Neuromuscular synapses form as a result of inductive interactions between motor neurons and muscle fibers. Following contact with the growth cone of a developing motor neuron, developing muscle fibers undergo a complex differentiation program in the synaptic region, and signals from the muscle in turn are thought to regulate the differentiation of the presynaptic terminals. Two different signaling pathways lead to the localization of acetylcholine receptors (AChRs) at synaptic sites. The signal for one pathway is agrin, a protein which is synthesized by motor neurons and which is secreted into the basal lamina at synaptic sites. Neither the receptor for agrin nor the mechanisms of agrin-mediated signaling, however, are known. The discovery of the muscle specific kinase , MuSK, and its role in agrin-mediated signaling provide an important advance in our understanding of how agrin signals to muscle. Current data support the idea that MuSK is a critical component of the agrin receptor complex but that another myotube-specific activity is required to bind agrin and to activate MuSK. The investigators will use minimal, functionally active forms of agrin, which stimulate MuSK but do not bind to alpha-dystroglycan, as affinity reagents to identify and isolate the functional agrin receptor from Torpedo electric organ postsynaptic membranes. In addition,they will determine whether the functional agrin receptor co-immunoprecipitates with MuSK, since co-immunoprecipitation may serve as an independent means to identify and purify the functional agrin receptor. These experiments will lead to the isolation of cDNAs encoding the agrin receptor. The molecules that are required for MuSK to respond to agrin and to initiate clustering of synaptic proteins are not known. They will adopt strategies that have been successfully applied to study other receptor tyrosine kinases to identify proteins that are tyrosine phosphorylated by agrin. Because proteins that are activated by MuSK may associate with MuSK, they will also identify proteins that co-immunoprecipitate with MuSK. They will identify and inhibit signaling pathways which are activated by agrin/MuSK to determine which, if any, signaling pathways are required for clustering synaptic proteins. They will determine whether AChRs are required to cluster other synaptic proteins and whether agrin-stimulated AChR tyrosine phosphorylation is required to cluster AChRs and other synaptic proteins. These studies will provide a better understanding of the mechanisms by which agrin signals through MuSK, regulates clustering of AChRs and other synaptic proteins, and organizes postsynaptic differentiation.
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