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• W2089035269 abstract "The nicotinic acetylcholine receptor in muscle is a ligand-gated ion channel with an ordered subunit arrangement of α-γ-α-δ-β. The subunits are sequestered in the endoplasmic reticulum (ER) and assembled into the pentameric arrangement prior to their exit to the cell surface. Mutating the Arg313–Lys314 sequence in the large cytoplasmic loop of the α-subunit to K314Q promotes the trafficking of the mutant unassembled α-subunit from the ER to the Golgi in transfected HEK cells, identifying an important determinant that modulates the ER to Golgi trafficking of the subunit. The association of the K314Q α-subunit with γ-COP, a component of COP I coats implicated in Golgi to ER anterograde transport, is diminished to a level comparable to that observed for wild-type α-subunits when co-expressed with the β-, δ-, and γ-subunits. This suggests that the Arg313–Lys314 sequence is masked when the subunits assemble, thereby enabling ER to Golgi trafficking of the α-subunit. Although unassembled K314Q α-subunits accumulate in the Golgi, they are not detected at the cell surface, suggesting that a second post-Golgi level of capture exists. Expressing the K314Q α-subunit in the absence of the other subunits in ubiquitinating deficient cells (ts20) results in detecting this subunit at the cell surface, indicating that ubiquitination functions as a post-Golgi modulator of trafficking. Taken together, our findings support the hypothesis that subunit assembly sterically occludes the trafficking signals and ubiquitination at specific sites. Following the masking of these signals, the assembled ion channel expresses at the cell surface. The nicotinic acetylcholine receptor in muscle is a ligand-gated ion channel with an ordered subunit arrangement of α-γ-α-δ-β. The subunits are sequestered in the endoplasmic reticulum (ER) and assembled into the pentameric arrangement prior to their exit to the cell surface. Mutating the Arg313–Lys314 sequence in the large cytoplasmic loop of the α-subunit to K314Q promotes the trafficking of the mutant unassembled α-subunit from the ER to the Golgi in transfected HEK cells, identifying an important determinant that modulates the ER to Golgi trafficking of the subunit. The association of the K314Q α-subunit with γ-COP, a component of COP I coats implicated in Golgi to ER anterograde transport, is diminished to a level comparable to that observed for wild-type α-subunits when co-expressed with the β-, δ-, and γ-subunits. This suggests that the Arg313–Lys314 sequence is masked when the subunits assemble, thereby enabling ER to Golgi trafficking of the α-subunit. Although unassembled K314Q α-subunits accumulate in the Golgi, they are not detected at the cell surface, suggesting that a second post-Golgi level of capture exists. Expressing the K314Q α-subunit in the absence of the other subunits in ubiquitinating deficient cells (ts20) results in detecting this subunit at the cell surface, indicating that ubiquitination functions as a post-Golgi modulator of trafficking. Taken together, our findings support the hypothesis that subunit assembly sterically occludes the trafficking signals and ubiquitination at specific sites. Following the masking of these signals, the assembled ion channel expresses at the cell surface. nicotinic acetylcholine receptors endoplasmic reticulum phosphate-buffered saline fluorescein isothiocyanate monoclonal antibody green fluorescent protein endo-β-N-acetylglucosaminidase H Multimeric transmembrane proteins, including complex ligand-gated ion channels represented by nicotinic acetylcholine receptors (nAchR),1 generally require subunit assembly to be transported beyond the endoplasmic reticulum (ER) into the secretory pathway leading to the cell surface (1Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1064) Google Scholar, 2Chen Y.T. Stewart D.B. Nelson W.J. J. Cell Biol. 1999; 144: 687-699Crossref PubMed Scopus (246) Google Scholar, 3Schwappach B. Zerangue N. Jan Y.N. Jan L.Y. Neuron. 2000; 26: 155-167Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). As integral membrane components of lipid trafficking vesicles, unassembled subunits are re-localized to the ER, stabilized by chaperones and either assembled with neighboring subunits or targeted for degradation by ubiquitination and cleavage in the proteasome (4Keller S.H. Lindstrom J. Taylor P. J. Biol. Chem. 1996; 271: 22871-22877Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 5Keller S.H. Lindstrom J. Taylor P. J. Biol. Chem. 1998; 273: 17064-17072Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Cellular mechanisms that distinguish whether a protein is folded and/or assembled and directed to the cell surface or misfolded and/or unassembled and shuttled into a degradative pathway are poorly understood, especially for the polytypic membrane proteins represented by ligand-gated ion channels. The importance for shedding light on this topic is borne out by several debilitating disorders associated with mutations that are believed to cause misfolding and inhibit the trafficking of physiologically important ion channels. Examples include inherited mutations in potassium channel subunits that increase the propensity to develop cardiac arrhythmias (6Furutani M. Trudeau M.C. Hagiwara N. Seki A. Gong Q. Zhou Z. Imamura S. Nagashima H. Kasanuki H. Takao A. Momma K. January C.T. Robertson G.A. Matsuoka R. Circulation. 1999; 99: 2290-2294Crossref PubMed Scopus (164) Google Scholar, 7Zhou Z. Gong Q. January C.T. J. Biol. Chem. 1999; 274: 31123-31126Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar) and amino acid substitutions in cystic fibrosis transmembrane conductance regulator that result in cystic fibrosis (8Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.C. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5925) Google Scholar, 9Denning G.M. Ostedgaard L.S. Welsh M.J. J. Cell Biol. 1992; 118: 551-559Crossref PubMed Scopus (158) Google Scholar, 10Welsh M.J. Denning G.M. Ostedgaard L.S. Anderson M.P. J. Cell Sci. (Suppl. ). 1993; 17: 235-239Crossref PubMed Google Scholar). In this study, we employ the nAChR α-subunits as a model to identify factors that modulate the trafficking of ion channel proteins into the secretory pathway. We approached this question by identifying mechanisms that restrict the placement of unassembled α-subunits at the cell surface and asked how subunit assembly abrogates these processes. The nAChR in muscle is a ligand-gated ion channel composed of two α-subunits and single β-, γ-, and δ-subunits which surround a central cation channel pore. As determined by subcellular fractionation, the unassembled α-subunits are confined primarily to the ER compartment (11Smith M.M. Lindstrom J. Merlie J.P. J. Biol. Chem. 1987; 262: 4367-4376Abstract Full Text PDF PubMed Google Scholar). Insignificant amounts of unassembled α-subunit are detected on the cell surface following transfection, transient expression in mammalian cells, and125I-α-bungarotoxin exposure (12Gu Y. Forsayeth J.R. Verrall S., Yu, X.M. Hall Z.W. J. Cell Biol. 1991; 114: 799-807Crossref PubMed Scopus (91) Google Scholar). In contrast, appreciable binding of 125I-α-bungarotoxin is detected on the cell surface when α-subunits are co-expressed with the β-, δ-, and γ-subunits, demonstrating that subunit assembly is a requirement for the cell surface expression (12Gu Y. Forsayeth J.R. Verrall S., Yu, X.M. Hall Z.W. J. Cell Biol. 1991; 114: 799-807Crossref PubMed Scopus (91) Google Scholar, 13Kreienkamp H.J. Maeda R.K. Sine S.M. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar). As the nAchR assembles into a pentamer, assembled α-δ and α-γ dimers and other intermediates are detected in intracellular pools of the cell (12Gu Y. Forsayeth J.R. Verrall S., Yu, X.M. Hall Z.W. J. Cell Biol. 1991; 114: 799-807Crossref PubMed Scopus (91) Google Scholar, 13Kreienkamp H.J. Maeda R.K. Sine S.M. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar, 14Green W.N. Claudio T. Cell. 1993; 74: 57-69Abstract Full Text PDF PubMed Scopus (122) Google Scholar). However, the α-δ and α-γ dimers are also sequestered intracellularly, as evidenced by the lack of125I-α-bungarotoxin binding to the surface of intact cells. (12Gu Y. Forsayeth J.R. Verrall S., Yu, X.M. Hall Z.W. J. Cell Biol. 1991; 114: 799-807Crossref PubMed Scopus (91) Google Scholar, 13Kreienkamp H.J. Maeda R.K. Sine S.M. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar). Co-expression of the β-subunit with the α-, γ-, and δ-subunits is required to transport the assembled subunits to the cell surface. We therefore hypothesized that trafficking signals positioned in the α-subunit at the interface that assembles with the β-subunit are enclosed when the subunits assemble to form a circular pentamer. The trafficking signals then become sterically occluded from the cellular machinery that otherwise would retrieve proteins back to the ER. The major aim of this study was to identify the ER retrieval sequences in the α-subunit that inhibit the trafficking of the unassembled subunit beyond the ER. Our experimental findings demonstrate that the adjacent basic amino acid signal Arg313–Lys314 in the large cytoplasmic loop of the α-subunit regulates trafficking of the α-subunit from the ER to the Golgi. This is revealed in the trafficking characteristics of the α-subunit with the K314Q mutation that proceeds from the ER and accumulates in the Golgi when expressed in the absence of the other receptor subunits. Although the altered α-subunit proceeds to the Golgi, it does not express at the cell surface. Inhibition of ubiquitination results in detecting the α-subunit with the K314Q alteration at the cell surface. We therefore conclude that ubiquitination is a modulator for the Golgi to cell surface trafficking of the α-subunit. Thus, we disclose two mechanisms that regulate the trafficking of nAChR. One of these involves masking of ER retrieval sequences by assembly of the heterologous subunits that regulate the ER to Golgi trafficking of the α-subunit. The second mechanism regulates Golgi to cell surface trafficking and is modulated by ubiquitination. These two mechanisms may operate together to provide the “quality control” of the completed receptor that progresses to the cell surface. Western blotting and immunofluorescence protocols aimed at detecting the α-subunit employed the rat monoclonal antibody (mAb) 210, which recognizes an epitope in the extracellular domain (15Das M.K. Lindstrom J. Biochemistry. 1991; 30: 2470-2477Crossref PubMed Scopus (37) Google Scholar). The antibody to α-mannosidase II used to detect the medial-trans-Golgi was a gift of Dr. M. Farquhar, University of California, San Diego. Antibody to γ-COP was provided by Dr. C. Harter, University of Heidelberg, Heidelberg, Germany, antibody to β-COP was purchased from Sigma, and antibody to calnexin was acquired from Stressgen (Victoria, British Columbia, Canada). The secondary antibodies employed for immunofluorescence were purchased from Jackson Laboratories (West Grove, PA), as were the peroxidase-labeled secondary antibodies used in Western blot detection. The cDNAs encoding the mouse nAChR subunits are inserted in the EcoRI site in the expression vector pBR4 (Invitrogen, San Diego, CA). Mutations were introduced in the wild-type cDNA template by employing the “Quickchange” method (Stratagene, San Diego, CA). Subsequently, double-stranded DNA subjected to mutagenesis was subcloned into plasmids not exposed to the polymerase chain reaction-based mutagenesis procedures, and the introduced fragment was checked by automated sequencing. HEK-293 and ts20 (16Kulka R.G. Raboy B. Schuster R. Parag H.A. Diamond G. Ciechanover A. Marcus M. J. Biol. Chem. 1988; 263: 15726-15731Abstract Full Text PDF PubMed Google Scholar) cells were employed in these studies, which do not express nAChR at detectable levels. The receptor subunits were transiently expressed in these cells following gene transfection. Calcium phosphate precipitation was used for transfection in HEK cells. For immunofluorescence, cells were grown in 35-mm glass bottom dishes to ∼80% confluency and transfected with 3 μg of plasmid DNA encoding the α-subunit. For immunoprecipitation and Western blotting, cells were grown in 10-cm dishes, and 15 μg of plasmid DNA encoding the α-subunit was added to each transfected plate of cells. Plasmids encoding the other subunits were transfected at one-half the mass employed for the α-subunit. Following transfection, cells were grown at 37 °C for ∼48 h and then prepared for immunofluorescence or immunoprecipitation. In ts20 cells, 1 μg of plasmid DNA was added to a 35-mm plate, and transfection employed a liposome incorporation method (LipofectAMINE, Life Technologies, Inc.). ts20 cells express a mutant temperature-labile ubiquitin-activating enzyme (16Kulka R.G. Raboy B. Schuster R. Parag H.A. Diamond G. Ciechanover A. Marcus M. J. Biol. Chem. 1988; 263: 15726-15731Abstract Full Text PDF PubMed Google Scholar). At 30 °C, the ts20 cells maintain their ability to ubuiquitinate proteins, but at 40 °C the mutant ubiquitin-activating enzyme is inactive, and cells lose this capacity. Following transfection, ts20 cells were grown for 24 h at 30 °C and then shifted to 40 °C and grown for another 15 h. ts20 cells were then fixed in paraformaldehyde and processed for immunofluorescence in the same manner as the HEK cells detailed below. Immunofluorescence methods were performed in Triton-permeabilized cells to detect intracellular protein or in non-permeabilized cells to detect α-subunits at the cell surface. Cells were fixed in 4% paraformaldehyde in PBS, rinsed, and quenched in PBS/glycine. Cells were then permeabilized and blocked in 0.1% Triton X-100, 1% fish gelatin (Sigma), and 1% bovine serum albumin in PBS. Exposure to primary antibody was for 1 h in the permeabilization solution diluted with an equivalent volume of PBS. Exposure to secondary antibody conjugated to fluorophores was also for 1 h in the same buffer. Non-permeabilized cells were processed in the same manner except Triton X-100 was omitted. Following exposure to antibodies, cells were preserved in gelvatol and stored in the dark under refrigeration. Confocal images were taken with a Bio-Rad MRC 1024 laser-scanning system attached to a Zeiss Axiovert microscope using a 40× oil NA 1.3 objective, and processed with Adobe Photoshop (San Jose, CA). The brightly fluorescent cells show evidence of gene transfection; other fainter appearing cells in the microscope field apparently do not express the transfected gene. Cells were washed in PBS adjusted to pH 8.0 and incubated in 0.5 mg/ml sulfo-NHS-biotin (Pierce) in PBS for 30 min at room temperature. The reaction was quenched in 50 mm glycine in PBS; cells were washed further and then lysed in 1% Triton, 150 mm NaCl, 5 mm EDTA, 20 mm Tris-HCl, pH 8.0, on ice with protease inhibitors (protease inhibitor mixture, Roche Molecular Biochemicals). Streptavidin-Sepharose beads (Pierce) were added to the mixture to isolate biotinylated protein. The beads were washed and sedimented, and Laemmli sample buffer was added to elute the bound protein. Other cells, transfected with the same calcium phosphate mixture, were lysed in the above buffer, and acetylcholine receptor subunits were co-immunoprecipitated with anti-β-subunit antibody (mAb 111). Similar numbers of harvested cells were rinsed in PBS, sedimented, and solubilized in 0.15m NaCl, 2 mm EDTA, 20 mm Tris-HCl, pH 7.5, 0.5% Triton with protease inhibitors (protease inhibitor mixture; Roche Molecular Biochemicals) on ice. The insoluble materials were removed by centrifugation, and a stoichiometric excess of antibody to γ-COP or β-COP (clone M3A5, Sigma) was added to the soluble fraction for 30 min, followed by addition of IgG-Sepharose beads for another 45 min. Equivalent volumes of sample were resolved in 10% SDS-polyacrylamide gels (NOVEX, San Diego, CA) and transferred to nitrocellulose. Western blots were developed with chemiluminescent techniques. Cells were transfected with the indicated subunit combinations, grown for 48 h, and blocked first with 10 mm carbamylcholine followed by exposure to 10 nm125I-α-bungarotoxin for 1 h; the resultant radioactive counts correspond to the residual nonspecific binding. Other cells, transfected with the same calcium phosphate mixture, were exposed directly to 10 nm125I-α-bungarotoxin. Acetylcholine receptor α-subunits are representative of the family of ligand-gated ion channels that display the general topology shown in Fig.1 A, consisting of one major extracellular domain, four transmembrane spans, and a large and small cytoplasmic loop (15Das M.K. Lindstrom J. Biochemistry. 1991; 30: 2470-2477Crossref PubMed Scopus (37) Google Scholar, 17Lindstrom J. Ion Channels. 1996; 4: 377-450Crossref PubMed Scopus (263) Google Scholar). The largest span of sequence is extracellular and is predicted to consist of the first 210 residues starting at the N terminus (17Lindstrom J. Ion Channels. 1996; 4: 377-450Crossref PubMed Scopus (263) Google Scholar). The major cytoplasmic loop is thought to be located between residues 299 and 408 in the α-subunit and includes the trafficking signals examined in this study. Conservation in the adjacent basic sequences corresponding to positions 313–314 in the α-subunit is observed among most receptor subunits when alignments of the major cytoplasmic loops are examined (Fig.1 B), suggesting that this sequence may encode an important conservation of function. Therefore, an adjacent basic sequence at position 313–314 in the α-subunit (18Boulter J. Luyten W. Evans K. Mason P. Ballivet M. Goldman D. Stengelin S. Martin G. Heinemann S. Patrick J. J. Neurosci. 1985; 5: 2545-2552Crossref PubMed Google Scholar) was selected as a candidate to be altered by site-directed mutagenesis from Arg313–Lys314 to Arg313–Gln314 (designated as the K314Q α-subunit) to examine whether subsequent changes occur in the trafficking characteristics. The potential trafficking signal RK was altered to RQ on the basis of its presence at an identical position in the β-subunit (Fig. 1 B), the subunit that facilitates transport of the assembled receptor to the cell surface (12Gu Y. Forsayeth J.R. Verrall S., Yu, X.M. Hall Z.W. J. Cell Biol. 1991; 114: 799-807Crossref PubMed Scopus (91) Google Scholar,13Kreienkamp H.J. Maeda R.K. Sine S.M. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar). For control and comparison, transfected wild-type α-subunits expressed in ts20 cells (at 30 °C) generally display the reticulate-diffuse pattern reminiscent of proteins deposited in the ER (Fig. 2 A, panels aand b, red) and appear to overlap in pattern with endogenous calnexin (Fig. 2 A, panels a andc, blue), a diagnostic marker for the ER. Wild-type α-subunits expressed in ts20 cells also display minimal co-localization (Fig. 2 A, panel a) with a GFP-Golgi protein marker (panel d, green, GFP linked to the Golgi localization signal of galactosyltransferase (19Llopis J. McCaffery J.M. Miyawaki A. Farquhar M.G. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6803-6808Crossref PubMed Scopus (922) Google Scholar)), which was expressed in these cells by co-transfection with the plasmid DNA encoding the α-subunit. Since the GFP-Golgi protein marker was expressed from a transfected gene, only a subset of cells display its expression. Moreover, wild-type α-subunits expressed in HEK cells display minimal overlap with the endogenous medial-trans-Golgi marker α-mannosidase II (Fig.2 B, panel e), further substantiating that the unassembled wild-type α-subunits are sequestered primarily in the ER. In support of our experimental observations on the trafficking characteristics of unassembled wild-type α-subunits as revealed by transfection and confocal microscopy, subcellular separations employing sucrose gradients of α-subunits expressed in muscle cells from the endogenous gene also demonstrated that unassembled α-subunits are restricted to the ER (11Smith M.M. Lindstrom J. Merlie J.P. J. Biol. Chem. 1987; 262: 4367-4376Abstract Full Text PDF PubMed Google Scholar). Under the numerous transfection and expression experiments employed over the course of our study, minimal overlap was observed between the unassembled wild-type α-subunits and endogenous α-mannosidase II. Thus, high levels of overexpression from the transfected gene did not appear to contribute to artificially induced trafficking patterns. In contrast to the wild-type α-subunits expressed alone, cells co-expressing α-subunits with β-, γ-, and δ-subunits display appreciable co-localization between the α-subunits and α-mannosidase II (Fig. 3 a), coinciding with the finding that co-transfection of plasmid DNAs encoding the entire complement of subunits in HEK cells results in detection of the receptor at the cell surface (13Kreienkamp H.J. Maeda R.K. Sine S.M. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar). Note that although significant co-localization between the α-subunits and α-mannosidase II is observed when the receptor subunits are co-expressed, a major fraction of α-subunits also display the reticulate-diffuse pattern suggestive of localization in the ER (Fig.3, a and b): this likely reflects the fraction of α-subunits not assembled with the other subunits or assembled receptor protein that did not exit the ER. As a further note, α-subunits co-expressed with the other receptor subunits in HEK cells yield glycosylated α-subunits that traffic to the cell surface and are fully cleavable with Endo-H in whole cell lysates (20Keller S.H. Kreienkamp H.J. Kawanishi C. Taylor P. J. Biol. Chem. 1995; 270: 4165-4171Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Thus, Endo-H cleavage was not employed in this study as a tool to characterize α-subunit trafficking. To examine whether cytoplasmically positioned adjacent basic amino acid sequences modulate the trafficking of nAChR α-subunits, the Arg313–Lys314 sequence was altered into Arg313–Gln314, with the aim of conserving side chain volume but neutralizing the charge of the lysine residue to remove the potential trafficking signal. HEK cells were transfected and processed for confocal microscopy in the same manner as the wild-type α-subunits displayed in Figs. 2 B and 3. The confocal microscope image of cells expressing K314Q α-subunits is displayed in Fig. 4, with the α-subunits exhibited by FITC emission (Fig. 4 b, green) and α-mannosidase II displayed by rhodamine emission (Fig. 4 c, red). Theyellow coloration corresponds to overlap between the α-subunits and α-mannosidase II (Fig. 4 a). Evident co-localization is observed between the K314Q α-subunits and α-mannosidase II (Fig. 4 a), suggesting spatial overlap in the cell and strongly indicating that the K314Q α-subunit traffics to and is retained in the Golgi compartment. To verify that the observed co-localization is due to spatial overlap in the three-dimensional field of the confocal image as opposed to artificial channel crossover caused by overly bright emission, wavelengths corresponding to FITC and rhodamine were individually blocked to examine whether images became apparent in the non-emitting channel. Since images were not observed in the non-emitting channel, crossover was excluded as the cause for the yellowish coloration observed in the merged images. Furthermore, since the steady state expression levels of the wild-type and K314Q α-subunits are similar (Fig. 5, lanes 3 and4), overexpression and saturation of the quality control machinery of the cell can be excluded as the cause for the acquired trafficking characteristics of the K314Q α-subunit. Wild-type α-subunits, when expressed in the absence of other subunits, sediment as monomers upon sucrose gradient centrifugation (13Kreienkamp H.J. Maeda R.K. Sine S.M. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar). Since residues in the extracellular region govern subunit assembly, the K314Q mutation is unlikely to promote assembly of the mutant α-subunit into a more complex structure such as a homopentamer. The above results therefore suggest that altering the adjacent basic amino acid signal Arg313–Lys314 into Arg313–Gln314 directly promotes the trafficking of the substituted α-subunit from the ER to the Golgi. The Arg313–Lys314 sequence therefore appears to be a major determinant that modulates the ER to Golgi trafficking of the α-subunit. Since this sequence is largely conserved among mouse nAChR subunits (Fig. 1 B), it can be postulated that it also plays a similar role in the trafficking of the other subunits. Proteins that have been retrieved back to the ER are incorporated into COP I vesicles that migrate in an anterograde direction from the intermediate compartment to the ER (1Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1064) Google Scholar). Exposed adjacent basic amino acid sequences are known to interact with the COP I components that incorporate the retrieved proteins into the COP I vesicles (21Harter C. Pavel J. Coccia F. Draken E. Wegehingel S. Tschochner H. Wieland F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1902-1906Crossref PubMed Scopus (79) Google Scholar, 22Harter C. Wieland F.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11649-11654Crossref PubMed Scopus (76) Google Scholar, 23Andersson H. Kappeler F. Hauri H.P. J. Biol. Chem. 1999; 274: 15080-15084Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Therefore, in contrast to the isolated wild-type α-subunit, K314Q α-subunits as well as wild-type α-subunits co-expressed with the full complement of receptor subunits should be expected to display minimal interactions with the COP I components, since these proteins traffic to the Golgi. Pull-down experiments were performed in HEK cell lysates employing an antibody to the COP I component protein, γ-COP. Cells were transfected to express K314Q α-subunits, wild-type α-subunits, or co-express wild-type subunits with the β-, γ - , and δ-subunits. Similar numbers of cells, volumes of buffer, and amounts of samples loaded into the gels were maintained in all steps. Proteins were subsequently resolved in 10% gels, transferred to nitrocellulose, and probed with an antibody to the α-subunit (mAb 210). The immunoprecipitated samples (Fig. 5,lanes 1 and 2) reflect the extent of COP protein recognition of the α-subunits when compared with the respective expression levels of the α-subunits displayed in the whole cell lysates (Fig. 5, lanes 3 and 4). Presumably, due to a low affinity interaction that becomes more evident with cross-linking (21Harter C. Pavel J. Coccia F. Draken E. Wegehingel S. Tschochner H. Wieland F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1902-1906Crossref PubMed Scopus (79) Google Scholar, 22Harter C. Wieland F.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11649-11654Crossref PubMed Scopus (76) Google Scholar), only a fraction of α-subunits remained associated with γ-COP after completion of the immunoprecipitation procedures employed in these experiments. The experimental observations indicate that wild-type α-subunits expressed alone have the most pronounced association with γ-COP (Figs. 5, A and B, lane 1), whereas the K314Q α-subunit (Fig. 5 A, lane 2) and wild-type α-subunits co-expressed with the β-, γ-, and δ-subunits (Fig.5 B, lane 2) display minimal associations. Note also that in the sample that displays the α-subunits co-expressed with the β-, γ-, and δ-subunits (Fig. 5 B, lane 2), a minor interaction of the α-subunits with γ-COP is apparent; this likely reflects the population of unassembled α-subunits present in these cells (13Kreienkamp H.J. Maeda R.K. Sine S.M. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar, 24Sugiyama N. Boyd A.E. Taylor P. J. Biol. Chem. 1996; 271: 26575-26581Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). In contrast to the interaction of the wild-type α-subunits with γ-COP, association with β-COP, another component of COP I coats (21Harter C. Pavel J. Coccia F. Draken E. Wegehingel S. Tschochner H. Wieland F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1902-1906Crossref PubMed Scopus (79) Google Scholar, 22Harter C. Wieland F.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11649-11654Crossref PubMed Scopus (76) Google Scholar, 23Andersson H. Kappeler F. Hauri H.P. J. Biol. Chem. 1999; 274: 15080-15084Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), was not detected. By employing the same buffer and conditions for immunoprecipitation, the β-COP antibody did not co-immunoprecipitate the α-subunits, although α-subunits were abundant in these cells, and β-COP was immunoprecipitated in the samples (data not shown). Taken together, the experimental findings suggest that recognition of unassembled α-subunits by the COP I complex is mediated through the Arg313–Lys314signal, which potentially directly interacts with γ-COP. Subunit assembly diminishes the interaction between the α-subunits and the COP 1 complex, which is reflected in the trafficking of the receptor beyond the ER compartment. To examine whether the K314Q α-subunit maintains the capacity to fold and assemble into the mature receptor pentamer, ligand binding assays were performed by protecting receptor-binding sites with carbamylcholine from 125I-α-bungarotoxin. Carbamylcholine recognizes and binds to the α-subunits associated with the γ- or δ-subunits but do" @default.
• W2089035269 created "2016-06-24" @default.
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• W2089035269 creator A5051156147 @default.
• W2089035269 creator A5084824937 @default.
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• W2089035269 date "2001-05-01" @default.
• W2089035269 modified "2023-09-29" @default.
• W2089035269 title "Adjacent Basic Amino Acid Residues Recognized by the COP I Complex and Ubiquitination Govern Endoplasmic Reticulum to Cell Surface Trafficking of the Nicotinic Acetylcholine Receptor α-Subunit" @default.
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