The goal of this project is to identify regulatory mechanisms controlling the expression of muscle surface proteins that mediate transmembrane signalling. Our studies 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 biogenesis and topogenesis, with emphasis on the contribution of cellular protein phosphorylation mechanisms. The studies to be performed are aimed at defining the significance of AChR phosphorylation to the regulation of AChR subunit assembly, as well as to the cell surface distribution and attachment to the cytoskeleton of oligomeric AChR. The assembly of AChR subunits will be measured by metabolic labeling and immunoprecipitation techniques, and AChR phosphorylation in situ will be assayed by the incorporation of (32P)i into immunoprecipitable AChR peptides, using pharmacological agents that alter cellular phosphorylation activities. The cell surface distribution of AChR will be monitored by fluorescence microscopy of cells labeled with a fluorescent ligand of AChR. Microinjection methods, and muscle cells transformed with Rous sarcoma viral mutants will be utilized to investigate the contribution of protein phosphorylation to AChR cell surface distribution. The attachment of AChR with 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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