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Assembly and intracellular trafficking of the B cell antigen receptor in a mutant B cell lymphoma, WEHI… Escribano, Jessica 2001

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Assembly and Intracellular Trafficking of the B Cell Antigen Receptor In a Mutant B Cell Lymphoma, WEHI 88.1 by JESSICA ESCRIBANO B.Sc, The University of British Columbia, 1994. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology; Cellular Biology Programme) We accept this thesis as conforming to the required standard: THE UNIVERSITY OF BRITISH COLUMBIA March 27,2001 © Jessica M. Escribano, 2001 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Department DE-6 (2/88) ABSTRACT Mutations that affect the synthesis of receptor subunits or that interfere with protein folding may lead to ER retention of unassembled receptors. This retention process is part of the secretory pathway's "quality control" mechanism, which consists of a variety of molecular chaperones and enzymes that assist nascent proteins in their folding, assembly, and post-translational modification. The B cell antigen receptor (BCR) is an oligomeric protein that undergoes a complex, stepwise, assembly process. The BCR consists of an antigen-binding component, mlgM (containing two immunoglobulin heavy (H) chains and two light (L) chains), and a signaling component, Ig-a/Ig-p. Both parts of the BCR must correctly assemble and associate with each other to leave the ER. If one of more subunits of the BCR is missing or defective, the entire receptor is retained and targeted for degradation (Matsuuchi et al., 1992). In the current study, we have characterized the BCR expressed by WEHI 88.1, a B lymphoma mutant. WEHI 88.1 expresses all four chains of the BCR, however, they are retained intracellularly in a pre-Golgi compartment. Our studies revealed that WEFfl 88.1 mlgM assembly was defective, likely due to limiting amounts of K L chain. As a consequence of limiting amounts of K L chain, completely assembled mlgM composed of two H chains and two L chains (H2L2) was not detected in the mutant. In addition, pulse chase analysis indicated that K L chain turnover in the mutant differed from that in wild type cells. In contrast, the Ig-cc/Ig-P part of the receptor assembled normally and associated with the mlgM present in the WEFfl 88.1 cell line. This study has identified a key defect in the assembly of the BCR in mutated WEHI 88.1 cells and this mutant can serve as a tool for the future studies of receptor: chaperone interactions during the assembly process. ii TABLE OF CONTENTS Abstract ii Table of Contents iii List of Tables v List of Figures vi Acknowledgements vii Dedication viii Introduction 1 I Quality Control and Protein Trafficking in the Secretory Pathway 1 1.1 Overview 1 1.2 Co-translational Translocation of Membrane Proteins into the ER 3 1.3 Membrane Protein Folding and Modification in the ER 6 1.4 Membrane Protein Oligomerization 7 II Molecular Chaperones 8 2.1 Overview 8 2.2 Bacterial Models of Molecular Chaperones .10 2.3 Protein Disulfide Isomerase (PDI) 11 2.4 Immunoglobulin Binding Protein (BiP) 12 2.5 Calnexin 13 2.6 Glucose Response Protein 94 (GRP 94) 14 2.7 Glucose Response Protein 170 (GRP 170) 14 2.8 Summary 15 III Models of Quality Control in the ER 15 3.1 Overview 15 3.2 Retention Model of Quality Control in the ER 16 3.3 Recycling Model of Quality Control in the ER 17 3.4 Summary 19 IV Degradation of Proteins in the ER 21 V The B Cell Antigen Receptor 25 5.1 BCR Expression During B cell Development 25 5.2 Structure of the mlgM BCR 28 5.3 Structural Homology between mlgM and slgM 30 5.4 Structure of the Ig-a/Ig- p heterodimer 32 5.5 Function of the BCR 33 VI Assembly and Intracellular Trafficking of Secreted and Membrane Immunoglobulins 38 6.1 Assembly of Secreted Immunoglobulins (slgs) 38 6.2 ER Retention and Intracellular Trafficking of slgs 40 6.3 Requirements for Surface Expression of the BCR 41 6.4 The BCR as a Model System for Studying Quality Control in the ER 44 Materials and Methods 47 I Antibodies 47 II Cell Culture 48 III Cell Extracts 49 IV Coimmunoprecipitation 49 V Gel Electrophoresis 50 iii VI Western Blotting 51 VII Metabolic Labeling of Proteins for Steady State and Pulse/Chase Analysis 52 VIII Surface Biotinylation of Plasma Membrane Proteins 52 IX Surface Iodination of Plasma Membrane Proteins 53 Results 54 I Expression of the BCR Chains in WEHI 231 and WEHI 88.1 54 II Gycosylation Differences Between WEHI 231 and WEHI 88.1 56 III Associations Between BCR Subunits 61 IV mlgM Assembly Intermediates 65 V Ig-a and Ig-p Assembly Intermediates 68 VI Protein Expression on the Surface of WEHI 231 and WEHI 88.1 72 Discussion 74 List of Abbreviations 103 References 104 Appendix 121 iv List of Tables Table 1. Molecular Weight of BCR Protein Bands (kDa) in WEHI 231 And WEHI 88.1 56 Table 2. Predicted Molecular Weight of mlgM Assembly Intermediates 65 Table 3. Apparent Molecular Weights and Band Intensities of Protein Protein Bands of mlgM Assembly Intermediates of Figure 23 68 v LIST OF FIGURES Figure 1. Schematic Model of Protein Transport through the Secretory Pathway 2 Figure 2. Diagrammatic Model of Co-translational Translocation of Secretory Proteins Through the Mammalian ER Membrane 4 Figure 3. Model for GroEL Mediated Folding of Unfolded Polypeptides 10 Figure 4. Retention Model of Quality Control in the ER 17 Figure 5. Recycling Model of Quality Control in the ER 18 Figure 6. Model of Quality Control of Protein Folding and Oligomerization intheER : 20 Figure 7. Schematic Representation of Protein Degradation via the 26S Proteasome/Ubiquitination Pathway 23 Figure 8. Summary of B Cell Development 27 Figure 9. Model of the B Cell Antigen Receptor in WEHI 231 cells 29 Figure 10. Schematic Representation of mlgM, slgM, and pentameric slgM 31 Figure 11. Schematic of the u. Heavy Chain Transmembrane Region 33 Figure 12. Model of BCR Signaling Events 37 Figure 13. Models of the WEHI 231 and WEHI 88.1 B Cell Lymphomas 45 Figure 14. Expression of BCR Proteins in the Murine B Lymphomas WEHI 231 and WEHI 88.1 55 Figure 15. Treatment of WEHI 231 and WEHI 88.1 with PNGase F andEndoH 59 Figure 16. Diagrammatic Representation of Endo H Sensitivity and Resistance Along the Secretory Pathway 60 Figure 17. Association of u. Heavy Chain and K Light Chain in WEHI 231 and WEHI 88.1 62 Figure 18. BCR Subunit Associations in WEHI 231 and WEHI 88.1 64 Figure 19. Assembly Intermediates of mlgM in WEHI 231 and WEHI 88.1 67 Figure 20. Ig-a and Ig-p Oligomers in WEHI 231 and WEHI 88.1 70 Figure 21. Deglycosylated Assembly Intermediates of Ig-a and Ig-p Do Not Differ Between WEHI 231 and WEHI 88.1 71 Figure 22. Surface Biotinylation of WEHI 231 and WEHI 88.1 Cells 73 Figure 23. Maturation Model of the BCR in WEHI 231 and WEHI 88.1 Cells 86 Figure 24. Diagrammatic Representation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) 94 Figure 25. Schematic of Human Insulin Receptor (HIR) Structure 100 Figure 26. Maturation Model for Human Insulin Receptor 102 VI ACKNOWLEDGEMENTS I sincerely thank my thesis advisor, Dr. Linda Matsuuchi, for her valuable guidance, patience, dedication and mentoring skills. Thank you so much Linda. I'm grateful to have received wonderful support and teaching from Dr. Hugh Brock and Dr. James Berger through the years. My eternal gratitude to my parents, Jose Luis and Alicia Escribano, my sister Alicia and my brother Javier for all their love, encouragement, and support. Many thanks to Shaun Foy and Sharon Hourihane for their support and friendship. I will miss Jacob Hodgson whose wisdom and conversations fed my fascination and inspiration for science even at two o'clock in the morning. I wish to thank everyone in the Matsuuchi lab, with whom I had the pleasure of working, for their contributions and for their incredible teamwork. vii / dedicate this thesis to my grandmother, Ester Olvera, my aunt, Elia Renteria, and my cousin, Nancy Olvera. INTRODUCTION I. Quality Control and Protein Trafficking in the Secretory Pathway 1.1 Overview Multimeric plasma membrane receptors, such as the B cell antigen receptor (BCR), provide responses to external stimuli which are essential for the proper function and development of cells. Mistakes in receptor folding or assembly may lead to the expression of incomplete and non-functional receptors on the cell surface. Such faulty receptors may disrupt the cell's ability to respond to external stimuli and may hamper the cell's function. Since maintaining cell function is critical for proper development, the assembly and surface expression of multi-subunit membrane receptors must be tightly regulated. A fundamental problem in cell biology is to understand how cells regulate the surface expression of their receptors, especially during development. Since surface receptors are synthesized, folded, assembled, and transported to the plasma membrane within the secretory pathway (figure 1), cells must have evolved a mechanism of regulating receptor quality along this pathway. The current belief is that there is a quality control system in the secretory pathway that identifies improperly folded or unassembled receptors and prevents their trafficking to the cell surface. Mistakes in receptor folding and assembly are likely to occur in the endoplasmic reticulum (ER), where most of the protein folding and oligomerization take place. To minimize such errors, an ER quality control system is necessary. Although it is unclear how all steps in the ER quality control system operate, resident ER proteins may bind to the receptors and assist in the proper folding and assembly of receptors and the retention of misfolded or unassembled receptor components. Thus, understanding the processes of protein synthesis, folding, assembly and transport in the ER should reveal the cellular mechanisms of quality control of multimeric surface proteins. 1 Figure 1. Schematic Model of Protein Transport Through the Secretory Pathway. Secretory proteins enter the secretory pathway through the Endoplasmic Reticulum (ER). ER resident proteins participate in the folding and maturation of secretory proteins. Some of these ER resident proteins may also monitor protein quality by regulating protein transport through the secretory pathway. Properly folded and assembled proteins may transit from the ER to the Golgi in vesicles along the forward pathway (red arrows). Faulty proteins may remain in the ER or may return to the ER from the Golgi along the return pathway (blue arrows) as part of the quality control mechanism. Proteins in the Golgi are modified further and sorted into vesicles for transport to their final destination such as the lysosomes, via the endosomes, or the plasma membrane. Adapted from Alberts et al, 1994. 2 1.2 Co-translational Translocation of Membrane Proteins into the ER Most membrane proteins enter the secretory pathway through the ER by a process known as co-translational translocation (figure 2) (reviewed in Hegde and Lingappa, 1997). During co-translational translocation, membrane protein insertion into the ER membrane occurs as the membrane protein is being translated from mRNA on the ribosome. The mRNA:ribosome complex forms in the cytosol and must first be targeted to the cytosolic face of the ER membrane before co-translational translocation of the polypeptide into the ER membrane can occur. This is accomplished by the signal peptide recognition particle (SRP). Although additional mechanisms of targeting proteins to the secretory pathway exist in organisms like yeast (Ng et al, 1996; reviewed in Schatz and Dobberstein, 1996), the majority of translocation in higher eukaryotes is SRP-mediated and is well understood (reviewed in Walter and Johnson, 1994). The SRP recognizes and binds to a specific signal peptide that emerges from the ribosome during the initial translation of the mRNA. The binding of the SRP to the signal peptide momentarily blocks or slows polypeptide elongation possibly to allow sufficient time for mRNA:ribosome:SRP complex to reach the ER membrane. The mRNA:ribosome:SRP complex docks at the ER membrane via the signal recognition particle receptor (SRPR), an ER membrane protein that binds to the SRP. The signal peptide transfers from the SRP to other ER membrane proteins such as the signal sequence peptidase (SSP), which cleaves the signal peptide (Randall and Hardy, 1986), and TRAM in mammals (Voigt et al, 1996) or the Sec62/63p complex in yeast (Rothblatt et al, 1989) which associate with the translocation apparatus, the translocon. 3 Lumen Figure 2. Diagrammatic Model of Co-translational Translocation of Secretory Proteins Through the Mammalian ER Membrane. The signal recognition particle (SRP) serves two main functions in the co-translational translocation process. A. First, the SRP binds to the signal peptide and momentarily slows protein translation of the nascent polypeptide emerging from the ribosome:mRNA complex. B. Second, the SRP targets the ribosome:mRNA:polypeptide complex to the ER membrane by binding to the SRP receptor (SRPR). C. The polypeptide is transferred from the SRP to an early translocating protein called TRAM and the signal peptide is cleaved by signal peptide peptidase (SPP). D. The ribosome:mRNA:polypeptide complex is stabilized by the ribosome receptor at the ER membrane where the complex becomes tightly associated with the translocon and protein translation resumes. NAC is involved in the ribosome:translocon interaction. E. The translating polypeptide is translocated through this channel to the ER lumenal side where it is greeted by various ER proteins that aid in the folding and maturation of the nascent polypeptide. The components of this process are discussed later in the text. F. After translation is complete the ribosome is released from the surface of the ER membrane to the cytosol where the process is repeated. 4 The release of the SRP allows polypeptide elongation to resume but for co-translational translocation to proceed, the polypeptide must be inserted into the translocon. Insertion of the growing polypeptide into the translocon requires the binding of the ribosome to the ribosome receptor on the ER membrane. The ribosome receptor stabilizes the mRNA:ribosome complex at the ER membrane and brings the mRNA:ribosome complex into contact with the translocon. The nascent polypeptide-associated complex (NAC), which associates with translating ribosomes, may also be involved in mRNA:ribosome targeting at the translocon (Wiedmann et al., 1994). At this time, the translocon is open at its cytoplasmic surface but temporarily sealed on its ER lumenal side. The elongating polypeptide inserts into the protein channel of the translocon, a 20 A° aqueous pore which consists of various Sec proteins including mSec61p in mammalian cells (Hanein et al., 1996). A tight seal forms between the large subunit of the ribosome and the cytoplasmic surface of the translocon which now opens to the ER lumen. This closes off the translocon channel from the cytosol exposing the growing polypeptide to an ER lumenal environment rather than a cytosolic one. Once the polypeptide is inserted into the translocon channel and sealed off from the cytosolic enviroment, polypeptide translocation through the channel can occur during protein translation. As the ribosome translates the mRNA, the elongating polypeptide is translocated through the translocon into the ER lumen and the polypeptide's transmembrane regions are embedded into the ER membrane. The driving force for protein translocation across the ER membrane may come from the energy of protein synthesis pushing the elongating polypeptide unidirectionally though the translocon towards the ER lumen. An additional motor for the translocation process is likely to be ATPase driven by ER resident proteins associated with the translocon machinery and with the emerging polypeptide (Sanders et al, 5 1992; Corsi and Schekman, 1997). In fact, numerous proteins residing in the ER aid in the folding, processing, and assembly of the growing polypeptide at the ER lumenal side of the translocon. 1.3 Membrane Protein Folding and Modification in the ER Membrane proteins are folded, modified, and assembled as they travel through the secretory pathway to their target destination. The process begins during protein translocation into the ER. Although the primary amino acid sequence largely determines the tertiary structure of the protein (Afinsen, 1973), the large concentration of proteins in the ER lumen facilitates the folding process. As the nascent polypeptide grows it encounters several resident ER proteins which aid in its proper folding and modification. In fact, various co-and post-translational modifications in the ER, including N-linked glycosylation (Schlesinger and Schlesinger, 1987), disulfide bond formation (Freedman, 1987), proline isomerization (Freedman, 1987), glycolipid tail additions (Rosenberry et al, 1986; Ferguson and Williams, 1988), proteolytic cleavage (Laemmli, 1970; Habener et al, 1981; Chaudhuri et al, 1992), and other amino acid modifications, occur during the folding process (reviewed in Hurtley and Helenius, 1989). Kinetically, this allows for the folding of proteins in 3-4 minutes or less (Hurtley and Helenius, 1989). After attaining their proper tertiary structure in the ER, most membrane proteins require oligomerization and/or further modification in the Golgi or other secretory pathway compartments on their way to their final destination. Membrane proteins are believed to be transported to the cell surface by a default process unless retained in subcellular compartments by specific signals (Pfeffer and Rothman, 1987). Thus, fully assembled membrane proteins in the ER may travel through the Golgi, where they receive further modification, to their final destination such as the Golgi itself, the lysosomes, or the plasma membrane. 6 1.4 Membrane Protein Oligomerization Plasma membrane proteins are often oligomers of more than one polypeptide subunit. Oligomeric subunits may associate via their cytoplasmic, transmembrane, or extracellular domains. These subunit associations are restricted to compatible surfaces of the polypeptide subunits which usually contain key amino acids side chains in the contact regions of the subunits (Wu et al., 1983). Oligomers can range from simple homo-dimers to complex hetero-oligomers joined together by covalent and/or non-covalent interactions. Although covalent interactions enhance the stability of the protein, non-covalent interactions are more common. Non-covalent interactions also provide greater stability within the protein. For example, when hydrophobic amino acid side chains protrude from the contact surfaces of the subunits, subunit association minimizes the exposure of their hydrophobic regions to the hydrophilic environment and thereby stabilizes the protein. Other molecules like water or ions may associate with amino acid side chains between two subunits and enhance their association. Glycosylation may help to further stabilize the structure by covering an area of the protein surface that spans one or more subunits (Varghese et al., 1983). Thus plasma membrane proteins are composed of individual subunits which oligomerize to form stable quaternary structures. Oligomeric plasma membrane proteins usually assemble and fold into their quaternary protein structure in the ER. Often, the localization and function of the final protein product depends on this quaternary structure (Pontow et al., 1996; Matsuuchi et al., 1992; Cheng et al., 1990; Venkitamarin et al., 1990; Bonifacino et al., 1989; reviewed in Hurtley and Helenius, 1989; Kreis and Lodish, 1986; Gething et al., 1986). As such, incomplete membrane proteins which fail to oligomerize properly in the ER can be detrimental to proper cell function if allowed to reach their target destination. To prevent 7 this, quality control in the ER ensures that individual subunits are prevented from trafficking further through the secretory pathway until they have oligomerized into complete and functional proteins. Unlike the folding process, the kinetics of receptor oligomerization can be quite variable from one receptor to the next and range from several minutes to several hours (Doms et al., 1987; Olson and Lane, 1987; Carlin and Merlie, 1986; reviewed in Hurtley and Helenius, 1989). The molecular basis for these kinetic differences in protein assembly have not yet been accounted for but may be related to the physical constraints of the proteins themselves or the protein regulation process involved. For example, while nascent chains cannot oligomerize co-translationally, they can interact and associate with complete chains during their co-translational translocation into the ER (Bergman and Kuehl, 1979; Y u et al., 1983). These interactions may be facilitated by ER resident proteins which speed up the folding and oligomerization reactions. Some receptors, therefore, spend a longer time in the ER than others. Thus, completion of oligomerization in the ER may vary kinetically from one receptor to the next but it is a prerequisite for intracellular trafficking and this ensures quality control of membrane receptors. II. Molecular Chaperones 2.1 Overview Although many steps of the ER quality control system are unknown, it is widely accepted that several resident ER proteins are involved in the proper folding and assembly of receptors and the retention of misfolded or unassembled receptor components (reviewed in Gething and Sambrook, 1992; Hammond and Helenius, 1995; Melnick and Argon, 1995). These ER resident proteins have been characterized via their transient associations with membrane and secretory proteins as putative molecular chaperones of the ER. Molecular chaperones are proteins that temporarily bind to and stabilize unfolded or partially folded 8 structures of normative proteins and maintain them in a competent state for subsequent folding and assembly by releasing them in a controlled manner (reviewed in Hartl et al., 1994). Chaperone proteins are non-specific and can associate with more than one type of polypeptide by recognizing structural elements, such as hydrophobic regions, and not necessarily specific amino acid sequences. Chaperones may facilitate protein folding by protecting these exposed hydrophobic residues from nonproductive inter- and intramolecular aggregation. There are several classes of molecular chaperones including enzymes and members of the HSP60, HSP70, and HSP90 heat shock protein families (reviewed in Georgopoulous and Welch, 1993; Hendrick and Hartl, 1993; Jakob and Buchner, 1994). In the ER, the enzyme Protein Disulfide Isomerase (PDI) and the heat shock proteins BiP (H chain binding protein), Calnexin, GRP94 and GRP 170 behave like chaperones (reviewed in Jakob and Buchner, 1994; Melnick and Argon, 1995). These molecules and others associate with nascent polypeptides in the ER until complete assembly occurs or the normative polypeptide is targeted for degradation. The manner in which this association and dissociation occurs is not completely understood but it is believed to occur in a fashion analogous to the well-studied bacterial systems, HSP60(GroEL) and HSP70(DnaK) (reviewed in Beissinger and Buchner, 1998), for which eukaryotic homologues such as TriC are known (Hartl, 1996). For example, bacterial HSP60 and HSP70 chaperones may act sequentially and in an ATP-dependent manner (reviewed in Bukau et al., 2000) and evidence of sequential action and ATP-dependence of mammalian chaperones is also accumulating (reviewed in Melnick and Argon, 1995). For comparison of prokaryotic and eukaryotic systems, the following sections provide a brief summary of the bacterial chaperone system and some of the key putative mammalian chaperones believed to operate in the ER of eukaryotes. 9 2.2 Bacterial Models of Molecular Chaperones In bacteria such as E. coli, two groups of chaperones, HSP60(GroEL) and HSP70(DnaK), may act sequentially and in an ATP-dependent manner to assist in the folding of nascent proteins (reviewed in Bukau et al., 2000). GroEL consists of two stacked heptameric rings of 14 identical 57kD subunits which form a hollow cylinder, 137 A in diameter and 146 A in height, with two binding chambers for unfolded proteins (reviewed in Hartl, 1994; Hartl, 1996; Bessinger and Buchner, 1998). An unfolded polypeptide, 10-55kD, binds into one chamber of the central cavity of the GroEL cylinder. The co-chaperone GroES, a dome-shaped ring of seven lOkD subunits, binds to the opening of the GroEL chamber and seals off the polypeptide from the cytosolic environment for folding to occur. GroEL with GroES, ADP, and unfolded protein bound on one side leads to the high affinity binding of a second normative protein at the other GroEL chamber. Subsequent association of ATP and a second GroES at the other GroEL chamber leads to ATP hydrolysis and release of GroES, ADP, and substrate from the opposite chamber. Several rounds of ATP-dependent release and rebinding aided by GroES result in correct folding (figure 3). GroES GroES GroES ADP ATP ADP GroEL 0 unfolded polypeptide polypeptide folded Figure 3. Model for GroEL Mediated Folding of Unfolded Polypeptides. GroES is not illustrated in the above figure but is proposed to act as a cap for GroEL to enclose the unfolded polypeptide in a protective environment so it can fold. This figure was taken from Foy, 1997. 10 Unlike GroEL, DnaK is a monomeric 70kD protein containing a 44kD N-terminal ATP-binding domain and a 27kD C-terminal substrate peptide-binding domain. DnaK binds short, linear hydrophobic sequences that protrude into a large hydrophobic pocket on the floor of the peptide-binding channel. The peptide-binding domain exists in one of two conformations, a low substrate affinity, ATP-bound, open state or a high substrate affinity, ADP-bound, closed state. Peptide binding to the open state of DnaK leads to ATP hydrolysis and a transition to the ADP-bound closed state of DnaK. The co-chaperone, GrpE, promotes the release of ADP from DnaK so a new ATP molecule can bind and shift DnaK back to its open conformation. After ATP-binding, peptide is released and a new round of peptide binding and release can occur. Although GroEL and DnaK differ in structure and substrate specificity, both chaperones require ATP binding and hydrolysis and co-chaperones for the association and dissociation of their substrates. Receptor folding and assembly in the ER may involve quality control systems like the GroEL/GroES and DnaK/GrpE systems in bacteria. ATP-dependent ER chaperones may bind to exposed hydrophobic regions of the receptor subunits and hold them in the ER until the chaperones are displaced by other receptor subunits that associate at the hydrophobic areas (Bonnerot et al, 1994). Once all of the chaperones are displaced and the receptor is fully assembled, it is free to traffick to the cell surface. Such chaperones may be part of an ER quality control mechanism. ER chaperones may operate according to the retention and/or recycling models mentioned in the next section. 2.3 Protein Disulfide Isomerase (PDI) Protein disulfide isomerase (PDI) is one of the earliest putative chaperones encountered by a nascent polypeptide entering the ER (Wang and Tsou, 1993; Weissman and Kim, 1993; Lamantia and Lennarz, 1993; Noiva et al, 1993; Otsu et al, 1994; Puig and Gilbert, 1994). 11 PDI is an abundant protein found ubiquitously in the lumen of the ER. It serves various functions (Noiva and Lennarz, 1992) but was originally and best characterized for its ability to catalyze the formation of disulfide bonds during protein biogenesis (Freedman ei al., 1989). This disulfide isomerase function of PDI is a major rate-limiting step in protein folding (Freedman 1992; Zapun et al., 1992). PDI may also assist in the folding of proteins that either contain disulfide bonds (Otsu et al., 1994; Puig et al., 1994) or lack them (Cai et al., 1994). However, the chaperone-like activity of PDI remains less understood than its disulfide isomerase activity. 2.4 Immunoglobulin Binding Protein (BiP) The chaperone-like activity of immunoglobulin binding protein (BiP) is better understood than that of PDI or any other ER protein (reviewed in Gething and Sambrook, 1992). BiP was originally identified through its coprecipitation with Ig heavy chains that accumulate in the ER of myeloma cells lacking Ig light chain (Morrison and Scharff, 1975) or in pre-B cells lacking Ig light chain (Haas and Wabl, 1983). BiP is a non-glycosylated soluble protein of approximately 78kDa that remains enriched in the lumen of the ER due to a KDEL recycling signal at its carboxyl terminus (Pelham, 1988) that associates with the hydrophobic region of the CHI domain of the H chain (Bole et al, 1986; Hendershot et al, 1987; Hendershot, 1990). BiP is believed to bind reversibly to various nascent polypeptide chains as they are translocated into the ER (Dorner et al, 1987; Vogel et al, 1990; Sanders et ah, 1992) and hold them in an appropriate configuration until proper folding and assembly are complete, perhaps even facilitating these processes (Bole et al., 1986; Gething et al, 1986; Pelham, 1986). BiP may also serve in the retrieval of misfolded proteins which have escaped from the ER to the cis-Golgi (Hammond and Helenius, 1994). BiP exhibits weak ATPase activity (Hendershot et al., 1988; Kassenbrock and Kelly, 1989) and it can be post-12 translationally modified by both phosphorylation and ADP ribosylation (Carlsson and Lazarides, 1983; Welch et al, 1983). Although BiP's ATPase activity is not required for protein binding (Gaut and Hendershot, 1993), dissociation from BiP is believed to be ATP dependent since adding excess ATP reduces association with BiP while ATP depletion leads to prolonged association with BiP (Frieden et al., 1992; Hendershot et al., 1995; Dorner et al., 1990). Thus, it is possible that BiP may undergo several rounds of association and dissociation from its ligands so that more than one BiP molecule may be associated with the same ligand over time. The BiP binding site is located in its carboxy-terminus (Munro and Pelham, 1984; Hendershot et al, 1995). BiP binds to two types of ligand (Munro and Pelham, 1986). One type of ligand is a subunit of a multimeric protein (Bole et al., 1986). This ligand usually interacts only briefly or transiently. The other type of ligand includes polypeptides that are unable to undergo post-translational modifications or proper oligomerization required for proper and complete assembly into multimeric proteins (Gething et al., 1986; Dorner et al., 1987). These ligands associate more stably or permanently with BiP and accumulate in the ER. Thus, BiP associates transiently with various nascent secreted and membrane proteins while forming more stable complexes with mutant, non-transported variants of these proteins (Gething et al, 1986; Dorner et al, 1987). 2.5 Calnexin Calnexin is an integral membrane protein that resides in the ER and acts as a chaperone molecule (Degen and Williams, 1991; Hochstenbach et al, 1992). It has been best categorized for its association with the T cell receptor (TCR) and major histocompatibility complex (MH chain) subunits during complex assembly (Rajagopalan et al, 1994; Rajagopalan and Brenner, 1994). Protein association with Calnexin is more 13 prolonged than with other ER chaperones and Calnexin is believed to be one of the last chaperones to dissociate from the membrane protein complex. Unlike BiP, Calnexin does not likely associate immediately with nascent polypeptides since it requires the presence of N-linked carbohydrates in order to associate (Hammond and Helenius, 1994b; Hammond et al, 1994; Ou et al, 1993; reviewed in Fiedler and Simons, 1995). Dissociation of hemagglutin from Calnexin requires glucose frirnming (Hebert et al, 1995). Also, unlike BiP, Calnexin is a membrane spanning protein (David et al, 1993) and its interactions with proteins are likely to involve interactions between transmembrane regions (Fiedler and Simons, 1995). Furthermore, studies of thyroglobulin and VSV G protein folding in the ER indicate a sequential interaction of BiP and Calnexin with thyroglobulin and VSV G assembly intermediates (Hammond and Helenius, 1994b; Kim and Arvan, 1995). Association with BiP is followed by BiP release and subsequent association with Calnexin. 2.6 Glucose Response Protein (GRP94) GRP94 is a homologue of the Hsp90 family of heat shock proteins (reviewed in Jakob and Buchner, 1994). Like BiP, GRP94 is an abundant ER lumenal protein containing an ammo-terminal ER targeting sequence and a carboxy-terminal ER retention motif, KDEL. Further, GRP94 possesses low ATPase activity similar to that of BiP but the purpose of this activity remains unclear. In addition to BiP, GRP94 has been shown to bind immunoglobulin H and L chains prior to their assembly in the ER (Melnick et al, 1992). In fact, recent evidence suggests a sequential interaction of BiP and GRP94 with immunoglobulin H chain occurs similar to that of BiP and Calnexin (Melnick et al, 1994). 2.7 Glucose Response Protein (GRP 170) Studies by Nilsson and Warren, 1994, indicate that another GRP protein localized in the ER associates with the H chains and L chains of both membrane and secretory 14 immunoglobulins. More recently, 170kDa protein, GRPT70, has been found to co-precipitate with both immunoglobulin chains and with GRP94 and BiP (Lin et al, 1993). Further studies to elucidate the role and mechanism of GRP170 as a chaperone of protein folding or assembly in the ER and its affiliation with other chaperones are necessary. 2.8 Summary Thus, the ER contains several different chaperone molecules that may act independently or in concert to allow nascent polypeptides to achieve their proper conformation. BiP, GRP94, and Calnexin are putative ER chaperones that associate with various proteins in the ER sequentially. ER chaperones may operate by binding transiently to nascent polypeptides, releasing them so they can fold/assemble into multimeric complexes and/or exit from the ER, but binding permanently to malfolded or incompletely assembled proteins (Gething and Sambrook, 1992; Hammond and Helenius, 1994a). The chaperones themselves are maintained in the ER by receptors that bind to specific ER retention sequences on the chaperones and either anchor the chaperone within the ER or recycle them continuously from the cis-Golgi to the ER (Pfeffer and Rothman, 1987; Pelham, 1988). As mentioned earlier, BiP contains a KDEL retention signal while Calnexin contains a di-lysine retention motif. These motifs retain the chaperones in the ER. The chaperones, in turn, retain the proteins they bind in the ER. Thus, chaperones may help control the quality of membrane proteins expressed at the cell surface. III. Models of Quality Control in the ER 3.1 Overview Only correctly assembled and fully functional membrane proteins typically reach their destination (Gelman et al, 1995; Hilton et al, 1995; Sagherian et al., 1994; Lederkremer and Lodish 1991; Amara et al, 1989; Rotundo, 1989; Esser and Russell, 1988; Doyle et al, 15 1986). Otherwise, mutations, unbalanced subunit synthesis or inefficient folding and assembly would lead to the expression of faulty membrane proteins which could prevent the cell from functioning properly (Cheng et al., 1990). Several lines of evidence suggest that the cell must possess a quality control system to ensure that only correct complexes are expressed at the desired destination (reviewed in Hammond and Helenius, 1995; Wei and Hendershot, 1996). Such a system would require the ability to recognize proteins unsuitable for transport and a method for eliminating or correcting them. Two models have been proposed to describe a possible quality control mechanism for the cell involving co-localized signals for assembly and retention/degradation (reviewed in Hammond and Helenius, 1995; Nilsson and Warren, 1994), one involving retention of non-functional proteins in the ER and the other involving recycling of non-functional proteins between the cis Golgi and the ER (figures 4 and 5). Both models depict the ER as the location for quality control and involve the presence of one or more resident ER proteins that act as chaperones. Although either model alone may account for quality control of membrane proteins, it is more likely that a combination of the two models occurs simultaneously. 3.2 Retention Model of Quality Control in the ER According to the retention model of quality control, shown in figure 4, an ER resident protein, such as BiP or Calnexin, recognizes and associates with an exposed internal or unfolded region on the ER lumenal domain of the membrane protein. This association serves three functions. First, it prevents the lumenal domain of the membrane proteins from improperly folding to hide hydrophobic regions necessary for interaction with other proteins. Second, it allows folding or assembly intermediates more time in the optimal environment of the ER for structural maturation into a multimeric complex to occur. Third, it prevents unmodified, misfolded, and unassembled proteins from leaving the ER. Upon proper 16 assembly, the ER resident protein is displaced and the membrane protein is free to exit the ER. Thus, one method of ER retention involves the reversible interaction of a nascent polypeptide with a protein that never leaves the ER. Figure 4. Retention Model of Quality Control in the ER. A resident protein of the ER acts as a chaperone molecule by associating with newly synthesized proteins in the ER until full assembly and folding occurs. The chaperone is released from the assembled protein which is free to transit through the secretory system to its final destination. The chaperone is also free to associate with another incomplete protein in the ER and prevent its further transport through the secretory pathway until it is properly assembled. 3.3 Recycling Model of Quality Control in the ER The recycling model of quality control, figure 5, is also based on the recognition of an amino acid sequence or an exposed internal region on the membrane protein by an ER resident protein. However, the ER retention signal is present in the cytoplasmic (Nilsson et ah, 1989) or transmembrane (Bonifacino et al., 1991) region of the protein and the signal is recognized by a transmembrane ER resident protein which can recycle between the ER and the cis Golgi compartment (Pelham, 1990; Jackson et al., 1993). This ensures that misfolded or unassembled membrane proteins that mistakenly escaped ER retention and transited to the 17 Golgi can be recognized and picked up in the Golgi and then transported back to the ER. Evidence for this model comes from studies of the KDEL and di-lysine retention motifs in mammalian ER resident proteins (Munro and Pelham, 1986; Jackson et al, 1990; Cosson and Letourneur, 1994; reviewed in Nilsson and Warren, 1994). The KDEL retention motif is made up of lysine (K), aspartic acid (D), glutamic acid (E), and leucine (L) amino acid residues. The presence of a KDEL motif at the COOH-terminal end of a soluble protein such as the immunoglobulin binding protein (BiP) can be recognized by a transmembrane protein, known as the KDEL receptor, that cycles between the ER and the Golgi (Munro and Pelham, 1987; Hardwick et al, 1990; Lewis et al, 1990; Semenza et al, 1990; Hammond and Helenius, 1994a). This allows efficient retention of the soluble protein in the ER. Similarly, Golgi/Intermediate compartment unassembled complex is returned to the ER \ ER resident proteins' mem proieins^ o o I Cell Surface Unassembled complex + ER resident proteins assembled complex Figure 5. Recycling Model of Quality Control in the ER. A membrane-bound ER resident protein associates with unassembled or unfolded proteins in the ER until they are completely folded and assembled. Upon release from the resident ER proteins, the complete protein is free to travel through the secretory system. Unreleased proteins are recycled back to the ER via their association with the ER resident proteins. These ER resident proteins act as chaperones which retreive improper proteins that might escape from the ER. 18 the di-lysine motif, which consists of two lysine residues at positions -3 and -4 from the COOH-terminus of the cytoplasmic domain of some membrane proteins including the K D E L receptor and Calnexin, allows the ER retrieval of di-lysine-tagged proteins from post-ER compartments. This retrieval involves the interaction of the di-lysine ER retention motif with the coatomer, a polypeptide complex froming the coat on recycling vesicles (Cosson and Letourneur, 1994; Letourneur et al., 1994). Thus, a second method of ER retention involves recycling of tagged proteins between the ER and cis-Golgi compartments. 3.4 Summary In either scenario, certain features are common and necessary for quality control in the ER (figure 6). Nascent polypeptides must associate with molecular chaperones during ER translocation and during each step of protein folding and oligomerization to hide exposed internal domains that contain co-localized signals for assembly and degradation. Polypeptide folding and release from the translocon allows the dissociation of some molecular chaperones. Failure to fold properly may prevent chaperone dissociation. Only properly folded polypeptides are free to assemble into complete receptors. Correct assembly allows complete dissociation from chaperones and packaging into transport vesicles. Mutations causing limiting amounts of subunits or the absence of subunits, or that cause alterations in the rate of protein folding could detain the release of nascent chains from the translocon and/or from chaperones. Misfolded and unassembled polypeptides thereby remain associated with chaperones and are retained in the ER until proper folding and assembly occur or until they are targeted for degradation. Thus, ER chaperones play a central role in the quality control models of protein regulation and a combination of both models is likely to exist. 19 A. AUG mRNA Ribosome B. SRP I Signal Peptide SPP C. Oligosaccharyl-Transferase Correct Folding and Oligomerization Incorrect Folding and Oligomerization 1 Release of Chaperones Association with Chaperones i Transport through the Secretory Pathway Retention/Recycling by E R Quality Control Mechanism Figure 6. Model of Quality Control of Protein Folding Oligomerization in the ER. A. Membrane proteins enter the secretory pathway by co-transla-tional translocation into the ER. B. In the ER lumen, the emerging polypeptide associates with various ER resident proteins involved in the folding, modification, and oligomerization of the nascent polypeptide. C. Some of these ER resident proteins are putative chaperones in the Quality Control System that prevents the transport of incomplete or faulty proteins (see text). 20 IV. Degradation of Proteins in the ER Quality control in the ER ensures that faulty proteins are retained intracellularly and prevented from trafficking further through the secretory system. Since cells are continuously synthesizing proteins, ER retention would result in the overwhelming accumulation of aberrant proteins. To avoid this, cells have evolved mechanisms for protein degradation to remove unnecesssary proteins as the final outcome of quality control by the cell. Such proteolytic systems must be able to distinguish between native and aberrant proteins in the ER (Lippincott-Schwartz et al., 1988). Unlike the degradation of soluble proteins containing sequences that direct them to the lysosome or vacuoles for destruction, the degradation of many ER proteins, including the unassembled components of the T cell antigen receptor (Bonifacino et al., 1990a,b; Shin et al., 1993), is not well understood (Klausner and Sitia, 1990; Bonifacino and Lippincott-Schwartz, 1991; Bonifacino and Klausner, 1994). In fact, how or where the ER quality control system targets proteins for disposal is unknown. Our understanding of ER protein degradation is further complicated by the fact that some proteins are retained in the ER longer than others prior to their destruction. The reason some proteins undergo rapid degradation while others do not is uncertain. It is possible that certain protein domains or modifications may confer an ability to remain in the ER longer to accommodate proteins whose folding and assembly may be kinetically slower. Eventually however, rhisfolded and incomplete proteins retained in the ER are targeted for degradation. It was previously assumed that exposed regions of the protein, which are normally hidden during correct folding and assembly or through interaction with chaperones, may be recognized by the degradation apparatus in the ER (Bonifacino and Lippincott-Schwartz, 1991). The existence of a degradation apparatus in the ER, however, is becoming increasingly unlikely for several reasons. Few proteolytic enymes, besides the ER 60 family 21 of cysteine proteases that are involved in the degradation of resident ER proteins (Otsu et al, 1995), have been identified as proteases responsible for the degradation of misfolded proteins in the ER (Urade et al, 1992). Unidentified serine proteases may be involved in the degradation of IgK (Gardner et al, 1993) and unassembled H2 subunits of the asialoglycoprotein receptor (Wikstom and Lodish, 1991). Nonetheless, the presence of proteolytic enzymes in the ER would likely interfere in the normal folding/assembly of proteins unless the proteases were confined to a special region of the ER. Further, there is mounting existence for the involvement of the 26S proteasome. The mechanism of ER protein degradation remains elusive but several lines of evidence suggest the involvement of the 26S proteasome (figure 7), (reviewed by Lord, 1996; Ward and Kopito, 1994, Kopito, 1997). Proteins are first marked for proteasomal degradation by the covalent addition of polymers of the 76 amino acid ubiquitin protein on exposed lysine residues of the targeted protein. Multi-ubiquitination acts as a protein tag that rapidly directs the doomed protein into the proteasome for degradation. The proteasome itself is a cylindrical complex consisting of a 19S cap which recognizes multiubiquitin-tagged proteins and directs them into a 20S catalytic core that cleaves proteins into peptides. The degradation of cytosolic proteins by the proteasome is well documented (reviewed by Coux et al, 1996; Weissman, 1997), however, the role of the proteasome in ER protein degradation has only recently been discovered (reviewed in Lord, 1996). 22 M u l t i " Peptides ubiquitin 20S Catalytic Core and Figure 7. Schematic Representation of Protein Degradation via the 26S Proteasome/Ubiquitination Pathway. Proteins are targeted for proteasomal degradation by the covalent attachment of multi-ubiquitin chains throught the action of ubiquitin-conjugating enzymes. The 26S proteasome is a cylindrical structure consisting of a central 20S catalytic core capped at one or both ends by a 19S complex. Multi-ubiquitinated proteins are recognized by the 19S cap. The 19S cap contains ATPase activity which unfolds the protein targeted for degradation and feeds them into the 20S catalytic core. The 20S catalytic core degrades the proteins into short peptides of 7-9 amino acid residues. Lactacystin, a proteasome inhibitor, blocks the degradation of a mutant cystic fibrosis transmembrane conductance regulator (CFTR), (Ward et al, 1995). This results in the accumulation of multi-ubiquitinated CFTR chains in the ER (Ward et al., 1995). Lactacystin also prevents the degradation of lumenal mutants in the ER, a i-antitrypsin and yeast prepro-a-factor (Qu et al., 1996; Werner et al., 1996). Similarly, loss of function mutations in the proteasome or other components of the proteasome degradation pathway block the ER protein degradation of several proteins. For instance, mutations in the 20S catalytic core (PRE1) or the 19S cap (CIM3) of the proteasome block the degradation of mutant Sec61p and yeast carboxypeptidase Y (CPY*) (Bierderer et al., 1996; Hiller et al, 1996). These studies also show that Sec61p and CPY* turnover requires UBC 6 and UBC 7, the ubiquitin conjugating components of the 26S proteasome degradation pathway (Bierderer et ah, 1996; Hiller et al., 1996). Similar results were seen for 3-hydroxy-3-methylglutaryl-CoA reductase 23 (HMG-R) under coexpression of mutants of putative components of the 26S proteasome (Hampton et al, 1996). Moreover, UbK48R, a dominant negative mutant of ubiquitin that fails to form multiubiquitin chains, and the conditional inactivation of the ubiquitin-conjugating enzyme, E l , cause the accumulation of mutant CFTR chains in the ER (Ward et al, 1995). UbK48R also disrupts the degradation of Sec61p and CPY* (Bierderer et al, 1996; Hiller et al, 1996). Thus, the collection of substrate degradation proteins in the ER during coexpression of mutants or inhibition of the proteasome, or components of the proteasome degradation pathway, suggests that the 26S proteasome is involved in ER protein degradation. Proteasomes are present in the cytoplasm and the nucleus but not in the ER. Therefore, ER protein degradation via the proteasome must occur in the cytoplasm or at the cytoplasmic face of the ER membrane. Consistent with this notion, components of the ubiquination process are associated with the cytoplasmc surface of the ER membrane and ubiquitination is required for dislocation to the cytosol and subsequent proteasomal degradation (Briederer et al, 1996). This suggests that ER proteins may be targeted for proteasomal degradation by ubiquitination and subsequently translocated across the ER membrane into the cytoplasm. Evidence for retrograde transport of proteins across the ER membrane comes from studies on toxins like ricin or Shiga which are endocytosed into the secretory pathway where they travel retroactively to the ER and are translocated into the cytoplasm (reviewed in Sandvig and van Deurs, 1994). More recently, studies by Wiertz et al, 1996a and b, demonstrate that MH chain class I molecules in the ER are associated with the Sec61 complex of the translocon immediately proceeding their transfer to the cytosol for proteasome degradation by human cytomegalovirus (CMV) proteins US2 and US11. The translocon may provide a mechanism for retrograde as well as anterograde translocation of 24 proteins across the ER membrane. A genetic interaction between Sec61 and UBC 6, an ubiquitin-conjugating enzyme associated with the cytoplasmic face of the ER, further implicates the translocon in the proteasomal degradation pathway of ER proteins (Ward et al., 1995). Other components of the ER quality control system have been identified in association with protein turnover including Calnexin (McCracken and Brodsky, 1996). Also, the ER chaperone BiP, which associates with the components of the translocon complex, contains substrate binding sites that co-localize with signals required for the degradation of misfolded proinsulin in the ER (Schmitz et al., 1995). Evidence is accumulating which supports the possibility that one mechanism of ER protein degradation occurs via an ubiquitin/proteasome pathway involving the retrograde transport of protein across the ER membrane, presumably through the translocon (see discussion). V. The B cell Antigen Receptor 5.1 BCR Expression During B Cell Development B cell development proceeds through several stages of differentiation (figure 8) (reviewed in Gold and Matsuuchi, 1994). Each stage of B cell differentiation is identifiable by changes in the expression of particular surface markers, including changes in the rearrangement and expression of immunoglobulin H and L chain genes. Other markers of B cell development include changes in growth factor dependence, cell size, and location. B cells arise from bone marrow stem cells which differentiate into early pro-B cells expressing the early marker CD45R. Although early pro-B cells occur prior to immunoglobulin heavy (H) chain gene rearrangement and therefore cannot express the complete BCR, the Iga/IgP complex is present on the cell surface of these pro-B cells (Koyama et al, 1997; Nagata et al., 1997). By the late pro-B cell stage though, the last step of H chain gene rearrangement has occurred and Ff chain expression follows. At the pre-B cell stage, H chain forms a pre-25 BCR complex expressed on the cell surface. Surface expression of the pre-BCR requires its association with the Iga/IgP signaling components of the BCR. Iga and IgP are expressed from the pro-B cell stage until cell death or its differentiation into a plasma cell. The pre-BCR complex also consists of surrogate light (L) chain, containing the X5 and v-preB proteins which precede regular L chain expression. Without the surrogate light chain, the H chain remains intracellular and further development does not occur suggesting that assembly into a pre-BCR complex is necessary for p H chain surface expression and subsequent signaling through the pre-BCR. These signals may lead to the progression from a pre-B cell to an immature B cell capable of producing L chains and expressing Iga/IgP and the membrane IgM (mlgM) form of the BCR. Later, mature B cells can express two forms of the BCR at the cell surface, one containing mlgM and the second containing membrane IgD (mlgD) which consists of 8 H chain. 26 Stem cell Early pro-B cell D H - J H rearrangement a Late pro-B cell FPr V H - D J H rearrangement Ig-a/ Ig-Large pre-BCR cell Plasma Cell and Memory B Cell Memory Cell mlgG I f Plasma Cell pre-BCR receptor Small pre-B cell V L - J L rearrangement Figure 8. Summary of B Cell Development. B cell development proceeds through various stages of differentiation characterized by the rearrangement and expression of the immunoglobulin genes. First, the heavy chain genes undergo rearrangement and then the light chain genes. B cell development begins in the bone marrow and by the mature B cell stage, the B cells move to the periphery. Adapted from Janeway, 1994. Signals from other lymphocytes and signaling through the BCR on mature B cells may give rise to antibody secreting plasma cells and longer-lived memory B cells or may result in cell death (reviewed in Reth, 1992). Plasma cells do not generally express membrane immunogloubulin on their cell surface while memory B cells do. Terminally differentiated plasma cells begin secreting IgM antibodies but can later switch to producing IgG, IgA or IgE antibodies by a process called isotype switching; IgM, IgD, IgG, IgA, and IgE consist of different H chain isotypes and may exist in either the membrane-bound or secreted form. For example, when memory B cells are produced they may express membrane IgG, IgA or IgE surface receptors instead of the usual mlgM and mlgD forms of the BCR found in mature B cells. The tight temporal regulation of the expression of 27 immunoglobulin genes and proteins associated with their expression, structure, or function suggests a progressive sequence of events take place during B cell differentiation. The expression of membrane and secretory Igs is regulated during B cell development not only at the levels of transcription and post-transcriptional processing but also at the protein level during intracellular transport. A comparison of the signals and mechanisms that influence retention and transport of secreted vs. non-secreted Igs offers a unique opportunity to study the process of quality control of protein assembly and intracellular trafficking. 5.2 Structure of the mlgM BCR In immature B cells, the BCR consists of four different polypeptide chains, p heavy (H) chain, K or X light (L) chain, Ig-ct and Ig-p (figure 9) (reviewed in DeFranco, 1993; Reth, 1992). Two disulfide-linked p H chains are each covalently associated with a L chain to form mlgM. Each L chain contains about 220 amino acids, weighs approximately 22kDa, and is not usually glycosylated. Each p H chain has approximately 440 amino acids and a molecular weight range of approximately 70-78kDa. mlgM is non-covalently associated with one or more disulfide-linked Ig-a/Ig-p heterodimers. Murine (mouse) Ig-a and Ig-8 range in molecular weight between 28-35kDa and 30-45kDa, respectively. Molecular weight ranges are due to the differential glycosylation of H chains, Ig-a and Ig-p. Thus, the BCR is a multimeric receptor made up of mlgM and Ig-a/Ig-P heterodimers. 28 Heavy Chain Protein Light Chain Protein Extracellular Cytoplasmic STOP* Heterodimer Ig-alpha/beta Figured Model of the B Cell Antigen Receptor in WEHI 231. Two p H chains are covalently attached to two K L chains to form mlgM. mlgM forms the antigen binding region of the BCR and associates non-covalently with one or more Ig-a/Ig-(3 heterodimers, the signal transduction components of the BCR. 29 5.3 Structural Homology between mlgM and slgM There are several structural similarities and differences between membrane IgM (mlgM) and secreted IgM (slgM) (figure 10). mlgM consists of two p H chains and two L chains covalently associated each other. The H and L chains contain variable and conserved constant regions. The H and L chain variable regions are located at the NH2-terminus of the chains and together they form a specific antigen binding pocket. Since each mlgM molecule contains two H/L chain dimers, there are two antigen binding pockets per molecule. Similarly, slgM consists of two H chains and two L chains with the same antigen binding specificity as mlgM. However, slgM differs from mlgM in that slgM forms a pentamer of IgM held together by a common joining chain (J chain) (figure 10). slgM also differs in that it lacks the C-terminus extracellular spacer, the 25 amino acid transmembrane domain and the short cytoplasmic domain of the p H chain found in mlgM. mlgM and slgM are generated by alternative mRNA splicing of different exons of the H chain gene that encode the additional membrane mlgM domains (Alt et al., 1980; Rogers et al, 1980; reviewed in Rogers and Wall, 1984). Due to these additional domains, mlgM is capable of association with the Ig-a/Ig-(3 heterodimer while sig is not. Thus, despite the structural differences between mlgM and slgM, mlgM shares extensive structural homology with the slgM produced by differentiated daughter cells. 30 Membrane IgM Secreted IgM Pentameric slgM Figure 10. Schematic Representation of mlgM, slgM, and pentameric slgM. mlgM contains the same variable (V) and constant (C) regions in its H and L chains as slgM. Both mlgM and slgM have one V region and four C regions in their p H chain. mlgM possesses in its carboxy-terminal an additional transmembrane region lacking in slgM. The cysteine residues at positions 337, 414, and 575 allow slgM to form a pentamer linked by a joining (J) chain. Adapted from Janeway, 1994. 31 5.4 Structure of the Ig-a /Ig-P Heterodimer Purification and N-terminal sequencing of Ig-a and Ig-p proteins indicate that they are the products of the mb-1 and B29 genes, respectively (Campbell et al, 1991; Hombach et al, 1990). B29 is expressed throughout the B-cell lineage from pro-B to plasma cells (Hermanson et al., 1988). Mb-1 is expressed in pro-B and mature B cell stages, but not in differentiated myeloma/plasma cells (Sakaguchi et al., 1988; ). Both gene products contain a single extracellular Ig-like domain and a single transmembrane domain. The cytoplasmic tail of Ig-a contains 61 amino acids while Ig-P contains 48 residues. Both proteins contain a 26 amino acid sequence motif called the intracellular tyrosine activation motif (ITAM) (Reth, 1989). This motif is important in coupling the Ig-a/Ig-P heterodimer to cytoplasmic signaling molecules containing SH2 domains such as Src kinases. Association with Ig-a and Ig-P is believed to be mediated at least in part via interactions between the transmembrane domains of H chains and Ig-a and Ig-B. The H chain transmembrane region is most likely an alpha helix in which all polar groups of amino acid side chains are hydrogen-bonded inside the helix for thermodynamic stability within the hydrophobic environment of the lipid bilayer. In the mouse, thirteen out of the 25 amino acids of the transmembrane H chain domain are highly conserved between the different isotypes and 11 of these conserved residues lie on one side of the a-helix (figure 11). Nine hydrophilic residues (serine and threonine) are present within the H chain transmembrane domain. These findings suggests that the conserved transmembrane residues are interacting with each other or with other transmembrane proteins. 32 A) B) Figure 11. Schematic of the u Heavy Chain Transmembrane Region. (A) The transmembrane region of n heavy chain drawn as an a-helix. The 13th residue of the ml domain of n heavy chain is at the lumenal end of the transmembrane region and the 38th residue is at the cytosolic end. (B) Top view of a-helix of the membrane spanning region. Circled residues are identical in the transmembrane regions of at least 7 of the 8 murine heavy chain isotypes (Blum, 1991). Polar residues lie on one side of the mlgM transmembrane region. The single amino acid code used is A, alanine; C , cysteine; D, aspartate; E, glutamate; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M , methionine; N , asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine. Taken from Foy, 1997. Contact between the H chain dimer and the Ig-a/Ig-P heterodimer is likely to be mediated via Ig-a since its transmembrane domain contains polar residues on both sides of the a-helix while Ig-P only contains polar residues on one side (Reth, 1992). Yet, the extracellular domains of these proteins may be important for complex formation since deletion of the CH3 and the CH4 domains of the p H chain prevents association of H chain with the Ig-a/Ig-P heterodimer. 5.5 Function of the B C R The humoral immune response involves the production of antibodies against foreign substances, called antigens, present in the bloodstream or on the surface of cells. This 33 response is initiated by the binding of a specific antigen to the B cell antigen receptor (BCR). The BCR is responsible for the recognition of and the response to antigens by B cells. The BCR serves two important functions in response to antigen binding (reviewed in Kishimoto and Hirano, 1988). First, it binds and internalizes specific antigens for processing and presentation to helper T cells. Second, it sends a signal to the nucleus leading to either cell growth or cell death. Mutation of the H chain cytoplasmic domain and certain residues in the transmembrane domain demonstrate that these regions are necessary for the two functions of the BCR and that they may be mediated by different protein interactions (Shaw et al, 1990). Further, without these two events the B cell is unable to produce the antibodies needed to eliminate the pathogens. Thus, the BCR is necessary for both endocytosis and signal transduction in response to antigen binding and the proper function of the BCR is critical in launching a humoral immune response. Antigen presentation involves receptor-mediated endocytosis of the entire antigen:BCR complex into endocytic vesicles. The antigen:BCR complex is then targeted to specialized endosomal compartments (CII) for processing, where the antigen is partially degraded into peptide fragments which associate with major histocompatability complex (MHC) class II molecules. The MHC:peptide complexes are transported back to the B cell surface where they are recognized by specific T cell antigen receptors on CD4+ helper T cells. These cell-to-cell interactions stimulate the T cells to secrete cytokines which promote B cell growth and differentiation into antibody-secreting effector B cells and memory B cells. The signaling capacity of the BCR is mediated via Ig-a and Ig-P (reviewed in Cambier et al, 1994; Gold and Matsuuchi, 1995; Reth, 1992). Ig-a and Ig-p each contain ITAM motifs. The ITAM motifs include two tyrosine residues that can be phosphorylated 34 by receptor-associated kinases upon receptor crosslinking with anti-BCR antibodies or upon antigen binding. Phosphorylation of the Ig-a/Ig-P ITAM motif allows the BCR to associate with the src homology 2 (SH2) domains of intracellular signaling molecules including Src family kinases, Syk kinases and families of adaptor proteins like SH chain. The kinases phosphorylate nearby Ig-a and Ig-p ITAMs, thereby allowing other signaling molecules to associate with Ig-a and Ig-p. This leads to a phosphorylation cascade of other signaling molecules and the activation of multiple signaling pathways (figure 12). 35 Figure 12. Model of B C R Signaling Events, (see next page) A . Initial signaling events involving the BCR. Few B C R molecules on the cell surface are tyrosine phosphorylated and associated with PTKs. Antigen cross-linking of the BCRs at the plasma membrane brings the ITAMs of the B C R close together. This allows tyrosines on other BCRs to become phosphorylated by the PTKs. Other signaling molecules are recruited to the plasma membrane by way of the interaction between their SH2 domains and phosphotyrosines on the BCR. B . Intial signaling events of the B C R trigger a cascade of downstream signaling molecules whose activities culminate in D N A expression and cell cyle regulation. 36 Src family IM K BCR cross-Unking by antigen 1 Tyrosine phosphorylation of BCR recruits additional PTKs to the BCR hospholipase C 4 Downstream Signaling Events 4 I"™Ca+2™T| release Downstream Signaling Events Ptdlns 3-kinase K C - el Gene expression Cell cycle regulation 37 VI. Assembly and Intracellular Trafficking of Secreted and Membrane Immunoglobulins 6.1 Assembly of Secreted Immunoglobulins (slgs) Most of our knowledge about immunoglobulin assembly comes from studies done to determine the assembly and folding of slgs. Unlike BCR complexes, slgs lack a transmembrane domain and association with Ig-a/Ig-(3 heterodimers. Consequently, secreted immunoglobulins only involve associations into monomers or polymers of H chains and L chains. However, understanding how these molecules are put together and the cellular proteins involved in this process could serve as a useful model of how BCR assembly occurs. Early studies in humans and mice show that the relative amounts of H and L chains synthesized and their assembly patterns vary among and even within the various Ig subclasses (Scharff and Laskov, 1970; Zolla et al., 1970). Some cell lines are balanced in H and L chain production while others secrete excess L chains (Zolla et al, 1970). Such differences in H and L chain subunit synthesis may influence the Ig assembly patterns observed. For example, in L chain excess, there would be a greater probability of a H chain interacting with a L chain instead of another H chain. Therefore, one might expect HL intermediates to occur more readily in cell lines expressing L chain excess while H2 intermediates may predominate in cell lines with limiting L chain quantities or balanced H and L subunit synthesis. Assembly intermediates and patterns in various human and murine cell lines have been studied for slgG, slgM, and to a lesser extent for slgA. HL intermediates occur in the assembly of IgG by some plasmacytomas while others are assembled through the H2 dimer (Askonas and Williamson, 1967; Scharff et al., 1967; Schubert, 1968). Murine IgGi (Schubert, 1968; Namba and Hanaoka, 1969) and rabbit IgG (Sutherland et al, 1970) both possess the HL intermediate. Some IgGi tumours may also exhibit some H2 and H2L intermediates (Buxbaum et al., 1971). Murine IgG2a assembles via 38 the H2 intermediate while IgG2b tumours assemble via either HL or H2 intermediates (Baumal and Scharff, 1970; Baumal et al, 1971). Therefore, IgG molecules may assemble via two different pathways. In murine myeloma cells, which contains L, H, H2, H2L, H2L2 assembly intermediates for slgG, slgG assembly is likely to occur in the following order: H + H -> H 2 H 2 + L - » H 2 L H2L + L -» H 2 L 2 In cells containing the HL intermediates, IgG assembles via: H + L -> HL HL + HL -> H 2 L 2 Other slgs assemble differently. For example, slgA assembles via H2 intermediates in some cell lines while in others its H and L chains associate non-covalently until final polymerization and secretion occur (Bevan, 1971; Bargellesi, 1972). Further, slgA undergoes further oligomerization into tetramers via inter-disulfide linkages and a joining (J) chain. Thus, for some murine cell lines, slgA assembly is depicted as: H + H -> H2 H 2 + L + L H 2 L 2 H2L2 + H2L2 + J chain -> (Fy^n n = 4 usually Similarly, slgM polymerizes into H2L2 pentamers (H2L2)s before it is secreted via its J chain and intermolecular-disulfide bonds (figure 10). However, like murine IgGi, murine IgM formation involves the production of L, H, HL, and H2L2 intermediates. This suggests that IgM assembles via the following pathway: 39 H + L -> HL HL + HL -» H 2 L 2 H 2 L 2 + H 2 L 2 + J chain ->(H 2L 2) 5 Despite their structural similarities and their association with common ER resident proteins, mlgs and slgs do differ and these differences are likely to lead to differences in assembly and quality control. For example, coimmunoprecipitation of immunoglobulin assembly intermediates from pre-B cells and plasma cells indicate that p H chain containing complexes (IgM) and y2b H chain containing complexes (IgG) are assembled differently (Haas and Wabl, 1983). In contrast, coimmunoprecipitation of assembly intermediates for mlgM yields the same pattern of assembly as for slgM (personal observations). In addition, pulse-chase analysis of human BCR formation indicates that Ig-a and Ig-P may associate independently and sequentially with the H2L2 tetramer within three minutes after biosynthesis (Brouns et al, 1995). Moreover, they found that Ig-a is the rate-limiting step in BCR complex fomation, while the remaining three BCR subunits are produced in excess. This suggests that Ig-a may control the exit of the BCR out of the ER. Studies are currently in progress to confirm/refute these results and to determine the nature of any associations of the BCR subunits with ER resident proteins. 6.2 ER Retention and Intracellular Trafficking of slgs Plasma cells secrete polymeric, pentameric and hexameric, IgM and degrade unassembled components including IgM monomers (Davis et al, 1989; Sitia et al., 1990; Randall et al., 1992). Two regions of the secretory p H chain act as sequential ER retention signals for unassembled IgM: the first constant domain (CHI ) and the C-terminal cysteine (Cys575). The C H I domain contains hydrophobic residues that are transiently masked during H chain folding by association with the ER resident protein BiP (Bole et al, 1986; 40 Hendershot et al., 1987; Hendershot, 1990). H chains of secreted immunoglobulins associate transiently with BiP in the absence of association with L chains and remain intracellular (Haas and Wabl, 1983). When BiP replaces L chain association with H chain, H chains remain stuck inside the cell in association with BiP which is never secreted. Thus, BiP may act as an ER chaperone which retains misfolded or unassembled proteins. In addition to its association with BiP, secreted H and L chains have been shown to interact sequentially with GRP94 which may also function as an ER chaperone (Melnick et al., 1992). Cys575 is necessary for both covalent polymerization and for the intracellular retention of IgM monomers by disulfide interchange reactions mediated by PDI (Alberini et al., 1990; Sitia et al., 1990). Further, mutating Cys575 to an alanine residue prevents ER degradation of the H chain whereas the 20 C-terminal residues of H chain are sufficient to confer ER retention and degradation of a cathepsin D:H chain chimera (Fra et al., 1993). These results suggest that ER retention is not sufficient for degradation but that it must be coupled with a degradation targeting signal which is Cys575 for the p H chain. Thus, sig H chains contain more than one quality control signal necessary for ER retention, one for assembly and one for degradation. 6.3 Requirements for Surface Expression of the BCR All four polypeptide chains of the BCR (p, light, Ig-a and Ig-P) are required for the proper assembly and transport of the BCR to the cell surface. In fact, transfection studies indicate that the four subunits are necessary and sufficient for plasma membrane expression of the BCR in non-B cells (Venkitaraman et al, 1991; Matsuuchi et al, 1992). In the absence of one or more BCR chains, the remaining chains are retained intracellularly in a pre-Golgi compartment, presumably in the ER (Venkitamarin et al., 1991; Matsuuchi et al, 1992; Bell and Goodnow, 1994). Initial studies using the J558L myeloma line, which only 41 produces L chain, transfected with p H chain showed that mlgM could be produced intracellularly but that H and L chains were insufficient to allow transport to the surface (Hombach et al., 1988; Sitia et al., 1987). A variant of these transfectants (J558Lpm3) which could express mlgM on the cell surface was found to coexpress a heterodimer non-covalently associated with mlgM (Hombach et al., 1990). This heterodimer was later found to be Ig-a/Ig-P and cotransfection of the mb-1 gene into the J558L cell line with p H chain allowed surface expression of the complete BCR complex indicating that Ig-a is the missing component in these p H chain transfected cell lines (Hombach et al., 1990; Sakaguchi et al., 1988). Thus, assembly of mlgM with the Ig-a/Ig-P heterodimer into a complete BCR is a requirement for surface transport of the BCR. In contrast, alteration of specific residues in the transmembrane region of the p heavy chain results in the ability of mlgM to reach the cell surface without the Ig-a/Ig-P heterodimer (Williams et al., 1990; Stevens et al., 1994). For example, replacing the p H chain transmembrane domain with the corresponding regions of the MHC I molecule rescues surface expression of the chimeric mlgM in the absence of Ig-a and Ig-P (Hombach et al., 1990; Williams et al., 1990). Also, altering four polar amino acids, the TTAST sequence, in the p H chain transmembrane domain to hydrophobic residues by site-directed mutagenesis allows mlgM to escape retention (Williams et al., 1990). These results not only suggest that the p transmembrane region is involved in the association of mlgM with Ig-a and Ig-P but also with the ER retention machinery. This ER retention machinery is part of a control mechanism that may recognize amphipathic transmembrane sequences containing hydrophilic or charged residues as well as hydrophobic amino acids. In fact, this hydrophilic patch is 42 patch is believed to interact with an ER resident molecule, probably calnexin, that functions as a chaperone and retains the H chain until the chaperone is displaced by the association of Ig-a/Ig-P with mlgM (Hochstenbach et al, 1992). This association alleviates the transmembrane specific retention signal. As for slgM, a second retention signal exists within the first constant ( C H I ) domain of the H chain (Hendershot et al, 1987; Cherayil et al, 1993). Deletion of the C H 1 allows the transmembrane p H chain to reach the surface in the absence of L chain or Ig-a/Ig-P (Cherayil et al, 1993). This retention signal is specific for interaction with the ER resident protein BiP (Haas and Wabl, 1983). BiP associates with the region of H chain that is also involved in interaction with L chain. Further, BiP associates with H chain only in the absence of association with L chain (Hendershot, 1990). This suggests that BiP may be displaced by L chain through (1) steric hindrance by L chain, (2) a configuration change of the H chain when it is covalently bound to L chain so that the binding site for BiP is altered, or (3) BiP and L chain might compete for the same binding site. In support of this, BiP also associates with L chain (Knittler and Haas, 1992). The nature of this association depends on the fate of the L chains. Light chains destined for secretion are bound by BiP only briefly whereas light chains retained in the ER, presumably for degradation, interact more stably with BiP. Thus, both heavy chain and light chain may associate with BiP prior to BCR assembly as for sig assembly. Thus, BCR chains which fail to associate properly in the ER may be targeted for degradation, retained in the ER until full assembly takes place, or recycled between intracellular compartments. These three processes are all likely to be mediated by protein chaperones. Several resident ER proteins (eg. BiP and grp94) associate sequentially and transiently with partially assembled molecules of the secreted form of IgM (slgM) and could 43 be chaperones (Hammond and Helenius, 1994; Hendershot, 1990; Kim and Arvan, 1995; Melnick et al., 1992; Melnick et al., 1994). These proteins may also interact with the mlgM since the only difference between mlgM and slgM is that mlgM has an additional 41 carboxy-terminal amino acids. Little is known about the association of Ig-a or Ig-P with these ER proteins. Thus, the role, if any, of resident ER proteins in BCR assembly, folding, and ER retention is uncertain but seems likely. 6.4 The BCR as a Model System for Studying Quality Control in the ER WEHI 231 is an immortalized, immature B cell expressing all four chains of a functional BCR on the cell surface (p H chain, K L chain, Ig-a, and Ig-P) (figure 13). WEHI' 231 cells display a biological response to stimulation of the BCR, cell death, which can be used to select for mutants that lack the receptor or that lack signaling components. One such mutant of the WEHI 231 B lymphoma, WEHI 88.1, was identified as a clone defective in signaling due to less than 7% of the p heavy chain being expressed on the cell surface (Page et ah, 1991) (figure 13). This variant serves as a useful tool in understanding how the BCR assembles and trafficks to the cell surface since the defect must affect some aspect of BCR assembly (ie. in a BCR chain or in the protein assembly machinery) or the transport process. The present study uses biochemical and immunological techniques to characterize the assembly and trafficking of the BCR in an immature B cell line, WEHI 88.1, that is defective in BCR transport to the cell surface. Biochemical evidence reveals that WEHI 88.1 contains all four chains of the BCR but few if any are expressed on the cell surface (Page et al, 1991; Jessica Escribano, personal observations). There are two possible explanations for this result. First, there may be defects in one or more chains of the BCR which prevent its expression on the cell surface. If so, then this mutant could be used to identify the regions of the BCR involved in protein-protein interactions with other components of the receptor and/or with 44 components of the secretory pathway. Second, the secretory pathway in WEHI 88.1 may be defective. This could be due to a defect in a chaperone protein that effects the assembly and and trafficking to the cell surface. This project was designed to test the hypothesis that a defect in one or more BCR components may be responsible for the BCR transport defect in WEHI 88.1. This working hypothesis requires characterizing the variant's BCR chains for abnormalities which may interfere with their assembly or transport function. Testing this hypothesis also requires eliminating the possibility that the alternate hypothesis, that a defect exists in the secretory machinery, is occuring. My first goal is therefore to distinguish between these two possibilities. WEHI 231 88.1 (wild-type) (mutant) Figure 13. Models of the WEHI 231 and WEHI 88.1 B Cell Lymphomas. Wehi 231 is the parental cell line expressing all four chains of the BCR both intracellularly and on the cell surface. WEHI 88.1 expresses all four chains of the BCR intracellularly but expresses less than 7% of parental amount on the cell surface. 45 To distinguish between these two hypotheses, I have used both biochemical and molecular techniques to determine the basis of defects in BCR assembly and/or trafficking in the WEHI 231 mutant, WEHI 88.1. One should keep in mind when using this approach that it takes time to fully analyze selected clones and that tissue culture cells are not ideal genetic systems; more than one mutation may be present and responsible for the phenotype. These limitations may present the following problems. Cultured cells do not always behave in the same manner or display the same properties that they do in live organisms. It is possible that observations made using cultured cells such as WEHI 231 and WEHI 88.1 differ from what is observed in the organism. To control for this, experiments conducted in cultured cells should be replicated in the organism. This may not be experimentally possible due to constraints in cell number or available methodology. Cultured cells are sometimes the only or best available means of conducting some studies. Further, the presence of more than one mutation in the cell may make it difficult to determine which mutation(s) is responsible for the failure of the BCR to traffick to the cell surface. For example, an inability of the BCR subunits to associate may be due to a combination of mutations in one or more of these subunits. Defects may also occur in both the BCR and in other parts of the cell. Thus, there are several limitations to my approach which may make distinguishing between my two hypotheses problematic. 46 MATERIALS AND METHODS I. Antibodies Antibodies for western blotting were diluted in Tris-buffered saline with 5% tween-20 (TBST) as described below. Polyclonal rabbit anti-mouse IgM antibodies, p heavy chain specific, from Jackson Immunochemicals (distributed by BioCan, Mississauga, ON, Canada) were diluted 1:1000 in TBST. Affinity purified goat anti-mouse p chain antibodies and goat anti-mouse p-HRP antibodies were obtained from Jackson Irnmunoresearch and used at a dilution factor of 1:20,000 (West Grove, PA). Polyclonal rabbit anti-mouse K light chain antibody was from ICN (Mississauga, ON, Canada) and diluted 1:500. Polyclonal rabbit anti-mouse X light chain antibody was from Bethyl Laboratories, distributed by Cedarlane Laboratories (Hornby, ON, Canada). Polyclonal rabbit anti-mouse Ig-a antibody, specific for a 34 amino acid peptide spanning residues 187-220 of the carboxyl terminal of Ig-a, was previously described by Gold et al., 1991. It was used at a dilution of 1:2000. The polyclonal rabbit anti-mouse Ig-P antibodies, SF2B5, specific for a 24 amino acid peptide spanning residues 76-96 of the amino terminal region of Ig~P, was developed and described by Foy, 1997. Anti-P antibodies were used at 1:500 dilution. Polyclonal rabbit anti-mouse actin antibody was also from ICN (Mississauga, ON, Canada. Protein A conjugated to horseradish peroxidase (PA:HRP), from Amersham Life Science (Oakville, ON, Canada), was used at a dilution of 1:10,000 in TBST. Antibodies for immunoprecipitation were added in excess as described under coimmunoprecipitation assays. Polyclonal rabbit anti-mouse IgM antibodies, p heavy chain specific, and polyclonal rabbit anti-mouse K light chain antibodies were from ICN 47 (Mississauga, ON, Canada). Anti-Ig-a and anti-Ig-p antibodies described previously under Antibodies were also used. II. Cell Culture The B cell lymphomas, WEHI 231 (Warner et al, 1979) and WEHI 88.1 (Page et al, 1991) were maintained in RPMI 1640 (Stem Cell Technologies, Vancouver, BC) supplemented with 10% fetal bovine serum (Intergen, Purchase, NY), 500 units/ml penicillin, 500 pg/ml streptomycin, 2 mM L-glutamine and 50 pM p-mercaptoethanol (Sigma, St. Louis, MO). Cells were incubated at 37°C in an atmosphere of 5% CO2 in a Forma Scientific model 3326 incubator (Marietta, OH). Suspensions of 5X106 cells of both the WEHI 231 and the WEHI 88.1 cell lines were grown separately in 10 cm tissue culture dishes (Falcon, Franklin Lakes, NJ) purchased through VWR Scientific (Edmonton, AB). WEHI 231 cells were split 1:5 every two to three days and grown to approximately 5X10 cells prior to experimentation. WEHI 88.1 cells were split 1:2 every three to four days and also grown to approximately 5X10 cells prior to experimentation. For long-term storage, WEHI 231 and WEHI 88.1 cells were frozen in liquid nitrogen as follows. Cells were removed from plates to 15ml Falcon 2097 conical polypropylene tubes and recovered by 4°C centrifugation at 1500rpm for 5 min in an IEC Centra-8R centrifuge. The media was removed and the cell pellet was resuspended in 1ml of normal media containing 10% dimethyl sulphoxide, DMSO (Sigma). Resuspended cells were then transferred to a pre-chilled 2 ml Nalgene cryogenic vial (Rochester, NY) and sequentially stored at -20°C for 30min and -80°C overnight prior to liquid nitrogen storage. Removal of cells from liquid nitrogen storage involved quickly thawing vials in a 37°C-water bath. The cells were transferred to 10 ml of media in a Falcon 2097 tube and centrifuged at 1500 r.p.m. in a 4°C IEC Centra-8R centrifuge to remove the DMSO and recover the cells in a pellet. 48 WEHI 231 cells were then resuspended in 10 ml of media and incubated at 37°C in a 10 cm dish. WEHI 88.1 cells were resuspended in 5 ml of media and incubated at 37°C in a Falcon 6-well dish and grown to 5X107 cells for transfer to 10 cm dishes. III. Cell Extracts Fresh cell extracts were prepared at 4°C for each experiment. Approximately 5X10 cells were collected in a pellet by 4°C centrifugation at 1500 r.p.m. The pellet was resuspended and washed twice in 1 ml of phosphate-buffered saline (PBS) and transferred to a sterile prechilled 1.5 ml eppendorf tube. Cells were then lysed on ice in the presence of 100-1000 pi of lysis buffer (LB) using MG LB (1% Triton X-100 detergent, 2 mM EDTA, 10% glycerol, 137 mM NaCl, 20 mM Tris pH 8.0), NDET LB (1% NP40, 0.4% DOC, 66 mM EDTA, 10 mM Tris pH 7.4), or Digitonin LB (1% digitonin, 10 mM triethanolamine pH 7.8, 150 mM NaCl, 1 mM EDTA). Lysates were centrifuged at 18,000 r.p.m. for 15-20 min at 4°C to remove nuclear material. Supernatants were transferred to a fresh prechilled 1.5 ml eppendorf tube and the nuclear pellet was discarded. SDS was added to a final concentration of 0.3% to MG LB and NDET LB extracts. DOC was added to a final concentration of 0.4% to MG LB extracts. Protease inhibitors (from Sigma) were added to retard protein digestion and used at a final concentration of 1 pM pepstatin A, 0.01 pM aprotinin, 1 mM PMSF, lOmM vanadate, and 1 pM leupeptin. The BCA protein assay (Pierce, Rockford, IL) was used to determine the relative protein concentration of each protein extract. For most experiments, lysates were adjusted to a final concentration of 1 pg protein/ ml. IV. Coimmunoprecipitation Standard coimmunoprecipitation procedures were used to recover BCR chains and to determine protein associations. Total cell extracts were prepared as previously described. Cell extracts were pre-cleared in 50-75 pi of protein A (PA)-conjugated Sepharose beads by 49 incubation at room temperature for 30 min with rocking. Extracts were recovered by centrifugation at 18,000 r.p.m. and transferred to a fresh eppendorf tube. Anti-p, anti-K, or anti-Ig-ct antibodies were added in a pre-determined excess of 20 pi of antibody to lOOpg of protein lysate in a final volume of 1ml. Lysaterantibody mixtures were rocked at 4°C overnight and then incubated with 100 pi of PA-Sepharose beads for 30 min at room temperature with rocking. Sepharose beads were recovered by centrifugation at 18,000 r.p.m. for 1 min and resuspended in 500 pi of LB. The entire sample was then layered over a 500 pi sucrose pad (30% sucrose in LB). The samples were centrifuged (18,000 r.p.m.) through the sucrose pad to remove debris. The pellet was washed three times with LB and once with PBS to minimize non-specific binding of proteins to the antibodies and to the Sepharose beads. Antibody complexes were dissociated from Sepharose beads by boiling samples in 30 ul of sample buffer and collected by centrifugation. Proteins in sample buffer were separated by SDS-PAGE as described. V. Gel Electrophoresis Protein samples prepared from total cell extracts were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using standard protocols for preparing and running 10% or 12% Tris-glycine (TG) gels (Laemmli, 1970) or 5% phosphate (PO4) gels (Melnick and Argon, 1995). Proteins were separated using either a mini-gel or large gel apparatus (CBS Scientific, Del Mar, CA). Sample buffer (at a final concentration of 5% SDS, 10% glycerol, 62.5 mM tris-H chainl pH 6.8, .001% bromophenol blue) was added to cell extracts. Protein extracts were reduced in reducing sample buffer containing 5% (3-mercaptoethanol (Sigma, St. Louis, MO) and boiled for five minutes prior to loading them onto gels. Non-reduced samples were incubated at 37°C for 30 min and then boiled for 5 min immediately before loading. Polyacrylamide gels of 5-12% in either PO4 or Tris 50 buffer were loaded with 5-25 pg of protein per lane. Mini-gels were run at 30 mAmps for approximately 30 min and large gels ran at 70 mAmps for approximately four hours. Gels were kept below 10°C with a small fan or with a water cooling system on the mini-gel apparatus to prevent plate cracking due to overheating. VI. Western Blotting Proteins in gels were transferred to Immobilon-P (Millipore Corporation, Bedford, MA) or nitrocellulose (Schleicher & Schuell, Keene, NH) filters at constant voltage by standard western blotting techniques. Transfer occurred at 100 V for lhour or 70 V for 2 hours at 4-10°C in a Transblotter (BioRad, Richmond, CA). This was sufficient to completely transfer proteins as detected by Coomassie staining of the gel and Ponceau staining of the filter after transfer. Blotted filters were incubated overnight at 4°C in Tris-buffered saline with 0.025% tween-20 (TBST) and 5% bovine serum albumin (BSA) to block non-specific binding of antibodies to the filter and sticky proteins on the filter (Boehringer Mannheim). The next day, filters were incubated, at room temperature, with rotation, for 1 hour, with a primary antibody and for 30 min with an HRP-conjugated secondary antibody or with PA:HRP at a dilution in TBST of 1:10,000. Filters were washed 3-5 times after each incubation, for 10 min each wash, with 5 ml of TBST for every 10cm2 of filter. Probed filters were treated with enhanced chemilumenescence (ECL) Amersham reagent for 1 min and exposed to Hyperfilm-MP autoradiography film (Amersham Life Science) for 10 sec to 5 min. A reactive chemical in ECL emits light in response to its oxidation by FfRP and H2O2 in the presence of phenol (Amersham Life Science Publication, 1993). 51 VII. Metabolic Labeling of Proteins for Steady-State and Pulse/Chase Analysis Proteins were metabolically labeled with 35S-methionine/cysteine translabel (ICN) for steady-state and pulse-chase analysis. For steady state analysis, 5X107 cells of WEHI 231 or WEHI 88.1 were incubated overnight with labeling media (methionine and cysteine double-depleted RPMI, 500 units/ml penicillin, 500 mg/ml streptomycin, 2mM L-glutamine, 50 pM P-mercaptoethanol) containing 5 mCi 35S-translabel (ICN) and supplemented with 10% FCS (Intergen). For pulse-chase analysis, 5X107 cells of WEHI 231 and WEHI 88.1 cells were starved for 20-30 min in labeling media. Cells were then pulsed for 15 min with 1 mCi of 35S-translabel (ICN). Translabel incorporation was stopped by adding 2mg/ml of cysteine and 2mg/ml of methionine directly to the labeling media and washed in 5ml of prewarmed chase media (labeling medium with 2 mg/ml of cysteine and 2 mg/ml of methionine). Cells were recovered by centrifugation at 1500 rpm and resuspended in 5 ml of prewarmed chase medium. Cells were chased for 0, 0.25, 0.5, 2, 4, 6, 8, 12, or 16 hours. For chases longer than two hours, supernatants were collected every two hours and pooled. Both steady state and pulse-chase radiolabeled cells were recovered by centrifugation and lysed to prepare detergent extracts as described. Total cell extracts were immunoprecipitated, separated by SDS-PAGE and visualized by autoradiography as previously described. Supernatants were also recovered and immunoprecipitated. VIII. Surface Biotinylation of Plasma Membrane Proteins The presence of membrane proteins on the surface of WEHI 231 or WEHI 88.1 cells were analyzed using the surface biotinylation procedure described in Lisanti et ah, 1988. Approximately 5X107 cells of WEHI231 and WEHI 88.1 were collected by centrifugation at 1500 rpm and transferred to 1.5 ml eppendorf tubes and washed three times with ice cold PBS. Cells were centrifuged at 1500rpm during transfer and between washes for recovery in 52 the pellet with minimal cell breakage. After the PBS washes, cells were resuspended in 1ml of PBS containing 1 mg of sulfo-NHS-biotin (Pierce, Rockford, Illinois) and rocked for 20 min at 4°C. Excess biotin was removed by washing cells twice in 1 ml of RPMI media containing 2mg/ml lysine and five times in 1 ml of PBS containing 2mg/ml lysine. Cells were recovered by centrifugation at 1500rpm. Detergent extract were prepared from biotinylated cells in MG Lysis buffer as described previously. Extracts were analyzed by SDS-PAGE and Western blotting with Streptavidin:HRP diluted 1:50,000 in TBST. Biotinylated protein bands on the filter were visualized using ECL and autoradiography. IX. Surface lodination of Plasma Membrane Proteins Various cell lines were radiolabeled with the membrane-impermeant Thompson's iodination reagent, 125I-sulfosuccmimidyl(hydroxyphenyl)propionate as noted previously (Thompson et al., 1987). Approximately 5X106 cells of each cell line were washed and resuspended in 0.5 ml PBS containing 1 mg/ml glucose prior to adding the radiolabel. The labeling reaction was quenched by adding 1 ml of PBS/glucose containing 1 mg/ml L-lysine. The cells were then lysed in NDET lysis buffer supplemented with 2mg/ml L-lysine as described above. 53 RESULTS I. Expression of the BCR Chains in WEHI 231 and WEHI 88.1 Using FACS analysis, Page et al, 1991, previously identified the WEHI 88.1 cell line as a randomly mutagenized variant of the WEHI 231 B cell lymphoma, unable to signal through its BCR, and expressing less than 7% of wild-type levels of the BCR on its cell surface. The decreased surface expression of the BCR on WEHI 88.1 cells may be due to a defect in the BCR and/or a defect in the secretory system. For surface expression to occur, all four chains of the BCR must assemble properly into a complete receptor (Venkitaraman et al., 1991; Matsuuchi et al., 1992). The absence of one or more BCR chains would prevent its transport to the plasma membrane. We were therefore interested in whether one or more BCR chains were absent or expressed in lower amounts in WEHI 88.1. We examined the ability of WEHI 88.1 to express all four BCR proteins (p heavy chain, K light chain, Ig-a and Ig-P) by western blotting. The WEHI 231 cell line was used as a positive control for BCR protein expression. Proteins from whole cell extracts of WEHI 231 and WEHI 88.1 were separated by 10% SDS-PAGE, transferred to nitrocellulose by western blotting, and probed separately with anti-p, anti-K, anti-Ig-a, and anti-Ig-P antibodies. As shown in figure 14, protein bands that reacted with the anti-BCR antibodies were present in the WEHI 88.1 mutant and migrated on SDS-PAGE gels at molecular weights (MWs) consistent with the predicted sizes of the four BCR chains. Although protein bands corresponding to all four BCR chains were present in the mutant, several differences between the WEHI 231 and WEHI 88.1 blots were seen. The two cell lines differed in the MW ranges of their p heavy chain, Ig-a and Ig-p proteins and in their protein expression levels of K light chain. WEHI 231 possessed additional protein bands of p heavy chain, Ig-a, and Ig-P of higher MWs that were absent in WEHI 88.1. The apparent MWs of the BCR 54 bands in each lane were calculated in relation to the mobility of protein standard markers and summarized in Table 1 . Also, protein expression of K light chain was qualitatively lower in WEHI 88.1 cells than in WEHI 231 cells even though equal amounts of total protein were loaded in each lane and cell extracts were prepared using approximately equivalent numbers of cells. Thus, WEHI 88.1 synthesizes all four chains of the BCR but p heavy chain, Ig-a and Ig-(3 are not expressed in the higher molecular weight ranges seen in WEHI 2 3 1 and WEHI 88.1 K light chain expression is decreased. " 0 # J>rAv ^ kDa ^ S r 97 — 70 — 43 — 28 — anti-it anti-K anti-Ig-a 4? 4? anti-Ig-f3 • u Heavy chain 3" Jlg-a ' K Light chain kDa 69 — 57 43 •Actin Figure 14. Expression of BCR Proteins in the Murine B Lymphomas WEHI 231 and WEHI 88.1. Whole cell extracts of WEHI 231 and WEHI 88.1 were prepared in M G lysis buffer which dissociates mlgM from Ig-a/p. Proteins from the cell extracts were separated by 1 0 % SDS-PAGE (lOpg of protein/lane) under reducing conditions and transferred to nitrocellulose by western blotting. The filters were probed with rabbit polyclonal antibodies against p heavy chain, K light chain, Ig-a and Ig-p followed by PA-HRP and developed using ECL. Molecular weight standards are indicated to the left of the filter. The arrows to the right identify the BCR protein bands present. 55 BCR Chain Molecular Weight of BCR Chains (kDa) WEHI 231 WEHI 88.1 p Heavy chain 70-72 70 K Light Chain 22 22 Ig-a 28-35 28 Ig-P 35-48 35 Table 1 - Molecular Weight of BCR Protein Bands (kDa) in WEHI 231 and WEHI 88.1 Cells. Calculated from values obtained from figure 14. II. Glycosylation Differences Between WEHI 231 and WEHI 88.1 PNGase Treatment of WEHI 231 and WEHI 88.1 Proteins Next, we investigated the higher MW forms of WEHI 231 p heavy chain, Ig-a and Ig-P proteins that were lacking in WEHI 88.1 (figure 14). These higher MW bands may represent differentially glycosylated versions of the p heavy chain, Ig-a and Ig-P proteins that are not present in WEHI 88.1. Alternatively, the absence of these bands in WEHI 88.1 cells may be due to structural defects in the core proteins of the WEHI 88.1 p heavy chain, Ig-a, and Ig-B. To distinguish between these two possibilities, we removed all the N-linked carbohydrates from the BCR proteins of each cell line using the enzyme PNGase F. The MWs of the stripped core proteins were then compared on SDS-PAGE. In figure 15A, WEFfl 231 and WEFfl 88.1 whole cell extracts were first immunoprecipitated with either anti-p or anti-Ig-a antibodies and then immunoprecipitates were treated with or without PNGase F and separated on 10% SDS-PAGE. In figure 15D, cell extracts treated with or without PNGase F were examined by western blotting with anti-Ig-P antibody. The WEFfl 231 and WEFfl 88.1 protein bands for the p heavy chain, Ig-a, and Ig-P components of the BCR drop to comparable MWs when treated with PNGase F. In both cell lines, the core 56 protein band for p heavy chain was 55 kDa, for Ig-a it was 25 kDa, and for Ig-p it was 28 kDa. As expected, PNGase F treatment has no effect on K light chains which are not normally glycosylated. Thus, the deglycosylated BCR proteins are the same MW in both WEHI 231 and WEHI 88.1 cells and there are no noticable differences in the core BCR proteins between these two cell lines. Endo H Treatment of WEHI 231 and WEHI 88.1 Proteins Since the WEHI 88.1 mutant might represent a defect in the trafficking process, we were curious to know how far along the secretory pathway these chains were able to travel. The PNGase F data in figure 15 suggested that WEHI 88.1 BCR chains do not undergo the glycosylation modifications that take place in the Golgi and later in the secretory pathway. We therefore examined whether the BCR chains were located in a pre-Golgi compartment in the mutant cell line using a deglycosylation technique indicative of protein progression through the secretory pathway. Most proteins that transit past the cis-Golgi undergo mannose trimming and modification rendering their carbohydrates resistant to treatment with Endoglycosidase H (Endo H), an enzyme that removes high mannose carbohydrates (figure 16). Thus, proteins demonstrating Endo H sensitivity are usually localized in a pre-Golgi compartment. In figure 15B, metabolically-labeled cells were treated with or without Endo H, immunoprecipitated with anti-p antibody and analyzed on SDS-PAGE. Endo H treatment in WEHI 231 results in the appearance of a lower MW band at 55 kDa in addition to the normally present bands of 70-72 kDa. In WEHI 88.1 Endo H-treated lanes, most of the p H chain dropped down to the same MW (approximately 55 kDa) as the PNGase F-treated WEHI 231 lane in figure 15A. The deglycosylated p H chain protein bands appear as a doublet in both cell lines. In figure 15C and D, whole cell extracts of WEHI 231 and WEHI 57 88.1, prepared in MGLB, were treated with or without Endo H or PNGase F. PNGase F digestion was used as a positive control for deglycosylation with which to measure the degree of Endo H digestion. Protein samples were separated by 10% SDS-PAGE and western blotted with anti-Ig-a and anti-Ig-f3 antibodies. In WEFfl 231 Endo Ff-treated lanes, some Ig-a and Ig-P remained the same MW as in the untreated lanes while some Ig-a and Ig-p dropped in MW to that seen in WEHI 231 PNGase F-treated lanes. In WEHI 88.1, Endo H treatment results in a decrease in the MW of all the Ig-a and Ig-P to that seen in WEHI 231 PNGase F- treated lanes. WEFfl 88.1 Endo H-treated Ig-a was 25 kDa and Endo H-treated Ig-P was 28 kDa. Thus, in WEHI 88.1 Endo H-treated lanes, p H chain, Ig-a and Ig-P were of comparable MW to the WEHI 231 p H chain, Ig-a, and Ig-P deglycosylated with PNGase F. 58 B kDa 108 — 70 — 43 — 28 — . + - + - + - + PNGase F It anti-u IP anti-Ig-a IP .4- |i Heavy chain deglycosylated \i Heavy chain p I g - « , K Light chain \ <li>i>|\ raw lati'il kDa 44 — 28 — 18— J$ J$ PNGase F - - + + — mm - - - - + + lg-u Biol Kudo II WEHI 231 WEHI 88.1 kDa - + - + 9 7 --4 3 -anti-u IP (« - - + + - -- - - - + + EndoH | i Heavy chain deylycosvlated u Heavy chain (doublet) - 4 - dcglycosylated Ig-a* 69 — 44 — 28 — M l Ig-B Blot PNGase F EndoH ^ ] l g | 5 m deglvcosvlated Ig-P Figure 15. Treatment of WEHI 231 and WEHI 88.1 B lymphoma Cell Lines with PNGase F and Endo H. A, Cell lines were metabolically labeled with Trans [ S]-label and solubilized in MGLB. Cell extracts were then immunoprecipitated with anti-p or anti-Ig-a antibodies followed by PA-Sepharose. Immuno-precipitates were stripped of all N-linked carbohydrates using PNGase F and separated by 10% SDS-PAGE under reducing conditions. Gels were dried and exposed to x-ray film by autoradiography for 3 days. B, (courtesy of Dr. Linda Matsuuchi1) Metabolically-labeled cells were solubilized in NDET lysis buffer, immunoprecipitated with anti-p. Followed by treatment with (+) or without (-) Endo H which strips high mannose carbohydrates. Immunoprecipitates were separated on 10% SDS-PAGE gels, dried and exposed on autorad. C and D, Whole cell extracts in MG lysis buffer were subject to PNGase F or Endo H treatment (+) or left untreated (-) and separated on 12% SDS-PAGE gels. Proteins on gel were transferred to nitrocellulose by western blotting and probed with anti-Ig-a or anti-Ig-P antibodies followed by PA-HRP and developed using ECL. 1 Dr. Linda Matsuuchi, Department of Zoology, University of British Columbia, 6 2 7 0 University Blvd, Vancouver, British Columbia V 6 T 1Z4, Canada. 59 Figure 16. Digrammatic Representation of Endo H Sensitivity and Resistance along the Secretory Pathway. Proteins undergo N-linked and/or O-linked glycosylation in the ER as they are co-translationally translocated into the ER. Such carbohydrates are sensitive to digestion by the Endo H enzyme. Upon exiting the ER, proteins enter the cis-Golgi and are still sensitive to Endo H. Proteins that transit to the medial and trans Golgi encounter enzymes that modify their carbohydrates such that they are resistant to Endo H activity. (Modified from Alberts et al, 1994). 60 III. Associations Between BCR Subunits ju H Chain and KL Chain Association All four chains of the BCR must associate and assemble properly into a complete receptor so that the BCR can escape ER retention and travel to the cell surface (Matsuuchi et al, 1989). We were therefore interested to see if the WEHI 88.1 BCR chains were capable of forming associations with each other and assembling properly. We first examined the types of associations the BCR subunits could form with each other. Three types of associations would be necessary for BCR formation, p H chain association with K L chain, p H chain association with Ig-a/Ig-p\ and Ig-a and Ig-P association. If any of these BCR interactions failed to occur in WEHI 88.1 cells, then complete BCR assembly would be hampered and this would prevent its trafficking to the cell surface. To explore whether H and L chains could associate with each other, whole cell extracts from metabolically-labeled WEHI 231 and WEHI 88.1 cells were immunoprecipitated with anti-mouse p H chain and anti-mouse K L chain antibodies. The immunoprecipitar.es were then separated on 10% TG gels by SDS-PAGE under reducing conditions and exposed on x-ray film. The molecular weights of bands were used to identify H and L chains. As shown in figure 17, anti-p H chain antibody co-immunoprecipitates both p H chain and K L chain in both cell lines. Similarly, anti-K L chain co-immunoprecipitates both H and L chain in both cell lines. The K L chain bands appear darker in anti-K IP lanes than in anti-p IP lanes. This may be due to the specificity for this antibody for the K L chain protein, insufficient immunoprecipitating antibody to remove all the H and L chains present in the extracts, or due to a greater protein concentration in extracts immunoprecipitated with anti-K L chain antibody. 61 kDa 108 — 70 — m m mm 43 — 28 — anti- anti-pIP KlP u Heavy chain K Light chain Figure 17. Association of u H Chain and K L Chain in WEHI 231 and WEHI 88.1. WEHI 231 and WEHI 88.1 cells were metabolically labeled overnight with Trans [ S] label and solubihzed in MGLB. The cell extracts were then immunoprecipitated with a pre-determined excess of either rabbit anti-mouse p H chain or rabbit anti-mouse K L chain antibodies. The immunoprecipitates were collected on PA-Sepharose beads and separated by SDS-PAGE on 10% tris-glycine gels under reducing conditions, which would separate H and L chains. Gels were dried on 3mm paper and examined by autoradiography. Molecular weight standards are shown on the left and the arrows on the right indicate the position of H and L chains identified by molecular weights. mlgM Association with Ig-a and Ig-fi Using a similar approach to investigate p H chain association with Ig-a and Ig-P, we immunoprecipitated p H chain and Ig-a proteins from metabolically-labeled cells solubilized in digitonin LB, a mild detergent that prevents the dissociation of non-covalent bonds between subunits such as those normally found between mlgM and Ig-a/Ig-p. As seen in figure 18A, protein bands corresponding in molecular weight to all four chains of the BCR co-immunoprecipitated with anti-p H chain or anti-Ig-a antibodies in both cell lines. Several attempts to immunoprecipitate using rabbit anti-mouse Ig-P antibody were unsuccessful due 62 to the instability of this antibody under immunoprecipitating conditions (personal observations). Ig-a Association with Ig-fi To confirm the identity of the co-immunoprecipitating proteins in figure 18A, we used western blotting. Whole cell extracts prepared in digitonin LB were immunoprecipitated as before, separated by gel electrophoresis under reducing conditions and transferred to nitrocellulose. The nitrocellulose filters were immunoblotted with anti-p H chain, anti-Ig-a or anti-Ig-P antibodies. Anti-p H chain antibody co-immunoprecipitated p H chain with both Ig-a and Ig-P with under mild detergent conditions (figure 18B). Similarly, co-immunoprecipitates collected using anti-Ig-a antibodies contained Ig-a, p H chain and Ig-P (figure 18C). Again, we were unable to perform immunoprecipitations with anti-Ig-P antibodies. 63 kDa 108 — 1 70 — 43 — 29 — « --• '•• 1 anti- anti-uTP Ig-a IP C •4- u Heavy chain K Light chain B kDa 99 — 69 — 43 — 29 — >^ 2p # § "»Sv ^ j j e a V y chain ^ Immunoprecipitating Ab — i « -anii-u IP anti- anti- anti-Ig-a Ig-P Blotting Ab kDa 99 — 69 — 43 — 29 — AMM^> /UHeavycbain Immunoprecipitating Ab Ig-a Blotting Ab -anti-Ig-a IP anti- anti- anti-it Ig-a Ig-P Figure 18. BCR Subunit Associations in WEHI 231 and WEHI 88.1. A, Metabolically labeled cells were solubilized in digitonin LB which does not disrupt the non-covalent interactions between mlgM and Ig-a/Ig-(3. The digitonin extracts were immunoprecipitated with either rabbit anti-mouse p H chain or rabbit anti-mouse Ig-a antibodies and collected on PA-Sepharose beads as described in Materials and Methods. Immunoprecipitates were separated on 10% tris-glycine gels by SDS-PAGE under reducing conditions. Gels were dried on 3mm paper and examined by autoradiography. B and C, Whole cell extracts of WEHI 231 and WEHI 88.1 were prepared in digitonin LB and immunoprecipitated with (B) rabbit anti-mouse p H chain or (C) rabbit anti-mouse Ig-a antibodies. Immunoprecipitates were collected on PA-Sepharose beads, reduced and separated by 12% SDS-PAGE. Proteins were transferred to nitrocellulose by western blotting, probed with the indicated antibody followed by PA-HRP, and developed using ECL. Ab = immunoprecipitating antibody control lane. Molecular weight markers are indicated on the left and arrows indicate the approximate positions of the BCR chains on the blot. 64 IV. mlgM Assembly Intermediates Next, we addressed the possible effects of lower L chain expression on BCR assembly and trafficking to the cell surface in WEFfl 88.1. Lower L chain expression in WEFfl 88.1 may interfere with mlgM assembly and prevent its escape from ER retention. As mentioned in the introduction, there are various pathways that the H and L chains can take to assemble into mlgM. In WEFfl 231 cells, HL is the common intermediate. However H2 and H2L intermediates are also possible and the predicted molecular weights of each possible assembly intermediate and complete mlgM (H2L2) are listed in Table 2. mlgM Assembly Predicted Molecular Weight (kDa) Intermediates WEHI 231 WEHI 88.1 H2L2 188-194 188 H2L 166-172 166 H2 144-150 144 HL 94-97 94 H 72-75 72 L 22 22 Table 2 - Predicted Molecular Weights of mlgM Assembly Intermediates. Molecular weights given are based on figures from Table 1 and are expressed in kilodaltons (kDa). To test the ability of WEHI 88.1 mlgM to assemble properly, we compared the mlgM assembly intermediates that form in both cell lines. WEHI 231 and WEHI 88.1 cells were 65 lysed in MGLB and the protein extracts were separated by SDS-PAGE under non-reducing conditions to maintain the covalent H and L chain interactions. One difficulty in analyzing mlgM assembly intermediates in WEFfl cells is that some of the intermediates may be of high MW, greater than 200kDa, and very difficult to resolve from one another using standard tris-glycine gels. To compensate for this difficulty, we used a low percentage (5%) phosphate gel to enhance the resolution of higher molecular weight bands and separated the protein bands on a larger gel so that the distances migrated would provide greater separation of bands of similar molecular weights. The proteins on the gels were then transferred to nitrocellulose by western blotting and probed with either anti-p H chain or anti-K L chain antibodies to reveal the presence of assembly intermediates. As expected, HL was the dominant assembly intermediate in both WEHI 231 and WEHI 88.1 cells. WEHI 88.1 cells however, expressed lower protein levels of HL intermediates. WEHI 88.1 cells also expressed H2L intermediates. H2 intermediates may be present in WEHI 88.1 and co-migrate with H2L intermediates. There was very little H2L2 present in WEHI 88.1 cells as compared to WEHI 231. The apparent MWs of the mlgM assembly intermediates in cell line were calculated in relation to the mobility of protein standard markers and are listed in Table 3. Thus, mlgM assembly intermediates were different in WEHI 88.1 cells compared to WEHI 231 cells. 66 Figure 19. Assembly Intermediates of mlgM in WEHI 231 and WEHI 88.1. Proteins from whole cell extracts of each cell line were prepared in MG LB and were separated on a 5% PO4 gel by SDS-PAGE under non-reducing conditions. Proteins on gels were transferred to nitrocellulose by western blotting and probed with (A) anti-p or (B) anti-K antibodies followed by PA-HRP and developed using ECL. Molecular weight markers are indicated to the left of the blot and arrows on the right indicate mlgM assembly intermediates. 67 mlgM Assembly Intermediates Apparent Molecular Weights (kDa) Band Intensity WEHI 231 WEHI 88.1 WEHI 231 WEHI 88.1 H2L2 210-225 210-225 +++ -/+ H2L 161-202 161-202 -/+ + H2 159-181 159-181 - ? HL 101-121 101-121 +++ ++ H 55-91 55-91 +++ ++ L 21-24 22-23 +++ + Table 3 - Apparent Molecular Weights and Band Intensities of Protein Bands of mlgM Assembly Intermediates in Figure 23. Apparent MWs of protein bands were calculated based on the relative mobility of protein molecular weight standards in kilodaltons (kDa). Relative band intensities are indicated by the number of (+) signs. Negligible band intensity is indicated by the (-) sign. F£2 intermediates may co-migrate with H2L in WEHI 88.1 as indicated by (?). V. Ig-a /Ig-p Assembly Intermediates To examine Ig-a/Ig-P intermediates, whole cell extracts of WEHI 231 and WEHI 88.1 were separated by 5% phosphate gel SDS-PAGE under non-reducing conditions, western blotted, and probed with anti-Ig-a and anti-Ig-P antibodies. Ig-a and Ig-P monomers and oligomers were present in both cell lines (figure 20). WEHI 231 Ig-a monomers range from 30-48 kDa whereas WEHI 88.1 Ig-a monomers range from 30-36 kDa. Also, WEHI 231 Ig-P monomers range from approximately 45-60 kDa while WEHI 88.1 Ig-P monomers range from 45-52 kDa. Ig-a and Ig-P monomers were of higher MW than those seen in 10% tris-glycine gels under reducing conditions (figure 14). This may be a consequence of using 5% phosphate gels of larger size under non-reducing conditions. In 68 both cell lines, Ig-a and Ig-B proteins formed oligomers with a similar range in MW, likely due to varying degrees of glycosylation (figure 20). In WEHI 231, Ig-a and Ig-p formed oligomers that ranged in MW from approximately 60-126 kDa. WEHI 231 Ig-P monomers and oligomers overlap in MW and appear as a continuous range of bands from 45-126 kDa. In WEHI 88.1, Ig-a and Ig-p formed oligomers that range in MW from approximately 55-85 kDa. In addition, fewer Ig-P oligomers formed compared to Ig-a oligomers. This may suggest a defect in Ig-P oligomerization. Although the range in MW of the oligomers may be due to differential glycosylation, it is possible that several different oligomers exist. Ig-a and Ig-P may be forming homodimers in addition to/instead of heterodimers. The nature of the components of Ig-a/Ig-P oligomers is complicated by the presence of differentially glycosylated bands which overlap in MW. One approach to avoid this problem is to remove all the N-linked carbohydrates on the Ig-a/Ig-p oligomers using the endoglycosidase PNGase F. PNGase F digestion of Ig-a and Ig-P oligomers should reveal the presence of homo- and/or heterodimers of Ig-a and Ig-P based on their MW. Deglycosylated Ig-a has a MW of 28kDa whereas Ig-P has a molecular weight of 32kDa on 10% TG-gels. Based on this, deglycosylated Ig-a homodimers would be 56 kDa, deglycosylated Ig-a/Ig-P heterodimers would be 60kDa, and Ig-P homodimers would be 64kDa. As predicted, whole cell extracts treated with or without PNGase F and examined by SDS-PAGE and western blotting under non-reducing conditions resulted in the presence of three bands (figure 21A, B, and C). Almost no Ig-a or Ig-P protein was detected in the supernatant after Ig-a was retrieved from extracts with anti-Ig-a antibodies prior to SDS-PAGE (figure 21A and B). Figure 21D shows that not all Ig-P protein immunoprecipitates with Ig-a under reducing conditions. Taken together, these Ig-a/Ig-P studies suggest that 69 there are no noticable differences between WEHI 231 and WEHI 88.1 in terms of their Ig-ct/Ig-p assembly intermediates. kDa 126 — 80 — B kDa Ig-a Oligomers Ig-a Monomers 126 — 80 — 48 — 28 — Ig-P Oligomers Ig-P Monomers Figure 20. Ig-a and Ig-p Oligomers in WEHI 231 and WEHI 88.1. Whole cell extracts were prepared in M G L B and run on 5% P04 gels by SDS-PAGE under non-reducing conditions. Proteins on gels were western blotted and probed with (A) anti-Ig-a or (B) anti-Ig-P antibodies. Molecular weight markers are indicated on the left. Monomeric and oligomeric species of Ig-a and Ig-p are indicated on the right. 70 B 100-65 — m • .v.: 45— i 29 — Sup Ig-a IP Ig-a Blot Ig-a Oligomers -IP Ab -Ig-a Monomers kDa ^ > > > 100-6 5 -45 — 2 9 -Sup Ig-a IP Ig-P Blot ] Ig-P Oligomers - Ig-p Monomers/ IP Ab i) 57-43-29-23-18-- - + + 1 + + - -in ai mm Ig-a Blot Ig-P Blot P N G a s e F kDa 69 — 43 — 28 — *~ anti- anti-Ig-a Ig-P -#— Immunoprecipitating Blotting Ab Figure 21. Deglycosylated assembly intermediates of Ig-a and Ig-P do not differ between WEHI 231 and WEHI 88.1. A and B, Whole cell extracts prepared in MGLB were immunoprecipitated 3x with anti-Ig-a antibodies. The immunprecipitates were removed by PA-Sepharose from the remainder of the cell extract (Sup) and each was treated with PNGase F and examined by SDS-PAGE under non-reducing conditions. Proteins on the gels were transferred to nitrocellulose by western blotting and probed with anti-Ig-a and anti-Ig-P antibodies. C, Whole cell extracts were treated -/+ PNGase F prior to protein separation by 12% SDS-PAGE under non-reducing conditions. Gels were blotted as in A and B. D, Whole cell extracts were depleted of Ig-a and loaded on 12% tris-glycine gels for protein separation by SDS-PAGE under reducing conditions. Proteins were transferred to nitrocellulose and immunoblotted with anti-Ig-a and anti-Ig-p antibodies. 71 VI. Protein Expression on the Surface of WEHI 231 and WEHI 88.1 The results of the previous experiments do not exclude the possibility that a cell defect exists in the quality control system of the secretory pathway. One would expect that a defective secretory pathway that fails to traffic the BCR to the cell surface would also fail to traffic other plasma membrane proteins. We therefore tested the secretory system's ability to express other proteins on the plasma membrane of WEHI 88.1 cells. WEHI 88.1 and WEHI 231 cells were surface biotinylated and then used to prepare whole cell extracts in MGLB. The extracts were loaded onto 10% tris-glycine gels and separated by SDS-PAGE. Proteins in the gel were transferred by western blotting to nitrocellulose and probed with streptavidin-HRP, which binds to biotin, to reveal the presence of biotinylated proteins (figure 26). The same biotinylation pattern was seen in both WEHI 231 and WEHI 88.1 lanes, two bands of approximately 218 kDa and 100 kDa. No differences between the two cell lines were observed in the presence of surface biotinylated proteins. This result is unusual in that one would predict the presence of additional protein bands in the WEHI 231 lanes corresponding to the biotinylation of BCR proteins present on the surface of WEHI 231 cells but lacking from that of WEHI 88.1. This result may be explained by the following. Surface biotinylation depends on the accessibility of biotin to exposed lysine residues in the extracellular regions of plasma membrane proteins. The lysine residues, however, may be inaccessible to biotinylation due to protein folding, the presence of neighbouring proteins, and obstruction by carbohydrate moieties and other protein modifications. In addition, surface proteins on WEHI 231 and WEHI 88.1 cells may contain few or no lysine residues making them difficult or impossible to tag by this method. Similar experiments in which WEHI 231 and WEHI 88.1 surface proteins were labelled with I25I instead of biotin demonstrate the presence of three protein bands migrating 72 • • • • • « 125 at the same MW in both cell lines (Appendix 1). There were no major differences in the I labelled protein bands between WEHI 231 and WEHI 88.1 lanes. One would also expect additional bands to be present in WEHI 231 lanes of the surface iodination studies representing the presence of the BCR on the surface of WEHI 231 cells which are absent on WEHI 88.1 cells. Figure 26 and Appendix 1 do differ in that there are three protein bands in some lymphoid and non-lymphoid cells in Appendix 1 and only two in Figure 26. Iodination may tag an additional protein and thereby be more useful than biotinylation for labelling surface proteins in these cell lines. Despite these discrepancies, both the biotinylation and iodination studies indicate that surface proteins are present in both WEHI 231 and WEHI 88.1 cells. MW 6 9 (kDa) 5 7 _ Actin Figure 22. Surface Biotinylation of WEHI 231 and WEHI 88.1 Cells. Cells incubated in NSF-biotin for 15min were washed repeatedly and lysed. Samples were run on 10% TG-gel SDS-PAGE and transferred to nitrocellulose by western blotting. Blots were probed with streptavidin-HRP to detect biotinylated proteins. Actin lanes serve as a control for internal biotinylation. 73 DISCUSSION Recent efforts to uncover the mechanism by which cells ensure only functional receptors are expressed on their surface has led to the discovery of a quality control system in the ER involving multiple chaperone proteins including BiP, Grp94, Calnexin, and Calreticulin (Melnick and Argon, 1995; Hurtley and Helenius, 1989). The interaction of chaperones with incompletely assembled receptor components has implicated chaperones in the role of intracellular retention of misfolded or unassembled proteins. This is consistent with the generally accepted model for ER quality control, in which misfolded and unassembled proteins are retained in the ER by molecular chaperones and degraded if they cannot be rescued. ER chaperones are also postulated to assist in the folding, assembly and transport of receptor proteins in the secretory system. Whether ER chaperones are responsible for assisting in the actual process of protein folding and assembly of receptor proteins as well as the intracellular retention of improperly folded or unassembled polypeptides remains uncertain. Our aim in the present study was to select and characterize a system that could be used to study the quality control role of chaperones that interact with folding and assembling receptor subunits. To establish such a system, mutations were introduced into the WEHI 231 B lymphoma cell line that expresses the well-characterized B cell antigen receptor or BCR. To serve as a useful system, the defect(s) in the mutant has to meet at least one of the following criteria: 1/ the defect hinders the ability of the BCR to fold correctly, 2/ the defect disrupts the ability of the receptor subunits to associate with one another and assemble, or 3/ the defect disturbs the ability of the quality control system/transport machinery to function properly. We selected and characterized the BCR proteins of the WEHI 88.1 mutant that fails to signal through its BCR due to a lack of BCR on its cell surface. Our goal throughout 74 the characterization of WEFfl 88.1 was to distinguish between a defect in its BCR and/or another cell defect as the underlying cause of the failure of its BCR to traffic to the cell surface. Although we did not rule out the possibility of a cell defect, the most likely explanation for the lack of BCR surface expression in WEHI 88.1 cells is a defect in the BCR that impairs its ability to assemble properly. The WEFfl 88.1 mutant cell line thereby provides a model system with which to study the fate of unassembled and intracellularly retained receptor subunits and their interactions with chaperones. Characterization of the WEHI 88.1 BCR In the present study, we identified WEFfl 88.1 as a B lymphoma mutant expressing all four chains of the BCR (including p H chain, K L chain, Ig-a, and Ig-P) as evidenced by the cross-reactivity of protein bands on a Western blot of total cell lysates with antibodies specific for each BCR chain (figure 14). This suggests that the BCR subunits are found intracellularly and that the presence of less than 7% of wild-type levels of BCR on the WEHI 88.1 cell surface (Page et al., 1991) is not due to the complete absence of one or more BCR chains. The quantity of K L chain and Ig-a protein, however, was reduced in WEHI 88.1 by approximately 10-fold (figure 14) and this lower subunit expression may account for the decreased surface expression of the BCR. There is a correlation in the quantity of protein expression between the small fraction (<10%) of surface BCR observed on WEHI 88.1 cells and the 10-fold decrease of K L chain and Ig-a protein. This suggests that the available K L chain and Ig-a protein may assemble a small amount of BCR that escapes intracellular retention or is transported through the secretory system. If so, the remaining BCR subunits would be unable to assemble complete BCR, due to the limiting quantities of K L chain and Ig-a protein, and would be retained or degraded. Thus, it is probable that WEHI 88.1 BCR transport is blocked due to a BCR defect that hinders BCR assembly and/or a cell defect. 75 A block in BCR transport would likely occur early in the secretory pathway where the quality control system exists and faulty or unassembled proteins are retained. To test this possibility, we used the Endoglycosidase H (Endo H) sensitivity assay to determine the intracellular location of the WEHI 88.1 BCR proteins (figure 16). One drawback to this approach is that it does not determine the exact location of a protein in the secretory system. Despite the inability to pinpoint the exact location of proteins, the Endo H assay has been extensively used to monitor the progress of proteins along the early secretory pathway (i.e. the ER to Golgi stacks). The Endo H assay was therefore sufficient to determine whether the WEHI 88.1 BCR chains were contained early in the secretory pathway. Also, since the method is based on changes in the glycosylated state of a protein as it travels through the Golgi, the Endo H assay is not useful on unglycosylated proteins or glycosylated proteins that do not undergo carbohydrate modification, such as K L chain. WEHI 231 p H chain, Ig-a, and Ig-P are glycosylated and previous studies (Matsuuchi, personal observations) have shown that they undergo carbohydrate modification in the Golgi that leads to Endo H resistance. We concluded that the WEFfl 88.1 p H chain, Ig-a, and Ig-P were held intracellularly in a pre-Golgi compartment since these chains were Endo H sensitive, an indication that they failed to transport past the cis-Golgi (figure 16). The K L chain is not glycosylated and could not be localized in this manner. We suspect that K L chain was also held intracellularly in association with the H chain or secreted from the cell in the absence of H chain association. The intracellular localization of the WEHI 88.1 BCR subunits to the ER is further supported by the lack of differential glycosylation on its p H chain, Ig-a and Ig-p subunits which results from carbohydrate modifications in the medial- and trans-Golgi (figure 16). Thus, WEHI 88.1 is a B lymphoma mutant that expresses its BCR proteins intracellularly, presumably in the ER. 76 We hypothesized that the block in transport of the BCR subunits beyond the cis-Golgi may be due to a defect in one or more chains resulting in their retention by the ER quality control system. To check for major structural abnormalities, such as deletions which would alter the protein's electrophoretic mobility through a gel, we completely deglycosylated the BCR proteins to compare the mobility shift of the core proteins of WEHI 88.1 with those of its parental cell line, WEHI 231. We did not see any major differences between the mobility shift of the deglycosylated BCR protein bands of WEHI 88.1 and WEHI 231 (figure 15) suggesting that no major structural defects occur although point mutations may be present. Eliminating the possibility that point mutations were present in one or more of the BCR proteins in WEHI 88.1 cells would require sequencing the WEHI 88.1 genes and comparing the sequences to those of WEFfl 231. We did not conduct a sequence analysis of any of the WEFfl 88.1 BCR genes to confirm the existence of point mutations in its BCR chains. Thus, a defect in one or more subunits of the WEHI 88.1 BCR may be present. We can also not rule out the possibility that many mutations occurred in other areas of the cell during the generation of the WEHI 88.1 mutant by random mutagenesis. This may include mutations in the ER retention machinery that could in turn be responsible for the block in BCR transport and the loss of BCR surface expression on WEHI 88.1 cells. There may be small mutations present in the BCR subunits that interfere with their ability to interact with one another and lead to the subsequent failure of the BCR to traffic to the cell surface. To test this possibility, we examined the ability of the various BCR chains to interact with one another using coimmunoprecipitation studies. We looked at the ability of H chain and L chain in WEHI 88.1 to associate into mlgM by coimmunoprecipitating H chain and L chain complexes with antibodies against either chain under reducing and non-77 reducing conditions. Our studies indicate that despite decreased K L chain protein levels in WEFfl 88.1 cells, covalent H chain and L chain interactions took place (figure 17). Since H chain and L chain can interact covalently we can reasonably expect that these chains have the capacity to associate into mlgM. Similarly, we found that Ig-a/Ig-P interactions occur in WEFfl 88.1 cells (figure 18). Further, we were able to show through coimmunoprecipitation studies that the H chain and L chain complexes were able to interact stably with Ig-a/Ig-P heterodimers (figure 18). Thus, all four BCR chains in WEFfl 88.1 were able to interact with each other indicating that some BCR assembly took place in the mutant cell line. These findings also support our previous conclusion that structural abnormalities were not present in the chains that would interfere with BCR assembly. In light of the absence of large abnormalities in the core proteins of the WEFfl 88.1 BCR and the ability of the BCR subunits to associate with one another we looked for another source of defect in the WEFfl 88.1 BCR. Early on, we had noticed a significant decrease (approximately 10-fold) in WEFfl 88.1 K L chain protein expression compared to that in WEFfl 231 (Figure 14 lane 2). Since all four chains of the BCR are required for complete assembly and trafficking to the cell surface (Venkitaraman et al, 1991; Matsuuchi et al., 1992), it seemed possible that the reduced level of K L chain in WEFfl 88.1 was interfering with BCR assembly and this would account for the subsequent failure of the BCR to traffic to the cell surface. Despite the ability of the H chain and L chain subunits to interact, the decreased K L chain protein levels in WEFfl 88.1 may interfere with the ability to form complete mlgM. To test whether these cells do indeed form complete mlgM we analyzed the assembly intermediates of mlgM (figure 19). According to the predicted molecular weights expected for the protein bands of mlgM assembly intermediates (Table II), WEFfl 88.1 cells possess almost no bands representing H2L2 intermediates despite the presence of 78 HL and H2L intermediates. The protein quantity of K L chain may be too low to allow for the completion of mlgM. We conclude that complete mlgM does not occur readily in these cells or is rapidly degraded and that the absence of mlgM formation represents the point at which BCR assembly is blocked and surface transport inhibited. The argument that the decreased protein levels of K L chain result in WEHI 88.1 may account for the lack of complete mlgM is based on the theoretical effects of limiting subunits on the rate of protein assembly. One would expect the kinetics and the order of receptor assembly to be influenced by the presence of a limiting subunit. If all receptor subunits were present in equal amounts then the rate and order of assembly would depend solely on the thermodynamic properties of the subunit interactions involved. In the presence of a limiting subunit, the rate of receptor assembly would also depend primarily on the availability of the limiting subunit. At low concentration, the amount of the limiting subunit available for complete assembly may be insufficient for the rate of receptor oligomerization to exceed the rate of protein degradation and the subunits would fail to oligomerize completely. We suspect that the presence of less K L chain in the WEHI 88.1 mutant may be the limiting factor in its BCR assembly and transport. Further, we believe that the rate limiting levels of K L chain present in WEHI 88.1 lead to a failure of the BCR to completely oligomerize, as indicated by the almost entire absence of H2L2 mlgM complexes in WEHI 88.1 cells (figure 19). Since all four chains of the BCR must associate into a complete BCR before it can exit the ER (Matsuuchi et al., 1992), the inability of H chain and L chain to form a complete mlgM in WEHI 88.1 cells suggest that the lower K L chain expression may represent the defect responsible for the failure of WEHI 88.1 BCR to traffic to the cell surface. In addition, we found that the mlgM assembly intermediates present in WEHI 88.1 differed from those of WEHI 231 (figure 19). The major assembly intermediates in WEHI 79 88.1 were H 2 and H2L instead of the HL complex found in WEFfl 231 cells. This suggests that an alternate route of mlgM assembly occurs in WEFfl 88.1. Since H 2 and H2L are the intermediates one would expect in a reaction where K L chain is limiting, our findings support the hypothesis that the decreased expression of K L chain in these cells interferes with mlgM assembly. Also, the presence of H 2 and H2L intermediates in figure 19 indicates that the lack of complete mlgM is not due to an inability of the WEFfl 88.1 H chains to associate with other H chains. It is possible that a structural defect in either one or both of the H chains or L chains may cause a misfolding of the HL intermediates, which could interfere with the ability of HL intermediates to associate into H 2 L 2 intermediates. Although, the analysis of the DNA sequence of H chain and L chain genes was not conducted to eliminate this possibility, the presence of more H2L intermediates than HL intermediates in WEHI 88.1 suggests that free H chains are more readily available and able to associate with HL intermediates than other HL intermediates. This is consistent with the presence of less K L chain than p H chain in WEHI 88.1 cells. Also, the number of protein bands and the intensity of the protein bands of the mlgM assembly intermediates present in WEHI 88.1 differ from those present in WEHI 231 (Table I). The assembly intermediates present in WEHI 88.1 (L, H. H 2 , H2L, and very little H2L2) are characteristic of what one would expect due to limiting L chain. The assembly intermediates present in WEHI 231 (L, H, HL, and H2L2) are consistent with the presence of equimolar concentrations of H chain and L chains. The presence of different BCR assembly intermediates in each cell line suggests that the mlgM assembly pathway in WEHI 88.1 differs from that of WEHI 231 (figure 19). Thus, the implication that the presence of limiting amounts of K L chain in the WEHI 88.1 cell line may interfere with the assembly of complete mlgM is supported by the almost complete lack of H 2 L 2 mlgM in WEHI 88.1, the presence of different mlgM assembly intermediates 80 compared to WEHI 231, and the consequent prediction of different mlgM assembly pathways between the two cells (figure 19). Although our results provide strong evidence that the protein decrease in K L chain interferes with mlgM assembly and thereby blocks BCR assembly and transport to the cell surface, we have not shown direct evidence that this is the case. One approach to determine if limiting K L chain interferes with mlgM assembly would be to analyze the ability of over-expression of normal K L chain in WEHI 88.1 to restore BCR expression on the cell surface or at least complete assembly of mlgM. Unfortunately, initial attempts to introduce wild-type K L chain into WEHI 88.1 cells were not successful due to the experimental design. In the future, the use of twice the number of cells, more radioisotope, shorter chase periods, and higher concentrations of cold methionine/cysteine during chase periods may help to overcome some of the problems faced. Alternatively, one could block transport from the ER with an inhibitor such as tunicamycin in WEHI 88.1 to allow the concentration of K L chain to build up in the ER and then compare the stability of mlgM assembly intermediates in WEHI 88.1 and WEHI 231. The build up of K L chain in WEHI 88.1 would allow more BCR to form and traffic to cell surface. We predict that differences in mlgM stability exist between both cell lines and that these differences are be attributable to the ability of complete mlgM molecules to escape targeting to the degradative pathway while incomplete mlgM molecules are targeted for degradation by the cell's quality control system. The reduced L chain expression may be the result of a general problem with the translation machinery. However, the protein concentrations of the total cell extracts used were determined in order to load equal amounts of total cellular protein per lane for this experiment and to control for differences in protein translation rates between the two cells. We found that the protein band intensities of the other BCR proteins were comparable to 81 those of the WEHI 231 parental cells. Further, protein bands of the ubiquitous actin molecule appeared similar in both cell lines (figure 14). This implies that other cellular proteins are also expressed normally in the mutant. We concluded that the protein translation machinery was operating normally in WEFfl 88.1 cells and that the decreased K L chain expression was likely due to a K L chain defect. Alternatively, the decreased protein levels of WEHI 88.1 K L chain could be due to rapid secretion or degradation. We looked for the presence of secreted K L chain in the supernatants of metabolically labeled cells by immunoprecipitation. In WEHI 231 cells, we would expect excess K L chain to be secreted since the remainder of the K L chain is used to build mlgM and is expressed as BCR on the cell surface. We found no secreted K L chain in the supernatants of either WEHI 231 or WEHI 88.1 cells (data not shown). The lack of secreted K L chain in supernatant cultures may have been due to an inability of the immunoprecipitating antibodies to interact with WEHI 88.1 L chain caused by the high dilution of antibody in the large volume of supernatant. This possibility is unlikely due to our antibody's usefulness in intracellular L chain coimmunoprecipitation studies. The observation that no K L chain were secreted in the supernatant and that lower amounts of K L chain were present in WEHI 88.1 at time zero in pulse chase experiments (Appendix 2) compared to that in WEHI 231 suggest that K L chain in WEHI 88.1 may be rapidly degraded or its synthesis impaired. It is possible that the lack of L chain in the supernatant might be due to prolonged retention in the ER. This explanation does not account for the low steady state levels of L chain present in WEHI 88.1 that one would expect to be higher if they reflected an accumulation of retained L chain. A more likely account for the decreased L chain protein levels is that L chain is rapidly degraded rather than secreted or retained for a prolonged period of time. Other studies have shown that L chain is produced in excess in 82 WEHI 231 cells and that in the absence of association with H chain, L chains homodimerize and are secreted. This evidence refutes the possibility that L chain may be retained in the ER for an extended period although L chain does associate with BiP prior to association with H chain or homodimerization. Further studies have shown that Ig-a, not L chain, may be the rate-limiting factor in WEHI 231 suggesting that prolonged ER retention of K L chain is not a requirement for BCR assembly. Thus, rapid degradation or decreased protein synthesis of the K L chain appear to be the most reasonable explanations for the apparent decrease in K L chain protein levels in WEHI 88.1 cells. We were unsuccessful in obtaining the optimal conditions necessary for pulse-chase analysis to be a useful technique with which to monitor the rate of H chain and L chain turnover in WEHI 88.1 Rapid degradation of K L chain would require that K L chain interact with ER resident chaperones that direct it to an ER- or proteasome-mediated degradation route. The rate at which proteins in the ER are folded, assembled, and transported out of the ER or degraded varies widely and the reasons for this are unknown. Chaperone interactions with proteins in the ER are believed to provide some stability to unfolded proteins to allow for proper folding and association with other proteins to occur. Chaperones such as BiP may also be involved in directing misfolded or faulty proteins towards degradation. WEHI 88.1 is a useful tool with which to study the biological stability of L chain in the ER. It is possible that in addition to the limiting K L chain present in WEHI 88.1, the formation and stability of the complete H2L2 form of mlgM may be dependent on its association with Ig-a and/or Ig-p during mlgM assembly. It is possible that Ig-a and Ig-P must first associate into a heterodimer before either chain can associate with mlgM. Alternatively, either chain alone may interact with mlgM prior to forming an Ig-a/Ig-P heterodimer. It is even possible that both cases occur with equal likelihood. A failure of Ig-83 a and/or Ig-P to associate with mlgM would also mean incomplete BCR assembly even in the presence of complete mlgM. This may occur if Ig-a protein amounts are indeed lower in WEHI 88.1 cells as suggested by figure 14. To analyze these possibilities we examined the ability of Ig-a to coprecipitate with Ig-P and p H chain (figure 18). Since Ig-a can coprecipitate with both of these proteins we can conclude that Ig-a can interact with both Ig-P and p H chain. The association of Ig-a with Ig-p would depend largely on the rate of formation of disulfide bonds between the two proteins by protein disulfide isomerase. This reaction may occur cotranslationally and increase the probability that Ig-a/Ig-P heterodimers form prior to their association with mlgM. If this assumption were correct then one would expect to see more Ig-a and Ig-P heterodimers than Ig-a or Ig-p in association with mlgM alone. Ig-a coprecipitates with Ig-P suggesting that Ig-a and Ig-P can form covalent interactions with each other and therefore Ig-a/Ig-P heterodimers are present in WEHI 88.1. An alternate explanation for the lack of BCR at the cell surface of WEHI 88.1 cells is that a defect in the cell's transport machinery may exist. Such a defect would affect the ability of other membrane proteins to reach the plasma membrane. Surface biotinylation of WEHI 88.1 indicated the presence of the same three protein bands in both cell lines and show no differences between WEHI 231 and WEHI 88.1 including the difference in BCR surface expression. This suggests that other plasma membrane proteins are able to transit to the cell surface in both cells. Since biotinylation depends on the presence of accessible lysine residues, this method may not tag all surface proteins on either cell. The presence of the Fc receptor was tested by immunofluorescence but the antibody did not react well on WEHI 231 and yielded fluorescence levels comparable to background (data not shown). Thus, it remains possible that the trafficking of some but not all surface proteins in both these cells may be disrupted due to a cell defect. Further, we did not exclude the possibility of a 84 cell defect in one or more of the chaperone molecules. It is possible that some or all of the BCR proteins are retained in ER by faulty chaperones that might bind irreversibly. Proteins not involved in the same ER regulatory pathway as BCR proteins may thereby escape ER retention. In such a scenario, the escaped proteins would traffic to the cell surface and account for the presence of surface biotinylated bands in the above experiment. In conclusion, our studies show that WEHI 88.1 is a B cell lymphoma with a defect in its protein expression of K L chain. The WEHI 88.1 mutant expresses all four chains of the BCR in a pre-Golgi compartment, presumably the ER, where they undergo partial assembly. However, we believe that the K L chain is the rate-limiting factor in mlgM assembly in WEHI 88.1 and that the rate of degradation of the BCR components exceeds the rate of mlgM assembly. The low protein expression of K L chain alters the mlgM assembly pathway and results in very reduced amounts of complete mlgM. In the absence of complete mlgM the remaining BCR chains are retained intracellularly by the cell's quality control mechanism. Consequently, the majority of mlgM assembly intermediates are degraded before they can assemble into complete mlgM and form BCR. The correlation between 10-fold decreased K L chain protein expression and 7% surface BCR expression suggests that most of the L chain present in WEHI 88.1 is able to assemble normally into BCR that can transport to the cell surface. This suggests that at higher expression levels of K L chain in WEHI 88.1 cells, i.e. by over-expressing wild-type K L chain gene, surface expression of BCR should be rescued. The results of this study suggest a model for BCR assembly which, together with recent experiments of chaperone interactions with immunoglobulins and other membrane proteins, can be used to predict BCR quality control and assembly in various mutants as depicted in figure 23. 85 A WEHI 231: H + L — • HL + HL —• H 2 L 2 -+> BCR <— Ig-a + Ig-fj Cytoplasm D J J l l U l l JUJL f = 0 : - M ER Lumen B WEHI 88.1: H + L — • HL + H — • HjL — • Incomplete <«— Ig-a + lg-|3 JCR _^ Cytoplasm ER Lumen KR CHAPERONES WJ Figure 23. Maturation Model of the BCR in WEHI 231 and WEHI 88.1 Cells. (A) In WEHI 231 cells, H chain and L chain are produced at similar concentrations and follow the H chain intermediate assembly pathway to produce complete mlgM that associates with Ig-a/Ig-p\ The complete BCR is freed from chaperones so that it can transit to the plasma membrane. (B) In WEHI 88.1 cells, the decreased protein quantity of K L chain is rate-limiting in the mlgM assembly pathway. The available L chain quickly associates with H chain to form H chain intermediates. Due to the low L chain concentration, free H chain associates with the H chain intermediates to form H2L. The stability of WEHI 88.1 mlgM assembly intermediates in the ER may be prolonged through their association with BiP (green) and other chaperones. During their extended stay in the ER, the WEHI 88.1 BCR subunits are marked for degradation. Due to insufficient L chain, BCR assembly is incomplete and it is targeted for degradation before it can fully oligomerize and transit to the cell surface. Recent Studies on BCR Assembly and Transport by Others Clues as to the fate of WEHI 88.1 K L chain may be present in recent studies involving L chain and H chain interactions with each other and with BiP chaperones. In pre-B cells, H chains form homodimers prior to the developmental expression of L chain (Kaloff 86 and Haas, 1995). These H chain homodimers are held intracellularly in association with BiP (Haas and Wabl, 1983; Bole et al, 1986) until L chains are developmentally expressed and H2L2 molecules are formed. The stable BiP binding site on unassembled H chains is the C H I constant domain that is also involved in both hydrophobic and covalent interactions with the constant domain of the L chain (Hendershot et al., 1987). The association of H chain Ig domains with BiP suggests that Ig domains of other proteins such as L chain may also interact with BIP. Several groups (Hendershot et al, 1996; Skowronek et al., 1998; Hellman et al., 1999) report that L chains with an unfolded variable region bind transiently to BiP and release BiP once the domain is folded. Leitzgen et al., 1997, show that in mutants lacking H chain expression some L chains homodimerize and are secreted. L chains that are not secreted bind to the ER chaperone BiP as partially folded molecules until they are degraded. These unsecreted L chain proteins only form one disulfide bond in their constant domain but lack the second disulfide bond in the variable domain and are subsequently unable to release BiP and homodimerize. This suggests that the variable region may be the site of L chain association with BiP and that proper folding is necessary for the secretion of L chains. Accordingly, Lee et al., 1999, show that the addition of ATP to unfolded H chain:BiP complexes in the absence of L chain results in the release of BiP and the folding of the C H I domain. They conclude that BiP maintains H chain in an unfolded state while L chain allows the release of BiP from the C H I domain so subsequent folding can occur. These findings indicate that BiP associates with unfolded H chains and L chains and maintains them in an unfolded state until they are able to associate. The release of BiP and subsequent folding of the CHI domain suggests that BiP is not required for H chain folding but rather to maintain H chain in a folding competent state until it becomes associated with L 87 chain. In WEHI 88.1 cells, the low K L chain expression may result in the association of H chain with HL intermediates or in the homodimerization of H chain and their intracellular retention by BiP as observed in pre-B cells. This would be consistent with the presence of H2 assembly intermediates co-migrating with H2L intermediates in WEHI 88.1 cells but not in WEHI 231. Please note, however, that we have not yet confirmed the presence of H2 intermediates although it would be possible by two-dimensional analysis of the H2L protein bands. The H2 intermediates that are unable to associate with L chain and proceed to the H2L and H2L2 may be targeted for degradation by the quality control system to prevent the accumulation of unassembled proteins. If so, the quality control system must possess some mechanism to monitor protein concentrations in the ER and distinguish between proteins that are newly made and those that have failed to fold or assemble completely. Additional studies show that the degradation rates of two unassembled Ig L chain mutants, kNSl and IFS62, are not identical when each is separately expressed in the same cell line. Each L chain mutant forms complexes with BiP but the kNSl mutant is less stable (half-life of one hour) than the 1FS62 L chain mutant (half-life of four hours). The unfolded variable domain of each L chain mutant formed the BiP binding site and determined the stability of the complex as evidence by swapping the variable regions of the two L chain isotypes. These results suggest BiP-release is a rate-limiting step for delivery of faulty proteins to the degradative pathway. Skowronek et al, 1998, find that the physical stability of BiP association with an unfolded region of a given L chain determines its half-life, suggesting a direct link between chaperone:unfolded protein interactions and their delivery to the degradation machinery. More recently, Hellman et al., 1999, show that the ability of the murine A.1 L chain to associate with BiP is based on the rate and stability of Ig domain folding. The two Ig 88 domains of the A, 1 L chain, the constant and the variable domains, fold separately (Goto et al., 1979; Goto and Hamaguchi, 1982) and both contain several potential BiP binding sites predicted by a phage display analysis algorithm (Blond-Elguindi et al., 1993; Knarr et al., 1995). The constant domain folds rapidly and stably even in the absence of an intradomain disulfide bond which provides structural stability to proteins (Hellman et al., 1999). Further, BiP does not bind to the XI L chain constant region despite the presence of potential BiP binding sites. This suggests that the presence of potential BiP binding sites alone is insufficient to predict BiP association with a protein region. BiP does bind to the unoxidized variable domain of the XI L chain. The XI L chain variable region folds more slowly and unstably than the constant region. This correlation between slow, unstable folding and BiP association suggests that the rate and stability of protein folding are determinants of BiP recognition/binding and not vice versa. As such, the folding pathway and the folding rate determine BiP association with a protein. A similar approach may be used to examine whether BiP association occurs with WEHI 88.1 K L chain. The K L chain oxidation state can be monitored by increases in mobility in SDS-PAGE due to compacting of protein structures by disulfide-bond formation in Ig domains. BiP ATPase mutants can be used to examine BiP binding indicated by an upper shift in mobility of L chain proteins under reducing conditions. In the presence of BiP ATPase mutants that bind irreversibly to substrate, L chain in WEHI 88.1 may be coimmunoprecipitated with an anti-BiP antibody. In such an experiment, a slow migrating L chain species would represent oxidized L chain in which both domains bind BiP. An intermediate mobility would represent L chain that binds BiP at only one domain or only one domain at a time. A fast migrating species would represent a reduced L chain state in which no BiP binding occurs. 89 Hellman et al., 1999, also find that disulfide bond formation in both domains is required for L chain secretion. All L chains contain a COOH-terminal cysteine that remains unpaired in L chains that have not formed homodimers or assembled with H chain. Although this suggests a role for the thiol-mediated retention mechanism possibly involving the ER-60 chaperone (Alberini et al, 1990; Fra et al., 1993; Lindquist et al., 1998), Hellman et al., find that other chaperones, namely BiP, may be involved. Based on Hellman's findings, some questions about the intracellular fate of WEHI 88.1 K L chain include: 1/ are K L chain homodimers present in WEHI 88.1?; 21 Are K L chains associated with PDI, BiP or other chaperones in the ER?; 3/ Does K L chain form appropriate disulfide bonds in WEHI 88.1 cells? Together, these studies and ours indicate that L chain is a simple protein with features, including the transient nature of the association between L chains and BiP and the secretion of L chain in the absence of H chain, that make it useful for the study of in vivo folding and quality control. Future Studies In the present study, we characterized the BCR subunits and their maturation in WEHI 88.1 cells and established a model system with which to address several remaining questions about BCR maturation in the ER and the quality control of surface receptors. The most significant question to address is which chaperones associate with the assembly intermediates of the BCR. The kinetics of subunit folding and the stability of the assembly intermediates may influence the ability of chaperones to associate with the BCR chains. We believe that in the presence of limiting quantities of L chain, mlgM assembly would occur more slowly. This would prolong the length of time that the BCR chains remain trapped in a pre-Golgi compartment during which they probably become destined for degradation by the quality control mechanism of the secretory pathway. As such, WEHI 88.1 would serve as a 90 useful system with which to study the kinetics of rate-limiting factors on receptor oligomerization and the association of chaperones with assembly intermediates. Such studies would involve the use of pulse chase experiments to follow the maturation of mlgM. The association of chaperones with assembly intermediates in the presence of a rate-limiting factor may reveal the mechanism of quality control of incomplete receptors. Of particular interest is how the quality control system distinguishes between proteins with slow folding and assembly steps vs. misfolded or unassembled proteins. Steady state labeling is not suitable for examining BiP association with proteins because interactions are transient and unstable. Pulse labeling under reducing conditions to inhibit domain folding and maximize BiP:protein interactions may be used instead. A protein's stability can also be examined by monitoring its fluorescence at increasing temperatures to establish a thermal unfolding curve since intrinsic fluorescence of a protein changes as it unfolds. One drawback to this approach is that establishing conditions where Ig domain remains unfolded (i.e. remains unoxidized in reducing conditions) may increase aggregate formation since BiP function may be reduced. The goal is to optimize conditions such that Ig remains unfolded and BiP is functional. Using our model system of the B cell antigen receptor, the present study could be expanded to address the roles of N-linked glycosylation and ubiquitination as signals for proteasome-mediated degradation of incompletely assembled BCR proteins in the ER. Our study indicated that membrane components of the BCR in WEHI 88.1 exist mostly in the core-glycosylated form and that these chains are maintained intracellularly for a longer period of time than wild-type cells. The increased life-span of the BCR chains in the ER may be accounted for by their inability to escape ER retention while the bulk of BCR chains in wild-type cells assemble properly and escape to the cell surface. N-linked glycosylation 91 may confer on BCR chains a greater stability in the ER to allow for slower folding and assembly steps to occur. This may involve chaperones, like Calnexin, that recognize carbohydrate moieties. Inhibition of N-linked glycosylation using tunicamycin or removal of their carbohydrates, would be expected to disrupt chain stability in the ER and result in the rapid degradation of the BCR chains. Since L chain lacks such glycosylation, its association with H chains may increase its longevity in the ER in WEHI 88.1. If so, one might expect L chain degradation in these cells to occur more rapidly in the absence of H chains. Conversely, adding sequences for N-linked glycosylation to the L chain gene may give it greater stability and a longer half-life in the ER. But how might carbohydrates prolong the stay of proteins in the ER? It is possible that the glycosyl groups block protein sites necessary for recognition by the quality control machinery. Alternatively, glycosylation may allow for recognition and increased stabilization by the quality control machinery. Since the BCR subunits in WEHI 88.1 cells fail to traffic to the cell surface, are localized intracellularly and eventually undergo degradation, WEHI 88.1 serves as a useful system in which to study protein degradation of faulty receptor proteins. We attempted to follow the fate of mlgM molecules in WEHI 88.1 by pulse chase analysis (data not shown) to determine the half-life and stability of the H chain and L chain subunits and to later examine the rate of assembly of the BCR under non-reducing conditions. We were unable to acquire consistent data using pulse chase analysis of mlgM due to technical difficulties. For the future, an alternate method to determine whether L chain was rapidly degraded or secreted from the cell is to block degradation using protease inhibitors and the proteasome inhibitor lactacystin and to block transport from the ER using Brefeldin A or tunicamycin. If K L chain in WEHI 88.1 is rapidly degraded then blocking degradation should result in the accumulation of K L chain in the ER. Similarly, ER accumulation of otherwise rapidly 92 secreted L chain would occur in the presence of Brefeldin A or tunicamycin. Alternatively, both the degradation or secretion of K L chains could be followed in live cells, in theory, by tagging the proteins with fluorescent green protein (FGP). This new approach might present a challenge given that the large size of the FGP tag which could interfere with K L chain folding and alter its normal fate in WEHI 88.1 cells. Since BCR subunits are retained in the ER of WEHI 88.1 cells, this cell line is a useful system with which to examine the association of BCR subunits with ER chaperones. To test for association of WEHI 88.1 BCR subunits with ER chaperone molecules, we could employ in theory chemical cross-linking to stabilize the transient association of chaperones with proteins followed by the coimmunoprecipitation of the BCR subunits with antibodies specific for ER chaperones. The disadvantage of using chemical cross-linking to stabilize proteimprotein interactions is that neighbouring but non-associated proteins may also be cross-linked. Chemical cross-linking experiments should be supported with other methods such as reconstituting the BCR assembly system in microsomes. By adding one chaperone at a time and later in combination, the microsome reconstitution system can be used to examine BCR:chaperone interactions. Quality Control of Other Receptors Much progress has been made in the field of quality control of membrane protein assembly in other systems. One of the best studied is the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR is a 1440 amino acid, glycosylated, multi-functional member of the ABC transporter family with a unique structure depicted in figure 28 based on Riordan's proposed model (Riordan et al., 1989). CFTR contains two potential sites for Asn-linked carbohydrate attachment at positions 894 and 900 (figure 24). CFTR also contains five domains: two membrane-spanning domains (MSDs), two nucleotide-93 binding domains (NBDs), and one regulatory (R) domain. The MSDs usually consist of six transmembrane-spanning regions and form the channel's pore. The NBDs contain putative ATP binding sites (Ames et al, 1990; Hyde et al, 1990). The R domain, which contains multiple consensus phosphoryaltion sites and many charged amino acids, links the two MSD-NBD motifs. MS.DJ MSD2 Extracellular Intracellular Figure 24. Diagrammatic Representation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The two membrane spanning domains (MSD1 and MSD2) are made up of six transmembrane segments each and together form . the Cl~ channel core. N-linked glycosylation occurs on the extracellular loop between transmembrane segments 7 and 8 and is proposed to facilitate interactions with Calnexin chaperones in the ER. The cytoplasmic side of the CFTR contains the nucleotide binding domains NBD1 and NBD2, which are joined by the regulatory (R) domain. The structure of the CFTR is important to its function. Located primarily in the apical epithelium, CFTR forms a phosphorylation dependent CF channel that mediates transepitheleal salt and liquid movement and regulates ion concentrations. It may also regulate the function of other ion channels. rDysfunctional CFTR disrupts transepitheleal ion transport of various organs lined by epithelia including the lungs, pancreas, intestine and the sweat glands causing the wide-ranging symptoms of cystic fibrosis. Many studies (reviewed 94 in Sheppard and Welsh, 1999) have begun to reveal the mechanism by which CFTR structural domains contribute to its function. Opening and closing of the CFTR channel is tightly controlled by a balance of kinase and phosphatase activity in the cytoplasm and by cellular ATP levels. Phosphorylation of the R domain determines channel activity while ATP hydrolysis of NBDs determines channel gating (reviewed in Sheppard and Welsh, 1999). Activation of cAMP-dependent protein kinase (PKA) leads to the phosphorylation of multiple serine residues within the R domain. Once the R domain is phosphorylated, channel gating is regulated by a cycle of ATP hydrolysis at the NBDs. Protein phosphatases dephosphorylate the R domain and return the channel to its resting state. Many CFTR mutations occurring naturally or by site-directed mutagenesis cause misfolding in the ER, decrease dissociation from chaperones, and prevent CFTR transport through the secretory pathway to the plasma membrane. One such mutant, AF508, encodes a phenylalanine deletion at position 508 in the NBD1 region, accounts for approximately 70% of cystic fibrosis, and is associated with the severe form of the disease. AF508 forms an unstable and inefficiently folded CFTR that is not transported to the plasma membrane. Pulse-chase analysis reveals that both CFTR and AF508 are initially synthesized as a 135-140 kDa core-glycosylated immature precursors containing high-mannose oligosaccharides that are Endo H sensitive (Lukacs et al, 1994; Ward and Kopito, 1994; AL-Awqati, 1995). On SDS-PAGE gels, mature CFTR migrates as a diffuse band of 150-160 kDa (Al-Awqati, 1995) and contains complex Endo H resistant carbohydrates processed by mannosidases in the cis/medial Golgi. The absence of AF508 from the cell surface could be due to either a defective intracellular delivery or protein maturation or due to instability of mature AF508 at the cell surface. Pulse-chase studies used to distinguish between these two possibilities show that only wild-type CFTR chased to the plasma membrane (AL-Awqati, 1995). This 95 indicates that the AF508 mutant allele interferes with a step in the maturation of immature CFTR from the ER to Golgi complex. CFTR maturation involves the interaction of nascent CFTR with various proteins both in the cytoplasm and in the ER lumen (Yang et al, 1993; Pind et al., 1994; Meacham et al, 1999; reviewed in Kopito, 1999). Nascent CFTR associates with the transmembrane chaperone Calnexin (Pind et al., 1994). Yang et al., 1993, show that immature CFTR and AF508 immunoprecipitate from pulse-labeled cells with antibodies to the cytoplasmic 70 kDa heat shock protein (HSP 70) but not with BiP or GRP 94. These findings suggest that HSP 70 and Calnexin are likely candidates for CFTR chaperones while BiP is not. Since little of the CFTR mass is accessible to the ER lumen, BiP association may be very unstable and the method used may not be sensitive enough to detect transient BiP binding. The association of immature CFTR with other cytoplasmic such as TriC, HSP 90, and HSP 40 chaperones or with other ER lumenal chaperones is also not excluded. More recently, Meacham et al, 1999, show that Hdj-2, an HSP 40, is localized with HSP 70 on the cytoplasmic face of the ER membrane and transiently associates with CFTR and AF508 translation intermediates. HSP 70 may associate with the cytoplasmic loops or the NBDs of CFTR whereas Calnexin may associate with the ER lumenal, high-mannose carbohydrates on immature CFTR in a lectin-like fashion. Thus, CFTR interacts with at least three molecular chaperones, HSP 70 and Hdj-2 in the cytoplasm and Calnexin in the ER. Several groups have examined the role of chaperone association with CFTR and AF508 in protein folding, trafficking and degradation (reviewed in Kopito, 1999). Meacham et al, 1999, show that the association of HSP70 and Hdj-2 with CFTR and AF508 coincides temporally with NBD1 expression in the cytoplasm. During CFTR biosynthesis, NBD1 forms a folding intermediate that is prone to self-aggregation. This suggests that Hdj-2 and 96 HSP 70 act together to suppress NBD1 aggregation of CFTR and AF508. Upon synthesis of the R domain, Hdj-2 and HSP 70 interaction with the ribosome-bound CFTR translation intermediates decreases with wild-type CFTR but not with AF508. Hdj-2 and HSP 70 association with AF508 may be prolonged due to the stabilization by AF508 of the folding intermediate of NBD1 that is prone to self-aggregation (Qu and Thomas, 1996; Qu et al., 1997a,b). In addition, mutations in NBD1 interfere more with CFTR presentation on the cell surface compared to mutations in homologous positions of NBD2. Nascent CFTR chains that lack correctly folded NBD1 are likely to interact with the quality control machinery before completion of synthesis than CFTR chains containing misfolded NBD2. The prolonged interaction of AF508 and other NBD1 mutants with chaperones leads to co-translational multi-ubiquitination and targeting to the proteasome for degradation (Sato et al, 1998) . Finally, the dissociation of immature CFTR from Calnexin correlates temporally with later steps of CFTR maturation and transport to the Golgi (Pind et al., 1994). Based on these finding and Meacham et a/.'s proposed model for NBD1 and HSP 70/Hdj-2 interaction, 1999, the evidence to date suggests the following model for CFTR/AF508 biosynthesis and maturation. Both CFTR and AF508 are synthesized in the rough ER as immature core-glycosylated 140 kDa precursors. The first step in CFTR folding occurs during the insertion and orientation of the transmembrane domains of MSD1 into the ER membrane (Lu et al., 1998). The second step is the synthesis of NBD1 and its extrusion into the cytoplasm where it associates transiently with Hdj-2 and then HSP 70. The interaction between NBD1 and Hdj-2/Hsp 70 is proposed to promote NBD1 folding (Strickland et al., 1997) or to stabilize NBD1 during R domain synthesis (Meacham et al., 1999) . In the third step of CFTR folding, the R domain is synthesized and putatively interacts with the N-terminal half of the CFTR in a way that reduces Hdj-2 binding. For 97 example, NBD1 and the R domain may interact (Winter and Welsh, 1997) to form a structural conformation that buries some of the binding sites on the CFTR from Hdj-2. The AF508 mutation present in the NBD1 may block CFTR maturation at this step by interfering with NBD1/R domain interaction and its subsequent stabilization in step four. In step four, MSD2 is synthesized and integrated into the ER membrane. This stabilizes NBD1 and R domain interactions and leads to the release of most Hdj-2 molecules from the CFTR. The stabilization of NBD1 and R domain interactions may involve the formation of MSD1/MSD2 complexes in the ER membrane and CFTR channel formation (Ostedgaard et al, 1997). Calnexin (Pind et al, 1994) may facilitate MSD1/MSD2 interactions. Finally, the fifth step involves the formation of the NBD2 domain and the ATP-dependent conformational maturation of the CFTR in the ER (Lukacs et al, 1994). Hdj-2 and HSP 70 may interact with NBD2 during the final steps of CFTR maturation. CFTR maturation is accompanied by a 20-fold increase in protein stability and Brefeldin A studies show that this stability is due to attainment of the correct tertiary structure accompanied by chaperone release. Once CFTR maturation in the ER is complete, the molecular chaperones dissociate and the CFTR traffics through the secretory system. AF508 remains partially unfolded or misfolded and chaperone association persists resulting in delivery to the degradation pathway. Thus, this model proposes that CFTR folding occurs by sequential interaction of its various domains as they are synthesized with molecular chaperones in the cytoplasm and in the ER and with other adjacent domains. There are several similarities between AF508 and the WEHI 88.1 BCR. Both AF508 and the BCR in the WEHI 88.1 mutant fail to transport efficiently to the cell surface. Unlike wild-type BCR, however, wild-type CFTR does not traffic to the cell surface efficiently. Only 20-40% of CFTR matures to the Golgi coincident with its dissociation from HSP 70 98 and its conversion to a stable (half-life=7.5h) 160 kDa, Endo-H resistant, mature form (Lukacs, et al, 1994; Ward and Kopito, 1994). This effect may be due to expression in established cell lines as the maturation N-acetylcholine receptor is almost entirely efficient in native tissue or primary cell lines but inefficient in established cell lines (Kopito, 1999). Both WEHI 88.1 BCR chains and AF508 (Cheng et al, 1990; Kartner et al, 1992; and Yang et al, 1993) mislocalize to the ER region. Both proteins interact with molecular chaperones in the ER region and appear to be intracellularly retained. One question of interest is whether misfolded AF508 or incompletely assembled BCR intermediates are actually retained by a quality control mechanism or whether they accumulate in the ER as degradation substrates at a rate greater than the cell's capacity to degrade the protein. A retention mechanism would be expected to increase the stability (half-life) of the protein in the ER that may be observed when the protein is present at low concentration. Conversely, the accumulation of degradation substrates would give the appearance of retention and may form aggregates (Kopito, 1999). The accumulation of degradation substrates may be mimicked by overexpression of the protein. Studies on the stability and trafficking of underexpressed and overexpressed proteins may help elucidate the mechanism by which the quality control machinery distinguishes between misfolded proteins and those that are in the process of folding and/or assembling. Another integral membrane protein, the human insulin receptor (HIR) has been used to explore the molecular basis of several sequential maturation steps during receptor biosynthesis and is depicted in figure 25. Like the BCR, HIR undergoes N-linked glycosylation (Ebina et al, 1985; Ullrich et al, 1985) and is a member of the family of receptor tyrosine kinases (RTK) involved in signal transduction from the cell surface to the interior of the cell. HIR is initially synthesized as a single chain proreceptor must undergo 99 homodimerization and form two symmetric interchain covalent disulfide bonds at cysteines 524-524 and 682-682 (Lu and Guidotti, 1996) before it is released from the ER. The N-linked oligosaccharides are modified in the Golgi and the proreceptor is cleaved by furin or related convertases in the Golgi (Robertson et al, 1993; Bravo et al., 1994). The HIR is then transferred to the plasma membrane as a heterotetramer composed of two a and two P subunits with a molecular mass of 350-400kDa (Olson et al., 1988). Thus, HIR is a plasma membrane receptor whose biosynthesis is well studied. HIR signal cysteine-peptide rich RKRR run tyrosine I M U kinase a 735 aa -P0194i P 620 i extracellular space cytoplasm Figure 25. Schematic of human insulin receptor (HIR) structure. (A) A linear representation of the — i human insulin proreceptor with functionally important regions shown. The proreceptor undergoes N-linked glycosylation at 17 sites and cleavage at the indicated tetrabasic site (RKRR). (B) The mature cell surface HIR is a 350-400 hormone ^ a heterotetramer composed of two binding a a n d two P subunits that are domain signalling organized into two separate modules: an extracellular domain consisting of the entire a and part of the P subunits, and an intracellular domain containing the tyrosine kinase region of the P subunit. Two symmetric interchain disulfide bonds, indicated with dark lines (cysteines 524-524 and 682-682) join the two a subunits.(TMD = transmembrane domain). Taken from Bass et al., 1998. Using pulse-chase labeling and non-reducing SDS-PAGE analysis, Bass et al, 1998, found that HIR the rate of HIR assembly determines the folding efficiency of the receptor and that the ER chaperones Calnexin and Calreticulin assist in HIR maturation by promoting folding prior to assembly. Using optimal detergent lysis and chemical cross-linking conditions, the group show that both BiP and Calnexin coimmunoprecipitate with HIR. They show that Calnexin associates with the proreceptor prior to dimerization and release 100 from the ER. When Calnexin association is blocked with castanospermine (CST), an inhibitor of glucose trimming, BiP association with the proreceptor and proreceptor dimerization increase. Surprisingly, the pulse chase analysis of surface expression of HIR shows that in the absence of Calnexin association with FAR proreceptor, functional HIR is transported to the cell surface although the rate of surface expression of HIR decreases. Taken together, these results suggest that BiP association serves as a retention mechanism while Calnexin association serves as a folding mechanism. A model for HIR maturation based on these findings is provided in Figure 26. Introduction of mutations into the HIR that disrupt folding, assembly or transport of the receptor to the cell surface can be generated to study the role of chaperones in these processes in a fashion similar to our WEFfl 88.1 model system. Thus, like the BCR, HIR serves as a model system for the study of the role of chaperones in the assembly and transport of receptor molecules. 101 Glucose Disulfide nim~r;*a*;n„ Removal Isomerization Dimenzation B i P CRT ER Lumen 150 min retained Oligosaccharide key -D - high-mannose glucose A - glucose trimmed 2 - capped c J „•• slow ' refolding Misfolded Capping Sugars & Cleavage CELL SURFACE Figure 26. Maturation Model for Human Insulin Receptor. Temporal sequence of ER folding of the insulin receptor showing synchronization of maturation and chaperone binding. The diagram illustrates that Cnx/Crt bind in complexes to newly synthesized proreceptor monomers (EM, early monomer) and that conversion to a second oxidative intermediate (S-S) occurs during binding to chaperones (LM, late monomer). Post-ER maturation involves proteolytic cleavage and addition of carbohydrate capping sugars to Asn-linked oligosaccharide chains. Addition of carbohydrates results in mobility shifts on non-reducing SDS-PAGE, whereas proteolytic cleavage does not alter the tertiary or quaternary structure of the receptor as detected by non-reducing SDS-PAGE. The dual pathways of maturation involve either Cnx/Crt or BiP (top route) with dimerization occurring at -75 min posttranslationally, whereas a second pathway occurs when Cnx/Crt are bypassed with rapid dimerization within ~10 min posttranslationally. Taken from Bass et al., 1998. 102 L I S T O F A B B R E V I A T I O N S aa - amino acid BCA - bicinchoninic acid protein assay kit BCR - B cell antigen receptor BiP - heavy chain binding protein CMV - cytomegalovirus DOC - deoxycholate ECL - enhanced chemiluminescence Endo F - Endoglycosidase F EndoH - Endoglycosidase H ER - endoplasmic reticulum FCS - fetal calf serum GRP - glucose response protein hr - hour H chainl - hydrochloric acid Igs - immunoglobulins ITAM - intracellular tyrosine activation motif kB - kilobase kDa - kilodalton LB - Lysis Buffer mA - milliAmp MG LB - Mike Gold's lysis buffer MH chain - Major Histocompatibility Complex mlgs - membrane immunoglobulins MW - molecular weight NAC - nascent polypeptide-associated complex NDET - Nonidet-P40 detergent PAS - Protein-A Sepharose PDI - Protein Disulfide Isomerase SAS - Staphylococcus-A Sepharose SB - Sample buffer SDS - sodium dodecyl sulphate SDS-PAGE - sodium dodecyl sulphate polyacrylamide gel electrophoresis slgs - secreted immunoglobulins SRP - Signal Recognition Particle SRPR - Signal Recognition Particle Receptor TBS - Tris buffered saline TBST - Tris buffered saline with Tween-20 TCR - T Cell Receptor TRAM - translocating chain-associating membrane protein VSV G protein - vesicular stomatitis virus 103 R E F E R E N C E S Afinsen, C.B. 1973. 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