BACKGROUND AND AIMS: Alpha-Dystrobrevin-1 (aDB1) is a component of the dystrophin-associated glycoprotein complex (DGC), which stabilizes the neuromuscular junction (NMJ) postsynaptic machinery. The goal of our research is to gain insight into the mechanism of aDB-1 function at the NMJ, with special focus on the role of aDB1 phosphorylation, which was previously shown to be critical for proper synaptic organization. METHODS: To determine the importance of each tyrosine phosphorylation site, we expressed mutant proteins lacking individual sites in myotubes. Next, we developed phospho-specific antibodies and used them to analyze the level of aDB1 phosphorylation during NMJ remodeling. We also performed a biochemical screen to identify general and phospho-specific aDB1-binding proteins. Finally, we performed RNAi experiments on cultured myotubes to demonstrate the importance of these proteins in the organization of AChR clusters. RESULTS: Our experiments identified Liprin-α1, α-Catulin and Usp9x as novel aDB1-interacting proteins. We have demonstrated that aDB1 phosphorylation is dynamically regulated during NMJ remodeling in development and upon denervation and that the phosphorylation at the most critical tyrosine Y713 triggers recruitment of aDB1 phospho-specific interacting proteins, including Grb2, SH3BP2, Arhgef5 and PI3K. Subsequently, we demonstrated that Liprin-α1, α-Catulin and Grb2 are associated with the postsynaptic NMJ machinery and are indispensable for proper AChR organization. CONCLUSIONS: Our research highlights the importance of aDB1 phosphorylation in the remodeling of neuromuscular synapses. We identify several novel aDB1-interacting components of the postsynaptic machinery, which play important roles in its organization. This research was supported by the grant 2013/09/B/NZ3/03524 from the National Science Centre (NCN).
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