Inhibition of glucose trimming with castanospermine reduces calnexin association and promotes proteasome degradation of the alpha-subunit of the nicotinic acetylcholine receptor.
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 delta-alpha-gamma-alpha-beta 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 alpha-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 alpha-subunits, and generally reverses the castanospermine induced loss of alpha-subunit protein. Co-transfection of cDNAs encoding the alpha- and delta-subunits, which leads to the expression of assembled alpha- and delta- subunits, also inhibits the loss of alpha-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.
Keller
S H
SH
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636, USA.
Lindstrom
J
J
Taylor
P
P
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D001704:Biopolymers / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D037282:Calreticulin; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary; D019281:Dimerization; D005947:Glucose / Q000037:antagonists & inhibitors*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007212:Indolizines / Q000378:metabolism*; D007535:Isomerases / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000378:metabolism*; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism; D054836:Indolizidines
D000111:Acetylcysteine; D001704:Biopolymers; D002135:Calcium-Binding Proteins; D037281:Calnexin; D037282:Calreticulin; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D005947:Glucose; D006360:Heat-Shock Proteins; D007212:Indolizines; D007535:Isomerases; D007976:Leupeptins; D009097:Multienzyme Complexes; D025821:Polyubiquitin; D046988:Proteasome Endopeptidase Complex; D019704:Protein Disulfide-Isomerases; D011978:Receptors, Nicotinic; D012261:Ribonucleoproteins; D014452:Ubiquitins; C067713:lactacystin; C085868:PDIA3 protein, human; C037806:castanospermine; D054836:Indolizidines; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde