Proper functioning of the nervous system requires highly ordered and tightly regulated synapse formation. The long-range goal of this project is to elucidate the molecular and cellular mechanisms that underlie postsynaptic differentiation. Agrin is an extracellular matrix protein that induces the clustering of acetylcholine receptors (AChRs) on myotubes in culture. At developing neuromuscular junctions in vivo, agrin is likely to direct the assembly of key elements of the postsynaptic apparatus. Agrin mRNA is alternatively spliced yielding agrin isoforms that have large differences in AChR clustering potency. During the previous funding period, a candidate agrin receptor from Torpedo electric organ postsynaptic membranes was identified and was shown to be a heteromeric complex whose subunits are homologous to alpha- and beta- dystroglycan. Preliminary experiments using alternatively-spliced agrin isoforms have indicated that myotubes and postsynaptic membranes express several classes of agrin receptors, each of which contains alpha-dystroglycan. The proposed experiments are designed to characterize these newly recognized classes of agrin receptors at the cellular, biochemical, and molecular levels. The first goal (Specific Aims #1 and #2) is to analyze agrin- isoform selective receptors at the cellular and biochemical levels. It is hypothesized that the different classes of agrin receptors perform distinct but interrelated roles during synapse development. The experiments in Specific Aim #3, are designed to establish the developmental profile of the expression of these agrin receptor classes, to localize them at synapses in vivo, and to study their regulation during key stages in nerve-muscle synaptogenesis. Further, it is hypothesized that other proteins that associate with the dystroglycans play important roles in mediating agrin's actions. Experiments in Specific Aim #4 are designed to characterize and to clone one such protein, a novel dystroglycan/dystrophin- associated protein discovered in this laboratory. The results of these studies will provide vital insights into the means by which synaptic differentiation proceeds during development and regeneration. In addition, the experiments proposed here focus on the dystroglycans, part of a protein complex that is defective in several muscular dystrophies. Knowledge of the function of this complex, and the factors that mediate its assembly, could provide new keys to understanding and eventually treating these diseases.
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