Roles of rapsyn and agrin in interaction of postsynaptic proteins with acetylcholine receptors

At the neuromuscular junction, aggregates of acetylcholine receptors (AChRs ) are anchored in the muscle membrane by association with rapsyn and other postsynaptic proteins. We have investigated the interactions between the AC hR and these proteins in cultured C2 myotubes before and after treatment wi th agrin, a nerve-derived protein that induces AChRs to cluster. When AChRs were isolated from detergent extracts of untreated C2 myotubes, they were associated with rapsyn and, to a lesser degree, with utrophin, beta-dystrog lycan, MuSK, and src-related kinases, but not with syntrophin. Treatment wi th agrin increased the association of AChRs with MuSK, a receptor tyrosine kinase that forms part of the agrin receptor complex, without affecting oth er interactions. Analysis of rapsyn-deficient myotubes, which do not form p rotein clusters in response to agrin, revealed that rapsyn is required for association of the AChR with utrophin and beta-dystroglycan, and for the ag rin-induced increase in association with MuSK, but not for constitutive int eractions with MuSK and src-related kinases. In rapsyn -/- myotubes, agrin caused normal tyrosine phosphorylation of AChR-associated and total MuSK, w hereas phosphorylation of the AChR beta subunit, both constitutive and agri n-induced, was strongly reduced. These results show first that aneural myot ubes contain preassembled AChR protein complexes that may function in the a ssembly of the postsynaptic apparatus, and second that rapsyn, in addition to its role in AChR phosphorylation, mediates selected protein interactions with the AChR and serves as a link between the AChR and the dystrophin/utr ophin glycoprotein complex.