IDENTIFICATION OF PHOSPHORYLATION SITES ON ACHR DELTA-SUBUNIT ASSOCIATED WITH DISPERSAL OF ACHR CLUSTERS ON THE SURFACE OF MUSCLE-CELLS

The innervation of embryonic skeletal muscle cells is marked by the re distribution of nicotinic acetylcholine receptors (AChRs) on muscle su rface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regul ation of AChR surface distribution, we have identified the sites on AC hR delta-subunits that undergo phosphorylation associated with AChR cl uster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser(378), Ser(393), and Ser(450), all located in the major intracellular domain of the AChR delta-subunit. A djacent to one of these sites is a PKA consensus target site (Ser(377) ) that was efficiently phosphorylated by purified PKA in vitro. The PK C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phospho protein phosphatase inhibitor okadaic acid (OA) produced increased pho sphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment o f these cells with the PKA activator forskolin, or with the cell-perme able cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation sta te of surface AChR, suggesting that PKA does not actively phosphorylat e the delta-subunit in intact chick myotubes. The effects of TPA and O A included an increase in the proportion of surface AChR that is extra cted in Triton X-100, as well as the spreading of AChR from cluster re gions to adjacent areas of the muscle cell surface. These findings sug gest that PKC-catalyzed phosphorylation on the identified serine resid ues of AChR delta-subunits may play a role in the surface distribution of these receptors.