7TMS receptor for large proteins	  844. Chemotactic receptors	  865. Neuropeptide receptors	  906. Humnyr3 and the immune response	  937. Conclusion	  94Xl. Bibliography	 96XII. Appendices	  105Appendix 1: Alignment of 12 classical neurotransmitterreceptors 	  105Appendix 2: Alignment for the receptors of threepituitary hormones 	  108Appendix 3: Alignment of the neuropeptide receptors	  110viIll. List of tables:pageTable I: 	 Relationship of the intercrine familymembers.	4Table II: 	 Human diseases characterized bypredominant neutrophil and/ormonocyte infiltration 	 4viiIV. 	 List of figures:PageA schematic representation of the topologicalorganization of G-protein coupled receptorsin the cell membrane 	 6Adenylyl cyclase signal transduction pathway 	 8Plasmid Bluescript 	 18Excision of recombinant pBluescript from theUni-ZAP cloning vector 	 20Alignment of the amino acid sequences from thehuman SP R, rabbit IL-8 R and the mouse SK R. 	 52PCR amplification of rabbit and human cDNA 	 53Purification of restriction digested DNA fromagarose gels 	 55Insertion of cDNA into pBluescript 	 57Alignment of the sequences from cloned insertsand the human IL-8 R (high affinity) 	 58PCR amplification of monocyte genomic DNA 	 59A) Xho I/Not I digestion of several isolatesfrom the clone s9a 	 61B) Mapping of clone s9a with various restrictionenzymes 	 61C) Scheme of recombinant s9a 	 62Partial sequence of the recombinant s9a 	 63A) cDNA sequence and translation of the ORFfor clone s9a 	 64B) Hydrophobicity plot for the predictedhumnyr3 protein 	 65C) Alignment of the bovine NPY R with theputative human homolog humnyr3	66Figure 1:Figure 2:Figure 3:Figure 4:Figure 5:Figure 6:Figure 7:Figure 8:Figure 9:Figure 10:Figure 11:Figure 12:Figure 13:viiiFaggFigure 14:	 Chromosomal localization	 69Figure 15: 	 Northern blots for various tissue and cellRNAs	 70Figure 16: 	 Alignment, from TMS V towards the C-terminus,of humnyr3 and the human IL-8 R (low affinity) 	 72Figure 17: 	 Comparison between the intracytoplasmic loopsil and 12 for the chemotactic factor receptorsand humnyr3 	 73Figure 18: 	 Alignment of the chemotactic factor receptorsand humnyr3	 75Figure 19: 	 Comparison between the extracellular loop elfor the chemotactic factor, the classicalneurotransmitter and the neuropeptide receptors 	 78Figure 20:	 Comparison of the N-terminal region in humnyr3,the chemotactic, classical neurotransmitters,and neuropeptide receptors 	 81Figure 21: 	 Alignment of the human cytomegalovirusencoded US28 and humnyr3 	 84Figure 22: 	 Alignment of humnyr3 and humintleu8	 89Figure 23: 	 Alignment of humnyr3 and the neuropeptidereceptors 	 91Figure 24: 	 Alignment of humnyr3 with the human NPY R(subtipe Y1) 	 92ixV. List of Abbreviations:A280	absorbance at 280 nmA260	absorbance at 260 nmAmp 	 ampicillinbp 	 base pair(s)!ARK 	 0-adrenergic receptor kinaseBSA 	 bovine serum albumincAMP 	 cyclic adenosine monophosphateDAG 	 diacylglyceroldATP	 2'-deoxyadenosine 5'-triphosphatedCTP 	 2'-deoxycytosine 5'-triphosphateDEPC 	 diethylpyrocarbamatedGTP 	 2'-deoxyguanosine 5'-triphosphated'TTP 	 2'-deoxythymidine 5'-triphosphatedH20 	 autoclaved, destilled waterDNA 	 deoxyribonucleic acid(s)ddNTP 	 dideoxynucleotide triphosphate(s)dNTP 	 deoxynucleotide triphosphate(s)DTT 	 dithiothreitolE. coli 	 Escherichia coliEDTA 	 ethylenediaminetetra-acetic acidfMLP 	 N-formyl-Met-Leu-PheGro/MGSA 	 melanocyte growth stimulating activityIPTG	 Isopropyl-6-D-thiogalactopyranosidekb 	 kilobaseskDa 	 kiloDaltonKOAc 	 potassium acetateI	 litre(s)M 	 molarmA 	 milliampereMilliQ 	 grade of water purity (resistivity ?_ 16 MO.cm)min 	 minute(s)ml	millilitre(s)mM 	 millimolarMOPS 	 3-(N-morpholino)-propanesulfonic acidnm 	 nanometer(s)OFF 	 open reading framepfu 	 plaque forming unit(s)PKA 	 cAMP-dependent protein kinasePKC 	 protein kinase CRNA 	 ribonucleic acidRNase 	 ribonucleaseRNAsin	 ribonuclease inhibitorrpm 	 revolution(s) per minuteRI 	 room temperatureSDS 	 sodium dodecyl sulfatesec 	 secondTEMED 	 N,N,N',N'-TetramethylethylenediamineTris 	 tris(hydroxymethyl)aminomethane2-ME 	 2-mercaptoethanolv/v 	 volume to volume ratiovv/v 	 weight to volume ratiox g 	 times gravityx i1-tg 	 microgram(s)111 	 microliter(s)oc 	 degree(s) CelsiusX-Gal 	 5-bromo-4-chloro-3-indoly1 13-D-galactosexiiVI. AcknowledgmentsFirst, I would like to thank my supervisor, Dr. Ian Clark-Lewis,for giving me the opportunity to work in his lab. Under his mix ofguidance, encouragement, independence and friendship I have learnedover scientific research and over what it means to be a scientist.I also offer my sincere gratitude to Dr. Frank Jirik, whointroduced me to the intricacies of molecular biology, for his soundadvice, steadfast encouragement, and his friendship. My thanks alsoto all the members of his lab, specially to Nicole Jantzen, NazMassoudi, Scott Pownall, Ian Meihado and Ken Harder, for sharingwith me their expertise and valuable discussions, friendship andlong hours in the lab.Dr. Allen Delaney not only simplified my life with his constanthelp and advice on the field of microcomputers, he also introducedme to this fascinating world.Finally, my thanks to all the members of the BiomedicalResearch Centre, specially my laboratory fellows, for valuableadvice, their friendship and kindness, which have made my stay inthe lab an enjoyable, profitable, and pleasant one.VII.- INTRODUCTION1. Inflammation:Inflammation is the first step in the immune response and intissue repair activities. It is due to the interaction of a host ofdifferent cells. The recruitment and co-ordination required are dueto specific mediators most of which are cytokines. These solublepolypeptide mediators are produced by accessory cells (fibroblasts,endothelial cells, keratinocytes, platelets, smooth muscle cells) andby leukocytes. The initial steps usually involves the production ofthe systemic pro inflammatory cytokines interleukin-1 (IL-1) and/ortumor necrosis factor (TNF).IL-1 is a pleiotropic cytokine which is produced by an array ofaccessory cells but mainly by activated macrophages (Oppenheim etal.,1986). IL-1 activates cells in a prothrombotic, pro inflammatorymanner. It is not chemotactic per se but elicits the extravasation ofleukocytes from the blood by changing the adhesive properties ofendothelial cells and inducing the secretion of chemotacticcytokines (Mantovani et al.,1989). TNF produces a set of responsesthat overlap with those from IL-1 (Old, 1985) but it also augmentsthe expression of the major histocompatibility complex (MHC) classI antigens whereas IL-1 has little effect on MHC expression (Poberet al., 1987). This action is also coincident with the activity of'y-interferon (IFN-y) but the latter does not induce a proinflammatory reaction. IFNI, also induces the expression of MHCclass ll antigens and the invariant chain, rather priming the cells toact as accessory cells (Pober et al.,1983; Collins et al., 1984).1Lymphotoxin (or 	 INF-13) is a cytokine related to TNF which isproduced by B- and T-lymphocytes. It acts through the same receptorand its activities are similar to TNF.The name "intercrine" has been suggested to separate thestructurally related chemotactic and activating cytokines from theexclusively pro inflammatory IL-1 and TNF (Oppenheim et al.,1991).Interleukin-8 (IL-8) is a pro inflammatory intercrine thatstimulates chemotaxis on neutrophils as well as the release oflysosomal enzymes and the respiratory burst (Oppenheim et al.,1991). It is produced by several cell lineages and its production isinduced by IL-1, TNF and Lipopolysaccharide (LPS). It has beenshown to have chemoattractant properties for neutrophils andbasophils (in addition, basophils release histamine upon IL-8stimulation). The counterpart of IL-8 for monocytes is the monocytechemotactic and activating factor (MCAF) (Leonard et al.,1990). Incontrast with the respiratory burst induced by formyl-peptidechemoattractants MCAF causes no respiratory burst in monocytes.Basophils but not eosinophils respond to MCAF. It is chemotactic andinduces the release of lysosomal enzymes in cells of the macrophagelineage. It may be also important in response to acute tissue injuryby participating in host defense mechanisms and in tissue repair.MCAF has also been shown to activate cultured monocytes to becytostatic for several tumor cell lines (Matsushima et al., 1989;Yoshimura et al., 1989).IL-8 and MCAF are representative of the two subfamilies inwhich the intercrines are divided. There are four half-cystines inthese molecules and they exist as disulfides. The IL-8 subset differs2from the MCAF subset in that the first two cysteines are separatedby a single amino acid residue forming the motif C-X-C (X being anyamino acid). In the MCAF subgroup this motif is C-C. Structuralanalysis have shown that these molecules have two disulfide bridgesformed by the first and the third and the second and the fourthcysteines in the sequence. The base of the bridges is therefore inclose proximity. Apart from these structural features the othercriterion to divide the intercrines in two subfamilies is theirchromosomal localization. Thus, the human subset localized inchromosome 4 and presenting the C-X-C motif belongs to thesubfamily a (i.e. IL-8). Those containing the C-C motif and located onthe chromosome 17 belong to the 13-subgroup (i.e. MCAF). Bothsubsets are in the molecular weight range of 8-10 kD and are basicpolypeptides.There is 20-40% homology between these subfamilies. BothIL-8 and MCAF are produced in a precursor form of 99 residues witha signal sequence that is cleaved by proteolysis. The mature formsare 72 amino acids long and are not glycosylated. IL-8 is active asan hydrogen bonded dimer whose structure has been resolved byx-ray diffraction analysis (Baldwin et al., 1990) and NMR (Clore etal.,1989, 1990). Even though IL-8 and MCAF display only 21%homology their tertiary structure can be superimposed. The tertiarystructure of MCAF was predicted having as a base the knownstructure of IL-8 (see tables I and II, next page).3Table I:Relationship of intercrine family membersIntercrine a-subfamily 	 Intercrine 13-subfamilychromosome 	 4 (q12-21) 	 17 (q11-32)structure	 C-X-C 	 C-Cmembers 	 IL-8 	 MCAFNAP-2 	 1-309ORD 	 LD-78PF-4 	 ACT-213TG 	 RAI1TESIP-10Table IIHuman diseases characterized by predominant neutrophiland/or monocyte infiltrationRheumatoid arthritisPsoriasisGoutImmune vasculitisGlomerulonephritisInflammatory bowel diseaseMyocardial infarctionAdult respiratory distress syndromeEmphysemaAsthmaArthritis associated with Mediterranean fever42. Seven-transmembrane segment receptors:Transmembrane signaling systems are the sensors that cellsevolved to detect changes in the environment and to adapt to them.The cell membrane is a physical barrier between the extra cellularspace and the tightly controlled intracellular matrix. As it is welldocumented this barrier is far from being inert, but the lipids of thebilayer form an effective hindrance to the movement of proteins andmost solutes across the membrane. For any signaling mechanism toinduce a productive response this obstacle has to be circumvented.The molecular switches that convey information inside the cellreside in a particular type of membrane proteins generically knownas receptors. This function, plus the specific recognition of theligand, places receptors in a key position within the processes ofcell-cell communication mediated by protein, and non-proteinfactors.One of the best characterized of such systems is a receptorfamily that shares significant homology and which overall topologyhas been established or can be inferred from predictive algorithms.This group is known as the seven-transmembrane segment (7TMS)receptors following the prediction that the seven hydrophobicsegments form membrane-spanning a-helices (figure 1). They arepresent in organisms so divergent as man and yeast and theymodulate a host of processes where the initial stimulus can be aneurotransmitter, a neuropeptide, a hormone or a cytokine. They arealso the molecular basis for light detection and odor recognition(Khorana, 1992; Buck and Axel, 1991).5Ext race IlularTMS IIntracellularC -terminusFigure 1: A schematic representation of the topologicalorganization of G-protein coupled receptors in the cellmembrane: segments I trough VII are predicted to span themembrane. The N-terminus and the connecting loops el, e2,and e3 would be located on the extra cellular surface of thebilayer. The C-terminus and connecting loops il, i2, and i3would lie in the cytoplasmic side.These molecular switches consist of a transmembrane protein(the receptor) coupled to an effector protein inside the cytoplasm ofthe cell. The effector system to which they are coupled is often anenzyme or an ion channel and the coupling is in turn mediated by thesystem of guanine-nucleotide binding proteins (G-proteins). Thisallows them not only to transmit a signal toward the cytoplasm, butalso to amplify the output response (figure 2). Growth factors,neuropeptides and cytokines can use a common signal transducing6mechanism but it is important to note that the final outcome for anygiven cell depends on the interaction of different stimuli and on thatcell phenotypic expression. The receptor-coupled G-proteins consistof three subunits called a, p and y (in order of decreasing molecularweight). They are present in the form of heterotrimeric complexeswith a stoichiometry of 1:1:1. Upon agonist binding the a-subunitexchanges bound GDP (which forms part of the complex) for GTP.This induces a conformational change and the a-subunit dissociatesfrom the complex and this is the active, effector modulating protein.The 13y complex enhances the efficiency of GTP/GDP exchange (Bourneet al.,1991) by driving the replacement of GDP for GTP.Amplification results from the multiplying effect of having a singleactivated receptor molecule interacting with several ternarycomplexes. There are more than 20 a-subunits in man and only foureach p- and 'y-subunits. The a-subunit is the GTPase and it is alsothe substrate for ADP-ribosylation by bacterial toxins. The ligandactivated receptor functions as a guanine nucleotide release protein(Bourne et al., 1991). Ligand binding and G-protein activating moietyreside on a single polypeptide. Binding sites for agonists andantagonists have both distinct and shared determinants, andimportant determinants for G-protein recognition are present in thetransmembrane segment V and VI as well as in the interconnectingcytoplasmic loop (Dohlman et al., 1991).3. Receptors for intercrine ligands:Scatchard analysis of IL-8 binding data showed that there are20,000 high affinity sites (Kd= 0.8 nM) per cell in human neutrophils.7Cross-reactivity has been observed between IL-8 and other membersof the a-subfamily, specially Gro and its murine homologue MIP-2.Figure 2: Adenylyl cyclase signal transduction pathway. R:receptor; a,Pand 7: the three subunits of a G-protein; AC:adenylyl cyclase; GDP: guanosine diphosphate; GTP: guanosinetriphosphate; H: hormone (or other ligand); ATP: adenosinetriphosphate; cAMP: cyclic adenosine monophosphate; PKA:protein kinase A.IL-8 receptor (IL-8 R) expression is dynamically regulated bythe ligand itself (Oppenheim et al.,1991). IL-8 causes a rapid8increase in cytosolic free calcium in human neutrophils that isinhibited by Bordetella pertussis toxin (Thelen et al., 1988; Dewaldet al., 1988). It also causes a respiratory burst that is inhibited by17-hydroxywortmannin and staurosporine (Thelen et al., 1988). Fromthese findings it was deduced that a G-protein is associated to theIL-8 R and that Protein Kinase C activation is needed for the actionof IL-8.The initial path for signal transduction appears to be the samefor all the chemotactic factor receptors which have beencharacterized up to now: they have the predicted topology of the7TMS family and they associate with G-proteins. Recently twocDNAs encoding IL-8 binding proteins have been cloned by expressioncloning or by hybridization (Holmes et al., 1991; Murphy et al., 1991).One of them is the high affinity site (Holmes et al.,1991) and theother is low affinity. The latter is an IL-8 functional receptor butits specificity is doubtful in light of the existing binding studiesand the cross-reactivity observed between some intercrines andtheir receptors (Moser et al. 1990). SDS-PAGE analysis of cross-linked 1251-IL-8 and receptor complexes showed two polypeptideswith receptor moieties estimated at 67 and 59 kD. As the cells fromthe promyelocytic lineage mature they express more IL-8 R, withmature neutrophils expressing the highest number. By contrast verylittle is known about MCAF receptors. Current estimation of theirnumbers in mature monocytes is 1,700 per cell with a Kd= 2 nM.94. Neuropeptides:In recent years a large development in the field ofneuropeptides took place (Hokfelt et el.,1980). Neuropeptides nowoutnumber the classical neurotransmitters in both central andperipheral nervous system. It has been thought that the main actionof neuropeptides was in the area of neurotransmission where theywould mainly mediate the depolarization of the cell. In this contextit is important to mention that is a change in membrane potentialwhich modulates the release of TNF in LPS-stimulated macrophages(Haslberger et al., 1992). Neuropeptides act as neurotransmittersthrough specific cell surface receptors but they can also act asmitogens for a variety of cell types. Even in the case of classicalneurotransmitters it has been observed that at least two muscarinicreceptor subtypes coupled to inositol metabolism can induce DNAsynthesis when stimulated with the stable muscarinic agonistcarbachol (Ashkenazi et al., 1989). This was observed in transfectedCHO cells and also with cell lines expressing the specificacetylcholine receptor subtypes. Furthermore, the transfection ofNIH 3T3 cells with the 5HT1c serotonin receptor results in theinduction of a transformed phenotype and tumorigenicity uponserotonin stimulation (Julius et al., 1989). It should be taken intoaccount that even though the neurotransmitter receptors arerestricted to neural cells, the signaling pathways that they activateare present in both neural and non-neural cells. It has also beenobserved that in the case of neuropeptides mitogenic activity ispossible. This is clearly the case for the mammalian mas oncogenethat encodes for an angiotensin receptor (Young et al., 1986; Jackson10et al., 1988). Growth factors or mitogens act in a diverse array ofevents including embryogenesis, tissue repair, immune response, andoncogenesis. In several of such cases sensory nerve endings are inclose apposition to the target cells.It has also been observed that platelet derived growth factor(PDGF) and epidermal growth factor (EGF) are able to inducecontraction in smooth muscle cells. All these findings stronglysuggest that the appropriate response is more determined byphenotypic factors rather than by the initial stimulus itself. Thus, inthe well characterized case of norepinephrine and epinephrine whichare both neurotransmitters in sympathetic neural cells butepinephrine also acts as a hormone for peripheral tissues.The tachykinins substance P (SP) and neurokinin A (NKA) alsopresent mitogenic activity on smooth muscle cells and fibroblasts(Nilsson et al., 1985). Comparing the effect on smooth muscle cellsof SP and NKA to that of PDGF it was found that PDGF decreases theamplitude of the response to the tachykinins. This suggests acompetition for the same intracellular messengers (both calciumand calmodulin antagonists inhibit the mitogenic effect) (Hultgaard-Nilsson et al., 1989). SP stimulates the proliferation of 1-lymphocytes and potentiates the response to mitogens (Payan et al.,1983).Beta-endorphin, vasoactive intestinal polypeptide (VIP) andsomatostatin both inhibit and stimulate lymphocytes in a dosedependent manner (Moore, 1984; O'Dorisio et al., 1985; Ottaway etal., 1984; Pawlikowski et al., 1985). It was shown that VIP11synergizes with EGF in stimulating the growth of keratinocytes butit was inactive by itself.Lung epithelial cells respond to growth stimulation bybombesin and its mammalian counterpart gastrin releasing peptide(GRP). GAP is an autocrine factor for small cell lung carcinoma(Cuttitta et al., 1985).Given all these findings it is interesting to speculate over thephysiological as well as the pathophysiological role ofneuropeptides apart from their usual function as neurotransmitters.Locally released neuropeptides could be associated to the woundhealing process. During development bombesin-likeimmunoreactivity is present in high concentration in fetal andneonatal lung, but it is absent in adults, indicating a possibleinvolvement of bombesin in lung embryogenesis. Some evidenceexists pointing to a role for gastrin, enteroglucagon, neuropeptidetyrosine (NPY) and GRP in the control of gastrointestinal epithelialcell proliferation. Neuropeptides could also be involved in thedesmoplasia associated with some tumors if the malignant cellsproduce NKA or SP, or they could be involved in some autocrine loop.SP and NKA are present in neurons surrounding large arteries. SPinduces dilatation when acting on endothelial cells but contractionwhen acting on smooth muscle cells. Thus, SP is more likely to causecontraction when acting on vessels with advanced atheroscleroticlesions.Sensory neurons may cause vasodilatation and edema(neurogenic inflammation) and this response involves SP (Dalsgaardet al., 1989). SP involvement has also been thought to be involved in12chronic inflammation and arthritis (Levine et al.,1984). SP alsoenhances the production of immunoglobulins by B-lymphocytes(Stasnitz et al., 1986).5. Neuropeptide tyrosine (NPY) and its receptor:The sympathetic nervous system innervates lymph nodes,spleen, bone marrow and thymus (Besedowski et al., 1979). Itsfunction is related to both vascular cells and parenchymal cells andits has been implicated in the modulation of the immune responseand inflammation (Felten et al., 1987). There are adrenergicreceptors in bone marrow derived cells such as macrophages butthere is no evidence for a specific role for catecholamines inmodulating immune function. Sympathetic nerves innervatingperipheral organs also possess a neuropeptide which is 36 aminoacids long and is called neuropeptide tyrosine (NPY) due to its C-terminal tyrosine amide (Tatemoto et al., 1982). NPY colocalizeswith noradrenaline and it is released in the same noradrenergicvesicles in some synapsis. (Fried et al., 1985; Stjarne et al., 1986).It is thought that NPY may potentiate noradrenaline evokedvasoconstriction and that it has a direct vasoconstrictory effect perse (Ekblad et al., 1984). There also exist intrinsic NPY-neurons inpancreatic tissue (Sheikh et al., 1988). It is believed that NPYparticipates in a negative loop at the pre-junctional release ofnoradrenaline (Lundberg et al., 1984) and it has been suggested thatthe pre- and post-synaptic effects are due to different types ofreceptors (Wahlestedt et al., 1986).13NPY is one of the most abundant neuropeptides in themammalian nervous system. In the central nervous system itcolocalizes with adrenergic and noradrenergic neurons in the brainstem (including the locus coerulus). It also colocalizes with theneurotransmitter 7-amino butyrate and somatostatin in the humanbrain cortex. In rats it has been observed to induce feeding behaviorand to produce hypothermia (Roscoe et al., 1991). It regulates bloodpressure, heart rate, respiratory rate and influences hypothalamic-neuroendocrine systems. In relation to the immune system NPYinduces the release of histamine from mast cells (Lundberg et al.,1982) therefore having a modulatory activity and it has also beenimplicated in depressed activity of natural killer cells (Irwin etal.,1991). It has been also found to up regulate the adhesiveness ofhuman neutrophils or U937 cells ( a human monocytic cell line) tohuman umbilical vein endothelial cells (Sung et al., 1991). This eventwas not associated with intercellular adhesion molecule-1 (ICAM-1)nor through the induction of cytokines such as IL-1 or TNF.NPY, peptide YY (PYY) and pancreatic polypeptide (PP), allbelong to the PP-fold family because of their characteristic andhighly conserved structural features. PYY and PP are intestinalhormones, PYY being present in endocrine cells of the gut and PP inpancreas. In contrast to other peptides of the same size (36residues) this family conserves a compact globular structure insolution (Glover et al., 1983). This structure has been resolved to aresolution of less than 1 A for avian PP by x-ray diffraction analysis(Wood et al., 1977). The structure consist of two anti parallela-helices: a N-terminal Pro rich helix and an amphipathic helix14joined by a (3-turn. The structure is stabilized by interdigitatinghydrophobic residues.High affinity NPY-binding sites (0.1 to 1 nM) had been observedin central nervous system, spleen and enterocytes, and in a ratpheochromocytoma cell line. It has been suggested, based on bindingcharacteristics, that there exist three NPY receptor subtypes termedY1, Y2 and Y3. Apparently post-synaptic receptors are of the Y1 andY2 subtypes while the pre-synaptic site belongs to the Y2 subtype.The Y2 receptor is the major one in central nervous system(Fuhlendorff et al., 1990). The Y1 receptor binds NPY and PYY withsimilar affinity but a "long" C-terminal fragment (e.g. NPY 13-36)does not bind. In contrast this fragment does bind to the Y2 subtype.The Y3 receptors are characterized because they bind NPY with muchhigher affinity than PYY. Recently three cDNAs have been cloned thatcode for NPY receptors. A human cDNA belongs to the putative Y1subclass. A Drosophila NPY receptor has been tentatively equated tothe Y2 subtype and a bovine protein which cDNA was cloned fromlocus coerulus appears to belong to the Y3 sub category (Larhamar etal., 1992; Li et al., 1992; Rimland et al., 1991).6. Project purpose:The identification and characterization of receptor proteinsfor two of the chemotactic and activating factors was the initialscope of this work. The factors in question were Gro/MGSA andMCAF. The biological activities of Gro are not well defined but it isinduced by IL-1 and TNF. It presents growth stimulatory activity onfibroblasts and is an autocrine growth factor for melanoma cells. It15also compete with IL-8 for the binding to the IL-8 R. MCAF is themonocytic chemotactic and activating factor. There are severalpathological processes where its involvement is suspected (seeTable II). During the course of this work a clone was isolated from ahuman spleen cDNA library. It was sequenced and analyzed, resultingin the identification of a human homologue of a bovine proteinisolated from locus coerulus that is a putative NPY R (subtype Y3).The cloning was done by hybridization with a probe obtained by thereverse transcriptase/polymerase chain reaction from humanmonocyte RNA. To prime both the reverse transcription and thepolymerase reaction a set of primers was designed on the basis ofhomology between conserved IL-8 R sequences. This neuropeptidereceptor is closely related to some of the intercrines receptors andappears to be expressed in cells of bone marrow origin.16VIII. MATERIAL AND METHODS:1. Materials:All chemicals were analytical or reagent grade. They wereeither from Sigma Chemical Co., BDH Inc., or Fisher Scientific. Waterwas processed with a Zenopure Laboratory water system (Mega 90)to 16 Macm. It was sterilized by autoclaving.Bacto-tryptone, yeast extract and bacto-agar were from DifcoLaboratories. Penbritin-1000 (ampicillin) was from AyerstLaboratories. Hybridization membranes were Hybond-N (0.45 gm)from Amersham. Enzymes were either from Boehringer-Mannhein orfrom New England Biolabs. DNA polymerase for sequencing wasSequenasee* Version 2.0 from United States Biochemical. Agarosewas from GIBCO Bethesda Research Laboratories. Films forautoradiography were from Kodak. For the radioactive labeling ofDNA two isotopes were used: 35S and 32P. Deoxyadenosine 51(a32P)triphosphate, triethylammonium salt, in aqueous solution containing5 mM 2-mercaptoethanol and at a concentration of 370 MBq/m1 (10mCi/m1; >3,000 Ci/mmole)(by the reference due date), anddeoxycytosine 5'(a-35S) triphosphate, triethylammonium salt, inaqueous solution containing 5 mM 2-mercaptoethanol and at aconcentration of 370 MBq/m1 (10 mCi/m1; >600 Ci/ml) (by thereference due date). Both were purchased from Amersham.Deoxynucleotide triphosphates, Ficoll-Paque andchromatographic gels (Sephadex; Sephacryl) were from PharmaciaP-L Biochemicals.17For sequencing, the LKB (Bromma), 2010 MacrophorElectrophoretic Unit was used.2. Plasmids:The pBluescripte II vector (Stratagene) is a phagemid derivedfrom pUC19 by replacing the polylinker with a syntheticmulticloning site containing 21 unique restriction sites. Its size is2.95 kb. It allows for blue/white selection because it contains theportion of the lacZ gene that provides a-complementation whenpropagated in cells containing the lacriZAM15 mutation on the F`.There are two possible orientations of the polylinker according tothe direction in which the 13-galactosidase transcription occurs. Thephagemid used was the SK in which the transcription proceeds fromSac I to Kpn I. The presence of a f1 origin of replication allows forsingle stranded DNA rescue upon co-infection with a helper phage(see figure 3 and 4).fl(Xmnl (2643)Scal (2526)Pvul (2415)blaColn onoriginPvul (502)Pvull (532)Kpnl (655)Apal (661)Xhol (670)Accl (676)Hind! (676)Clal (685)Hindi!! (691)coRV (697)EcoR1 (703)Smal (715)BamH1 (721)Xba 1 (733)Not! (739)Eagl (740)Sac!! (749)Sac! (757)Pvul (977)110Figure 3: Scheme of the plasmid Bluescript.183. Bacteria:A E.Coli derivative, XL1-Blue strain was used trough this work(Bullock et al., 1987). Its genotype is: ndA1, hsdR17 (rk-,nik+),supE44, thi-1, X-, recA1, gyrA96, relA1,(lac-),[F,proAB,lacicIZAM15,Tn10, (tetr)]. This strain was propagated in 2xYT medium (16 g/I bacto-tryptone, 10 g/I yeast extract, and 5 g/INaCl) at 37°C with vigorous shaking, or on YT plates (YT medium plus15 g/I bacto-agar) incubated 16 hours at 37°C. Other media usedwere NYT (16 g/I casein hydrolysate, 2 g/I MgSO4.7H20, 5 g/I yeastextract) and LB ( bacto-tryptone 10 g/I, yeast extract 5 gIl, NaCI 10g/I).The lacicIZAM15 mutation contained in the F' episome allowsfor blue/white color selection and the episome bearing cells can beselected with tetracycline. These cells are restriction negative, andboth endonuclease and recombination deficient. Aftertransformation, cells bearing the pBluescript were selected byadding ampicillin to the plates or to the media (at a concentration of50 to 100 p,g/m1).4. The human Fetal Spleen cDNA library:This premade library was purchased from Stratagene. It wasconstructed in a A-vector (Uni-ZAPTm XR). It was derived frompoly(A)+ enriched RNA using oligo(dT) primers. The origin of the RNAwas pooled tissue.All inserts were cloned with the 5'-end closest to the LacZpromoter allowing the expression of 13-galactosidase fusion proteins(the cDNA is unidirectionally inserted in the sense orientation). Each19colE1fl (-)originInitiatorTerminatorT7insert is flanked by specific 13 and T7 promoters that can be used togenerate end-specific RNA transcripts. (see figure 4)ccT3 '10"1" 	 -44* Ticos1 Excision of the pBluescript containing the clonedDNA insert by co-infection with helper phage.Figure 4: The Uni-Zap vector arms have been digested withEcoR I and Xho I and dephosphorylated, therefore, it allowsconstruction of unidirectional cDNA libraries.20The average insert size was 1.0 kb. The possibility of in vivoexcision of the plasmid Bluescript from the Unilap vector using ahelper phage is convenient because a plasmid system containing theinsert of interest for characterization purposes can be obtainedwithout having to subclone. The cloning sites are produced in theUnilap vector by Xho I and EcoR I double digestion. The insert sizecan be anywhere from 0 to 10 kb.5. Synthesis of oligonucleotides:Oligonucleotides were synthesized with an automatic ABImodel 391B DNA synthesizer by John Babcook (Biomedical ResearchCentre, University of British Columbia) following thephosphoramidite protocol according to the instructions of themanufacturer. After synthesis the resin was treated withconcentrated ammonia (fresh solution) to release and deprotect thebound oligonucleotides. Briefly, the cartridge containing the resinand the bound oligonucleotide was incubated for 15 min at roomtemperature with approximately 0.5 ml of ammonia. After thisincubation the aliquot was eluted in an screw cap eppendorf tube.The treatment was repeated three times to increase the yield andthe aliquots were pooled. The tubes were incubated at 55 °Covernight for deprotection. Then the samples were concentrated in aspeed-vacuum for 3-4 hours (until total dryness). Driedoligonucleotides were redissolved in 0.5 ml of destilled water andfurther purified by desalting on a Sephadex G50 spin column (2 ml ofpacked beads equilibrated against destilled water). Theconcentration of the purified oligonucleotides was estimated by21measuring the A at 260 nm. The usual concentration obtained was100 mM. The aqueous solution was aliquoted and stored at - 80°Cuntil needed.6. Purification of rabbit or human leukocytes:Anticoagulant-treated blood (200 ml) was centrifuged for 10min at 3,500 rpm (2,000 xg) at room temperature. The interphasebetween the plasma and the red blood cells contains the leukocytesand the platelets ("buffy" coat). This fraction was recovered with aPasteur pipette, together with some of the plasma but avoiding theerythrocytes, and recentrifuged. The upper layer containing plasmawas discarded and the leukocyte pellets, still contaminated with redblood cells, were resuspended in red blood cells (RBC)-lysis buffer (N H4CI 6.57 g/I and Tris base 2.59 g/I adjusted to pH 7.2 with HCI)and then incubated for 7 1/2 min at 37°C in a water bath. Theosmolarity was normalized by adding an equal volume of ice-coldPBS ( NaCI 8 gil, KCI 0.2 g/I , Na2HPO4 1.44 g/I, KH2PO4 0.24 g/Iadjusted to pH 7.4 with HCI) and the leukocytes were recovered bycentrifugation. Cell pellets were resuspended in GT bufferimmediately to isolate the RNA (see below).7. Purification of Monocytes:200 ml of heparinized blood were collected from a singlehuman volunteer donor. After centrifuging for 5 min at 2,000 xg the"buffy" coats containing mainly leukocytes and platelets were pooledand recentrifuged. The pooled leukocytes were transferred to a newtube and underlayered with 10 ml of Ficoll-Paque. (Boyum, 1968).22The tube was then centrifuged for 20 min at 650 x g. The interphasecontaining monocytes and lymphocytes was collected and spun down.The supernatant was discarded and the cells were resuspended in100 ml of RPMI-1640 containing 2-mercaptoethanol (2ME) andsupplemented with Gin (2 mM) and 10% FCS. The suspension wastransferred into a 200 ml Falcon tissue culture flask and incubatedovernight at 37 °C in a tissue culture incubator. After thisincubation the flask was gently shaken to remove non-adherent cellsand the supernatant was discarded. The remaining adherent cellswere washed 10 times with 50 ml aliquots of ice-cold PBS. Thecells were lysed in situ with 2 ml of GT solution (25 mM sodiumcitrate buffer pH 7.5 containing 4 M guanidine thiocyanate, 0.5%sarcosyl and 0.1 M 2-ME added just before using). This solution wasprepared with diethylpyrocarbamate (DEPC)-treated water. An extra2 ml were used to rinse the flask and this wash was pooled with thelysis solution.8. Extraction of total cell RNA:The basic molecular cloning techniques used here can be foundin Sambrook et al. (1989). All the modifications are indicated in therespective protocols.This procedure was performed on ice unless otherwiseindicated and all the material was treated with DEPC to minimizethe contamination with ribonucleases. The lysed samples werehomogenized by 25 passages through a 22G1-bore needle to shear thegenomic DNA (avoiding foaming). Per 500 gl aliquots 50 jil of 3 Msodium acetate pH 4.0 were added. After a short vortexing to mix23500 p.1 of phenol/water (50:50) were added and the tubes werevortexed again. 100 pi of chloroform/isoamyl alcohol (24:1) wereadded and mixed by inverting the tubes; then, they were incubatedon ice for 20 min. The samples were centrifuged for 20 min at14,000 rpm at 4°C in an Eppendorf microcentrifuge. The upper layerwas transferred to a new tube and 600 p.1 of ice-cold isopropanolwere added and mixed by inverting the tubes. After incubating at-20°C for 1 hour the precipitate was collected by centrifugation at14,000 rpm for 20 min at 4°C. The supernatant was aspirated andthe pellet was resuspended in 200 gl of GT solution. Three of suchaliquots were pooled and re-precipitated with 600 p.1 of ice-coldisopropanol. The resulting pellets were resuspended in 200 p.1 ofDEPC-treated double destilled water and 20 p.1 of 3 M sodium acetatepH 4 were added followed by 500 p,1 of 100% ethanol cooled at-20 °C. After 1 hour incubation at -20 °C, the precipitated RNAwas recovered by centrifugation for 20 min at 14,000 rpm (at 4°C).The supernatant was aspirated and the pellet was dried in adessicator for 10 min under vacuum. Each pellet was resuspended in50 p.1 of DEPC-treated water. The A260 nm and A280 rim weremeasured for a 1/250 dilution and the remaining sample was storedat -80°C.Normally, 0.5 to 1 lig of total cell RNA is enough to amplify byPCR even rare mRNA sequences. Considering that the RNA content percell is approximately 10 pg, 1 p.g is the amount obtained from 1 x105 cells. Thus, the number of messenger copies per 1 gg RNA is atleast the same as the number of cells. Therefore preparation ofpoly[A]+RNA is usually not required.24The A260nm/A280nm ratio (which is indicative of the quality ofthe preparation with respect to protein contamination) wasconsistently close to 2.0 with this technique.9. Extraction of RNA from tissues:The tissue was removed, quickly frozen in liquid nitrogen andgrinded into pieces of about 2 g. For each 2 g of tissue 20 ml ofguanidine solution (without sarkosyl) were added. The tissues werehomogenized by three burst of 10 seconds each with an Ultraturrax.After centrifuging for 10 min. at 10,000 rpm in a Sorvall SS34 rotor(at 4°C), the supernatant was decanted and 1/10 volume of 20 13/0sarkosyl was added. Total RNA was isolated using the guanidinethiocyanate-acid phenol method with the addition of a cesiumchloride ultracentrifugation step to purify the RNA.10. Reverse Transcription:All the materials and labware were either treated with DEPCor free of ribonuclease contamination. The following reagents werecombined in a 20 III final volume: reaction buffer: 50 mM Tris.HCI(HCI-neutralized Tris[hydroxymethyl]aminomethane) pH 8.3 at roomtemperature containing 75 mM KCl, 3 mM MgC12 and 10 mM DTT (theDTT solution was stored at -20°C as a 10x solution and it wasadded just before starting the reaction), 1 mM of eachdeoxynucleotide triphosphate (dNTPs) (from a stock solution atneutral pH), 1 unit/ill RNAsin (Promega), 100 pmole of reverseprimer oligonucleotide, 0.5 gg of RNA and 200 units of MoloneyMurine Leukemia Virus RNAse H- reverse transcriptase (GIBCO BRL)25(Kotewics et al., 1988). The RNA sample and the primer were firstmixed together and the reaction tube was heated at 65°C for 5 min.After a short spin the mixture was incubated on ice for 3 min. Thiswas intended to disrupt secondary structure elements that couldhinder the cDNA synthesis. After this, the reaction mixture wascompleted as indicated and the reverse transcription was allowed toproceed for 1 hour at 42 °C. At the end of this incubation thereaction was heated at 95°C for 5 min in a water bath, briefly spunto collect condensed water from the tube walls, and then quick-chilled on ice. This treatment denatures RNA-cDNA hybrids andinactivates the enzyme (Veres et al., 1987)11. Polymerase Chain Reaction (PCR) amplification:The heat-treated reverse transcriptase reaction was scaled upto 100 p,I by adding 100 pmole of direct primer oligonucleotide, 10ill of 10x PCR buffer ( 250 mM Tris.HCI pH 8.3 at 25 °C containing 20mM MgCl2, 500 mM KCI and 10 mM DTT in MilliQ grade water) 5 unitsof Thermus aquaticus (Taq) DNA polymerase and MilliQ water. Aftermixing by vortexing and a short spin to collect the sample, 60 p,I ofliquid paraffin were layered on top of the PCR reaction mix toprevent liquid evaporation during the thermal cycling. The number ofcycles for amplification was 35 and the thermal cycle profile wasas follows: 1) denaturing for 30 seconds at 96°C, 2) cooling over 1min to 55°C, 3) annealing primers for 30 seconds at 55°C, 4) heatingover 30 seconds at 72°C, 5) primer extension for 90 seconds at 72°C.After the number of cycles was completed the reaction mix was kept26at 4°C until further analysis or stored at -20°C for prolongedperiods of time.12. Electrophoretic analysis of Nucleic Acids:The electrophoresis was performed in horizontal submarinegels. The desired agarose (w/v) concentration was added to theappropriate volume of TAE buffer ( 40 mM Tris.HCI pH 7.5 containing1 mM EDTA and 5 mM Sodium acetate) and dissolved by microwaveradiation. The gel was poured and allowed to cool down either atroom temperature or at 4°C to accelerate the solidification process.Two types of agarose were employed, either low melting pointagarose for preparative or electrophoretic grade agarose foranalytical electrophoresis. The samples were applied in a loadingbuffer mix (25% glycerol, 0.25% xylene cyanol FF, 0.25% bromophenolblue) diluted 1/10 and electrophoresed at 100 mA constant current,until the marker dyes reached the desired reference points. A 1 kbDNA-ladder from BRL was used as size marker (1 1.1g/lane).After electrophoresis the gel was stained by soaking in aethidium bromide solution (10 jig/m1 freshly made in tap water) forat least 10 min. Photographs were taken on a short wave UV (254nm) transilluminator using an orange photographic filter and a highspeed polaroid film with the camera set at f=5.6 and an exposuretime of 1/2 second.13. Purification of DNA from agarose gels:After electrophoresis and staining as described the gel wastransilluminated with a low energy UV lamp and the bands of the27desired size were excised using new surgical blades. Excess agarosewas trimmed and the agarose plug was transferred to pre-weightedmicrofuge tubes. The SephaglasTh BandPrep kit from Pharmacia wasemployed according to the manufacturer instructions. Briefly, foreach 250 mg of agarose plug 250 gl of gel solubilizer were added andvortexed for 1 min. The tube was heated at 67°C for 5 min. Then 5 1.11per pig of DNA of a uniform suspension of Sephaglas BP were added tothe dissolved gel slice and the tube was vortexed gently. Thesuspension was centrifuged at 14,000 rpm in a microcentrifuge andthe supernatant was removed by aspiration. The pellet was washedwith a buffer containing ethanol (8x times the volume of Sephaglasadded). After a spin at high speed for 1 min the supernatant wasremoved. This wash was repeated three times. The tube was invertedon a paper towel on the bench top and left to dry for 10 min. Theadsorbed DNA was eluted with a minimum of 10 g I of elution buffer(after vortexing gently to resuspend the pellet) and incubated for 5min at room temperature. After a high speed centrifugation thesupernatant was recovered (with care not to resuspend the glasspellet). This step was repeated once more to improve the final yield.14. Restriction enzyme digestion of plasmid DNA andamplified cDNA:To obtain a recombinant plasmid the amplified cDNA waseither restricted using the EcoR I sites engineered in the primers(for cohesive end ligation) or was left uncut (for blunt-end ligation).The phagemid plasmid Bluescript SK (-) was restricted accordingly.10 gl containing approximately 1 pig of purified cDNA were mixed28with 2 ill of 10x buffer (lx = 50 mM NaCI,100mM Tris.HCI pH 7.5, 5mM MgCl2, 100 gg/m1 BSA) and 1 gl (5 U) of EcoR I. The volume wasbrought up to 20 pi with MilliQ water and the reaction was allowedto proceed at 37°C overnight. Plasmid DNA (10 pig) was digested in50 gl of a mixture containing 5 RI of 10x buffer and 25 units ofEcoR I in MilliQ water for complementary ends or in 50 gl of amixture containing 5 IA of 10x buffer (lx = 25 mM Tris.HCI pH 7.7,10 mM MgC12, 1 mM DTI, 100 pg/m1 BSA) and 25 units of Sma I forblunt ends. In both cases the reaction was allowed to proceed at37°C overnight.15. Ligation:For complementary-ends ligation 0.5 gg of amplified cDNA(EcoR I restricted) were mixed with 1 ptg of EcoR I restrictedpBluescript in a final volume of 10 ptl containing ligase buffer (10xconcentrate: 0.5 M Tris.HCI pH 7.6, 100 mM MgCl2, 100 mM DTT, 500gg/m1 BSA Fraction V) and 400 units of T4 DNA-ligase (NEB). Thereaction mix was incubated overnight at 16 °C.For blunt ends ligation the same amount of amplified cDNA(unrestricted) was admixed with 1 gg of Sma I restrictedpBluescript in a final volume of 10 gl containing ligase buffer and400 units of T4 ligase (NEB). This reaction was incubated overnightat room temperature.Both reactions were heat inactivated by incubating the tubesat 70 °C for 15 min and briefly spun to collect condensed water. Theligation volume was brought up to 80 III with 10.10.10 buffer (10 mM2 9Tris.HCI pH 7.5, 10 mM EDTA, 10 mM NaCI). 40 gl were used fortransformation and the remaining was stored at -80°C.16. Preparing competent cells:A single colony of XL1-blue was chosen from a fresh agarplate and used to inoculate 2 ml of 2x YT medium. The cellsuspension was incubated at 37°C in a shaker for approximately 2hours (until the cells reached log phase). Then the culture wasexpanded into 200 ml of 2x YT medium and incubated for a further 2hours. After reaching confluence it was transferred into an icebucket, aliquoted in 50 ml conical Falcon tubes and centrifuged for10 min at 3,000 rpm (1500 xg) at 4 °C. The supernatant wasdiscarded and the pellets were resuspended in 20 ml of ice-cold,sterile 100 mM CaCl2. The resulting suspension was incubated on icefor 30 min. and then centrifuged as described. The supernatant waspoured off and the pellets were resuspended in 5 ml of 15% glycerolin 100 mM CaCl2. Aliquots of 200 III were transferred to pre-chilledeppendorf tubes and the tubes were quickly frozen in a dry ice-methanol bath and stored at -80°C.17. Transformation:To a 200 gl aliquot of competent cells that was quickly thawedat 37 °C and transferred into ice, 40 41 of the ligation mix (in10.10.10 buffer) were added. After mixing by gently inverting thetubes the reaction mixture was incubated on ice for 15-30 min. Thecells were then heat-shocked for 2 min at 42 °C. After this thetubes were incubated for 5 min on ice. 750 j.tl of NZY medium were30added and the bacterial suspension was incubated at 37 °C for 20min. without shaking.For blue-white selection 2x YT agar plates containingampicillin (100 gig/m1) were spread till dryness first with 40 III ofX-gal (20 mg/ml in dimethylformamide), and then with 10 lU of IPTG(1 M in dH20 ). The cells were plated at two concentrations: 200 glof transformed cells were spread on the surface of the agar and theremaining cells were spun down resuspended in 200 J.Ll of mediumand then plated as above. In both cases the plates were incubatedovernight at 37 °C in an incubator. After overnight incubation theplates were stored for at least 2 hours at 4°C to increase the bluecolor.18. Minipreps for plasmid DNA:To purify plasmid DNA for further characterization 3 ml of 2xYT medium containing 100 lig/m1 ampicillin were inoculated with asterile toothpick containing a single white colony. The cultures wereincubated for 6 hours in a shaker at 37 °C and the tubes were placedon ice at the end of this incubation. Two aliquots of 750 pi for eachculture were transferred to pre-chilled eppendorf tubes andcentrifuged at high speed for 30 seconds in a microcentrifuge. Thesupernatant was aspirated carefully and the pellets wereresuspended by up and down pipetting in 110 p1 of STET buffer (0.1 MNaCI, 10 mM Tris.HCI pH 8.0, 1 mM EDTA pH 8.0, 5% Triton X-100).The tubes were incubated for 5-10 min at room temperature.Samples were heated at 95 °C for precisely 3 min to burst open thecells, and the cell debris was collected by centrifugation at 14,00031rpm for 5 min. Pellets were pulled out with sterile toothpicks anddiscarded. Tubes were allowed to reach room temperature and 110121 of isopropanol at room temperature were added and mixed.Plasmid DNA was pelleted and the supernatant was aspirated(exercising care not to aspirate the small pellets) and the sampleswere dried under vacuum for 3 min. Plasmid DNA was resuspended in40 p.I of TE buffer (10 mM Tris.HCI pH 8.0, 1 mM EDTA pH 8.0) andcentrifugated at high speed for 10 min. Pellets were discarded andthe samples were stored frozen at - 20°C until further processing.19. Mapping of recombinant plasmids:To confirm the presence of the insert in the recombinantplasmids an aliquot of 5 p.I of plasmid DNA ( 300 ng) was incubatedin a final volume of 20 pi containing 0.5 p.1 of RNAse A (20 mg/ml), 1gl of Eco RI and 2 gl of 10x buffer (lx = 50 mM NaCl, 100 mM Tris.HCIpH 7.5, 5 mM MgC12, 100 pg/ml BSA) for 90 min at 37 °C. To analyzethe composition of the plasmids the digested samples wereelectrophoresed in 1.5% agarose as described.20. Plasmid DNA purification for sequencing insert:Recombinant cultures (as analyzed by restriction digestion andelectrophoresis) were plated on ampicillin/2x YT agar plates andincubated overnight to obtain single colonies. These single colonieswere used to start 20 ml cultures in NZY containing 100 p9/m1ampicillin (50 ml Falcon conical tubes with secured but loose capswere used for this purpose). After 16 hours incubation in the shakerat 37°C the cells were collected by centrifugation at 2,800 rpm32(1,400 xg) for 10 min. at 4°C. The supernatant was decanted and theresulting cell pellets were thoroughly resuspended in 1 ml ofGlucose solution (50 mM glucose, 25 mM Tris.HCI pH 8.0, 10 mM EDTApH 8.0) at 4°C. Then, 2 ml of a solution containing 0.2N NaOH and 1%SDS were added to each tube and the contents were mixed byswirling. After incubating for 5 min on ice, 1 ml of 3M potassiumacetate buffer, pH 5.7, was added and mixed. The precipitate wascollected by centrifugation at 3,500 rpm (2,000 xg) for 10 min (at4°C). The supernatant was saved by decanting into Falcon 2059tubes to which one volume (approximately 3.5 to 4 ml) ofphenol/chloroform were added. The two phases were thoroughlymixed by shaking and then separated by centrifugation at 3,500 rpm(2,000 xg) for 10 min at room temperature. The aqueous (upper)layer was transferred to new tubes. One volume (3.5 ml) ofisopropanol at -20°C was added and, after mixing by gentle inversionof the tubes, the plasmid DNA was precipitated at -20°C for 1 hour.The precipitate was collected by centrifugation at 3,500 rpm (2,000xg) for 10 min (at 4°C), the supernatant was poured off and thepellet let to dry (but not completely). The white, gelatinous pelletwas resuspended in 200 p.I of STE (0.1 M NaCl, 10 mM Tris.HCI pH 8.0,1 mM EDTA pH 8.0) by vortexing after which 200 m.1 more of STEwere added. On occasion the pellet did not solubilize, in this casethe samples were incubated for 5 min at 55°C. To eliminatecontaminating RNAs the solubilized samples were treated with 200jig of RNase A at 37°C for 20 min to 1 hour. The sample wasextracted with 1 volume of phenol/chloroform. The aqueous phasewas transferred to clean tubes and further purified by desalting on33Sephacryl S300 spin columns (3 ml of packed beads equilibratedagainst TAE buffer). The excluded volume was spun off bycentrifuging the columns for 1 min at 1100 rpm (250 xg), thesamples were then applied and the DNA recovered by centrifugationfor 2 min at 1100 rpm (250 xg). The salt content was increased byadding 40 pi of 3 M potassium acetate, pH 5.7, and the DNA wasprecipitated with 2.5 volumes of 100% ethanol (at -20°C) for 1 hourat -20°C. Precipitated material was recovered by high speedcentrifugation at 4°C in a microcentrifuge. The supernatant wasaspirated, the plasmid DNA was dried under vacuum in a dessicatorfor 10 min., and then resuspended in 50 pi of TE buffer. The purityand concentration were estimated from electrophoresis andethidium bromide staining.21. Sequencing reactions:The chain termination method involves the synthesis ofDNA by DNA polymerase. It occurs only at the site where a primeroligonucleotide is annealed to the template. The synthesis proceedsuntil a nucleotide analog, that does not allows further elongation ofthe DNA strand, is incorporated (Sanger et al.,1977). These analogsare the 2',3'-dideoxynucleoside 5'-triphosphates (ddNIPs) that lackthe 3'-OH group necessary for the synthetic reaction to continue.When mixtures of dNTPs with one of the ddNTPs are employedelongation will be terminated in a fraction of the DNA strands ateach site where the ddNTP could be incorporated. Hence, fourseparate reactions each with one of the four ddNTPs will render34the complete sequence information. Pyrophosphatase was used inconjunction with Sequenase®Version 2.0 (USB). The former is usedto avoid the slow, sequence dependent reversal of the DNApolymerase reaction (Tabor and Richardson, 1990). The latter ischaracterized by high processivity, lack of 3' to 5' exonucleaseactivity and the efficient use of the nucleotide analogs.The reaction is carried out in two steps. First, the primer isextended in the presence of limiting concentrations of dNTPs andradioactive labeled dATP. The labeled chains synthesized in this stephave a random length distribution. Then, the concentration of thedNTPs is increased and one of the four ddNTPs is added. This is theactual chain-termination step. Finally the reactions are halted byadding EDTA and formamide and heating up to 85°C for two minutes.The samples are immediately applied to the gel and electrophoresedat 55°C and 35 W (constant power). The gel is dried as described andthe sequence pattern is revealed by autoradiography. The use of thelow energy [cc-35S]-dATP (instead of 32P) increases the resolution atthis step. Double stranded templates work well provided that theplasmid DNA is free of RNA and salts. As described above, thealkaline lysis method, if combined with a RNase treatment stepfollowed by desalting on a molecular sieve (e.g. with spin columns),gives templates of enough purity for sequencing. The templates arethen alkali denatured , and after neutralization and ethanolprecipitation, the annealing to the primers can be performed as itwould with single stranded DNA.3521.1. Alkaline denaturation of double stranded DNA:To 8 pi of DNA containing 1-3 jig of plasmid 2 pi of 2 M NaOHwere added (when a smaller volume of DNA was used the volume wascompleted up to 10 p.I with MilliQ water). After mixing by vortexingand a short spin to collect the solution from the tube walls thesamples were incubated for 10 min at room temperature. Themixture was neutralized by adding 3 ill of 3 M sodium acetate pH 4.8and the balance up to 20 ill was made up with MilliQ water. The DNAwas precipitated with 3 volumes of 100% ethanol (at -20°C) byincubation at -20°C for 1 hour, and recovered by high speedcentrifugation in a microcentrifuge. The supernatant was discardedand the pellet was washed with ice-cold 70% ethanol. Aftercentrifuging and discarding the washing solution, the final pelletwas dried briefly and resuspended in 10 III of MilliQ water.21.2. Annealing reaction:For every four sequencing lanes a single annealing and labelingreaction was performed. To 10 RI of template DNA 2 ptl of annealingbuffer (5x concentrate: 200 mM Tris.HCI pH 7.5 containing 100 mMMgC12 and 250 mM NaCI) and 2 pi (0.5 pmole411) of either T7 or T3primer were added before mixing and incubating at 37°C for 20 min.This gives a primer:template molar stoichiometry close to 1:1. Theannealing reaction was allowed to proceed for 10 min at roomtemperature. After a short spin the samples were either stored at-20°C or used immediately for the labeling reaction.3621.3. Labeling reaction:For each template set the following was added: 3 RI of labelingmix diluted 1/3 in water (5x concentrate containing 7.5 [tM dGTP,7.5 1.1M dCTP and 7.5 gM d'TTP), 0.5 p.I of 0.1 M DTT and 0.5 gl ofpyrophosphatase (USB). The DNA-polymerase (Sequenase version 2.0,USB) was diluted 1/8 in 10 mM Tris.HCI, pH 7.5, containing 5 mM DTTand 0.5 mg/ml BSA. Per template, 0.5 pi ( 5 IX') of 35S-dATP wereadded immediately followed by 2 gl of diluted enzyme. Tubes werequickly vortexed and •spinned and incubated for 10 min at roomtemperature.21.4. Termination reactions:The composition of the termination mixes was as follows: 8011,M each of dGTP, dATP, dCTP, dTTP and 8 p.M of the correspondingddNTP and 50 mM NaCl. 2.5 p.1 of the appropriate termination mixwere placed in the wells of microtitration plates (the plates werewarmed at 37°C just prior to initiate the reaction). When thelabeling incubation was complete, 4.5 III of labeled template (pertermination reaction) were mixed by up and down pipetting resultingin four sets (G,A,T, and C) of reactions per template. The incubationswere continued for 5 min at 37°C. At the end of this incubation 4 glof stop solution (98% formamide, 20 mM EDTA pH 8.0, 0.05%Bromophenol Blue and 0.05% Xylene Cyanol FF) per reaction wereadded. Just before loading the samples in the gel they were heated at85°C. If the samples were stored at -20°C the final heating stepwas repeated before the electrophoresis.3721.5. Casting of polyacrylamide gels for sequencing:The acrylamide stock solution was prepared by dissolving100 g of ultrapure acrylamide (ION) and 5 g of N,N'bismethyleneacrylamide in 150 ml of MilliQ water. Once dissolved the volume wasbrought up to 250 ml and the solution was deionized by stirring with5 g of mixed-bed resin (Dowex XG8) for 2 hours in the cold room .The resin was filtered out through Whatman #2 and the resultingsolution was stored wrapped in tin foil at 4°C .The LKB system (2010 Macrophor - Electrophoresis Unit) wasused for sequencing. The thermostatic plate was siliconized bythoroughly wiping 6 ml of Repel-silane (LKB) until dryness twotimes. The front glass plate was siliconized with 5 ml of a solutioncontaining 20 of Bind-silane (LKB A-174), 5 ml of 95% ethanoland 2.5 ml of 10% acetic acid. The solution was wiped on the surfaceuntil dryness followed by 2 quick rinses with 95% ethanol. When theplates were ready, a polymerizing solution was prepared by mixing 7ml of acrylamide stock solution with 20 ml of MilliQ water, 5 ml of10x TBE (45 mM Tris.borate pH 8.0, 1 mM EDTA H 8.0) and 21 g ofurea. After stirring to dissolve the urea the volume was brought upto 50 ml with water and the solution was filtered through a 0.45 pmfilter. To initiate the polymerization 400 pi of 10% ammoniumpersulfate freshly made, and 40 j.tl of TEMED, were added. Aftermixing thoroughly, the gel (0.2 mm thickness) was poured accordingto the instructions for this particular apparatus.3821.6. Denaturing gel electrophoresis:Before applying the samples the gel was pre-run for 30 min to1 hour at 55 °C and 45 W (constant power). 3 1.1,1 for eachtermination reaction were loaded (after heat denaturing them for 2min. at ?.. 85°C; see above). The electrophoresis was allowed toproceed using the dyes as references. After the run was completedthe gel was soaked for two periods of 15 min with 1 liter each of10% acetic acid (in a shaker). The gel was dried with a hair drier andexposed with a fast film (Kodak XAR-5) overnight at roomtemperature.22. Large scale preparation of plasmid DNA:This protocol was performed at 4°C or on ice unless otherwiseindicated. A fresh overnight culture of transformed bacteria in 2aliquots of 500 ml NZY containing 100 lig ampicillin per ml wasprepared. Cells were pelleted by centrifuging for 10 min at 5,000rpm in a Sorvall GS3 rotor. The supernatant was discarded and thewalls were dried with a Kimwipe. The pellets were vortexed andflicked till they formed a thick slurry. To each pellet 7 ml of coldglucose solution (50 mM glucose, 25 mM Tris.HCI pH 8.0, 10 mM EDTApH 8.0) were added and mixed thoroughly. Each slurry was divided intwo pre-chilled 50 ml conical tubes and 14 ml of a 0.2N Na0H/1%SDS solution were added to lyse the cells. After mixing by inversion,tubes were incubated for 10 min on ice (with occasional gentlyshaking). 7 ml of potassium acetate, pH 5.7, were added and the mixwas transferred to pre-chilled 50 ml Sorvall tubes and spun for 10min at 10,000 rpm in a SS34 Sorvall rotor. The supernatants were39then transferred to pre-chilled 50 ml conical tubes and extractedwith an equal volume of phenol/chloroform. The phases wereseparated by centrifugation in a benchtop centrifuge for 10 min at3,000 rpm (at room temperature). The top layer was carefullyremoved and transferred to new tubes and the nucleic acids wereprecipitated with 1 volume of isopropanol (at -20°C) for 1 hour at-20°C. After centrifuging for 15 min at 10,000 rpm in the SS34rotor the supernatant was discarded and the pellets were dried for10 min in a dessicator. They were resuspended and pooled in 800 glSTE buffer, and treated with 200 gg of RNAse A for 1 hour at 37°C.The reaction mix was extracted with 1 volume of phenol/chloroformand the top aqueous layer was precipitated with 2.5 volumes of100% ethanol (at -20°C) for 1 hour at -20°C. The DNA was pelletedby spinning at 14,000 rpm for 20 min in a microcentrifuge, thesupernatant was removed and the pellet was dried in a dessicatorfor 10 min. The DNA was resuspended in 500 gl of TE buffer andeither stored at -20°C or processed immediately.23. Purification of insert cDNA from recombinant plasmids:Plasmid DNA obtained through the large prep procedure wasdigested with EcoR I and purified by preparative agaroseelectrophoresis as described above and stored in TE buffer at aconcentration of 100 ng/gl.24. Screening of the cDNA library:A premade human fetal spleen cDNA library was purchasedfrom Stratagene (Vector Uni-ZAPThXR). Upon arrival the library was40thawed, diluted 1/4 in SM buffer (5.8 g/I NaCI, 2 g/I MgSO4, 50 m1/ITris.HCI, pH 7.5, 5 m1/I 2% (w/v) gelatin) and 40 pi of chloroform/mlwere added (250 !al aliquots were stored at -80°C until needed).24.1. Preparation of host cells:An isolated single colony of XL1-Blue cells was used toinoculate 50 ml of NZY medium containing 500 RI of 20% maltoseand 500 pi of 1 M MgSO4. After 16 hours in the shaker at 37°C thecells were collected by centrifugation at 2,000 rpm (750 xg) for 10min. The supernatant was discarded and the resulting pellet wasresuspended as a slurry in 15 ml of 10 mM MgSO4. This suspensionwas stored at 4°C and made fresh every week.24.2. Titration procedure:An initial 1/1,000 dilution of the library was prepared in SMbuffer. Different aliquots of this dilution were used to inoculate 500gl of host cells. Two extra dilutions (1/10,000 and 1/100,000) werealso tested. After a brief incubation of 20 min at 37°C in the shakerto initiate the infection cycle, the cells were mixed with 6.5-8 mlof melted top agar at 45°C and poured on top of pre-warmed 2x YTagar plates. The plates were maintained for 10 minutes at roomtemperature and then incubated overnight at 37°C. The plaques fromthe best dilution were counted and the titer expressed as number ofplaque-forming units (pfu) per ml.4124.3. Screening procedure:A dilution corresponding either to 5,000 pfu/150 mm platewas employed for overnight or 10,000 pfu/150 mm plate for 6-8hours incubation. After the incubation period was completed theplates were refrigerated for 2 hours at 4°C to prevent the top agarfrom sticking to the nylon filters (Hybond-N). The filters were seton top of the plates for transfer during 2 min, and marked fororientation purposes. Filters were denatured after lifting by settingthem on a Whatman 3 MM soaked in 1.5M NaCl, 0.5M NaOH for 2 min.Excess agar was removed at this stage. Then, the filters wereneutralized by incubating on a Whatman 3 MM soaked in 1.5M NaCI,0.5M Tris.HCI pH 8.0 for 5 min. After that, they were rinsed for 30sec. in 0.2M Tris.HCL pH 7.5 prepared in 2x SSC (20x concentrate:175.3 g/I NaCl, 88.2 g/I sodium citrate pH 8.0, adjusted with 10NNaOH) and blotted on Whatman 3 MM to absorb excess humidity butwithout let them dry completely. To cross-link the DNA to thefilters the Stratalinker-1'm UV crosslinker with a setting of 1,200 Win 60 sec. was employed. A duplicate filter was obtained in whichthe transfer time was extended to 7 min., the remaining steps werekept the same.24.4. DNA labeling reaction:To radioactive label the fragment the method of Feinberg andVogelstein (1983; 1984) was used. It is based on the hybridizationof a mixture of all possible hexanucleotides to the DNA to be labeled.The synthesis of the second strand proceeds from the 3' end of therandom primer. The large fragment of DNA polymerase (Klenow42enzyme) was used for this step. Analogs of the dNTPs present in themixture are going to be incorporated. Thus radioisotopically orotherwise labeled dNTPs form part or the replicated strands.After purification as described, insert cDNA (388 bp) wasradioisotopically labeled with 32P. Briefly, 100 ng cDNA in a final10 pi (in water) were heat denatured by boiling for 5 min andincubated on ice for 3 min after a short spin. To this were added inthe following sequence: 2 p.I of 10x Klenow buffer (400 mMTris.HCI pH 7.5, 66 mM MgC12 and 10 mM 2-mercaptoethanol), 2 p.Iof ATG mix (containing 25 mM each of dATP, dTTP, dGTP), 1 p.I ofrandom hexamers (equivalent to 100 pmole), 1 p,1 of the Klenowfragment of DNA polymerase and quickly afterwards 5 p.I of 32P -dCTP. The mixture was incubated for 60 min. at 37°C. The reactionwas halted by adding 60 pi of 10 mM Tris.HCI pH 7.5 containing 10mM EDTA and 0.5% SDS, mixing and adding 80 p.I of 5 M ammoniumacetate followed by 10 p.I of a solution containing 10 pg/g1 tRNA(as a carrier). The resulting radiolabeled probe was precipitatedwith 400 RI of 100% ethanol and collected by centrifugation for 15min at 14,000 rpm in a microcentrifuge. The supernatant wasdiscarded and the pellet dissolved in 100 pi of dH20. The solutionwas boiled for 5 min and after a short spin transferred onto ice. Therandom-primed labeled cDNA was prepared always shortly beforeuse.24.5. Hybridization:The excess of binding sites on the filters were quenched byincubating them in 20 ml of 0.33M phosphate buffer, pH 7.5,43containing 1% (w/v) BSA, 30% (v/v) deionized formamide and 7% (w/v)SDS. The filters were incubated in this solution from a minimum of1 hour at 55°C to overnight at room temperature. After this thehybridization bag was opened and the contents replaced with 10 mlof fresh hybridization solution. To this, 100 ill of 32P-labeled probewere added (see above), and the incubation was allowed to proceedfor 12-16 hours at 55°C in a water bath with agitation. Afterdiscarding the hybridization solution the filters were taken out ofthe bag, and transferred into 500 ml of washing solution (150 mMphosphate buffer, pH 7.5, containing 0.1% SDS) at room temperature.After a short wash this solution was discarded and replaced with500 ml of fresh buffer warmed up to 60°C and incubated in theshaker for 10 min. Filters were blotted dry on Whatman 3 MM paperand the radioactivity was measured with a Geiger-Mueller tube. Thiswas repeated every 10 min until the measured back ground was low.Then the wash was interrupted, the excess of humidity was blottedon Whatman 3 MM paper and the filters transferred, one at a time,onto Saran wrap, wrapped, positioned onto used film and securedwith tape. For orientation purposes the film was marked with 35S -ink. Autoradiographic replicas were obtained by overnight exposureto Kodak fast film (XAR-5) at -80°C. Usually 20 filter at a timewere processed using this protocol. All manipulations wereperformed with gloved hands and forceps.24.6. Rescue protocol:The positive plaques were cored from the master agar plateand transferred into a sterile eppendorf tube containing 500 RI of44SM buffer and 20 !al of chloroform. The tube was vortexed to releasethe phage particles and incubated overnight at 4°C. Assuming a phageconcentration of 0.5 to 1 x 106 phage particles per core, threedifferent dilutions were tested around a desired titer of 5 x 102plaques per plate. Incubation, lifting and hybridization wereperformed as described. Positive plaques in this secondary screeningthat were well isolated from neighboring plaques were cored and theresulting phage solution was used in the subsequent rescueprocedure.An aliquot of 200 ill of competent XL1-Blue were infectedwith an aliquot of 1 x 105 phage particles (from the stock obtainedafter secondary screening of the plaques of interest) and 1 pi ofR408 helper phage (1 x 1011 pfu/ml). Negative controls wereperformed with helper phage alone. This mixture was incubated at37°C for 15 min after which 5 ml of 2x YT medium were added andthe incubation was allowed to proceed for 3 hours with shaking.Then, the cultures were heated at 70°C for 20 min.; unlysed cells andcell debris were removed by centrifugation for 5 min at 1,200 rpm(4,000 xg). The supernatant containing the phagemid particles wasdecanted on a sterile tube and stored at 4°C (for up to one month)after use. Different aliquots of this stock were used to infect 200III aliquots of competent XL1-Blue. These cultures were incubated at37°C for 15 min.; 10 gl were then plated on LB/ampicillin plates andincubated overnight at 42°C. Colonies that grew under ampicillinselection were streaked on new plates and/or expanded for furtheranalysis.4524.7. Mapping of phagemids:The rescued pBluescript phagemids containing the hybridizingcDNA insert were expanded in 20 ml cultures and plasmid DNA wasisolated by the alkaline-lysis method as described above. To confirmthe rescue of the recombinant phagemid, an excision of the clonedinsert was performed with a combination of Xho I/ Not LTo map restriction sites in the insert different enzymes wereemployed to digest the recombinant plasmid (see results).25. Northern blot analysis of tissue and cell RNAs:A hybridization membrane containing total RNA from variousRhesus monkey tissues was purchased from BIOS. Cell lines and micetissues RNA were prepared as described (see Methods 8 and 9) andprocessed according to the following protocol.The gel was prepared by first melting the agarose in DEPCtreated MilliQ water. To 1 volume of this solution (when reachedapproximately 60°C) 1/10 volume of 10x MOPS (200 mM MOPS pH 7.0containing 50 mM Na0AC, 10 mM EDTA and 0.1% DEPC; autoclaved)and 1/10 volume of 37% formaldehyde were added. The componentswere quickly mixed and the gel was poured while this solution wasstill hot (in a fume hood). In the meanwhile, 20 lag of RNA wereethanol precipitated. Briefly, to 1 volume of RNA sample 1/10volume of 3 M Na0Ac, pH 5.0, (DEPC treated) and 2.5 volumes of100% ethanol (at -20°C) were added and these mixtures wereincubated for ?_ 20 min. at -20°C. The RNA precipitate was collectedby centrifugation at 14,000 rpm for 15 min. in a microcentrifuge.The excess of ethanol was aspirated and the pellet was dried in a46dessicator for 5 min. The RNA was redissolved in 25 IA of DEPCwater to which an equal volume of loading buffer (1,000 ill ofdeionized formamide plus 250 ill of 10x MOPS and 250 ill of 37%formaldehyde) was added. The RNA was denatured by heating thesample at 70°C in a water bath for 10 min. Ideally the gel should beallowed to polymerize for 1 hour. The buffer chambers in theelectrophoresis system were filled with running buffer (MOPS buffercontaining 6% formaldehyde) but without flooding the gel. To thesamples, 10 111 of formaldehyde loading buffer were added, mixed,spinned for a few seconds, and loaded onto the gel. Samples wereapplied in duplicates to make a replica for staining. The gel was runat a constant voltage of 5 V/cm for 3 hours or until the BromophenolBlue had migrated halfway down. The section to be transferred wascut and rinsed several times with dH20 to wash out theformaldehyde. The gel was incubated in 0.5 I of 10x SSC in a rotatingplatform for 45 min. Transfer to Hybond-N membranes was doneovernight in 20x SSC using a stack of paper over the membrane topromote the movement of buffer plus solutes upward, and throughthe gel and the filter. The duplicate gel was stained with ethidiumbromide. Prehybridizing, hybridizing and washing of the filters wasdone as described for library screening.26. Chromosomal Localization:These experiments were performed in the laboratory of Dr.Alessandra M.V. Duncan, Department of Pathology, Queens University,Kingston, Ontario K7L 2V7. The recombinant phagemid containingthe insert DNA (388 bp) was labeled to a specific activity of 7.8 x47107 cpm/mg of DNA with [3Fl]dTTP and [31-1]dATP using a multiprimeDNA labeling kit (Amersham). in situ hybridization to BrdU-synchronized peripheral blood lymphocytes was performed accordingto Jirik et al. (1992). After washing and dehydration, the slides werecoated with a Kodak NTB/2 emulsion, exposed, and developed. Thestaining of chromosomes was performed by a modified fluorescence,0.25% Wright's stain procedure (Lin et al., 1985).48IX. RESULTS;1. Experimental strategy:The experimental design employed to isolate cDNAs encodingchemotactic factor receptors was based on the fact that theseproteins are likely to belong to a subclass of the 7 TMS superfamily,thereby transducing intracellular signals by coupling to G-proteins.Given the variety of ligands that act as chemotactic and activatingfactors, this subclass should exhibit significant diversity. On theother hand, because of the overlap in the activities of the factors,the structural relatedness for some of them, and the cross-competition for binding to the receptors observed in others, thereceptors should also exhibit more or less extensive areas ofidentity.To identify, in peripheral-blood leukocytes, proteins 	 thatbelong to the family of receptors for chemotactic factors, primersdesigned either on conserved areas of these molecules or primerspatterned in specific sequences were used in the amplification ofwhite blood cell cDNA by PCR. To ascertain whether the PCRproducts consisted of a mixture or a single species the isolatedfragments were cloned and sequenced. All the clones analyzed werethe amplification of the IL-8 R (high affinity). This was so either inthe rabbit or in the human. Neutrophils are the predominant whitecells in unfractionated blood and the sets of primers tested werepreferentially amplifying the IL-8 R mRNA. The possibility that theprimers would solely target the IL-8 R message could not bediscarded at this stage.49RNA from highly purified cell fractions was then used as thestarting material. When monocyte RNA was tested and the PCRproducts were sequenced it was found that the IL-8 R and thematerial amplified from monocytes presented only partial homology,likely being closely related but divergent 7 TMS receptors. To clarifywhether this protein was a novel chemotactic receptor, a cross-hybridizing, full-length cDNA was isolated from a spleen library,subcloned and sequenced. Alignments from this sequence with knownsequences deposited in data banks established that it is a humanhomologue of the putative bovine neuropeptide Y receptor (Y3subtype; bovnyr). It also presents a high degree of homology with thehuman IL-8 R (low affinity).2. Primer oligonucleotides:When this work was initiated only one of the chemotacticfactor receptors had been isolated: the rabbit interleukin-8 receptor.This protein had some homology with two of the tachykinins (thesubstance P and substance K) receptors. Only a few long clustersappeared conserved between these proteins with the interveningsequences presenting higher variability (see figure 5 ). Theseclusters were also partially conserved in other 7 TMS proteins, theless divergent were the proteins the more extended the sequencehomology around the cluster area. They were targeted foroligonucleotide primers design because of the possibility that thesame conserved pattern could be present in other members of thechemotactic factor family of receptors. Given the high homology ofthe intercrines themselves this could be somewhat mirrored in the50structure of the receptors (this assumption proved to be correctwhen more of these sequences became available). According to thepredicted topology the chosen fragment encompasses the secondcytoplasmic loop, transmembrane domains IV, V and part of the VI,and the second extracytoplasmic loop. The forward primer is in anarea of the molecule that is thought critical for interaction with G-proteins and therefore tends to be conserved. The reverse primer isin one of the transmembrane domains around a conserved Cys. Inboth cases the selection was biased toward those clusters whichresidues could indicate important structural determinants for theprotein.MEV-NVWNMTDLWTWFEDEFANATGMPPVEKDYS PC LVVTQTLNKYVVVVI YALVF L LS LLGNS LVMLVI 69 IL-8 RMDN-VLPVDS D LS PN I STNT SEPNQFVQPAWQ IV- - LWAAAYTVIVVTSVVGNVVVMW I I LAHKRMRTVT 67 Substance P RMGTRA I VS DAN I L SG LE SNATGVTAF SMPGWQLA - -  LWATAYLALVLVAVTGNATV IWI I  LAHERMRTVT 68 Substance R RTMS ILY SRSNRSVTDVY LLNLAMADLLFALTMP IWAVSKEKGW IFGTPLCKVVS LVKEVNFY SG I LLLAC I SVD 139 3NYFLVNLAFAE- - -ASMAAFN	 TVVNFTYAVHNEWYYGLF YCKFHNFF P IAAVFAS I Y SMTAVAFD 129 1NYF I IN 	 4 	 - 	 4 4 	 	  *NIWYFGRAFCYFONLF PITAMFVS I YSMTAIAAD 130 2* 	 * 	 * 	 *TMS II 	 TMS III RYLAIVHAT RTLTQKRHLVKFICLGIWALSLILSLPFFLFRQVFSPNNSSPVCYEDLGHNTAKWRMVLRI 209 3RYMAI I HPLQPRLSATATKVVI C -VIWVLALLLAFPQGYYSTTETMPSRVVCM I EWPEH PNK I YEKVYH I 198 1RYNAIVHPFQPRLSAPSTKA T TA -G I WLVA LA LAS POCF Y ST I TVDEGATKCVVAWPNDNGGKMLLLY H T., 199 2** 	 ** 	 * 	 * 	 * 	 * 	 * 	 *TMS IVL PHTFGF I LP LLVMLFCYGFTLRTL 	 FQAHMGQKHRAMRVI FAVVL I F LLCWLPYNLV 266 3CVTVL I YF LP L LVI GYA YTVVG I T LWASEI PG-DS SDRYHEQVSAKRICVVKMM IVVVCTFA I CWLPPIII F 267 1YSVIGLTLWKRAVPRHQAHGANLRHLQAKKKFV • g 	 C 	 Y 269 2** * ****TMS V	TMS VILLADTLMRTHVIQETCQRRNDIDRALDATEI LGFLHSCLNPI IYAF I GQNF RNGF	321 3FL LPY 	 INPDLYLKKFIQQVYLAIMWLAMSSTMYNPI IYCCLNDRFRLGFKHAFRCCPF I SAGDY 331 1EILLGT 	 FQEDI YYHKF I QQVY LALFWLAMS S TMYNPI YCCLNHRFRSGFRLAPRCC PWVTPTEE 333 2* 	 * 	 *****	 * 	 *TMS VII51- - LKMLAARGL I SKEF - - - LTRHRVTSYTSSSTNVP	  352 3EGLEMKSTRYLQTQGSVYKVSRLETTISTVVGAHEEEPEDGPKATPSSLDLTSNCSSRSDSKTMTESFSF 401 1DRLELTHTPSLSRRVN- - -RCHTKETLFMTGDMTHSEATNGQVGSPQD	GEPAGP 384 2- - -SNL 355 3SSNVLS 407 1ICKAQA 390 2Figure 5: Alignment of the amino acids sequences from thehuman substance P receptor, rabbit IL-8 receptor and themouse substance K receptor. The underlined sequences are thetransmembrane domains as defined by hydrophobicity plots. Thealignment was produced by the HEIN program (Hein, 1990). Thebold residues indicate the position of the primers used inthe cloning of the human neuropeptide Y receptor.A survey of 12 proteins belonging to the 7TMSsuperfamily demonstrate that the combination for the upstreamprimer is present only in the 131-adrenergic receptor and thedownstream primer is absent in 7 of them (see appendix I). Theproteins involved were some of the receptors for the classicalneurotransmitters in all their different subtypes: dopamine,acetylcholine (muscarinic), a- and 0-adrenergic.To facilitate subcloning procedures an EcoR I site was createdupstream in each of the primers. A (G+C) content 50% was reachedby manipulating the codons when necessary but taking into accountthe codon usage. The complete sequence for the forward primer was5'-CGGAATTCGACCGCTACCIGGCCATT-3', which translate as Asp-Arg-Tyr-Leu-Ala-lle (DRYLAI), and for the antisense primer 5'-ACGAATTCGTTGTAGGGCAGCCAGCA-3', which translate as Cys-Trp-5 2Leu-Pro-Tyr-Asn (CWLPYN). Both contain an extra complement of 8nucleotides to accommodate the restriction site.The average yield for the production and purification of the26-mers was 35 gmole/batch.3. The Polymerase Chain Reaction:The amplified products of the PCR reaction were analyzed byagarose gel electrophoresis. A single band was seen with thedescribed primers combination and it was within the expected sizerange (figure 6). The sequencing of the monocytes cDNA, and of thefragments obtained by PCR of either human or rabbit leukocytes,proved that the primers were targeting legitimate 7TMS sequences.A1 234  5678 1 2 3 4 5 6 7 8Figure 6: PCR amplification of rabbit and human cDNA: 2%agarose gel electrophoresis; (A) lanes 1,4,5, and 8: 1 kb DNAladder (1 gg/lane), lanes 2 and 3: human white blood cellsRNA to cDNA (0.4 gg/lane), lanes 6 and 7: rabbit white bloodcells RNA to cDNA (0.4 gg/lane). (B) The same gel as in (A)after excising the bands for cloning.53Leu-Pro-Tyr-Asn (CWLPYN). Both contain an extra complement of 8nucleotides to accommodate the restriction site.The average yield for the production and purification of the26-mers was 35 gmole/batch.3. The Polymerase Chain Reaction:The amplified products of the PCR reaction were analyzed byagarose gel electrophoresis. A single band was seen with thedescribed primers combination and it was within the expected sizerange (figure 6). The sequencing of the monocytes cDNA, and of thefragments obtained by PCR of either human or rabbit leukocytes,proved that the primers were targeting legitimate 7TMS sequences.A-.41 2 3 4 5 6 7 8	 1 2 3 4 5 6 7 8Figure 6: PCR amplification of rabbit and human cDNA: 2%agarose gel electrophoresis; (A) lanes 1,4,5, and 8: 1 kb DNAladder (1 gg/lane), lanes 2 and 3: human white blood cellsRNA to cDNA (0.4 gg/lane), lanes 6 and 7: rabbit white bloodcells RNA to cDNA (0.4 gg/lane). (B) The same gel as in (A)after excising the bands for cloning.53The possibility of using the thermostable enzyme Taq DNA-polymerase over the Klenow fragment of E.coli has improved thespecificity and the yield , on top of simplifying the whole PCR cycle.The fact that high temperatures can be used has a direct bearing onthe elimination of non-specific amplification. Furthermore, long andshort PCR products can be amplified from genomic DNA due to thedisruption of secondary structure obtained at high temperature.Taq DNA-polymerase does not have proof reading capabilities(no 3' to 5' exonuclease activity) which has a bearing on themisincorporation rate. The appropriate PCR conditions, such as theright concentration of dNTPs and Mg2+, high annealing temperaturesand short extension times have brought the misincorporation ratebelow than 10-5 per cycle (Eckert and Kunkel, 1990; Gelfand andWhite, 1990). For library screening it is the whole of the amplifiedproducts that counts and rare misincorporation go unnoticed. Theamplified 388 bp of cDNA used as the probe for library screeningcorresponded unequivocally to the sequence between bases 486 and858 for the full length sequence (see figure 10). In vitrorecombination or template switching is a problem that only occurswith genomic amplifications and therefore being an unlikely eventhere.The strategy of modeling the primers on areas of highhomology to search for cDNAs encoding related but novel proteinshas its rationale in the highly conserved structural and functionalfeatures among the subfamilies in this particular group. There issome intrinsic flexibility inherent to the system : note that theantisense primer matches only five codons from the original six5 4intended but obviously it did not hinder the annealing of this 26-merto only 15 bases in the template. The destabilizing effect that non-annealed termini have was probably compensated by the high (G+C)content. The 3'-end of the primer was the properly annealed side andthereby synthesis could progress unhindered.4. Purification of DNA from agarose gels:The SephaglasTm BandPrep kit consistently gives a high yield ofpurified DNA. It is based on the specific adsorption of DNA to glassmatrices in the presence of sodium iodide that also functions as gelsolubilizer. The matrix is then washed from contaminants withbuffered ethanol that promotes the binding of DNA to the glasspowder. The DNA is eluted in low ionic strength buffer and furtherprocessed (see figure 7).1 2 3 4 5 234Figure 7:2% agarose gel electrophoresis. (A) lane 1: 1 kb DNAladder (1Rg). Lanes 2 and 3: Sephaglas purified cDNA, 80ng/lane. Lanes 4 and 5: EcoR I digested cDNA, Sephaglasspurified, 80 ng/lane. (B) Lane 1: 1 kb DNA ladder (1 Rg).Lane 2: Supercoiled pBluescript (80 ng). Lane 3: pBluescriptlinearized with Sma I (80 ng). Lane 4: pBluescript linearizedwith EcoR I (80 ng).555. Insertion of cDNA into plasmid Bluescript:To create the sequencing and cloning vector two strategieswere attempted: cohesive ends ligation and blunt ends ligation.Digestion of pBluescript with EcoR 1 produces a linear duplex withshort single stranded tails (this site occurs only once and solely inthe multicloning site). The same result is obtained when digestingthe cDNA with this enzyme because of the EcoR 1 sites engineeredupstream of the primers used in this amplification (and providedthat it lacks internal EcoR 1 sites). Because of the possibility that asmall fragment of the cDNA could be actually digested without beingdetected by size fractionation on agarose gels, blunt ends ligationwas also performed using Sma 1 to digest pBluescript. This enzymecreates blunt termini that can associate with the undigested cDNA.The ligation reaction was performed in both cases using T4 DNA-ligase and for blunt ends was carried out at 25 °C overnight. This isa compromise between the rate of enzymatic action and stability ofassociation of the termini. To favor the creation of recombinants thereaction was performed at high cDNA concentration therebyincreasing the probability of recombinants to form, andsimultaneously decreasing the chances of recircularization for theplasmid DNA. The T4-DNA ligase does catalyze blunt end ligation.After ligation the mixtures were used to transform XL-1 Bluecells. The selection was done with ampicillin, the chromogenicsubstrate X-gal and IPTG. When recombination took place there isinsertional inactivation of the 1 a cZ a -peptide anda-complementation does not occur. Therefore the colonies of cellsbearing recombinants appear white. These colonies were expanded in5 61 3 5 79 11 13 15 17 19liquid cultures containing amp and processed to isolate recombinantplasmid DNA. An aliquot of these preparations was digested withEcoR I to verify the insertion (see figure 8).2 4 6 8 10 12 14 16 18ar 41.111104%Figure 8: 2% agarose gel electrophoresis. Lanes 1, 10 and 19:1 kb DNA ladder (lgg) Lanes 2 to 9: recombinant plasmidDNA/EcoR I digested, obtained from cohesive ends ligation(240 ng). Lane 11 to 18: recombinant plasmid DNA/EcoR Idigested, obtained by blunt ends ligation (240 ng). Lanes 9and 18 are originated in rabbit cDNA. The remainingrecombinants contain a human insert.576. Results from total white blood cell cDNA:6.1. Primers specificity:Total cell RNA was prepared from rabbit and human whiteblood cells and reverse transcribed using the antisenseoligonucleotide to prime the reaction. The cDNA so obtained was PCRamplified after adding the sense primer (Methods, 10 and 11). Afragment of the expected size was amplified by the polymerase.To screen the clones for putative members of the chemotacticfactor receptor group plasmid DNA was isolated from several clonesand used as a template to sequence the insert. In all the casesanalyzed the sequence corresponded to the human IL-8 R (highaffinity) (figure 9).t3se7per 	 VHATRTLTQKRHLVKFVCLGCWGLSMNLSLPFFL 	t3be3per 	 VHATRTLTQKRHLVKFVCLGCWGLSMNLSLPFF 	t7se3per_p 	 VHATRTLTQKRHLVKFVCLGCWGLSMNLSLPFFLFRQAYHPt3se2per 	 VHATRTLTQKRHKVKFVCLGCWGLSMNLSLPFF 	t3se2lr 	 AYHPhumilSra 	 FYSGILLLACISVDRYLAIVHATRTLTQKRHLVKFVCLGCWGLSMNLSLPFFLFRQAYHPt3se7pert3be3pert7se3per_p 	 NNSSPVCYEVLGNDTAKWRM 	t3se2pert3se2lr 	 NNSSPVCYEVLGNDTAKWRMVLRILPHTFGFIVPLFVMLFCYGFTLRTLFKAH 	humil8ra 	 NNSSPVCYEVLGNDTAKWRMVLRILPHTFGFIVPLFVMLFCYGFTLRTLFKAHMGQKHRAFigure 9: Alignment of some of the plasmid sequences obtainedfrom human white blood cells. Humil8ra is the humaninterleukin-8 receptor (high affinity), the remainingsequences are some of the PCR-amplified cDNA clones.587. Results from Monocytes cDNA:7.1 Amplification of cDNA and genomic DNA:Genomic DNA was also used as template in PCR. The resultingamplifications were clean and appear specific (see figure 10). Theband size is identical to that obtained by amplification of cDNA. Theexplanation for this could be that the whole fragment is located in asingle exon. It is more likely that the gene encoding for this proteinis intronless. The genes of the a2- and p2-adrenergic receptors(Kobilka et al., 1987a and b respectively), as well as each of themuscarinic cholinergic receptors (Bonner et al., 1987) do notpossess introns, be it in the coding or in the 3'-untranslated region.Some of them do but in the 5'-unstranslated region. The more likelyexplanation for this is that this protein is also intronless in thecoding region. It has been suggested that this superfamily hasevolved by a gene-processing event from an ancestral gene (O'Dowdet al., 1989).Figure 10: Ethidium bromide stained 2% agarose gel showingthe PCR amplification of human monocyte cDNA (lane 1), humanmonocyte genomic DNA (lane 2) and the size standard, 1 kb DNAladder (lane 3)597.2. Library Screening:The library was plated at 5,000 to 10,000 plaques per plate.This dilution gave a convenient plaque size to facilitate the transferof the DNA to the filters in spots that could be clearly individualizedat the end of the procedure. Positives plaques were replated for asecond and even a third time to ensure that the proper purity wasobtained. The clone on which this work is based (s9a) was present ata frequency of 1/200,000 plaques and a total of 400,000 plaqueswere screened. The library is expected to have a minimum of 1 x 106different clones.7.3. In vivo excision of the recombinant s9a:The UniZAP vector has been constructed to allow the in vivoexcision of the cloned insert contained within the pBluescript whichis in turn contained within the X-vector. pBluescript containssequences that belong to the filamentous phage f1 origin ofreplication and termination signals. Bacteriophage f1 (or M13)encode proteins that recognize the origin of replication for positivestrand synthesis. For this to happen a single cell has to besimultaneously infected with the helper filamentous phage and the2-vector. Inside the cell the proteins of f1 will bind to the f1initiation sequences which are present in the pBluescript. Theseproteins then nick one of the strands and synthesis begins,duplicating all the sequences that are down-stream from the nickingsite. Once the termination signal is reached duplication stops andthe single strand DNA is circularized by a protein that is a gene IIproduct from the phage f1. This includes all the sequences of the60pBluescript phagemid and the insert that is contained in it. In theterminator sequences are also signals for packaging the phagemid.The cells can be killed by heat treatment and removed bycentrifugation. The supernatant containing the packed singlestranded phage is used to infect new cells. In this step the phage isconverted to the double stranded form. The phage used in this workwas the R408 interference-resistant helper phage. It does not use aselectable marker, thus, bacteria infected with helper phage alonewill not grow under ampicillin selection. Single-stranded R408 isapproximately 4 kb. It was developed by Russel et al. (1986) (seefigure 11).(A) Xho I/Not I double digestion of several isolatesfrom clone s9a2 4 6 8101 3 5 7 9 11Figure 11 (A): Agarose gel electrophoresis of 200 ng ofrecombinant s9a. Lane 1: 1 kb DNA ladder; odd lanes are theundigested controls, with supercoiled and nicked plasmidDNAs; even lanes are the digested samples. A fragment ofapproximately 1.6 kb was excised with this enzyme combinationand the linearized plasmid is 2.95 kb (arrowheads).61LI(-) origi.XmnI (4251)ScaI (4134)Sac! (653)Ilmi6*7)Bam141 (689)Pstl (701)coRI (707)amHI (1278)in.ort .9aColE1 onKionI (2365)Apal (2359)Xhol (2350)bl62(B)Mapping of clone s9a with various restrictionenzymes:11 2 4 5 67 8 9Figure 11 (B):Gel 1; lane 1: 1 kb DNA ladder (1 gg), lane 2:clone s9a supercoiled and nicked circular (0.4 Mg);the sameamount of plasmid DNA was digested with the following enzymes(Methods 24.7): lane 3: Kpn I; lane 4: Apa I; lane 5: Xho I;lane 6: Sal I; lane 7: Hind III; lane 8: EcoR V; lane 9: EcoRI. In gel 2: lanes 1 and 2 were the same as in gel 1.Remaining lanes were as follows: lane 3: Pst I; lane 4: SmaI; lane 5: BamH I; lane 6: Spe I; lane 7: Xba I; lane 8: NotI; and lane 9: Sac I. Those enzymes that did not cut weretested again to confirm.(C)Schematic of the recombinant clone s9a:7.4. Sequence Information of the clone s9a:The clone s9a was excised in vivo by co-infection with thephage R408. Cells containing pBluescript carrying the insert wereselected with ampicillin. Plasmid s9a was prepared for sequencingas described in methods (figure 12).GGC ACG AGC GGC ACA GGG TAG 	 27CAA AGT GAC GCC GAG GGC CTG AGT GCT CCA GTA GCC ACC GCA TCT	 72GGA GAA CCA GCG GTT ACC ATG GAG GGG ATC AGT ATA TAC ACT TCA 117MEGISIYTSGAT AAC TAC ACC GAG GAA ATG GGC TCA GGG GAC TAT GAC TCC ATG 162DNYTEEMGSGDYDSMAAG GAA CCC TGT TTC CGT GAA GAA AAT GCT AAT TTC AAT AAA ATC 207KEPCFREENANFNKITTC CTG CCC	216F L PFigure 12: Partial sequence with best predicted translationof the insert in s9a.Homology searches of this fragment revealed that it was anovel protein possibly related to the chemotactic factor receptors.XL-1 Blue cells carrying the plasmid s9a were streaked onampicillin/2x YT-agar plates, and this material was sent for fulllength sequencing to the DNA sequencing core facility of theCanadian genetic diseases network ( Dr. Keith Schappert) (Figure 13).The 352 residue predicted protein was called humnyr3 after the highhomology found with the bovine neuropeptide Y receptor (subtype 3)(see figure 13 C).63(A)cDNA sequence and translation of the ORF for clone89a:GAATTCGGCACGAGCGGCACAGGGTAGCAAAGTGACGCCGAGGGCCTGAGTGCTCCAGTACCGCATCTGGAGAACCAGCGGTTACC 	 90ATG GAG GGG ATC ACT ATA TAC ACT TCA GAT AAC TAC ACC GAG GAA	 135M 	 EGISIYTSDNYTEEATG GGC TCA GGG CAC TAT GAC TCC ATG AAG GAA CCC TGT TTC CGT 	 180MGSGDYDSMKEPCFRGAA GAA AAT GCT AAT TTC AAT AAA ATC TTC CTG CCC ACC ATC TAC	225E ENANFNKIFLPTTYTCC ATC ATC TTC PTA ACT GGC ATT GTG GGC AAT GGA TG GTC ATC	270S TIFLTGTVGNOLVT CTG GTC ATG GGT TAC CAG AAG AAA CTG ACA AGC ATG ACG CAC AAG 	 315L VMGYQKKLRSMTDKTAC ACC CTG CAC CTG TCA GTG GCC CAC CTC CTC TTT GTC ATC ACG	360YRLHLSVADLLFVTT CTT CCC TTC TGG GCA GTT GAT GCC GTG GCA AAC TGG TAC TTT GGG 	 405L PFWAVDAVANWYFGAAC TTC CTA TGC AAG GCA GTC CAT GTC ATC TAC ACA GTC AAC CTC 	 450N FLCKAVHVIYTVNI,TAC ACC ACT GTC CTC ATC CTG GCC TTC ATC ACT CTG CAC CGC TAC 	 495YSSVLILAFISLDRYCTG GCC ATC GTC CAC GCC ACC AAC ACT CAC ACC CCA ACC AAG CTG 	 540L AIVHATNSQRPRKLTTG GCT CAA AAG GTG GTC TAT GTT GGC GTC TGG ATC CCT GCC CTC 	 585L AEKVVYVGVWIPALCTG CTG ACT ATT CCC GAC TTC ATC TTT GCC AAC GTC ACT GAG GCA 	 630L LTIPDFTFANVSEAGAT CAC ACA TAT ATC TGT CAC CGC TTC TAC CCC AAT CAC TTG TGG 	 675D DRYICDRFYPNDLWGTG GTT GTG TTC CAC TTT CAC CAC ATC ATG GTT GGC CTT ATC CTG 	 7203 VVFQFOHTMVGLIT,CCT GGT ATT GTC ATC CTG TCC TGC TAT TGC ATT ATC ATC TCC AAG	765P GIVILSCYCIIISKCTG TCA CAC TCC AAG GGC CAC CAC AAG CGC AAG GCC CTC AAG ACC	 810L SHSKGHQKRKALKTACA GTC ATC CTC ATC CTG GCT TTC TTC GCC TGT TGG CTG CCT TAC	 855TVTLILAFFACWLPY TAC ATT GGG APT ACC ATC CAC TCC TTC APT CTC CTG GAA ATC ATC	900YIGISTDSFILLEIIAAG CAA GGG TGT GAG ITT GAG AAC ACT GTG CAC AAG TOG ATT TCC	945K QGCEFENTVHKWIS64	ATC ACC GAG GCC CTA GCT TTC TTC CAC TGT TGT CTG AAC CCC ATC 	 990TTEALAFFHCCLNPT 	CTC TAT GCT TTC CTT GGA GCC AAA TTT AAA ACC TCT GCC CAG CAC 	 1035L YAPLGAKFKTSAQH	GCA CTC ACC TCT GTG AGC AGA GGG TCC AGC CTC AAG ATC CTC TCC 	 1080ALTSVSRGSSLKILS	AAA GGA AAG CGA GGT GGA CAT TCA TCT GTT TCC ACT GAG TCT GAG 	 1125K GKRGGHSSVSTESE	TCT TCA AGT TTT CAC TCC AGC TAA CAC AGA TGT AAA AGA CTT TTT 	 1170S SSFHSS#	TTTATACGATAAATAAC=TTTTAAGTTACACATTTTTCAGAT 	 1215	ATAAAAGACTGACCAATATTGTACAGCTTTTATTGCTTGTTGGAT 	 1260	TTTTGTCTTGTGTTTCTTTAGTTATTGTGAAGTTTAATTGACTTG 	 1305	ATTTATATAAATTTTTTTTGTTTCATATTGACGTGTGTCTAGGCA 	 1350	GGACCTGTGGCCAAGTTCTTGATGCGGATGCTCTGTGGTAGGACT 	 1395	GTAGAAAGGGAACTGAACATTCCAGAGCTGTAGTGAATCACGTAA 	 1440	AGCTAGAAATGATCCCCAGCTGTT1ATGCATAGATAATCTCTCCA 	 1485	TTCCCGTGGAACGTiaTICCTGTTCTTAAGACGTGATTTTGCTGT 	 1530	AGAAGATGGCACTTATAACCAAAGCCCAAAGTGGTATAGAAATGC 	 1575	TGGTTTTTCAGTTTTCAGGAGTGGGTTGATTTCAGCACCTACAGT 	 1620GTACAGTCTGTATTAAGTTG 	 1641(B)Hydrophobicity plot of the predicted humnyr3 protein.100	200	300— 4— 3— 2— 10- -1- -2- -3100	200	300residue number65(C)Alignment of the bovine and human homologues of theNPY R (type Y3)bovnyr 	 MEGIRIFTSDNYTEDDLGSGDYDSMKEPCFREENAHFNRIFLPTVYSIIFLTGIVGNGLV 60humnyr3 	 MEGISIYTSDNYTEE-MGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLV**** *.*******. .******************.**.*****.***************bovnyr 	 ILVMGYQKKLRSMTDKYRLHLSVADLLFVLTLPFWAVDAVANWYFGKFLCKAVHVIYTVN 120humnyr3 	 ILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVN*****************************.****************.*************bovnyr 	 LYSSVLILAFISLDRYLAIVHATNSQKPRKLLAEKVVYVGVWLPAVLLTIPDLIFADIKE 180humnyr3	 LYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSE**************************.***************.**.******.***.. *bovnyr 	 VDERYICDRFYPSDLWLVVFQFQHIVVGLLLPGIVILSCYCIIISKLSHSKGYQKRKALK 240humnyr3 	 ADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALK*.*********.***.********.***.********************** *******bovnyr 	 TTVILILTFFACWLPYYIGISIDSFILLEIIQQGCEFESTVHKWISITEALAFFHCCLNP 300humnyr3	 TTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNP*******.***********************.******.*********************bovnyr 	 ILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS 	 352humnyr3 	 ILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS*****************************************************Figure 13:(A) Sequence of s9a and translation of its major open readingframe. DNA sequencing was done on both sense and anti-sensestrands. Positions discussed in the text (see below) are inbold. Putative hydrophobic transmembrane domains areunderlined. Potential N-glycosylation site is marked with astar. The sequence has been deposited in GenBank under theaccession #M99293.(B) Average hydrophobicity values were determined for spansof 9 residues using the method and values of Kyte andDoolittle (1982).(C) Alignment obtained by comparing the protein sequence ofthe protein encoded by s9a (humnyr3) and a NPY receptorisolated from bovine brain (Rimland et al, 1991). Thehomology is 93%. Positions differing in both sequences wereverified.7.5. ORF for the protein humnyr3:Previous analysis of eukaryotic 5'-noncoding sequencesrevealed that those sequences surrounding the initiation codon arenot random. These analysis have permitted to identify the expandedconsensus sequence for initiation: (GCC)GCCA/G CC ATG  G (Kozak,661987). Site-directed mutagenesis experiments have demonstratedthat the position -3 and +4 have the strongest influence; hence, aninitiator codon can be considered weak or strong according to thesetwo positions. In the s9a sequence the first in frame Met has thesecharacteristics. It has been theorized that the initiator methionineand flanking sequences function as a stop signal for the 40sribosomal subunit. Therefore, efficient translation is only achievedwith an ATG triplet in the right context (Kozak, 1983). Therepetition of G at the positions -3, -6 and -9 have been signaled asplaying a role by helping the ribosomes to stay in frame duringtranslation. This is also the case with the first ATG codon in s9a.Upstream initiator codons are uncommon in vertebrate RNA,except for the case of oncogenes, but they are an observed feature inadrenergic and muscarinic receptors (Kobilka et al., 1987c). In thisrespect s9a and the chemotactic factor receptors follow the generalrule. The length of the s9a leader sequence falls in thecommunicated range for vertebrate mRNA (20 to 100 bp).At the 3'-end there is a long stretch of about 500 bp after thefirst termination codon that interrupts the OAF. The mammalianconsensus for addition of the poly(A) tail is either AAUAAA orAUUAAA (Wickens, 1990). No consensus sequence for polyadenylationwas observed in s9a. Neither was a poly(A) tail detected. This couldindicate that even if the cDNA contains the full length informationfor the ORF, it actually is an incomplete fragment of the originalmRNA. A strong argument for this is that the mRNAs of othermembers of the 7 TMS family of proteins have long, sometimes up to671 kb, 3'-untranslated sequences. Also, the band detected by Northernblots have a size of 1.8 kb to 2.0 kb, while the insert in s9a is 1.6 kb.The alignment of the human and bovine proteins confirms theassumptions made about the initiation codon, and the extendedreading frame has a termination codon that coincides in these twohighly homologous proteins.8. Chromosomal localization:In situ hybridization localized the gene for humnyr3 in the2q21 region of chromosome 2 (human). The loci that have been giventhe same regional assignment do not seem particularly relevant (inthe context of this superfamily). The fact that there is a unique siteshows that there is only one copy of the gene per haploid genome andthat there are not pseudogenes or highly homologous sequences forthis receptor (figure 14).9. Northern blots:Primate (Rhesus) tissues RNA northern blot showed differentlevels of an approximately 2 kb hybridizing band (figure 15 A and B).The pattern of expression is similar to that from the bovinehomologue (Rimland et al., 1991) on bovine and rat tissues with theexception of kidney. The intensity of the signal in brain and heart isnot surprising: NPY itself is highly expressed in both tissues. Thelow expression on spleen contrast with the results in the micewhere spleen and thymus gave prominent signals; it might reflectspecies differences. NPY has been identified in spleen innervation byin situ hybridization. NPY-like immunoreactivity has been68demonstrated in various areas of the heart and the gastrointestinaltract (Dumont et al., 1992).Transcripts have also been detected in the human Burkittlymphoma cell lines (Ramos, Daudi and Raji), where humnyr3 appearsto be highly expressed, and in the promyelocytic leukemia cell lineHL-60 (without inducing differentiation) (figure 15 B).iunir It ,11111111 .1 all 1 odi NAM fi/it 	 •111'1inialial1 	 2 	 3 	 4 	 5Nith 	 lifft hisIl 	 mai 	 ittall'A 4.1.41 win 	 Moult !Miry 	 ?it iIL parr Hir,13 	 14 	 15 	 16 	 17 	 18 	 19 	 20 	 21 	 22 	 XFigure 14: The position of grains on metaphase chromosomeswere mapped to an ideogram. Analysis of the distribution ofsilver grains revealed a cluster (p<0.0001) in the 2q21region of chromosome 2.691 	 2 	 3 	 4 	 5 	 6 	 7A1 	 2	4 	 5Figure 15: Autoradiography of humnyr3 expression from varioussources. Arrowhead indicates the position of the 28Sribosomal band.(A)Rhesus total RNA; 1) skeletal muscle, 2)liver, 3) colon, 4) kidney, 5) brain, 6) heart, and 7)spleen. Panel (B) lanes: 1)Ramos, 2) Daudi, and 3) Raji humanBurkitt lymphoma cell lines; 4) murine 3T3 fibroblasts, and5) HL-60 human promyelocytic leukemia cell line.70X. DISCUSSION: 1. Structural and functional determinants of the 7 TMSsuperfamily:The adrenergic and related receptors, together with the visualpigment rhodopsin, have been the best characterized of the 7 TMSsuperfamily and they became the model for understanding the molecularbasis of functioning in G-protein coupled receptors. To study thestructural determinants of bioactivity several approaches have beenattempted. The a2- and 132-adrenergic receptors are specially wellsuited to be studied by the construction of chimeras. They have a highdegree of similarity and have ligand binding characteristics that can bedifferentiated. Also, they are coupled to distinct G-proteins. It wasobserved that the binding of the antagonists was dependent on theorigin of the TMS VII (Kobilka et al., 1988). This was also the case forchimeras of two cc-factor receptors for two different but related yeastspecies: the TMS VII was determinant of the pheromone specificity(Marsh and Herkowitz, 1988) Homology analysis of this TMS for thehumnyr3 protein shows that there exist a high degree either of identityor conservative substitutions when this area is compared with both thereceptors for IL-8 (see Figure 16).Following the same approach it was found that TMS V, TMS VI andthe interconnecting loop seem to specify the binding to a particulartype of G-protein (Cotecchia et al., 1990; Kubo et al., 1988). The TMS VIin particular contains a motif that is conserved in several members ofthis superfamily: Cys-Trp-Leu-Pro (CWLP) and that is prominent in thechemotactic, neuropeptide, and some of the adrenergic and71dopaminergic receptors. On the extended motif Cys-Trp-Leu-Pro-Tyr-Asn (CWLPYN) was designed the 3'-end primer used in the cloning ofhumnyr3.humnyr3 	 FQHIMVGLILPGIVILSCYCIIISKLSHhumintleu8 	 ILPOSFGFIVPLLIMLFCYGFTLRTLFK*.*.* ...* ** . . 	 *TMS V	 i3SKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEAHMGQKHRAMRV 	 I • 	 DTLMRTQVIQETCERRNHIDRALDATE*. ***** •i3 	 TMS VI 	 e3ALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSILGTLHSCLNPLIYAFIGQKFRHGLLKILA-IHGLISKDSLPKDSRPSFVGSSSGHTSTT*...* ****..***.* **. • *. . *.*. * .TMS VII 	 C -terminusFHSS putative human NPY R (Y3)L--- Human IL-8 R (low affinity)Figure 16: Alignment of humnyr3 and humintleu8: identicalresidues are marked by stars and conservative substitutions bydots. Note the high degree of overall homology in areas thoughtto participate in the interaction with G-proteins as well as inligand binding. The program used was CLUSTAL (Higgins and Sharp,1989).In the intercrine receptors and in humnyr3 there is anotherfeature conserved in TMS V and that is likely to have a strong effect onthe helical structure: a Pro (P) in a very hydrophobic context (likely toinduce a bend in the a-helix) and a Cys-Tyr (CY) pair at the end of themembrane spanning domain, facing the cytoplasmic side.The intracytoplasmic loop (i3) is very divergent amongstdifferent subgroups: again, the subfamily of adrenergic and muscarinicreceptors appears unrelated to humnyr3 (because of both the lack ofidentity and the differences in length) (see Appendix 1). Nonetheless,there is a motif composed of conservative substitutions plus identitieslocated at the C-terminal side of i3, where the TMS VI begins. It is72composed of (+)(A/P)(hydrophobic)(+)(hydrophobic/T). This motif, or thevariations thereof, are present in most of the proteins analyzed andmay be important for the interaction with some well conserved featureof G-proteins. Specificity determinants for the interaction with G-proteins in the i3 have been also demonstrated with chimeras. This isthe longest loop and the more variable in length and together with boththe N- and C-termini shows the least sequence homology. It is ingeneral the ends of the loop which renders its specificity (Strader etal., 1987) but at least in one case (the al -adrenergic receptor) themiddle of the loop is also important for G-protein binding specificity.The second intracytoplasmic loop, i2, also appears to be involvedin maintaining the appropriate surface for G-protein binding(see figure 17).humil8ra 	 LSLLGNSLVM-LVILYSRVORSVTDVYLLN-LALADLLFALTLPIW--AASKVNGWI-FGTFLhumintleu8 	 LSLLGNSLVM-LVILYSRVORSVTDVYLDN-LALADLLFALTLPIW--AASKVNGWI-FGTFLrabil8c 	 LSLLGNSLVM-LVILYSRSNRSVTDVYLLN-LAMADLLFALTMPIW--AVSKEKGWI-FGTPLhumc5aar	 VGVLGNALVV-NME=EAMMINAlffELN-LAVADFLSCLALPILFTSIVQHHHWP-FGGAArabfmlp 	 LSLLGNSLVM-LVILYSRSNRSVTDVYLDN-LAMAPA-FCPDHAYL--GRLQGKRLD-FRTPLhumfmlpy 	 FGVLGNGLVI-WVAGF-RMTRTVNTICYDN-LALADFSFSAILPFRMVSVAMREKWP-FASFLhumfmlpx 	 LGVLGNGLVI-WVAOF-RMTRTVTTICYLN-LALADFSFTATLPFLIVSMAMGEKWP-FGWFL** **	 * 	 .humnyr3 	 GIVGNGLVI-LVMGYOKKLRSMTDKYRLH-LSVADLLFVITLPFWAVDAVANWYFGNFLCKAiihumil8ra 	 CKVVSLLKEVNFYSGILLLACISV-DRYLAIVHATRTLTOKRH-LVK-FVCLGC-WGLSMNLShumintleu8 	 CKVVSLLKEVNFYSGILLLACISV-17RYLAIVHATRTLTOKRY-LVK-FICLSI-WGLSLLLArabil8c 	 CKVVSLVKEVNFYSGILLLACISV-DRYLAIVHATRTLTOKRH-LVK-FICLGI-WALSLILShumc5aar 	 CSILPSLILLNMYASILLLATISA-DRPLLVFKPIWCONFRGAGLAW-IACAVA-WGLALLLTrabfmlp 	 CKVVSLVKEVNFYSGILLLACISV-DRYLAIVOSTRTLTOKRH-LVK-FICLGI-WALSLILShumfmlpy 	 CKLVHVMIDINLFVSVYLITIIALAACICVLHPAWAONHRTMSLAK-RVMTGL-WIFTIVLThumfmlpx 	 CKLIHIVVOINLFGSVFLIGFIALHULQWZORWAMBRIYOLAM-KVIVGP-WILALVLT• 	 **humnyr3 	 VHVIYTVNLYSSVLILAFISL-DRYLAIVHATNSORPRKL-LANK-VVYVGVWIPALLLTi2Figure 17: Comparison between the intracytoplasmic loops il andi2 for chemotactic factor receptors and humnyr3. Stars showidentity and dots conservative substitutions. See name code infigure 18.73Where 12 joins TMS III there is always the sequence Asp-Arg (DR) thatis highly conserved between 7TMS receptors and that seems to play animportant role in creating the G-protein binding domain. Variationssuch as Glu-Arg (ER) have been observed in the thyrotropin receptorsubfamily. It may also be totally absent, as in the human PlateletActivating Factor receptor. In some cases also the remaining of loop i2(and loop 14, only when there is a Cys modified by palmitoylation, seebelow) have been found to be determinants of the coupling specificity.There is a dissociation between the binding and the activation of thespecific G-protein: some amino acids in loops i2 and i3 are necessaryfor activation but do not affect binding. Synthetic peptidescorresponding to loops i1 and 12 inhibit the coupling of G-proteins tothe receptors. For the chemotactic receptors and humnyr3 these twoloops are fairly short and have a high degree of conservation amongrelated proteins (see figure 17 and appendixes)Another 131-adrenergic receptor peptide (C-terminal segment ofloop i3) activates adenylyl cyclase as it were the ligand occupiedreceptor (Palm et al., 1990). This region is also conserved betweenclosely related proteins. In humnyr3 this area appears as a blendbetween the chemotactic receptors and some of the catecholaminereceptors.For rhodopsin and other 7 TMS receptors for small molecules ithas been proposed that the TMSs assemble in a ring structure definingin this way a ligand binding pocket (Dohlman et al., 1987). Thereforeseveral determinants contribute to the ligand binding site. Usually themembrane spanning domains display the highest degree of homology.Those amino acids conserved among the adrenergic and muscarinic74receptors are located in the half cytoplasmic side of thetransmembrane a-helices. On the other hand, the extracytoplasmic halfcontain those residues showing the more variation. This is inagreement with the fact that G-proteins, that couple to the receptor bybinding to its cytoplasmic surface, have highly conserved primarystructures. The external halves contain the determinants accounting forligand binding specificity (O'Dowd et al., 1989). This is also true forsome of the chemotactic receptors and humnyr3 (see figure 18).N -terminushumfmlpx 	 MET-N	 FSTP 	 LNEYEEVSYESAGYTVLRILPLVVLGVTFhumfmlpy 	 MET-N 	 FSIP 	 LNETEEVLPEPAGHTVLWIFSLLVRGVTFrabfmlp 	 M----EVNVWNMTDLWTWFEDEFANATGMPPVEKDYSPCLVV-TQTLNKYVVVVIYALVFhumc5aar 	 MNSFN 	 YTTPDYGHYDDKDTLDLNTPVDKTSNTLRVPDILALVIFAVVFrabil8c 	 M----EVNVWNMTDLWTWFEDEFANATGMPPVEKDYSPCLVV-TQTLNKYVVVVIYALVFhumintleu8 	 MESDSFEDFWKGEDL 	 SNYSYSSTLPPFLLDAAPCEPE-SLEINKYFVVIIYALVFhumil8ra 	 MSNITDPQMWDFDDL	NF---TGMPPADEDYSPCMLE-TETLNKYVVIIAYALVFhumnyr3 	 MEGIS	IYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIF* 	 *TMS Ihumfmlpx 	 VLGVLGNGLVIWVAGF-RMTRTVTTICYLNLALADFSFTATLPFLIVSMAMGEKWPFGWFhumfmlpy	VFGVLGNGLVIWVAGF-RMTRTVNTICYLNLALADFSFSAILPFRMVSVAMREKWPFASFrabfmlp 	 LLSLLGNSLVMLVILYSRSNRSVTDVYLLNLAMAPA-FCPDRAYL--GRLQGKRLDFRTPhumc5aar 	 LVGVLGNALVVWVTAF-EAKRTINADTFLNLAVADFLSCLALPILFTSIVQHHHWPFGGArabil8c 	 LLSLLGNSLVMLVILYSRSNRSVTDVYLLNLAMADLLFALTMPUN--AVSKEKGWIFGTPhumintleu8 	 LLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIW--AASKVNGWIFGTFhumil8ra 	 LLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIW--AASKVNGWIFGTFhumnyr3 	 LTGIVGNGLVIDVMGYQKKLRUMTDRYRLHLSVADLLFVITLPFW--AVDAVANWYFGNF** ** * 	 .,• 	 ... 	 • 	 • 	 • 	 *	 • 	 •TMS IIhumfmlpx 	 LCKLIRIVVDINLFGSVFLIGFIALDRCICVLHPVWAQNHRTVSLAMKVIVGPWILALVLhumfmlpy 	 LCKLVHVMIDINLFVSVYLITIIALDRCICVLHPAWAQNHRTMSLAKRVMTGLWIFTIVLrabfmlp	 LCKVVSLVREVNFYSGILLLACISVDRYLAIVQSTRTLTQKRRLV-RFICLGIWALSLILhumc5aar 	 ACSILPSLILLNMYASILLLATISADRFLLVFKPIWCQNFRGAGLAWIACAVANGLALLLrabil8c 	 LCKVVSLVKHVNFYSGILLLACISVDRYLAIVHATRTLTQKRHLV-KFICLGIWALSLILhumintleu8 	 LCKVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLV-RFICLSIWGLSLLLhumil8ra 	 LCKVVSLLICEVNITSGILLLACISVDRYLAIVHATRTLTQKRHLV -KFVCLGCVIGLSMNLhumnyr3 	 LCKXVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLIAllYMMYKIEALLL* 	 *	TMS III	TMS IV75humfmlpxhum fmlpyrabfmlphumc5aarrabil8chumintleu8humil8rahumnyr3humfmlpxhumfmlpyrabfmlphumc5aarrabil8chumintleu8humil8rahumnyr3humfmlpxhumfmlpyrabfmlphumc5aarrabil8chumintleu8humil8rahumnyr3TLPVFLFLTTVTIP-NGDTYCTFNFASWGGTPEERLKVAITMLTARGIIRFVIGFSLPMSTLPNFIFWTTISTT-NGDTYCIFNFAFWGDTAVERINVFITMARVFLILHFIIGFTVPMSSLPFFLFRQVFSPN-NSSPVC---YEDLGHNTAKWCMVL 	 RILPHTFGFILPLLTIPSFLYRVVREEYFPPKVLCGVDYSH--DKRRER 	 AVAIVRLVLGFLWPLLSLPFFLFRQVFSPN-NSSPVC---YEDLGHNTAKWRMVL 	 RILPHTFGFILPLLALPVLLFRRTVYSS-NVSPAC---YEDMGNNTANWRMLL 	 RILPQSFGFIVPLLSLPFFLFRQAYHPN-NSSPVC---YEVLGNDTAKWRMVL 	 RILPHTFGFIVPLFTIPDFILPANVSEAD-DRY-IC---DRFYPNDL--WVVVF 	 OFOHIMVGLILPGI* 	 *TMS VIVAICYGLIAAKIHKKGMIKSSRPLRVLTAVVASFFICWFPFQLVALLGTVWLKEMLFYGIITVCYGIIAAKIHRNHMIKSSRPLRVFAAVVASFFICWFPYELIGILMAVWLKEMLLNGVMLFCYGFTLRTLFQAHMGQKHRAMRVIFAVVLIFLLCWLPYNLV-LLADTLMRTHVIQETLTICYTFILLRTWSRRATRSTKTLKVVVAVVASFFIFWLPYQVTGIMMS-FLEPS--SPVMLFCYGFTLRTLFQAHMGQKHRAMRVIFAVVLIFLLCWLPYNLV-LLADTLMRTHVIQEIMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLV-LLADTLMRTQVIQEVMLFCYGFTLRTLFKAHMGQKHRAMRVIFAVVLIFLLCWLPYNLV-LLADTLMRTQVIQEVILSCYCIIISKLSHSKGHQKRKALKTTVILILAFFACWLPYYIG-ISIDSFILLEIIKQ. ** .TMS VIKYKI---IDILVNPTSSLAFFNSCLNPMLYVFVGQDFRERLIHSLPTSLERALSE--DSAKYKI---ILVLINPTSSLAFFNSCLNPILYVFMGRNFQERLIRSLPTSLERALTEVPDSATCQRRNELDRALDATEILGFLHSCLNPIIYAFIGQNFRNGFLKMLAA--RGLISKEFLTRTFLL---LNKLDSLCVSFAYINCCINVIIYVVAGQGFQGRLRKSLPSLLRNVLTE-ESVVTCQRRNDIDRALDATEILGFLHSCLNPIIYAFIGQNFRNGFLKMLAA--RGLISKEFLTRTCERRNHIDRALDATEILGILHSCLNPLIYAFIGQKFRHGLLKILAI--HGLISKDSLPKTCERRNNIGRALDATRILGFLHSCLNPIIYAFIGQNFRHGFL GCEFENTVHRWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLK-ILSKTMS VIIhumfmlpxhumfmlpyrabfmlphumc5aarrabil8chumintleu8humil8rahumnyr3PTNDTAANCASPPAETELQAMQTSNTHTTSASPPEETELQAMHRVTSYTSSST-NVPSNL---RESKSFTRSTVDTMAQKTQAVHRVTSYTSSST-NVPSNL---DSRPSFVGSSSGHTSTTL---human fMLP-related receptor IIhuman RMLP-related receptor Irabbit fMLP receptorhuman C5a anaphylatoxin receptorrabbit IL-8 receptorhuman IL-8 receptor (low affinity)human IL-8 receptor (high affinity)putative human NPY Y3 receptorGKRGGHSSVSTESESSSFHSS  C-terminus  Figure 18: Clustal alignment of the chemotactic factor receptorsand humnyr3. Identities and conservative substitutions arelabeled with stars and dots respectively. Predictedtransmembrane segments are underlined and labeled.76A possible functional role for cysteine palmitoylation at theC-terminal region of the protein has been indicated (O'Dowd etal.,1989). Cys palmitoylation in this area has been observed in both thep2-adrenergic receptor and rhodopsin. The C-terminus of humnyr3 lacksCys eliminating the possibility that such a modification could occur.Four cysteines important for both ligand binding and cell surfaceexpression have been identified in the p2-adrenergic receptor,indicating that disulfides are an important structural feature. Theprediction is that the two disulfides are extracellular and maystabilize the hydrophilic extracytoplasmic loops (Doh!man et al., 1990).This does not appear to be a universal requisite for these receptors: theonly two cysteines in yeast a-factor receptor can be replaced by site-directed mutagenesis without affecting the function of the receptor.Intramolecular disulfides bridges have been also thought to take part inreceptor activation processes (Malbon et al., 1987). In humnyr3 there isa Cys in position 109 (in el) and another in position 186 (in e2). Bothare conserved in adrenergic and chemotactic receptors and could fulfillthe role of forming a stabilizing disulfide. It is noteworthy that el,which is a short loop, has two conserved residues (Trp and Cys) inalmost all the adrenergic, dopaminergic, muscarinic and the moredistant chemotactic receptors. It may contain structural determinantthat affect the overall alignment of the TMSs in the membrane andtherefore the effect on binding specificity would be indirect (seeFigure 19). This does not appear to be the case for those pituitaryhormone receptors in which the ligand is of appreciable size (seeAppendix II).77humil8rahumintleu8rabil8crabfmlphumfmlpyhumfmlpxhumnyr3LLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIW--AAS--KVNGWIFGTFLCK-VLLSLLGNSLVMLVILYSRVGRSVTDVYLLNLALADLLFALTLPIW--AAS--,KVNGWIFGTFLCK-VLLSLLGNSLVMLVILYSRSNRSVTDVYLLNLAMADLLFALTMPIW--AVS--. -VLLSLLGNSLVMLVILYSRSNRSVTDVYLLNLAMAPA-FCPDHAYL--GRLQG--RLDFRTPLCK-VVFGVLGNGLVIWVAGF-RMTRTVNTICYLNLALADFSFSAILPFRMVSVAMRE--KWPFASFLU-LVLGVLGNGLVIWVAGF-RMTRTVTTICYLNLALADFSFTATLPFLIVSMAMGE--KWPFGWFLCK-LLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFW--AVDAV--ANWYFGNFLCK-A	** ** 	 .* 	 * .* 	 *	 * 	 • 	 * 	 ***. 	 . 	 . 	  elacm2_humanblar_humand4dr_humana2aa_humana2ab_humanacm5_humanb2ar_humand5dr_humana2ac_humanb3ar_humand2dr_humandldr_humanTV----IGYWPLGPVVCD--LWLALDYVVSNASVMNLLIISFDRYFCVTKPLTYPVKRTTKMAG-VV----WGRWRYGSFFCE--LWTSVDVLCVTASIETLCVIALDRYLAITSPFRYQSLLT-RARAREVQ---GGAWLLSPRLCD--ALMAMDVMLCTASIFNLCAISVDRFVAVAVPLRYN-RQGGSRRQLEV----MGYWYFGKAWCE--IYLALDVLFCTSSIVHLCAISLDRYWSITQAIEYNLKRTP-RRIKEL----MAYWYFGOVWCG--VYLALDVLFCTSSIVHLCAISLDRYWSVTQAVEYNLKRTP-RRVKIL----MGRWALGSLACD--LWLALDYVASNASVMNLLVISFDRYFSITRPLTYRAKRTPKRAG-IL----MEMKTEGNEWCE--FWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLT-KNKAREV----AGYWPFGAF-CD--VWVAFDIMCSTASILNLCVISVDRYWAISRPFRYKRKMT-QRMALEL----LGYWYFRRTWCE--VYLALDVLFCTSSIVHLCAISLDRYWAVSRALEYNSKRTP-RRIKAL----TGHWPLGATGCE--LWTSVDVLCVTASIETLCALAVDRYLAVTNPLRYGALVT-KRCAREV----VGEWKFSRIHCD--IFVTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTEI----AGFWPFGSF-CN--IWVAFDIMCSTASILNLCVISVDRYWAISSPFRYERKMT-PKAAF* 	 * 	 **•elmussubkrecrat krbosskrguipigsprehumsubpragpisprecHERMRTVTNYFIINLALADLCMAAFNATFNFIYASH-NIWYFGSTFCY-FQNLFPVTAMFVSHERMRTVTNYFIINLALADLCMAAFNATFNFIYASH-NIWYFGRAFCY-FQNLFPITAMFVSHQRMRTVTNYFIVNLALADLCMAAFNAAFNFVYASH-VIWYFGRAFCY-FQNLFPITAMFVSHKRMRTVTNYFLVNLAFAEASMAAFNTVVNFTYAVH-NEWYYGLFYCK-FHNFFPIAAVFASHKRMRTVTNYFLVNLAFAEASMAAFNTVVNFTYAVH-NEWYYGLFYCK-FHNFFPIAAVFASHKRMRTVTNYFLVNLAFAEASMAAFNTVVNFTYAVH-NEWYYGIRYCK-FHNFFPIAAVFAS* *********..***.*. *****• ** ** * * **•* •* *.*.**..*.* *elFigure 19: Comparison of extracellular loop el in humnyr3,chemotactic factor, classical neurotransmitter and neuropeptidereceptors. C onvention to mark homology positions is as describedpreviously. The name code is in the appendices an figure 18.78Analyzing the phenomenon of desensitization for p-adrenergicreceptors it was found that phosphorylation is a transient covalentmodification that modulates receptor activity (Bouvier et al., 1988). Itappears to cause the uncoupling or attenuated coupling to G-proteins.Therefore the presence of consensus sequences for different kinases isa very important feature (Protein Kinase cAMP dependent, PKA, forheterologous desensitization; PKA and p—Adrenergic Receptor Kinase,pARK, for homologous desensitization). Clusters of Ser and Thr arecommon at the C-terminus of the 7TMS receptors. In mutants of the02-adrenergic receptor where the hydroxy-amino acids had beenreplaced or the C-terminus has been truncated by deletion there exist asignificant attenuation of receptor desensitization and of ligand-induced phosphorylation. The findings implicate pARK and because theyare only observed at high agonist concentration they point to the factthat PKA is active on the receptor even at low agonist concentration(Hausdorff et al., 1989). pARK appears to have a broad substratespecificity, phosphorylating in vitro other 7TMS proteins (Kwatra etal., 1989). It has been demonstrated that for maximal desensitizationat high agonist concentration both kinases are needed (Lohse etal.,1990). Because the action of pARK is manifest at high agonistconcentration it is currently thought that this enzyme is important inhighly innervated tissues at the synaptic level (Benovic et al.,1990).Dephosphorylation seems to play a role in regenerating activereceptors but it needs more investigation. The action of thephosphatases has been linked rather to receptor sequestration.In the C-terminus of humnyr3 there are 15 Ser and 3 Thr over 46residues, suggesting that modulation by phosphorylation is also79important for this protein. Some of these hydroxy-amino acids arewithin a consensus frame for PKA phophorylation (Kennelly et al.,1991).From the different studies performed with adrenergic andmuscarinic receptors it was concluded that: 1) the same ligand maybind and activate two functionally distinct receptors, 2) Different G-proteins with similar receptor and effector binding characteristicsexist, 3) receptors can couple to different G-proteins, mainly whenexpressed at high levels, and 4) individual G-proteins can activatedifferent effector systems. All this complexity is further increased bythe existence of receptor subtypes. It has been speculated thatsubtypes reflect different expression pattern in different tissues orduring development or that different patterns of desensitization areinvolved (Doh!man et al., 1991).The Drosophila mutant ninaA displays a marked reduction in thelevel of its rhodopsin. This mutant is defective in a cis-transprolylisomerase. (Shieh et al., 1989). In these mutants as well as in oneform of the degenerative disease retinitis pigmentosa (with themutation Pro23His) rhodopsin accumulates in the endoplasmicreticulum. The equivalent Pro in humnyr3 is located in position 27, 13residues downstream of the sole N-glycosylation consensus site in theprotein. In humnyr3 and the intercrine receptors (but not in thecatecholamine receptors) this Pro is associated to a Cys forming thepair Pro-Cys (see figure 20).80(A)humil8ra 	 MSNITDPQMWDFDDL 	 NF---TGMPPADEDYS--PCM-LE-TETLNKYVVIIAYALVFhumintleu8	 MESDSFEDFWKGEDL 	 SNYSYSSTLPPFLLDAA--PCE-PE-SLEINKYFVVIIYALVFrabil8c 	 M----EVNVWNMTDLWTWFEDEFANATGMPPVEKDYS--PCL-VV-TQTLNKYVVVVIYALVFrabfmlp	 M----EVNVWNMTDLWTWFEDEFANATGMPPVEKDYS--PCL-VV-TQTLNKYVVVVIYALVFhumfmlpy 	 MET 	 NFSI 	 PLNETEEVLPEPAGHTVLWIFSLLVHGVTFhumfmlpx 	 MET 	 NFST 	 PLNEYEEVSYESAGYTVLRILPLVVLGVTFhumnyr3	 MEGIS 	 IYTSDnITEEMGSGDYDSMKE--PCF-REENANFNKIFLPTIYSIIFN-terminus(B)acm2_human 	 MNNSTNSSNNSLAL 	 TS 	 PYKTFEVVblar_human 	 MGAGVLVLGASEPGNLSSAAPLPDGAATAARLLVPASPPASLLPPASESPEPLSQQWTAGd4dr_human 	 MGNRSTA 	 DADGLLAGRGPAAGASAGASAGLAGQGAAALVGa2aa_human 	 m 	 GSLQPDAGNASWNGTEAPGGGARATP---YSLQVTLa2ab_human 	 MASPALAA	 ALAVAAAAGPNASGAGERGSGGVANASGASWGPPRGQYSAGAVAacm5_human 	 MEGDSYHNATTVNG 	 TPVNHQPLERHRLWEVIb2ar_human 	 MG 	 QPGN 	 GSAFLLAPNRSHA----PDHDVTQQRDEVWVVGd5dr_human 	 MLPPG-SNGTAYPGQFALYQQLAQGNAVGGSAGAPPLGPSQV	a2ac_human 	 MD 	 HQDPYSVQATAb3ar_human 	 m 	 APWPHENSS----LAPWPDLPTLAPNTANTSGLPGVPWEAAd2dr_human 	 MDPLNLS	WYDDDLERQNWSRPFNGSDGKADRPHYNYYATLLTdldr_human	 M 	 RTLNTSAMDGTGLVVERDFSVRI 	*N-terminus(C)rat skr 	 MGTRAIVSDANILSGLESNATGVTAFSMPGWQLA---LWATAYLALVLVAVTGNATVIWIILAhumsubprec 	 MDN-VLPVDSDLSPNISTNTSEPNQFVQPAWQIV---LWAAAYTVIVVTSVVGNVVVMWIILAmussubkrec 	 MGAHASVTDTNILSGLESNATGVTAFSMPGWQLA---LWATAYLALVLVAVTGNATVIWIILAguipigspre 	 MDN-VLPVDSDLFPNISTNTSEPNQFVQPAWQIV---LWAAAYTVIVVTSVVGNVVVMWIILAhumsubpra 	 MDN-VLPVDSDLSPNISTNTSEPNQFVQPAWQIV---LWAAAYTVIVVTSVVGNVVVMWIILAgpisprec 	 MDN-VLPVDSDLFPNISTNTSEPNQFVQPAWQIV---LWAAAYTVIVVTSVVGNVVVMWIILAhumsprlong 	 MDN-VLPVDSDLSPNISTNTSEPNQFVQPAWQIV---LWAAAYTVIVVTSVVGNVVVMWIILAbosskr 	 MGACVVMTDINISSGLDSNATGITAFSMPGWQLA---LWTAAYLALVLVAVMGNATVIWIILAhumsubprec 	 MDN-VLPVDSDLSPNISTNTSEPNQFVQPAWQIV--- A4k4 11 _AA • IJAAJu IILA*. 	 * 	 *.**. 	 **..** 	 * ** 	 * *****N-terminus 	 TMS IFigure 20: Comparison of the N-terminal region in humnyr3 andthe chemotactic (A), classical neurotransmitter (B), andneuropeptide receptors (C). The N-glycosylation site in humnyr3and the TMS I in neuropeptide receptors are underlined. For thename code see appendices and figure 18.81Mutations at the N-glycosylation sites on the N-terminus have amore deleterious effect: in some cases no perturbation was observedbut in Drosophila rhodopsin, and in another form of retinitispigmentosa, mutations at these sites proved to be impairing.In some tumors a constitutive activation of adenylyl cyclase islinked to a defect in the Gs component (Landis et al., 1989).Proliferative effects are described for the serotonin (5HT1c),angiotensin (mas oncogene) and adrenergic receptors. They stimulatethe phosphatidyl inositol and the cAMP pathways.Until now most of the work performed in the area of structure-function relationship has been with receptors for the classicalneurotransmitters, receptors for yeast pheromones and the visualpigment proteins, rhodopsins.2. Virally encoded 7TMS proteins:It has been found that there are virally-encoded G-protein coupledreceptors. The genome of human cytomegalovirus (HCMV) has threeORFs that encode putative 7TMS receptors (US27 ,US28 and UL33). Twoof these genes are arranged consecutively suggesting gene duplication;the third is divergent and is separated from the other two by 180 bp.Features of gene organization seem to indicate that these genes aretranscribed. No specific ligand for these HMCV proteins is known butthey lack an Asp residue in helix III that is characteristic of thecationic amine receptors. By the other hand US27 and US28 have an Aspin position 96 which is important for ligand binding in the p-adrenergicreceptor. All three HCMV sequences have a Lys residue in i3 in aposition that is critical for the binding to G-proteins. This Lys is82conserved in the intercrine receptors and humnyr3. This loop (13) isshort in the viral sequences as it is in the mas oncogene, in humnyr3and in the chemotactic receptors. The viral proteins also containphosphorylation consensus sequences in the C-termini (Chee et al.,1990). Some of the primary metabolic changes in HCMV-infected cellsare typical of G-protein mediated signaling, such as increased Ca2+intracellular and elevation of cAMP and DAG levels. All US27, US28 andUL33 have ORFs with TATA-like promoter elements and stop codons inthe appropriate frame but only US28 has a polyadenylation signal. WhenmRNA was prepared from HCMV infected fibroblasts and was probedwith specific oligonucleotides (29- or 30-mers) it was found thatUS27 and US28 are expressed as a long cotranscript and that US28alone is also expressed. The same was observed with UL33 and otherprotein of the unique long repeat UL34, the latter having a nearbypolyadenylation signal. This was shown to occur late during theinfection (Welch et al., 1991).Human homologues have not been identified yet. It is not knownwhich role they have in the virus life cycle. Similarity between US 28and humnyr3 exist and it is spread out over the entire sequence, but ismost noticeable in the TMSs (see figure 21).MTPTT - - TTAELTTEF - - -DYDEDATPCVFTDVLNQSKPVTLFLYGVVFLEGSIGNFLVIFTITNIRRR IQ 65 US 2 8MEG IS I YTS DNYTEEMGSGDYDSMK EPCFRE ENANFNK  I FLPT IYS I IFLTGIVGNGLVILVMGYQKKLR 70 humnyr3* 	 * 	 * * 	 ** * 	 * * 	 * 	 ** * ** ***TMS IC SGDVYF INLAAADLLFVCTLPLWMQYLLDHNS LASVPCTLLTACFYVAMFASLCFI TS IALDRYYA IVY 135  1SMTDKYRLHLSVADLLFVITLPFINAVDAVANWYRGNFLCKAVHVIYTVNLYSSVLILAF I S LDRYLA IVH 140  2* * 	 ft ****** *** *	 * 	 *	 * 	 * **** ***Pus ix	TM I I I83MRYRFVKQACLFSIFW----WIFAVIIAIPHFMV-VTKKDNQCMTDYDYLEVSYFIILNVELMLGAFVI 199 1ATNSQRFRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYICDRFYFNDLWVVVFQFOHIMVGLIL 210 2** *	 ** * 	 * 	 * 	 * 	 *TMS IV 	 TMS VPLSVISYCYYRISRIVAVSQSRHKGRIVRVLIAVVLVFIIEWLPYHLTLFVDTLKLLKWISSSCEFERSL 269 1PGIVILSCYCII1SKLSHSKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTV 280 2* 	 ** 	 ** 	 * 	 * * 	 **** 	 * 	 **	 * 	 ****TMS VIKRALILTESLAFCHCCLNPLLYVEWTICFRKNYTVCWPSFASDSFRAMYFGT	321 1HKWISITEALAFFHCCLNPILYAFLGAKFKTSAQUALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFH 350 2** *** ****** ** * * **TMS VIITA 323 1SS 352 2Figure 21: Clustal alignment of the human cytomegalovirusencoded US28(1) and of humnyr3 (2). Identities are indicated bystars.Most of these traits are present in the majority of the membersof this superfamily, except perhaps the pairs Pro-Cys (PC) in the N-terminus, Cys-Glu (CG) in the third external loop (e3), and Cys-Tyr (CY)in the TMS V that are conserved only in some of the chemotactic factorreceptors and humnyr3. The N-terminus and external loops are poorlyconserved which speaks against a similar ligand for both, the viral andthe human receptors. The homology in internal loops is also poor,therefore, they likely bind to different types of G-proteins.3. 7TMS receptors for large proteins:The receptors for pituitary and placental glycoprotein hormonesdiffer from other members of the 7TMS superfamily in having largeextracellular domains (see Appendix II). This is consistent with thelarger size of their cognate ligands. Site-directed mutagenesis of thethyrotropin receptor involving substitutions and deletions have beenperformed. This receptor upon activation induces a rise in cAMP levels.84Mutations in i1 anulate the ability to activate adenylate cyclase but didnot alter the binding affinity for TSH. When the second loop 12 wastargeted a different pattern was observed depending on which regionthe mutations were clustered. For those mutants with altered N-terminal region within the loop, again the ligand binding wasunaffected but the receptor was no longer functional. This confirms therole of the Asp-Arg pair in creating a generic G-protein binding motif.This is the basis for delineating an extended consensus in this area forrelated proteins. So, in both human IL-8 Rs , the rabbit IL-8 R, therabbit formyl-peptide receptor and humnyr3 the motif is Asp-Arg-Tyr-Leu-Ala-lle (DRYLAI) (that was precisely the forward primer in thecloning of humnyr3).For mutations clustering in the C-terminal region of this loopboth the affinity for the ligand and the coupling to G-proteins wereabolished. As the N-terminal portion of i3 was mutated, both affinityand coupling were impaired. However, when the C-terminal region wasmodified no difference between this mutant and the wild type wasfound. This loop is the more heterogeneous across the wholesuperfamily, presenting variation in both length and composition. Itlikely imparts specificity for the binding to G-proteins.For cytoplasmic tail mutants total truncation affected both thebinding affinity and the coupling. When two thirds of the C-terminusproximal sequence were deleted leaving a short tail which is homologueto the C-terminus in the TSH, and lutropin/chorionic gonadotropinreceptors both parameters remained unchanged (Chazenbalk et al.,1990). Again, this region presents more variation and tends not to beconserved even in related proteins. One prediction is that the85desensitization mechanisms should be affected because truncationremoves most of the phosphorylation sites. These receptors are doublein size of the remaining 7TMDS proteins. This difference is mainly dueto the N-terminus which is 412 residues long in the case of thethyrotropin receptor.4. Chemotactic Receptors:Recently, several cDNAs encoding chemotactic and activatingfactor receptors have been cloned. No studies of structure-functionrelationship have been performed on these proteins up to now. Thetranslated sequence follows the predicted topology for the 7TMSreceptors: they all contain seven stretches of 20-28 hydrophobic aminoacids that likely form membrane spanning domains. Identical residuesor conservative substitutions are clustered in these regions. Otherregions are less, but still to a significant extent, conserved. These arethe two first intracytoplasmic loops. The extracellular domains and theC-terminal tail are quite divergent. Not surprisingly, the greatestsimilarity is found in those proteins that bind to closely relatedligands.The high-affinity IL-8 R (humil8ra) was isolated by expressioncloning from human neutrophils mRNA (Holmes et al., 1991). Cross-reactivity with Gro/MGSA, MIP-1 and NAP 2 but not with fMLP or withother chemotactic factors had been observed (both, by binding assaysand by functional studies in human neutrophils) (Moser et al., 1991).There are two glycosylation sites at the N-terminus and this regioncontains several acidic residues. The third cytoplasmic loop is shorterthan in the adrenergic or muscarinic receptors and is thought to contain86determinants for G-protein binding (see above). The C-terminuscontains several Ser (S) and Thr (T) residues that could be substratesfor phosphorylation.A second IL-8 binding protein (humintleu8) which displays 74%identity with the previous one was obtained from a cDNA isolated of apromyelocytic leukemia cell line (HL-60) that was induced todifferentiate (Murphy et al., 1991). The affinity with which thisreceptor binds IL-8 is 20 times lower than humil8ra. The proteinbelongs to the 7TMS superfamily. The C-terminal region contains 11 Sor T that could be phosphorylation sites for kinases. It has a single N-glycosylation site in the N-terminus and two in the second externalloop. The former is also enriched in acidic residues.The anaphylatoxin C5a is a 74 amino acids glycoprotein derivedfrom C5 during activation of the complement cascade. It is a potentactivator of neutrophils and monocytes apart from inducing spasms invarious tissues, stimulating smooth muscle contraction, inducinghistamine release from basophils, serotonin from platelets and alsoinducing vascular permeability. All these activities are due to theinteraction of C5a with high affinity receptors in the cell membrane.These receptors are of the 7TMS type and bind G-proteins, and they arerelated to other chemotactic factor receptors (Boulay et al.,1991). Asin the two previous cases the N-terminal domain is enriched in acidicresidues, it contains one N-glycosylation site at the N-terminaldomain, and the C-terminus is rich in S and T. They have even moresimilarity with the human N-formylpeptide receptors.87Another trigger for the locomotion and activation of phagocyticcells are the N-formyl peptides, which are thought to be derived frombacterial degradation or injured tissue mitochondria degradation. Thisgroup also includes the platelet activating factor (PAF), and thearachidonate metabolite leukotriene B4. The human N-formyl peptidesreceptor has been characterized. A cDNA has been cloned and sequenced(Boulay et al., 1990). The hydrophobicity profile indicates a 7TMStopology in agreement with previous functional studies that revealedthe coupling to G-proteins. There are three putative sites for N-glycosylation but previous studies with endoglycosidase F have showedthat only 2 are actually glycosylated. The third intracytoplasmic loopis short and contain a site for PKA phosphorylation (KSSSR). Also, thereare several hydroxy-amino acids in the C-terminus that could functionas phosphate acceptors.From all these proteins the humintleu8 (IL-8 R, low affinity)first and then the humil8ra (IL-8 R, high affinity) are the ones with thehighest degree of homology with the humnyr3 receptor. Identicalresidues or conservative substitution are found all over the length ofthese proteins (see figure 22).humnyr3 	 MEGISIYTSDNY- -TEEMGSGDYDSM 	 KEPCFREENANFNKIFLPTIYSIIFLThumintleu8 	 MESDSF---EDFWKGEDLSNYSYSSTLPPFLLDAAPC-EPESLEINKYVVVIIYALVFLL** , *. .	 *. .	 * *	 **	 *. , .** *. 	 *Ir. ..**. 	 . 	 .N-terminus 	 TNS IGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKSLLGNSLVNIVILYSRVGRSVTDVYLLNLALADLLFALTLPIWAASICVNGWIFGTFLCK...**.**.**. * . 	 **.** * *.* ..  	 .***.** 	 * * ** ****11 	 TMS II 	 el88AVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDVVSLLKEVNFYSGILLLACISVDRYLAIVHATRTLTQKRYLV-KFICLSINGLSLLLALPV* **.**..*.** **.********** . * *TMS III 	 12 	 TMS IVFIFAN-VSEADDRYIC--DRFYPNDLWVVVFQFOHIMVGLILPGIVILSCYCIIISKLSHLURRTVYSSNVSPACYEDMGNNTANWRMLLRILPOSFGFIVPLLIMLFCYGFTLRTLFK* *	 *.*.* ...* **e2 	 TMS V 	 13SKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEAHMGQKHRAMRVIFAVVLIFLLCWLPYNLVLDADTLMRTQVIQETCERRNHIDRALDATB13 TMS VI e3ALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSILGILHSCLNPLIYAFIGQKFRHGLLKILA-IHGLISKDSLPKDSRPSFVGSSSGHTSTT*..•* ****.•***•* **. •• • * *•*• * *• *TMS VII C-terminusFHSSL---Figure 22: Alignment of humnyr3 and humintleu8 (the low affinityIL-8 R) Notice the high degree of homology, extended over theoverall length, between both moleculesThis would indicate a rather close relationship in both functionand structure but until the structural determinants of bioactivity startto emerge nothing definitive to that respect can be said Both, humnyr3and chemotactic receptors N-termini are enriched in acidic residues. Inthe case of the intercrine receptors it was thought that their functionwas to participate in the binding to the chemotactic factors which arebasic polypeptides. But NPY is a rather neutral molecule. The C-terminal side of the loop 13 is conserved but the N-terminal side is not.It would appear that both receptors interact with the same or a similartype of G-protein. The external loop e2 is the least conserved of themall. The high degree of similarity between both proteins (35% identity;8964% similarity counting the conservative substitutions) is suggestive.The affinity of humintleu8 for IL-8 is 20 times lower than the affinityof humil8ra for IL-8. This cast doubts about the specificity of theformer. Perhaps, once the specific ligand of humintleu8 is defined therelationship with humnyr3 will also be clarified.Other chemotactic factor receptors (with the exception ofhumil8ra, and the rabbit IL-8 R and fMLP receptors) share lesshomology with humnyr3. This is also true among the intercrinereceptors themselves.5. Neuropeptide receptors:When the sequence of the rat substance K receptor, mousesubstance K receptor and human substance P receptor are compared tohumnyr3 the homology is restricted to a few basic features ofG-proteins coupled receptors (figure 23 and appendix 3).*. 	 *. 	 * •  * 	 *  • 	 * 	 *.**. **•*** 	 .*• .*.** 	 *•*****bosskr	 MGACVVMTDINISSGLDSNATGITA---FSM--PGWQLALWTAAYLALVLVAVMGNATVIWIILAhumsubpra 	 MDN-VLPVDSDLSPNISTNTSEPNQ---FVQ--PAWQIVLWAAAYTVIVVTSVVGNVVVMWIILAhumnyr3 	 MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSTIFLTGTWNGLVILVMGY** 	 * 	 ** *	•	 . 	 . 	 . 	 • 	 . 	 . 	 . 	 . 	 . 	 . 	 ... 	 . 	 ..N-terminus 	 TMS Ibosskrhumsubprahumnyr3*.*********.****.*• 	 *****. 	 ** 	 ** ** **.* 	 •* *•*•***.*.* *HQRMRTVTNYFIVNLALADLCMAAFNAAFNF-VYASHNIWYFGRAFCYFQNLFPITAMFVSHKRMRTVTNYFLVNLAFAEASMAAFNTVVNF-TYAVHNEWYYGLFYCKFHNFFPIAAVFASQKKLRSMTDKYRLHLSVADL---_LFVTTLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSS* 	 * 	 **.* 	 * 	 *11 	 TMS II 	 el 	 TM'S III******.* ******.**•****** 	 • *• ** 	 **	 ** ***** 	 *** 	 *••bosskr	 IYSMTAIAADRYMAIVHPFQPRLSAP--GTRAVIAGIWLVALALAFPQCFYSTITTDEGATKhumsubpra 	 IYSMTAVAFDRYMAIIHPLQPRLSAT--ATKVVICVIWVLALLLAFPQGYYSTTETMPSRVVhumnyr3 	 VLTLAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIFANVSEADDRYI	*** ** * 	 * 	 * ** * *.. 	 .. 	 • 	 • 	 • 	 ... 	 • 	 • 	 • 	 . 	 . 	 .. 	 •	12 	 TMS IV90*.. ***.. 	 .**. * 	 *******.*. 	 **.*.*.*** 	 .**. 	 .bosskr 	 CVVAWPEDSGGKMLLLYHLIVIALIYFLPLVVMFVAYSVIGLTLWRRSVPGHQAHGANLRhumsubpra 	 CMIEWPEHPNKIYEKVYHICVTVLIYFLPLLVIGYAYTVVGITLWASEIPGDSSDRYH-Ehumnyr3 	 CDRFYPND---LWVVVFOFOHTMVGLTLPGIVTLSCYCIIISKLSHS 	*..e2 	 TMS V 	 13**•* ** *..** ********•*. * * • * •* ********* 	 *******• 	 • 	 • bosskrhumsubprahumnyr3HLQAKKKFVKTMVLVVVTFAICWLPYHL 	YFILGTFQEDIYCHKFIQQVYLALFWLAMSSTFLLPYINPDLYLKKFIQQVYLAIMWLAMSSTKGHQKRKALKTTVILILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHK 	 WISIMEL* * * 	 * ****QVSAKRKVV lulu AA	 II13 	 TMS VI 	 e3***********•*** **. ****** * 	 * *. bosskr 	 MYNPIIYCCLNHRFRSGFRLAFRCCPWVTPTEEDKMELTYTPSLSTRVNRCHTKEIFFMShumsubpra 	 MYNPIIYCCLNDRFRLGFKHAFRCCPFISAGDYEGLEMKSTRYLQTQGSVYKVSRL----humnyr3 	 L--AFFHCCLNPIL 	YAFLGAKFKTSAQHALTSVSRGSSLK 	 ILSK... **** 	 . 	 ***.. 	 . 	 .TM'S VII 	 C-terminus* 	 * 	 * * 	 * 	 • *•. .bosskr	GDVAPSEAVNGQAESPQAGVSTEP 	humsubpra 	 -ETTISTVVGAHEEEPEDGPKATPSSLDLTSNCSSRSDSKTMTESFSFSSNVLShumnyr3 	 GKRGGHSSVSTESESS 	 SFHS---S* 	 *Figure 23: Alignment of two of the neuropeptide receptors withhumnyr3. Identities and conservative substitutions between thetwo neuropeptide (SP and NKA) receptor proteins are showed bythe stars and the dots respectively on top of the sequences. Thesame convention is used for the comparison among the threesequences, but the homology is marked at the bottom of thesequencesEssentially a five residues cluster at the boundary of TMS III and13 is conserved. Two more clusters of 4 residues each are conserved inTMS VI and TMS VII. The loop el is short and have some conservedfeatures. The overall homology for the four proteins is 13% which is byfar below that of the humintleu8 and humnyr3. The putative human NPYreceptor subtype Y1 (humneypepy) shows 20% homology with humnyr3(figure 24).91humnyr3 	 MEGISIYTSDNYTEEMGSGDYDSMKEP--CFREENANF--NKIF-LPTIYSIIFLTGIVGhumneypepy 	 MNS-TLFS--QVENHSVHSNFSEKNAQUAFENDDCHLPLAMI 	 G 	 SG* . . 	 .... 	 . 	 .. 	 ... 	 . 	 * 	 ... 	 .. 	 ** *. 	 * 	 * 	 *TMS Ihumnyr3 	 NGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVIT-LPF-WAVDAVANWYFGNFLCKAVHhumneypepy 	 NLALIIIILKQKEMANVTNILIVNLSFSDLLVAIMCLPFTFVYTLMDUWVFGEAMCKLEf* 	 .* 	 ** * * 	 ** *** * *** 	 * ** 	 **TMS IIhumnyr3 	 VIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGV---WIPALLLTIPhumneypepy 	 FVOCVSITVSTFSLVT.TaVERHQLIINPRGW-RPNN----'.4 	 P**	 • 	 *TMS III 	 TMS IVhumnyr3 	 DFIFANVSEA 	 DDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYChumneypepy	 FLIyQVMTDEPFQNVTLDAYKDKYVCFDQFPSDSHRLSYTTLLLVLOYFGPLCFIFICYF* * * 	 * * 	 * 	 * **. 	 . 	 . 	 . 	 . 	 . 	 . 	 . 	 .TMS Vhumnyr3 	 IIISKLSHSKG	 HQKRKALKTTVIL---ILAFFACWLPYYIGISIDSFILLEhumneypepy 	 KIYIRLKRRNNMMDKMRDNKYRSSETKRINIMLLSIVVAFAVCWLP--LTTFNTVFDWNH**	 **** 	 . * 	 * 	 .TMS VIhumnyr3 	 IIKQGCEFENTVHKWISITEALAFFHC	CLNPILYAFLGAKFKTSAQ 	humneypepy 	 QIIATCN 	 HNLLFLLCHLTAMISTCVNPIFYGFLNKNFQRDLQFFFNFC* 	 *. 	 * *. * 	 * *** * ** 	 *. 	 . 	 . 	 . 	 .TMS VIIhumnyr3 	 HALTSVSRGSSLKILSKG--KRGGHSSVSTESESSSFHSShumneypepy 	 DFRSRDDDYETIAMSTMHTDVSKTSLKQASPVAFKKINNNDDNEKI.** 	 *... 	 .Figure 24: Alignment of the human NPY receptor type 1 and theputative human receptor type 3.TMS II and el are the most conserved regions. In TMS V, VI and VIIthe homology clusters in the cytoplasmic side of the predicted helices.Both N- and C-termini are highly divergent, and e2 is conserved but e3is not. These two proteins have distinct binding characteristics: thesubtype Y1 binds both NPY and PYY with similar affinities; the subtypeY3 is more specific for NPY, binding PYY very weakly.926. Humnyr3 and the immune response:Substance P and substance K induce the release of IL-1, TNF-aand IL-6 from human blood monocytes (Lotz et al.,1988). This effect isspecific. Because monocyte-derived cytokines are key mediators ininflammation and immunity, and, neuropeptides can be released fromperipheral nerve endings into adjacent tissues, this constitutes a clearmechanism of interaction of the nervous system and cells of theimmune response. Neuropeptides are released from unmyelinated axonsin response to trauma or inflammation, inducing the production andrelease of cytokines that mediate host defensive responses in aparacrine and/or endocrine way. Substance P also regulates othermonocyte functions such as arachidonic acid metabolism, chemotaxisand respiratory burst (Hartung et al., 1986). IL-1 and TNF initiatecellular and humoral responses and produce systemic changes such asthe synthesis of acute-phase proteins and fever. It is likely that asimilar, or the same inductive effect, exist in other substance Presponsive cells, like fibroblasts and endothelial cells.Preprotachykinin A is the precursor of both substance P andneurokinin A. It was shown by Northern blot that these twoneuropeptides are expressed in the thymic medulla. By the sametechnique NPY mRNA was detected also in the thymic medulla (Ericssonet al.,1990). Several other neuropeptides have been found in thymus,with some differences according to the species but providing evidencefor the intrathymic synthesis of these neuropeptides. It is not clearwhich is the role of these neurotransmitter/neuromodulators, but ithas been suggested that they may act in T-cell differentiation and/orearly activation that occurs mainly in the thymus.93By immunocytochemistry for NPY and lymphoid markers the NPYinnervation in the rat spleen have been mapped. NPY positive nerveswere present along the vasculature, trabeculae, and capsula, and alsowere found associated with specific lymphoid compartments in closecontact with lymphocytes and macrophages (Romano et al.,1991).Activated B-cells, like those represented in the Burkitt lymphomacell lines, express high levels of this protein (figure 15 B). The findingsreported here, that mRNA for the type 3 receptor has been detected inboth thymus and spleen, complements the findings about the expressionof the NPY itself in those organs. At this stage nothing can be saidabout the meaning of the expression of the Y3 subtype, both because theexpression of the other two subtypes is not ruled out and because morepharmacological characterization of humnyr3 is desired. Nonetheless,the presence of mRNA for a NPY receptor in organs where theexpression of the factor itself has already been established is in goodagreement with the hypothesis that this neuropeptide and its receptorcould participate in a neuro-immunomodulatory loop, but to clarifythese aspects more investigation is needed. The significant homologyof humnyr3 with some of the intercrine receptors is another importantquestion that should be addressed.7. Conclusion:NPY can induce a variety of physiological responses through theactivation of specific pre- and post-synaptic receptors. Differentbinding characteristics for agonists in several model systems indicatethat there are multiple types of NPY receptors. This is being confirmedby the cloning studies. The receptor isolated here could be the putative94Y3 subtype based on the high homology to the characterized bovine Y3receptor. To complete its pharmacological definition more experimentsare needed. The coupling of NPY receptors to specific signaltransduction pathways (like inhibition of adenylyl cyclase andstimulation or inhibition of intracellular Ca2+ increases) has not beenestablished. The pathophysiological role of NPY and its receptors is notknown. The availability of a human cDNA for the Y3 subtype is a tool fordifferent lines of experimentation from in situ hybridization toexpression of the receptor in stable cell lines.NPY is one of the most abundant peptides in the human brain andappears as an integrator of endocrine, metabolic and behavioralprocesses (Leibowitz, 1991). 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TMSs are underlined.acm2_human 	 MNNSTNSSNNSLAL 	 TS 	 PYKTFEVVblar_human 	 MGAGVLVLGASEPGNLSSAAPLPDGAATAARLLVPASPPASLLPPASESPEPLSQQWTAGd4dr_human 	 MGNRSTA	 DADGLLAGRGPAAGASAGASAGLAGQGAAALVGa2aa_human 	 M 	 GSLQPDAGNASWNGTEAPGGGARATP---YSLQVTLa2ab_human 	 MASPALAA 	 ALAVAAAAGPNASGAGERGSGGVANASGASWGPPRGQYSAGAVAacm5_human 	 MEGDSYHNATTVNG 	 TPVNHQPLERHRLWEVIb2ar_human 	 MG 	 QPGN 	 GSAFLLAPNRSHA----PDHDVTQQRDEVWVVGd5dr_human 	 MLPPG-SNGTAYPGQFALYQQLAQGNAVGGSAGAPPLGPSQV 	a2ac_human 	 MD 	 HQDPYSVQATAb3ar_human 	 M 	 APWPHENSS----LAPWPDLPTLAPNTANTSGLPGVPWEAAd2dr_human 	 MDPLNLS 	 WYDDDLERQNWSRPFNGSDGKADRPHYNYYATLLTdldr_human 	 M 	 RTLNTSAMDGTGLVVERDFSVRI 	*acm2_human 	 FIVLVAGSLSLVTIIGNILVMVSIKVNRHLQT-VNNYFLFSLACADLIIGVFSMNLYTLYblar_human 	 M-GLLMALIVLLIVAGNVLVIVAIAKTPRLQT-LTNLFIMSLASADLVMGLLVVPFGATId4dr_human 	 CV 	 LLIGAVLAGNSLVCVSVATERALQTP-TNSFIVSLAAADLLLALLVLPLFVYSa2aa_human 	 TLVCLAGLLMLLTVFGNVLVIIAVFTSRALKAP-QNLFLVSLASADILVATLVIPFSLANa2ab_human 	 GLAAVVGFLIVFTVVGNVLVVIAVLTSRALRAP-QNLFLVSLASADILVATLVMPFSLANacm5_human 	 TIAVVTAVVSLITIVGNVLVMISFKVNSQLKT-VNNYYLLSLACADLIIGIFSMNLYTTYb2ar_buman 	 M-GIVMSLIVLAIVFGNVLVITAIAKFERLQT-VTNYFITSLACADLVMGLAVVPFGAAHd5dr_human 	 VTACLLTLLIIWTLLGNVLVCAAIVRSRHLRANMTNVFIVSLAVSDLFVALLVMPWKAVAa2ac_human 	 AIAAAITFLILFTIFGNALVILAVLTSRSLRAP-QNLFLVSLAAADILVATLIIPFSLANb3ar_human 	 LAGALLALAVLATVGGNLLVIVAIAWTPRLQT-MTNVFVTSLAAADLVMGLLVVPPAATLd2dr_human 	 LLIAVIVFGNVLVCMAVSREKALQTT-TNYLIVSLAVADLLVATLVMPWVVYLdldr_human		 LTACFT,W01,STTJ,GNTLVCAAVIRFRHLRSKVTNFFVISLAVSDLLVAVINMPWKAVA** **	TMS I	 TMS IIacm2_human 	 TV-IGYWPLGPVVCDLWLALDYVVSNASVMNLLIISFDRYFCVTKPLTYPVKRTTKMAG-blar_human	VV-WGRWEYGSFFCELWTSVDVLCVTASIETLCVIALDRYLAITSPFRYQSLLT-RARARd4dr_human 	 EVQGGAWLLSPRLCDALMAMDVMLCTASIFNLCAISVDRFVAVAVPLRYN-RQGGSRRQLa2aa_human 	 EV-MGYWYFGKAWCEIYLALDVLFCTSSIVHLCAISLDRYWSITQAIEYNLKRTP-RRIKa2ab_human 	 EL-MAYWYFGQVWCGVYLALDVLFCTSSIVHLCAISLDRYWSVTQAVEYNLKRTP-RRVKacm5_human 	 IL-MGRWALGSLACDLWLALDYVASNASVMNLLVISFDRYFSITRPLTYRAKRTPKRAG-b2ar_human 	 IL-MKMWTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLT-KNKARd5dr_human 	 EV-AGYWPFGAF-CDVWVAFDIMCSTASILNLCVISVDRYWAISRPFRYKRKMT-QRMALa2ac_human 	 EL-LGYWYFRRTWCEVYLALDVLFCTSSIVHLCAISLDRYWAVSRALEYNSKRTP-RRIKb3ar_human 	 AL-TGHWPLGATGCELWTSVDVLCVTASIETLCALAVDRYLAVTNPLRYGALVT-KRCARd2dr_human 	 EV-VGEWKFSRIHCDIFVTLDVMMCTASILNLCAISIDRYTAVAMPMLYNTRYSSKRRVTdldr_human 	 EI=AGFWPFGSF-CNIWVAFDIMCSTASTLNLCVISVDRYWAISSPFRYERKMT-PKAAF**TMS III 	 TMS IVacm2_human 	 MMIAAAWVLS-FILWAPAILFWQFIVGVRTVEDG 	 ECYIQ 	blar_human	GLVCTVWAISALVSFLPILMHWWRAES 	 D-EARRCYNDPKCCDd4dr_human 	 LLIGATWLLSAAVAA-PVLCGLNDVRGRDPAV 	 CRa2aa_human	AIIITVWVISAVISF-PPLISIEKKGGGGGPQPAEPR 	 CEa2ab_human	ATIVAVWLISAVISF-PPLVSLYRQPDG----AAYPQ 	 CGacm5_human 	 IMIGLAWLIS-FILWAPAILCWQYLVGKRTVPLD 	 ECQIQ 	b2ar_human 	 VIILMVWIVSGLTSFLPIQMHWYRATH 	 Q EAINCYANETCCDd5dr_human	 VMVGLAWTLSILISFIPVQLNWHRDQAASWGGLDLPNNLANWTPWEEDFWEPDVNAENCDa2ac_human 	 CIILTVWLIAAVISL-PPLIY---KGDQGPQPRGRPQ 	 CKb3ar_human	TAVVLVWVVSAAVSFAPIMSQWWRVGA 	 DAEAQRCHSNPRCCAd2dr_human	VMISIVWVLSFTISC-PLLFGLNNA---DQNE 	dldr_human	TiT9yAWLSVLISFIPVQLSWHKAKPTS 	 PSDGNATSLAETIDNCD105a cm2_human 	 FFSNAAVTFGTAIAAFYLPVI I MTVLYWH I SRASKS RI KK 	 DKKEP- -blar_human 	 FVTNRAYAIASSVVSFYVPLC IMAFVYLRVFREAQKQVKKIDSCERR- - F LGGPARP PS Pd4dr_human 	 LEDRD- YVVYSSVCSFFLPCPLMLLLYWATFRGLQRW- - - -EVAR 	a 2 aa_human	INDQICWYVI S SC I GSFFAPC L I MI LVYVRIYQIAICARTRVPPSRRGPDAVAAPPGGTERRa 2 ab_human 	 LNDETWY I LSSC I GS FFAPC L I MGLVYARI YRVAKRRTRTLSEKRAP - - -VGPDGASPTTacm5_human 	 F LSE PT I TFGTA IAAFY I PVSVMT I LYCRIYRETEKRTKDLADLQGSDSVTKAEKRKPAHb2 ar_human 	 FFTNQAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGR- -F 	d5dr_human 	 SSLNRTYA I SSSL I SF YI PVAIMIVTYTRI YR IAQVQ I RRI SS 	 LERAAEHAQSa 2 ac_human 	 LNQEAWY I LASS I GSFFAPC L IMI LVYLRIYLIAKRSNRRGPRAKGGPGQGESKQPRPDHb 3 ar_human 	 FASNMPYVLLSSSVSFYLPLLVMLFVYARVFVVATRQLRLLRGELGR- -F - PPEESPPAPd2 dr_human	IANPA-FVVY SS IVSF YVPF IVTL LVY I KI Y IVLRRRRKRVNTKRSSRAF 	 RAH Ldldr_human 	 SSLSRTYAI SSSVT SFYT PVA TMTVWTRT YR  TAQKQ IRR IAA	LERAAVHAKN* 	 * 	 * 	 .TM Vacm2_human 	 VANQDPVSPSLVQGR 	 IVICPNNNNMPS SD- - - -DGLE 	blar_human 	 SP 	 SPVPAPAPP 	d4dr_human 	 RA 	 KLHGR 	 AP 	a2 aa_human 	 PN 	 GLGPERSAGPGGAEAEPLPTQLNGAPGEPAPAGPRDT - - - -a 2 ab_human 	 EN 	 GLGAAAGEARTGTARPRPPTWSRTRAAQRPRGGAP 	acm5_human 	 RALFRSC LRC PRPTLAQRERNQASWS S SRRSTSTTGKPSQATGPSANWAKAEQLTTC SS Yb2 ar_humand 5 dr_human 	 CR 	 S 	a2 ac_human 	 GG 	 ALASAKLPALASVASAREVNGHSKSTGEKEEGETPED- - - -b 3 ar_human 	 SR 	 SLAPAPVGT 	d2dr_human 	 RA 	 PLKGNCTHPEDMKLCTVIMKSNGSFPVNRRRVEAA 	dldr_human 	 CQ 	 T 	a cm2_human 	 HNK I QNGKAPRDPVTENCVQGEEKES SNDSTSVSAVASNMRDDE I TQDE-NTVSTSLGHSblar_human 	 PGPPRPAAAAATA 	 PLd4dr_human 	 RRP 	 SGPGPP 	 SPTPPAPR- LPQDPCGPDCAPPAPGLPPDPCa 2 aa_human 	 - - - -DALDLEESSSSDHAERPPGPRRPERGPRGKGICA 	 RASQVKPGa2ab_human 	 - - - -GPLRRGGRRRAGAEGGAGGADGQGAGP- - -GAA 	 QSGALTASa cm5_human 	 PSSEDEDKPATDPVLQVVYKSQGKESPGEEFSAEETEETFVKRETEKSDYDTPNYLLSPAb2ar_human 	 HVQNLSQ 	 VEd5dr_human 	 SAA 	a 2 ac_human 	 - - - -TGTRALPPSWAALPNSGQGQKEGVCGASPEDEAEEEEEEEEEEEECEPQAVPVSPAb 3 ar_human 	 CAPP 	d2dr_human 	 RFtAQELEMEMLS STS PPERTRYS P I PPSHHQ LTL PDPSHHGLHSTPDS PAKPEdldr_human 	 TTGNGK 	 PVacm2 _human 	 KDENSK-QTC I - - - - RIGTKTPKSDSCTPTNTTVEVVGSS - -GQNGDEKQNIVARKIVKMblar_human 	 ANGRAGK- - RRPSRLVAL 	d 4 dr_human 	 GSNCAPPDAVRAAALPPQTPPQ 	 TRRFtRRAKITGR 	a 2 aa_human 	 DS LRGAGRGRRGS - - - -GRR 	 LQGRGRSASGLPRRRAGAGa 2 ab_human 	 RS PGPGGRLSRAS - - - - SRSVE 	 FF LSRRRRARS SVC RRKVA- -acm5_human 	 AAHRPKSQKCVAYKFRLVVICADGNQETNNGCHKVK I MPC PF PVAKEPSTKGLNPNP SHQMb2 ar_human 	 QDGRTGHGLRRSSKFC -L 	d 5 dr_human 	 -CA- PDTS LRAS I K 	a 2 ac_human 	 SAC S PPLQQPQGSRVLATLRGQ 	 VLLGRGVGA I GGQWWRRRAHVb 3 ar_human 	 - EGVPACG-RRPARLLPL 	d2 dr_human 	 KNGHAICDH PK IAK IF EI QTMPN 	 GKTRTSLKTMSRRKLS- -dldr_human 	 EC SQ PESSF KMSFK 	106acm2_human	 TKQPAKKKPPPSREKKVTRTILAILLAFIITWAPYNVMVLINTFCA	 PC-Iblar_human 	 REQKALKTLGIIMGVFTLCWLPFFLANVVKAFH-REL 	 Vd4dr_human 	 ERKAMRVLPVVVGAFLLCWTPFFVVHITQALCPA	CSVa2aa_human 	 GQN	 REKRFTFVLAVVIGVFVVCWFPFFFTYTLTAV- -G 	 CSVa2ab_human	 -QA 	 REKRFTFVLAVVMGVFVLCWFPFFFIYSLYGICREA 	 CQVacm5_human 	 TK---RKRVVLVKERKAAQTLSAILLAFIITWTPYNIMVLVSTFCD 	 KC-Vb2ar_human 	 KEHKALKTLGIIMGTFTLCWLPFFIVNIVHVIQ-DNL 	d5dr_human 	 KETKVLKTLSVIMGVFVCCWLPFFILNCMVPFCSGHPEGPPAGFPC-Va2ac_human 	 REKRFTFVLAVVIGVFVLCWFPFFFSYSLGAICPKH 	 CKVb3ar_human 	 REHRALCTLGLIMGTFTLCWLPFFLANVLRALGGPSL 	 Vd2dr_human 	 -QQ 	 KEKKATQMLAIVLGVFIICWLPFFITHILNIHC-D 	 CNIdldr_human 	 RETKVLKTLSVIMGVFVCCWLPFFTLNCTLPFCGS---GETQPF-C-I* .* 	 * *. 	 .TMS VIacm2_human 	 PNTVWTIGYWLCYINSTINPACYALCNATFKKTFKHLL-MCHYKNI 	blar_human 	 PDRLFVFFNWLGYANSAFNPIIYC-RSPDFRKAFQGLLC-CARRAARRRHATHGDRPRASd4dr_human	 PPRLVSAVTWLGYVNSALNPVIYTVFNAEFRNVFRKAL 	 RACC	a2aa_human	 PRTLFKFFFWFGYCNSSLNPVIYTIFNHDFRRAFKKILCRGDRKRIV	a2ab_human 	 PGPLFKFFFWIGYCNSSLNPVIYTVFNQDFRPSFKHILFRRRRRGFRQ	acm5_human 	 PVTLWHLGYWLCYVNSTVNPICYALCNRTFRKTFKMLL-LCRWKKKKVE 	b2ar_human	 RKEVYILLNWIGYVNSGFNPLIYC-RSPDFRIAFQELLC--LRRSSLKAYG 	d5dr_human 	 SETTFDVFVWFGWANSSLNPVIYA-FNADFQKVFAQLLG-CSHFCSRT--PVETVNISNEa2ac_human 	 PHGLFQFFFWIGYCNSSLNPVIYTIFNQDFRRAFRRILCRP-WTQTAW	b3ar_human 	 PGPAFLALNWLGYANSAFNPLIYC-RSPDFRSAFRRLLCRCGRRLPPEP 	d2dr_human 	 PPVLYSAFTWLGYVNSAVNPIIYTTFNIEFRKAFLKIL 	 H- -Cdldr_human 	 DSNTFDVFVWFGWANSSLNPIIYA-FNADFRKAFSTLLG-CYRLCPATNNAIETVSINNN**. ** 	 *. 	 . TMS VIIacm2_humanblar_human 	 GCLARPGPPPSPG-AASDDDDDDVVGATPPA 	 RLLEPWAGCNGGAAd4dr_humana2aa_humana2ab_humanacm5_human 	 EKLY 	b2ar_human 	 GYSSNGNTGEQSGYHVEQEKENKLLCEDLPG 	 T--EDFVGHQGTVPd5dr_human 	 ---LISYNQDIVFHKEIAAAYIHMMPNAVTPGNREVDNDEEEGPFDRMFQIYQTSPDGDPa2ac_humanb3ar_human 	 CAAARP 	 ALFPS 	 GVPAd2dr_humandldr_human 	 GAAMFSSHHEPRGSISKECNLVYLIPHAV--GSSEDLKKEEAAGIARPLE--KLSPALSVacm2_human 	GATR-- Acetylcholine Muscarinic receptor(M2)blar_human A DSDSSLDEPCRPGFASESKV Pl-adrenergic receptord4dr_human   dopamine receptor (D4)a2aa_human   a2-adrenergic receptora2ab_human   al-adrenergic receptoracm5_human  WQ GNSKLP Acetylcholine Muscarinic receptor (M5)b2ar_human 	 S 	 DNIDSQGRNCS---TNDSLL P2-adrenergic receptord5dr_human 	 VAESVWELDCEGEISLDKITPFTPNGFH-- dopamine receptor (D5)a2ac_human   02-adrenergic receptor (type C2)b3ar_human 	 A 	 RSSPAQPRLCQR-LDG---- P3-adrenergic receptord2dr_human   dopamine receptor (D2)dldr_human 	 I 	 LDYDTDVSLEKIQPITQNGQHPT dopamine receptor (Dl)The names used correspond to the names in the swiss protein data bank107Appendix 2: Clustal alignment for the receptors for three pituitaryhormones. Stars indicate matches across all sequencesand dots are conservative substitutions. Name code isat the end of the alignment.tshr_human	 MR----PADLLQLVLLLD--LPRDLGGMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTlshr_human 	 MKQRFSPLQLLKLLLLLQAPLPRALRRL-CPEP-CNCVPDGALR 	 APAPRPSfshr_rat 	 M 	 ALLLVSLLAFLGT 	 GSGCHHWLCHCSNRVFLCQDSKVTEIPTDLPRNA*.*tshr_human	 QTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLlshr_human 	 TRLSLAYLPVKVIPSQSFRGLNEVIKIEISQIDSLERIEANAFDNLLNLSEILIQNTKNLfshr_rat 	 IELRFVLTKLRVIPKGSFAGFGDLEKIEISQNDVLEVIEADVFSNLPKLHEIRIEKANNL	** 	 * 	 * 	 * 	 * 	 * 	 * ** 	 * *	 **	. 	 . 	 . 	 . 	 . 	 .tshr_human 	 TYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGlshr_human 	 RYIEPGAFINLPRLKYLSICNTGIRKFPDVTKVFSSESNFILEICDNLHITTIPGNAFQGfshr_rat 	 LYINPEAFQNLPSLRYLLISNTGIKHLPAVHKIQSLQKVL-LDIQDNINIHIVARNSFMG**.*.*. .** *..* * ***.. .*.. *. * 	 * * *** .* *- 	 . 	 . . tshr_human	LCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSlshr_human 	 MNNESVTLKLYGNGFEEVQSHAFNGTTLTSLELKENVHLEKMHNGAFRGA-TGPKTLDISfshr_rat 	 LSFESVILWLSKNGIEEIHNCAFNGTQLDELNLSDNNNLEELPNDVFQGA-SGPVILDIS* 	 * * 	 ** 	  ***** * 	 * 	 * 	 * 	 * * 	 ** 	 **.*tshr_human 	 QTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGlshr_human 	 STKLQALPSYGLESIQRLIATSSYSLKKLPSKQTFVNLLRATLHYPSHCCAFRN 	fshr_rat 	 RTKVHSLPNHGLENLKKLRARSTYRLKKLPNLDKFVTLMEASLTYPSHCCAFANLKRQIS*.. .**. *** 	 * * 	 ***** 	 .*. * 	 *.* ******** *tshr_human 	 ILESLMCNESSMQSLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVlshr_human 	 LPTK 	 ELNFSHSISEN 	fshr_rat 	 ELHPI-CNKSIL 	 RQDIDDMT 	 QIGDQRVSLID 	 DEPSY--...tshr_human 	 FFEEQEDEIIGFGQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMGYlshr_human 	 -FSKQCESTV 	 RKSELSGWD--YEYGFCLPKTPR-CAPEPDAFNPCEDIMGYfshr_rat 	 GKGSDMMY 	 NEFDYDLCNEVVDVTCSPKPDAFNPCEDIMGX* 	 * 	 * . * 	 * .**********tshr_human 	 KFLRIVVWFVSLLALLGNVFVLLILLTSHYKLNVPRFLMCNLAFADFCMGMYLLLIASVDlshr_human 	 DFLRVLIWLINILAIMGNMTVLFVLLTSRYKLTVPRFLMCNLSFADFCMGLYLLLIASVDfshr_rat 	 NILRVLIWFISTLAITGNTTVLVVLTTSQYKLTVPRFLMCNLAFADLCIGIYLLLTASVD..**...*....**. ** 	 ** •* **•***.*********.***•*•*•*********	TMS I	 TMS IItshr_human 	 LYTHSEYYNHAIDWQTGPGCNTAGFFTVFASELSVYTLTVITLERWYAITFAMRLDRKIRlshr_human 	 SQTKGQYYNHAIDWQTGSGCSTAGFFTVLASELSVYTLTVITLERWHTITYAIHLDQKLRfshr_rat 	 IHTKSQYHNYAIDWQTGAGCDAAGFFTVFAELSVYTLTATTLERWHTITHAMQLECKVQ*...*.*.*******.**..******.**********.******..** *..*. *..TMS IIItshr_human 	 LRHACAIMVGGWVCCFLLALLPLVGISSYAKVSICLPMDTETPLALAYIVFVLTLNIVAFlshr_human	LRHAILIMLGGWLFSSLIAMLPLVGVSNYMKVSICFPMDVETTLSQVYILTILILNVVAFf shr_rat	 LRHAASVMVLGWTFA F AA A UP T FG I  SSYMKVSICLPMDIDSPLSQLYVMALLVLNVTAF**** 	 •*• ** 	 *..*. *•*•* *****.***....*. 	 *•. .*.**..**TMS IV 	 TMS Vtshr_human 	 VIVCCCHVKIYITVRNPQYNPGDKDTKIAKRMAVLIFTDFICMAPISFYALSAILNKPLIlshr_human 	 LIICACYIKIYFAVRNPELMATNKDTKIAKKMAILIFTDFTCMAPISFFAISAAFKVPLIfshr_rat 	 VVICGCYTHIYLTVRNPTIVSSSSDTKIAK 	 KVPLI* * •** 	 **** 	 • 	 ****** ** ****** ******* * **• • . 	 . . . 	 . . 	 . . 	 . 	 . 	 • • 	 ***TMS VI108tshr_human 	 TVSNSKILLVLFYPLNSCANPFLYAIFTKAFQRDVFILLSKFGICKRQAQA-YRGQRVPPlshr_human 	 TVTNSKVLLVLFYPINSCANPFLYAIFTKTFQRDFFLLLSKFGCCKRRADPLYRRKDFSAfshr_rat 	 TVSKAKILLVLFYPINSCANPFLYATFTKNFRRDFFILLSKFGCYEMQAQ-IYRTE----•** 	 * ******* ************** * ** * ****** 	 * 	 **•TM VIItshr_human 	 KNSTDIQVQKVTHDMRQGLHNMEDVYELIENSHLTPKKQGQISEEYMQTVLlshr_human 	 YTSNCKNGFTGSNKPSQSTLKLSTLH--CQGTALLDKTR 	 YTEC--fshr_rat 	 -TSSATHNFHARKSHCSSAPRVTNSYVLV---PLNHSSQN 	The names used are those of the swiss protein data bank.tshr= thyrotropin receptorlshr= lutropin-choriogonadotropic hormone receptorfshr= follicle stimulating hormone receptor109Appendix 3: Alignment of the neuropeptide receptors. Stars indicateidentity and dots conservative substitutions. Name codeis at the end of the alignment.MGTRAIVS--DANILSGLESNATGVTAFSMPGWQLAL 	 WATAYLALVLVAVTGNATMGAHASVT--DTNILSGLESNATGVTAFSMPGWQLAL	 WATAYLALVLVAVTGNATMDN-VLPV--DSDLFPNISTNTSEPNQFVQPAWQIVL	 WAAAYTVIVVTSVVGNVVMDN-VLPV--DSDLSPNISTNTSEPNQFVQPAWQIVL	 WAAAYTVIVVTSVVGNVVMNSTLFSQVENHSVHSNFSEKNAQLLAFENDDCHLPLAMIFTLALAYGAVIILGVSGNLAMDN-VLPV--DSDLFPNISTNTSEPNQFVQPAWQIVL 	 WAAAYTVIVVTSVVGNVVMDN-VLPV--DSDLSPNISTNTSEPNQFVQPAWQIVL 	 WAAAYTVIVVTSVVGNVVMGACVVMT--DINISSGLDSNATGITAFSMPGWQLAL	 WTAAYLAJNIVAVMGNAT** 	 * **TMS IVIWIILAHERMRTVTNYFIINLALADLCMAAFNATFNFIYASHNIWYFGRAFCYFQNLFPVIWIILAHERMRTVTNYFIINLALADLCMAAFNATFNFIYASHNIWYFGSTFCYFQNLFPVMWIILAHKRMRTVTNYFLVNLAFAEASMAAFNTVVNFTYAVHNEWYYGLFYCKFHNFFPVMWIILAHKRMRTVTNYFLVNLAFAEASMAAFNTVVNFTYAVHNEWYYGLFYCKFHNFFPLIIIILKQKEMRNVTNILIVNLSFSDLLVAIMCLPFTFVYTLMDHWVFGEAMCKLNPFVQVMWIILAHKRMRTVTNYFLVNLAFAEASMAAFNTVVNFTYAVHNEWYYGLFYCKFHNFFPVMWIILAHKRMRTVTNYFLVNLAFAEASMAAFNTVVNFTYAVHNEWYYGLFYCKFHNFFPVTWTILAHQRMRTVTNYFIVNLALADLCMAAFNAAFNFVYASHNIWYFGRAFCYFONLFP* * 	 * 	 * 	 *. 	 . 	 . 	 . 	 . 	 . 	 .TMS IIITAMFVSIYSMTAIAADRYMAIVHPFQPRLSAPSTKAIIAGIWLVALALASPQCFYSTITVTAMFVSIYSMTAIAADRYMAIVHPFQPRLSAPSTKAVIAVIWLVALALASPQCFYSTITIAAVFASIYSMTAVAFDRYMAIIHPLQPRLSATATKVVICVIWVLALLLAFPQGYYSTTEIAAVFASIYSMTAVAFDRYMAIIHPLQPRLSATATKVVICVIWVLALLLAFPQGYYSTTECVSITVSIFSLVLIAVERHQLIINPRGWRPNNRHAYVGIAVIWVLAVASSLPFLIYQVMTIAAVFASIYSMTAVAFDRYMAIIHPLQPRLSATATKVVICVIWVLALLLAFPQGYYSTTEIAAVFASIYSMTAVAFDRYMAIIHPLQPRLSATATKVVICVIWVLALLLAFPQGYYSTTETTAMPVSTYSmTATAADRYMAIVHPFQPRLSAPGTRAVIAGIWLVALALAFPOCFYSTIT* 	 ** 	 * 	 * 	 *. 	 . 	 . 	 . 	 . 	 .TMS III 	 TNS IVVDE 	 GATKCVVAWPNDNGGKMLLLYHLVVFVLIYFLPLLVMFGAYSVIGLTVDQ 	 GATKCVVAWPNDNGGKMLLLYHLVVFVLIYFLPLVVMFAAYSVIGLTTMP 	 GRVVCMIEWPSHPDKIYEKVYHICVTVLIYFLPLLVIGYAYTVVGITTMP 	 SRVVCMIEWPEHPNKIYEKVYHICVTVLIYFLPLLVIGYAYTVVGITDEPFQNVTLDAYKDKYVCFDQFPSDSHRL---SYTTLLLVLQYFGPLCFIFICYFKIYIRTMP 	 GRVVCMIEWPSHPDKIYEKVYHICVTVLIYFLPLLVIGYAYTVVGITTMP 	 SRVVCMIEWPEHPNKIYEKVYHICVTVLIYFLPLLVIGYAYTVVGITTDE 	 GATKCVVAWPEDSGGKML • V 	 YSVIGLT*ratskrmussubkrecguipigsprechumsubprahumneypepygpisprechumsprlongbosskrratskrmussubkrecguipigsprechumsubprahumneypepygpisprechumsprlongbosskrratskrmussubkrecguipigsprechumsubprahumneypepygpisprechumsprlongbosskrrats krmussubkrecguipigsprechumsubprahumneypepygpisprechumsprlongbosskr.* ** **. TMS V110rats krmussubkrecguipigsprechumsubprahumneypepygpisprechumsprlongbosskrratskrmussubkrecguipigsprechumsubprahumneypepygpisprechumsprlongbosskrratskrmussubkrecguipigsprechumsubprahumneypepygpisprechumsprlongbosskrLWKRAVPRHQAHGANLRHLQAKKKFVKAMVLVVLTFAICWLPYHLYFILGTFQEDIYYHKLWKRAVPRHQAHGANLRELQAKKKFVKAMVLVVVTFAICWLPYHLYFILGTFQEDIYYRKLWASEIPGDSSDRYH-EQVSAKRKVVKMMIVVVCTFAICWLPFHIFFLLPYINPDLYLKKLWASEIPGDSSDRYH-EQVSAKRKVVKMMIVVVCTFAICWLPFHIFFLLPYINPDLYLKKLKRRNNMMDKMRDNKYRSSETKRINI-MLLSIVVAFAVCWLPLTIFNTVFDWNHQIIATCLWASEIPGDSSDRYH-EQVSAKRKVVKMMIVVVCTFAICWLPFHIFFLLPYINPDLYLKKLWASEIPGDSSDRYH-EQVSAKRKVVKMMIVVVCTFAICWLPFHIFFLLPYINPDLYLKKLWRRSVPGHQAHGANLRHLQAKKKFVKTMVLVVVTFATCWLPYRLYFIi,GTFQEDIYCHK.	* 	 ** ****. 	 . 	 . 	 . 	 .TMS VIFIQQVYLALFWLAMSSTMYNPIIYCCLNHRFRSGFRLAFRCCPWVTPTEE-DRLELTHTPFIQQVYLALFWLAMSSTMYNPIIYCCLNHRFRSGFRLAFRCCPWGTPTEE-DRLELTHTPFIQQVYLAIMWLAMSSTMYNPIIYCCLNDRFRLGFKHAFRCCPFISAA-DYEGLEMKSTRFIQQVYLAIMWLAMSSTMYNPIIYCCLNDRFRLGFKHAFRCCPFISAG-DYEGLEMKSTRNHNLLFLLCHLTAMISTCVNPIFYGFLNKNFQRDLQFFFNFCDFRSRDDDYETIAMSTMHFIQQVYLAIMWLAMSSTMYNPIIYCCLNDRFRLGFKHAFRCCPFISAA-DYEGLEMKSTRFIQQVYLAIMWLAMSSTMYNPIIYCCLNDRFRLGFKHAFRCCPFISAG-DYEGLEMKSTRFIQQVYLALFWLA 	 WVTPTEE-DKMELTYTP** ** 	 *** * 	 ** 	 ** 	 *	. 	 . 	 ......	 .TMS VIISLSRRVNRCHTKETLFMTGDM--THSEATNGQVGSPQDGEPAGPIC	SISRRVNRCHTKETLFMTGDM--THSEATNGQVGGPQDGEPAGPx	YF 	 QTQGSVYKVSRLETTISTVVGAHEEDPEEGPKATPSSLDLTSNGSSRSNSKYL 	 QTQGSVYKVSRLETTISTVVGAHEEEPEDGPKATPSSLDLTSNCSSRSDSKTDVSKTSLKQASPVAFK 	 KINNNDDNEK 	YF 	 QTQGSVYKVSRLETTISTVVGAHEEDPEEGPKATPSSLDLTSNGSSRSNSKYL 	 QTQGSVYKVSRLETTISTVVGAHEEEPEDGPKATPSSLDLTSNCSSRSDSKSLSTRVNRCHTKEIFFMSGDV--APSEAVNGQAESPQAGVSTEP	ratskrmussubkrecguipigsprechumsubprahumneypepygpisprechumsprlongbosskr KAQA rat substance K receptormouse substance K receptorguinea pig substance P receptorhuman substance P receptor protein mRNAhuman neuropeptide Y receptor (type Y1)porcine substance P receptorhuman substance P receptor long formbovine substance K receptor TVTESSSFYSNMLSTMTESFSFSSNVLS IxTVTESSSFYSNMLSTMTESFSFSSNVLS   The names used are those from the gp data base111

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