Involvement of the chaperone protein calnexin and the acetylcholine receptor beta-subunit in the assembly and cell surface expression of the receptor.
The nicotinic acetylcholine receptor at the neuromuscular junction is a ligand-gated ion channel assembled in the endoplasmic reticulum from four distinct glycoprotein subunits into the pentameric configuration of alpha2betagammadelta. The individual homologous subunits form specific contacts at interfaces with neighboring subunits to achieve the appropriate orientation and order of each subunit in surrounding the ion channel. Assembly is thought to proceed through the formation of intermediates composed of dimers of the alphadelta and alphagamma subunits which are eventually joined by the beta-subunit to achieve a circular structure enclosing the gated ion channel. In this study, we transfect cDNAs encoding receptor subunits in various combinations into HEK-293 cells to identify intracellular factors that influence the assembly and cell surface expression of the receptor. Our data derived from brefeldin A-treated cells indicate that intracellular association of the receptor subunits with the beta-subunit increases the pool of fully assembled receptors available for transport to the cell surface, presumably by protection from degradation. In addition, we determined that the chaperone protein calnexin is associated with the isolated alpha-, beta-, and delta-subunits of the receptor, but calnexin is not detected in association with assembled alphadelta subunit dimers. Calnexin is also detected in association with maturely folded, unassembled alpha-subunits, as observed by the recognition of this complex by the monoclonal antibody mAb 35, believed to be specific for correctly folded alpha-subunits. Thus, calnexin appears to associate with the individual nascent subunits, thereby facilitating their assembly into the mature pentameric receptor.
Keller
S H
SH
Department of Pharmacology 0636, University of California, San Diego, La Jolla, California 92093, USA.
Lindstrom
J
J
Taylor
P
P
D000818:Animals; D015153:Blotting, Western; D020126:Brefeldin A; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003517:Cyclopentanes / Q000494:pharmacology; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism*; D011487:Protein Conformation; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014162:Transfection
D002135:Calcium-Binding Proteins; D003517:Cyclopentanes; D018832:Molecular Chaperones; D011950:Receptors, Cholinergic; D037281:Calnexin; D020126:Brefeldin A