The objective of this proposal is to identify the molecular mechanisms responsible for neuron-induced clustering of muscle nicotinic acetylcholine receptors (AChRs) in postsynaptic membranes during formation of neuromuscular junctions. The focus is on the signaling elicited by the extracellular matrix components agrin and laminin and the role of a specific family of regulatory proteins - the Rho GTPases - as crucial mediators of AChR clustering. This project stems from recent findings that the coupling of both agrin and laminin signaling to AChR clustering is critically dependent on the activities of three Rho GTPases - Cdc42, Rac, and Rho - that couple extracellular signaling to localized changes in peripheral actin-based cytoarchitecture. The molecular mechanisms by which agrin/laminin elicit RhoGTPase activation as well as the means by which this activation is transduced into localized AChR clustering are not understood at present. The proposed studies will utilize cultured muscle cell lines, transfection techniques, biochemical assays and fluorescence imaging to analyze the mechanistic basis for the induction of AChR redistribution into high density clusters on the muscle cell surface in response to these extracellular cues. ? ? ?
