The goal of this project is to identify regulatory mechanisms controlling the expression of muscle surface proteins that mediate transmembrane signalling. Our study will focus on the nicotinic acetylcholine receptor (AChR), a well-characterized transmembrane oligomeric glycoprotein that functions as a ligand gated ion channel. During muscle differentiation and innervation, AChR undergoes marked changes in biogenesis and cell surface distribution. Using cultured embryonic muscle cells, we will study the regulation of AChR assembly from its constituent subunits, and the control of its cell surface distribution. The studies to be performed are aimed at defining the significance of AChR phosphorylation state to the regulation of AChR subunit assembly, as well as to the cell surface distribution and attachment to the cytoskeleton of oligomeric AChR. A related objective is to characterize the cellular mechanisms that direct AChR phosphorylation and dephosphorylation, using pharmacological agents that alter cellular phosphorylation activities. The assembly of AChR subunits will be measured by metabolic labeling and immunoprecipitation techniques, and AChR phosphorylation will be assayed by the incorporation of [32P]i into immunoprecipitable AChR subunits, and further characterized by analysis of phosphopeptides and phospho amino acids. The cell surface distribution of AChR will be monitored by fluorescence microscopy of cells labeled with a fluorescent ligand of AChR. The anchorage of AChR to underlying cytoskeletal structures will be measured by mild detergent fractionation of muscle monolayers. It is anticipated that these studies will identify the regulatory significance of phosphorylation in AChR expression, and elucidate the role of post-translational modifications in the regulation of surface properties of specialized membrane ionic channel proteins.
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