The Agrin/Lrp4/MuSK/Dok-7 signal transduction cascade is critical for synaptogenesis. Binding between Agrin and Lrp4 stimulates tyrosine phosphorylation of MuSK and recruitment and tyrosine phosphorylation of Dok-7. Hypomorphic mutations in human Agrin, MuSK or Dok-7, which impair their function, cause congenital myasthenia, characterized by structurally and functionally defective synapses, leading to muscle weakness and fatigue, emphasizing the clinical relevance of this signaling pathway. Once phosphorylated, Dok-7 recruits Crk and Crk-L, two related adapter proteins. The Agrin/Lrp4/Dok-7/Crk/Crk-L signaling pathway ultimately intersects with the cytoskeleton to cause accumulation of AChRs and other proteins in the postsynaptic membrane. The experiments in this proposal are designed to reveal the molecules and mechanisms that link recruitment of Crk/Crk-L to the redistribution and anchoring of acetylcholine receptors (AChRs) at developing and adult synapses. The proposed experiments will identify proteins that associate with Rapsyn, an AChR-associated protein that is essential to cluster AChRs, and analyze how these newly identified synaptic proteins, including Vezatin, regulate synaptic differentiation. These studies are clinically relevant, as mutations in Agrin, MuSK, Dok-7 and Rapsyn cause congenital myasthenia, so understanding how these proteins work will contribute to a better understanding of these diseases and may lead to novel therapeutic strategies. Moreover, mutations in genes that are downstream from Crk/Crk-L may likewise cause congenital myasthenia, so identifying the pathway downstream from Crk/Crk-L may reveal new genes responsible for congenital myasthenia.
The formation of neuromuscular synapses requires a complex exchange of signals between motor neurons and developing muscle fibers leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. The signals and mechanisms responsible for this complex differentiation program are poorly understood but require the neurally-derived ligand, Agrin, the receptor for Agrin, Lrp4, the transducing receptor tyrosine kinase, MuSK, and the adapter protein, Dok-7, which is recruited to phosphorylated MuSK. Defects in this signaling pathway are responsible for a variety of congenital neuromuscular disorders and could underlie or contribute to neurodegenerative diseases such as ALS. The experiments described here are designed to determine how Dok-7 mediates Agrin responsiveness and stimulates synaptic differentiation.