Phosphorylation and assembly of nicotinic acetylcholine receptor subunits in cultured chick muscle cells.
The assembly of the nicotinic acetylcholine receptor (AChR), an oligomeric cell surface protein, was studied in cultured muscle cells. To measure this process, the incorporation of metabolically labeled alpha-subunit into oligomeric AChR was monitored in pulse-chase experiments, either by the shift of this subunit from the unassembled (5 S) to the assembled (9 S) position in sucrose density gradients, or by its coprecipitation with antisera specific for the delta-subunit. We have found that AChR assembly is initiated 15-30 min after subunit biosynthesis and is completed within the next 60 min. The alpha-subunit is not overproduced, as all detectable pulse-labeled alpha-subunit can be chased into the oligomeric complex, suggesting that AChR assembly in this system is an efficient process. The rate of AChR assembly is decreased by metabolic inhibitors and by monensin, an ionophore that impairs the Golgi apparatus. We have observed that the gamma- and delta-subunits of AChR are phosphorylated in vivo. The delta-subunit is more highly phosphorylated in the unassembled than in the assembled state, indicating that its phosphorylation precedes assembly and that its dephosphorylation is concomitant with AChR assembly. These findings suggest that subunit assembly occurs in the Golgi apparatus and that phosphorylation/dephosphorylation mechanisms play a role in the control of AChR subunit assembly.
Ross
A F
AF
Department of Anatomical Sciences, State University of New York at Stony Brook 11794.
Rapuano
M
M
Schmidt
J H
JH
Prives
J M
JM
D000818:Animals; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D006056:Golgi Apparatus / Q000378:metabolism; D007106:Immune Sera / Q000276:immunology; D009132:Muscles / Q000032:analysis; D010766:Phosphorylation; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D007106:Immune Sera; D011978:Receptors, Nicotinic