Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells.
We have investigated the mechanisms regulating the clustering of nicotinic acetylcholine receptor (AChR) on the surface of cultured embryonic chick muscle cells. Treatment of these cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, was found to cause a rapid dispersal of AChR clusters, as monitored by fluorescence microscopy of cells labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin. The loss of AChR clusters was not accompanied by an appreciable change in the amount of AChR on the surface of these cells, as measured by the specific binding of [125I]Bgt. Analysis of the phosphorylation pattern of immunoprecipitable AChR subunits showed that the gamma- and delta-subunits are phosphorylated by endogenous protein kinase activity in the intact muscle cells, and that the delta-subunit displays increased phosphorylation in response to TPA. Structural analogues of TPA which do not stimulate protein kinase C have no effect on AChR surface topography or phosphorylation. Exposure of chick myotubes to the cholinergic agonist carbamylcholine was found to cause a dispersal of AChR clusters with a time course similar to that of TPA. Like TPA, carbamylcholine enhances the phosphorylation of the delta-subunit of AChR. The carbamylcholine-induced redistribution and phosphorylation of AChR is blocked by the nicotinic AChR antagonist d-tubocurarine. TPA and carbamylcholine have no effect on cell morphology during the time-course of these experiments. These findings indicate that cell surface topography of AChR may be regulated by phosphorylation of its subunits and suggest a mechanism for dispersal of AChR clusters by agonist activation.
Department of Anatomical Sciences, State University of New York, Stony Brook 11794.
A
A
Ross
M
M
Rapuano
J
J
Prives
D000818:Animals; D001345:Autoradiography; D002217:Carbachol / Q000494:pharmacology*; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D003201:Computers; D004591:Electrophoresis, Polyacrylamide Gel; D007118:Immunoassay; D009132:Muscles / Q000032:analysis / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011978:Receptors, Nicotinic / Q000032:analysis / Q000187:drug effects* / Q000378:metabolism; D012044:Regression Analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D011978:Receptors, Nicotinic; D002217:Carbachol; D013755:Tetradecanoylphorbol Acetate