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• W2038857470 abstract "To identify factors involved in the expression of ligand-gated ion channels, we expressed nicotinic acetylcholine receptors in HEK cells to characterize roles for oligosaccharide trimming, calnexin association, and targeting to the proteasome. The homologous subunits of the acetylcholine receptor traverse the membrane four times, contain at least one oligosaccharide, and are retained in the endoplasmic reticulum until completely assembled into the circular arrangement of subunits of δ-α-γ-α-β to enclose the ion channel. We previously demonstrated that calnexin is associated with unassembled subunits of the receptor, but appears to dissociate when subunits are assembled in various combinations. We used the glucosidase inhibitor castanospermine to block oligosaccharide processing, and thereby inhibit calnexin's interaction with the oligosaccharides in the receptor subunits. Castanospermine treatment reduces the association of calnexin with the α-subunit of the receptor, and diminishes the intracellular accumulation of unassembled receptor subunit protein. However, treatment with castanospermine does not appear to alter subunit folding or assembly. In contrast, co-treatment with proteasome inhibitors and castanospermine enhances the accumulation of polyubiquitin-conjugated α-subunits, and generally reverses the castanospermine induced loss of α-subunit protein. Co-transfection of cDNAs encoding the α- and δ-subunits, which leads to the expression of assembled α- and δ- subunits, also inhibits the loss of α-subunits expressed in the presence of castanospermine. Taken together, these observations indicate that calnexin association reduces the degradation of unassembled receptor subunits in the ubiquitin-proteasome pathway. To identify factors involved in the expression of ligand-gated ion channels, we expressed nicotinic acetylcholine receptors in HEK cells to characterize roles for oligosaccharide trimming, calnexin association, and targeting to the proteasome. The homologous subunits of the acetylcholine receptor traverse the membrane four times, contain at least one oligosaccharide, and are retained in the endoplasmic reticulum until completely assembled into the circular arrangement of subunits of δ-α-γ-α-β to enclose the ion channel. We previously demonstrated that calnexin is associated with unassembled subunits of the receptor, but appears to dissociate when subunits are assembled in various combinations. We used the glucosidase inhibitor castanospermine to block oligosaccharide processing, and thereby inhibit calnexin's interaction with the oligosaccharides in the receptor subunits. Castanospermine treatment reduces the association of calnexin with the α-subunit of the receptor, and diminishes the intracellular accumulation of unassembled receptor subunit protein. However, treatment with castanospermine does not appear to alter subunit folding or assembly. In contrast, co-treatment with proteasome inhibitors and castanospermine enhances the accumulation of polyubiquitin-conjugated α-subunits, and generally reverses the castanospermine induced loss of α-subunit protein. Co-transfection of cDNAs encoding the α- and δ-subunits, which leads to the expression of assembled α- and δ- subunits, also inhibits the loss of α-subunits expressed in the presence of castanospermine. Taken together, these observations indicate that calnexin association reduces the degradation of unassembled receptor subunits in the ubiquitin-proteasome pathway. The nicotinic acetylcholine receptor is a prototype molecule of a family of ligand-gated ion channels, which include GABAA, glycine, and 5HT3 receptors (1Changeux J-P. Biochem. Soc. Trans. 1995; 23: 195-205Crossref PubMed Scopus (24) Google Scholar, 2Karlin A. Akabas M. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (566) Google Scholar). Following binding by agonists to these receptors, a conformational change increases cation permeability through the central pore of the receptor, eliciting depolarization of the cell membrane (1Changeux J-P. Biochem. Soc. Trans. 1995; 23: 195-205Crossref PubMed Scopus (24) Google Scholar, 2Karlin A. Akabas M. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (566) Google Scholar). Peptide backbones of each of the four subunits of the acetylcholine receptor transverse the membrane four times, and possess at least one Asn-X-Ser/Thr glycosylation signal (2Karlin A. Akabas M. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (566) Google Scholar, 3Noda M. Furutani Y. Takahashi H. Toyosato M. Tanabe T. Shimizu S. Kikoyotani S. Kayano T. Hirose T. Inayama S. Numa S. Nature. 1983; 305: 818-823Crossref PubMed Scopus (297) Google Scholar). The subunits are thought to undergo a maturation pathway which includes oligosaccharide attachment (4Smith M.M. Schlesinger S. Lindstrom J. Merlie J.P. J. Biol. Chem. 1986; 261: 14825-14832Abstract Full Text PDF PubMed Google Scholar), formation of disulfide bonds (5Merlie J.P. Lindstrom J. Cell. 1983; 34: 747-757Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 6Kao P.N. Karlin A. J. Biol. Chem. 1986; 261: 8085-8088Abstract Full Text PDF PubMed Google Scholar, 7Fu D.-X. Sine S.M. J. Biol. Chem. 1996; 271: 31479-31484Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), proline isomerization (8Helekar S.A. Char D. Neff S. Patrick J. Neuron. 1994; 12: 179-189Abstract Full Text PDF PubMed Scopus (99) Google Scholar), and intersubunit contacts at specific interfaces (9Gu Y. Camacho P. Gardner P. Hall Z.W. Neuron. 1991; 6: 879-887Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 10Blount P. Merlie J.P. J. Biol. Chem. 1988; 263: 1072-1080Abstract Full Text PDF PubMed Google Scholar, 11Kreienkamp H-J. Maeda R.K. Sine S. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar). Members of this family of receptors are composed of a multisubunit complex of glycoproteins which are retained and assembled in the endoplasmic reticulum prior to transport to the cell surface (9Gu Y. Camacho P. Gardner P. Hall Z.W. Neuron. 1991; 6: 879-887Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 12Smith M.M. Lindstrom J. Merlie J.P. J. Biol. Chem. 1987; 262: 4367-4376Abstract Full Text PDF PubMed Google Scholar). Acetylcholine receptor subunits at the neuromuscular junction assemble into a circular orientation of subunits of δ-α-γ-α-β, to enclose the central ion channel (2Karlin A. Akabas M. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (566) Google Scholar, 13Tsigelny I. Sugiyama N. Sine S.M. Taylor P. Biophys. J. 1997; 73: 52-66Abstract Full Text PDF PubMed Scopus (69) Google Scholar; but see Ref. 14Unwin N. J. Mol. Biol. 1993; 229: 1101-1124Crossref PubMed Scopus (717) Google Scholar). The endoplasmic reticulum localized protein, calnexin, is associated with unassembled subunits of the acetylcholine receptor (15Gelman M.S. Chang W. Thomas D.Y. Bergeron J.J.M. Prives J.M. J. Biol. Chem. 1995; 270: 15085-15092Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 16Keller S.H. Lindstrom J. Taylor P. J. Biol. Chem. 1996; 271: 22871-22877Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 17Chang W. Gelman M.S. Prives J.M. J. Biol. Chem. 1997; 272: 28925-28932Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), but calnexin appears to be absent with combinations of assembled subunits (16Keller S.H. Lindstrom J. Taylor P. J. Biol. Chem. 1996; 271: 22871-22877Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Connolly et al. (18Connolly C.N. Krishek B.J. McDonald B.J. Smart T.G. Moss S.J. J. Biol. Chem. 1996; 271: 89-96Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar) demonstrated that calnexin is associated with subunits of the GABAA receptor, indicating that calnexin association might be involved in the biogenesis of other multisubunit ion channels. Numerous investigations have established that calnexin associates primarily with monoglucosylated oligosaccharides, which are intermediates in the processing of nascent oligosaccharides or products of reglucosylation by the enzyme UDP-glucose:glycoprotein glucosyltransferase, and that treatment with the glucosidase inhibitor castanospermine can disrupt the interaction (reviewed in Refs. 19Helenius A. Trombetta E.S. Herbert D.N. Simons J.F. Trends Cell Biol. 1997; 7: 193-200Abstract Full Text PDF PubMed Scopus (345) Google Scholar and 20Van Leeuwen J.E. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Using transient expression of acetylcholine receptor subunits in HEK cells, we find a role for calnexin association in reducing degradation of unassembled subunits by the proteasome, since treatment with castanospermine reduces the subunit-calnexin association and increases the polyubiquitination of the α-subunit. Additionally, co-treatment with proteasome inhibitors blocks the degradation. Although unassembled α-subunits are degraded at a rapid rate (5Merlie J.P. Lindstrom J. Cell. 1983; 34: 747-757Abstract Full Text PDF PubMed Scopus (127) Google Scholar), treatment with glucosidase inhibitors substantially promotes degradation (4Smith M.M. Schlesinger S. Lindstrom J. Merlie J.P. J. Biol. Chem. 1986; 261: 14825-14832Abstract Full Text PDF PubMed Google Scholar). Our data also indicate that castanospermine treatment does not cause the α-subunit to misfold, as detected by a conformationally sensitive antibody. Therefore, increased degradation by the proteasome appears to be independent of the nascent peptide undergoing misfolding in this system. In contrast, a recent study indicated that treatment of chick myotubes with castanospermine disrupts α-subunit folding and assembly (17Chang W. Gelman M.S. Prives J.M. J. Biol. Chem. 1997; 272: 28925-28932Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Our data also indicate that calreticulin and ERp57, the two other proteins known to have glycoprotein-associating properties similar to calnexin (19Helenius A. Trombetta E.S. Herbert D.N. Simons J.F. Trends Cell Biol. 1997; 7: 193-200Abstract Full Text PDF PubMed Scopus (345) Google Scholar, 21Elliot J.G. Oliver J.D. High S. J. Biol. Chem. 1997; 272: 13849-13855Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), do not appear to be bound to the α-subunit. Assembly with the δ-subunit reduces the degradation of the α-subunit when α- and δ-subunits are co-expressed in the presence of castanospermine, indicating that glucose trimming, calnexin association, assembly and entrance into the proteasome are linked in the expression of the receptor. Connections among these phenomena are also likely to be critical to the fidelity of expression of other multisubunit glycoproteins. Castanospermine (Calbiochem, San Diego, CA) was solubilized in Dulbecco's modified Eagle's medium at a concentration of 100 μg/ml, and immediately added to 10-cm plates of cells. Cells were treated with castanospermine 2 h prior to transfection, transfected with receptor subunit cDNAs, allowed to grow for 16 h, or replenished with fresh castanospermine in Dulbecco's modified Eagle's medium and raised for another 24 h. Transfections employed the calcium phosphate precipitation method, as described in Keller et al. (16Keller S.H. Lindstrom J. Taylor P. J. Biol. Chem. 1996; 271: 22871-22877Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Generally, 15 μg of plasmid DNA encoding each receptor subunit were added to plates of cells, unless noted otherwise. In transfections where α- to δ-subunit ratios were varied, α-subunit cDNA was transfected at 15 μg of plasmid DNA/plate, and the mass of plasmid DNA encoding the δ-subunit was varied (Figs. 5 and 6).Figure 6The influence of castanospermine on the accumulation of α-δ subunit dimers depends on the transfection ratios of plasmid DNAs encoding the α- and δ-subunits. The transfected α:δ-subunit ratios refer to the masses of plasmid DNAs present in the transfection mixture, where the amount of plasmid DNA encoding the α-subunit is kept constant at 15 μg of DNA/plate and plasmid DNA encoding for the δ-subunit is varied. A,lanes 1 and 2 display samples transfected at a 8:1 ratio and lanes 3and 4 display samples transfected at a 3:1 ratio of plasmid DNAs encoding the α- and δ-subunits. The numbers of cells, antibody dilutions for immunoprecipitations, and volumes loaded in each lane were the same among samples, and consisted of approximately 5% of the total sample. The Western blot was developed first with mAb 210 to detect the α-subunit and then reprobed with mAb 137 to detect the δ-subunit, and the exposures on films were overlaid to show α- and δ-subunits together. The δ-subunits displayed in the Western blot are assembled with α-subunits, since the immunoprecipitating antibody recognized the α-subunit (mAb 61). B, Western blot revealing assembled δ-subunits from a similar experiment as displayed inA, developed with mAb 137.View Large Image Figure ViewerDownload (PPT) In experiments involving immunoprecipitations with antibodies to calnexin, calreticulin, ERp57, and the receptor subunits, the solubilization buffer consisted of 0.5% CHAPS, 1The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; Bgt, bungarotoxin; CST, castanospermine; ER, endoplasmic reticulum; HEK, human embryonic kidney; Me2SO, dimethyl sulfoxide; LAC, lactacystin; Z-LLF-CHO, benzyloxycarbonyl-Leu-Leu-phenylalaninal; mAb, monoclonal antibody. 150 mmNaCl, 1 mm CaCl2, 20 mm HEPES, pH 8.0, and the protease inhibitors: benzamidine, aprotinin, leupeptin, and pepstatin A. The characteristics of the antibodies used for these immunoprecipitations, mAb 35 or mAb 61 to precipitate the α-subunit, mAb 111 to precipitate the β-subunit and mAb 137 to precipitate the δ-subunit, were described previously (22Tzartos S.J. Rand D.E. Einarson B.L. Lindstrom J.M. J. Biol. Chem. 1981; 256: 8635-8645Abstract Full Text PDF PubMed Google Scholar). Anticalnexin, antipolyubiquitin, and anti-calreticulin were purchased from Stressgen (British Columbia, Canada). Solubilization of cells and immunoprecipitations using anti-polyubiquitin were in 50 mmTris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 0.4% deoxycholate, 1% Nonidet P-40, 0.1% SDS, phenylmethanesulfonyl fluoride, N-ethylmaleimide, and the other protease inhibitors listed above. The ratio of solubilization buffer volume relative to the number of cell plates were the same for each sample within an experiment. Following solubilization, samples were centrifuged at 10,000 × g for 5 min. Dilution ratios for antibody in all immunoprecipitations were approximately 1:100. Equivalent volumes of immunoprecipitated samples, consisting of approximately 5% of the total immunoprecipitated material, were loaded in each gel lane. All samples were resolved on 10% Novex gels (San Diego, CA) and transferred to nitrocellulose for Western blot detection. The antibodies used to detect the receptor subunits were mAb 210 for α, mAb 111 for β, and mAb 137 for δ. The antibody to ERp57 was a gift from Dr. Jordan Holtzman (University of Minnesota). Calreticulin was detected on Western blots with the same antibody used in immunoprecipitations. All primary antibodies were diluted 1:1000 to probe Western blots, which were developed by chemiluminescent techniques (Pierce). Immunoprecipitation and Western blot experiments were replicated usually three or more times. Proteasome inhibitors were solubilized in Me2SO and added 3 h after transfection. Equivalent concentrations of Me2SO were added to untreated cells. The final concentration of Me2SO in a plate of cells was at most 0.3%. Cells were grown for an additional 16 h and then solubilized. The proteasome inhibitor benzyloxycarbonyl-Leu-Leu-phenylalaninal (Z-LLF-CHO) was obtained from Dr. F. Mercurio (Signal Pharmaceuticals, San Diego, CA) and used at a final concentration of 20 μm. MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) and lactacystin were purchased from Calbiochem (San Diego, CA) and used at 50 and 10 μm, respectively. Calpain inhibitor I (N-Ac-Leu-Leu-norleucinal; Calbiochem, San Diego, CA) was used at final concentration of 100 μm. The snake toxin α-bungarotoxin (α-Bgt) binds to both unassembled and assembled α-subunits, whereas carbamoylcholine recognizes only α-subunits assembled with γ-, δ-, or ε-subunits (10Blount P. Merlie J.P. J. Biol. Chem. 1988; 263: 1072-1080Abstract Full Text PDF PubMed Google Scholar). Transfected and untransfected cells were permeabilized in a 0.1% saponin buffer (11Kreienkamp H-J. Maeda R.K. Sine S. Taylor P. Neuron. 1995; 14: 635-644Abstract Full Text PDF PubMed Scopus (92) Google Scholar), exposed to an excess of125I-α-bungarotoxin (125I-α-Bgt) for 3 h, washed, and γ-radiation was counted. The binding of125I-α-Bgt to untransfected cells was subtracted from its binding to transfected cells. Data were standardized to the average of125I-α-Bgt bound to cells untreated with castanospermine. Bands on Western blots were scanned with Deskscan (Hewlett Packard) and integrated with the program Collage (Fotodyne, New Berlin, WI). Cells were treated with the glucosidase inhibitor castanospermine, to identify roles for glucose trimming in the biogenesis of acetylcholine receptors expressed in HEK cells. Treatment with castanospermine, prior to and during the transfection period, inhibits the glucosidase I and glucosidase II enzymes, leaving oligosaccharides capped with three glucose residues. Castanospermine treatment, therefore, maintains larger sized oligosaccharides which are attached to glycoproteins confined to the endoplasmic reticulum. Since unassembled receptor subunits and α-δ dimers are retained intracellularly (23Saedi M.S. Conroy W.G. Lindstrom J.M. J. Cell. Biol. 1991; 112: 1007-1015Crossref PubMed Scopus (51) Google Scholar, 24Sine S.M. Claudio T. J. Biol. Chem. 1991; 266: 19369-19377Abstract Full Text PDF PubMed Google Scholar), treatment with castanospermine should maintain larger oligosaccharides attached to the receptor subunits. A side reaction, which may result from a residual fraction of glucosidase enzymes remaining active, is that, nascent oligosaccharide chains whose glucosyl residues have been trimmed, in turn, become reglucosylated by UDP-glucose:glycoprotein-dependent glucosyltransferase. Reglucosylated receptor subunits may accumulate transiently because most glucosidase enzymes are inhibited (20Van Leeuwen J.E. Kearse K.P. J. Biol. Chem. 1997; 272: 4179-4186Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Cells were untreated or treated with castanospermine prior to transfection with cDNAs encoding the α-, β-, or δ-subunits. Following detergent solubilization, receptor protein was immunoprecipitated, resolved on gels, transferred to nitrocellulose and detected with appropriate antibodies. The Western blot in Fig. 1 A demonstrates that castanospermine decreases the migration in gels of the α- (comparelanes 1 to 2), β- (lanes 3 to4), and δ-subunits (lanes 5 and 6), indicating untrimmed oligosaccharides predominate. The δ-subunit often appears as a doublet when resolved toward the bottom of a gel, which is presumably due to partial phosphorylation. Similarly, the α-subunit often appears as a doublet, with a faint unglycosylated lower molecular weight band and a more abundant higher molecular weight protein, which is glycosylated (25Blount P. Merlie J.P. J. Cell Biol. 1990; 111: 2613-2622Crossref PubMed Scopus (93) Google Scholar). γ-Subunits were not included in these experiments due to the unavailability of the appropriate antibodies. The banding densities in Western blots of α-, β-, and δ-subunits are significantly diminished when expressed in castanospermine (Fig. 1 A), indicating that the intracellular accumulations of these unassembled subunits are reduced. Although treatment with castanospermine at 100 μg/ml diminishes the accumulation of receptor protein, and presumably other glycoproteins, it does not influence the accumulation of most cellular proteins detected with a general stain or antibody (Fig. 1 B). To establish this, untransfected cells were treated or untreated with castanospermine, and grown in a similar manner to transfected cells. Cells were harvested, washed 3 × in phosphate-buffered saline, and solubilized in the 0.5% CHAPS buffer as described earlier. Samples were resolved in gels, which were then stained with Coomassie Blue (Fig. 1 B, lanes 1 and2). Other samples were resolved in gels, transferred to nitrocellulose, and exposed to an excess of anti-rat IgG-conjugated peroxidase to detect cellular proteins nonspecifically (Fig. 1 B, lanes 3 and 4). Proteins of similar banding densities align in the gels (Fig. 1 B), indicating that the loss of acetylcholine receptor protein observed in Fig. 1 A, is not due to a general reduction in protein synthesis caused by castanospermine treatment. To identify mechanisms which regulate receptor subunit degradation, experiments were designed to ascertain whether treatment with castanospermine increases conjugation of polyubiquitin to α-subunits. Cells were treated or untreated with castanospermine, transfected with cDNA encoding the α-subunit, and maintained in the presence of proteasome inhibitors: calpain inhibitor I and benzyloxycarbonyl-Leu-Leu-phenylalaninal (Z-LLF-CHO; 26Vinitsky A.C. Michaud C. Powers J.C. Orlowski M. Biochemistry. 1992; 31: 9421-9428Crossref PubMed Scopus (196) Google Scholar). Calpain inhibitor I and Z-LLF-CHO were used in this experiment, because other investigations have demonstrated their efficacy for detecting the conjugation of polyubiquitin chains to proteins (27DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 28DiDonato J., A. Mercurio F. Caridad R. Wu-li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar). Detergent extracts were immunoprecipitated with an antibody to polyubiquitin, and Western blots were developed with mAb 210 to detect α-subunits (Fig. 2). The appearance of a characteristic ladder pattern near the top of the gel indicates that the proteins are polyubiquitinated (27DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 28DiDonato J., A. Mercurio F. Caridad R. Wu-li J. Suyang H. Ghosh S. Karin M. Mol. Cell. Biol. 1996; 16: 1295-1304Crossref PubMed Google Scholar, 29Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Abstract Full Text PDF PubMed Scopus (1133) Google Scholar, 30Yeung S.J. Chen S.H. Chan L. Biochemistry. 1996; 35: 13843-13848Crossref PubMed Scopus (170) Google Scholar). A high molecular weight ladder pattern is observed when α-subunits are expressed in the presence of castanospermine (Fig. 2 A, lane 2), indicating that the α-subunits become polyubiquitinated. In comparison, α-subunits expressed in the absence of castanospermine are also polyubiquitinated, but the high molecular weight ladder pattern is fainter (Fig. 2 A, compare lanes 1 and2). The high molecular weight pattern is absent when Z-LLF-CHO is omitted in the experiment (Fig. 2 B); this finding indicates that treatment with Z-LLF-CHO inhibits the degradation of polyubiquitinated α-subunits in the proteasome, thereby allowing the conjugated intermediates to accumulate. Treatment with calpain inhibitor I alone does not lead to the detection of polyubiquitinated α-subunits. In other experiments with proteasome inhibitors, treatment with 50 μm MG-132 resulted in the detection of a distinct, but fainter high molecular weight ladder pattern compared with that observed with Z-LLF-CHO treatment; the density of the high molecular weight ladder pattern was also enhanced when cells were treated with castanospermine (data not shown). To further characterize the mechanisms of degradation, cells were treated with the proteasome inhibitors lactacystin or MG-132, to examine whether degradation of α-subunits is reduced. Lactacystin is a irreversible inhibitor which binds specifically to the proteasome, as demonstrated by affinity labeling and peptide sequencing (31Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1504) Google Scholar). MG-132 is a congener of Z-LLF-CHO, also a peptide-aldehyde that blocks the proteolytic activities of the proteasome (32Lee D.H. Goldberg A.L. J. Biol. Chem. 1996; 271: 27280-27284Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar). Cells were treated or untreated with castanospermine, transfected with the same plasmid DNA transfection mixture in all plates, and then treated or untreated with a proteasome inhibitor. Following an incubation period, cells were solubilized, and α-subunits were immunoprecipitated and detected on Western blots. As displayed in Fig. 3, lanes 3and 4, and in previous experiments, treatment with castanospermine results in a loss of α-subunit protein; however, inclusion of lactacystin diminishes the loss of α-subunit protein (Fig. 3, lanes 1 and 2). Similar results were obtained with MG-132 (Fig. 4 C, lanes 3 and 4). These data indicate that α-subunits, which may have been polyubiquitinated and subjected to isopeptidase activity (33Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1602) Google Scholar), are degraded in the proteasome in cells treated with castanospermine.Figure 4Characteristics of calnexin recognition of α-subunits. A, cells were treated or untreated with CST and transfected (TFT) with 15 μg of plasmid DNA encoding the α-subunit or also co-transfected with 15 μg of plasmid DNA encoding the δ-subunit. Cells were not treated with a proteasome inhibitor. Sequential immunoprecipitation (IP to:) was first to calnexin (c), followed by immunoprecipitation of the unbound material with anti-α (mAb 61). Lanes 1 and3, 2 and 4, 5 and 7, and, 6and 8 are pairs of sequentially immunoprecipitated samples. The Western blot was developed with mAb 210 to detect α-subunits. The density ratios were calculated by scanning and integrating the density of α-subunit bands on the Western blot and dividing the CST-treated sample by the CST-untreated sample for α-subunits precipitated by one of the antibodies. B, similar to A, except cells were transfected with 3.0 μg of plasmid DNA encoding the α-subunit to reduce expression levels: cells were also treated with lactacystin.C, similar to A, except cells were treated with 50 μm MG-132. Blot was exposed to film 10 times longer than in A and B.View Large Image Figure ViewerDownload (PPT) Treatment with lactacystin does not appear to enhance the accumulation of α-subunits expressed in the absence of castanospermine (Fig. 3, compare lanes 1 and 3), at a concentration of 10 μg/ml and 16 h of incubation. The degradation rate for α-subunits may be substantially lower when expressed in the absence of castanospermine, preventing detection of an enhanced accumulation in cells not treated with castanospermine. In support of a substantially lower degradation rate for α-subunits expressed in the absence of castanospermine, the conjugation of polyubiquitin chains is substantially reduced when α-subunits are expressed without castanospermine (Fig. 2 A, compare lane 1 to2). To examine whether α-subunits are more prone to aggregate in cells treated with castanospermine, and whether this contributes to the loss of α-subunits, we analyzed insoluble fractions following detergent solubilization of cells. Cells were solubilized in the 0.5% CHAPS buffer described above, subjected to low speed centrifugation (5 min, 2,000 × g), and the resulting supernatant was subjected to high speed centrifugation (30 min, 16,000 ×g). Western blots, of insoluble materials in the final fraction, displayed equivalently dense α-subunit bands in samples treated or untreated with castanospermine (data not shown). Therefore, we were unable to establish aggregation as a factor contributing to the loss of α-subunit protein expressed in the presence of castanospermine. To ascertain whether glucose trimming influences interactions between calnexin and α-subunits, cells were treated with castanospermine, and then transfected with various amounts of plasmid DNA encoding the α-subunit (Fig. 4). In some experiments, cells were also treated with proteasome inhibitors to reduce degradation and to facilitate the distinction between altered recognition by calnexin from increased degradation. Sequential immunoprecipitations were conducted with an antibody to calnexin (c), followed by immunoprecipitation of the unbound material with an anti-α- subunit antibody (mAb 61), to assess the extent of calnexin-α-subunit recognition. In Fig. 4, sequentially precipitated α-subunits are displayed, with lane 1 in sectionsA-C displaying α-subunits bound to calnexin, andlane 3 exhibiting the α-subunits not cleared with calnexin; likewise, lane 2 in sections A-Cdisplays α-subunits bound to calnexin and lane 4 exhibits α-subunits not cleared with calnexin. Densities of α-subunit bands were quantified, and density ratios for α-subunits expressed in the presence relative to the absence of castanospermine were calculated. Each histogram bar was calculated separately for α-subunits precipitated with anti-calnexin or anti-α-subunit antibodies; for example, in Fig. 4, A-C, the histogram bars designatedTFT: α and IP to: c, were calculated for α-subunits co-immunoprecipitated with calnexin by anti-calnexin antibody, by dividing the band densities for α-subunits expressed in the presence (shown in lane 2) and absence of castanospermine (shown in lane 1" @default.
• W2038857470 created "2016-06-24" @default.
• W2038857470 creator A5004460355 @default.
• W2038857470 creator A5084824937 @default.
• W2038857470 creator A5085382062 @default.
• W2038857470 date "1998-07-01" @default.
• W2038857470 modified "2023-10-10" @default.
• W2038857470 title "Inhibition of Glucose Trimming with Castanospermine Reduces Calnexin Association and Promotes Proteasome Degradation of the α-Subunit of the Nicotinic Acetylcholine Receptor" @default.
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