The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR δ-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR δ-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13- acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR δ-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell- permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the δ- subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X- 100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR δ-subunits may play a role in the surface distribution of these receptors.
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