Interactions of the rapsyn RING-H2 domain with dystroglycan

Rapsyn, a peripheral membrane protein of skeletal muscle, is necessary for the formation of the highly organized structure of the vertebrate neuromusc ular junction. For mice lacking rapsyn, there is a failure of postsynaptic specialization characterized by an absence of nicotinic acetylcholine recep tors (nAChRs) and other integral and peripheral membrane proteins such as b eta -dystroglycan and utrophin, Dystroglycan is necessary for the formation of the mature neuromuscular junction and has been shown to interact direct ly with rapsyn, Previous studies with rapsyn fragments and mutants, express ed in 293T cells along with nAChRs, establish that the rapsyn tetratricopep tide repeat (TPR) domain is involved in self-association and its coiled-coi l domain is necessary for nAChR clustering, The function of the rapsyn RING -MS domain, which is not necessary for rapsyn self-association or nAChR clu stering, is unknown. To further characterize these domains, we have used a yeast two-hybrid assay to test for interactions at the plasma membrane betw een rapsyn domains and a nAChR beta -subunit fragment, the beta -dystroglyc an cytoplasmic domain, or rapsyn domains. The rapsyn coiled-coil domain int eracts with the nAChR beta -subunit cytoplasmic domain, but not with itself , other rapsyn domains, or beta -dystroglycan, The RING-MS domain interacts only with the beta -dystroglycan cytoplasmic domain. Furthermore, when exp ressed in 293T cells, a rapsyn construct containing as few as two TPRs and the RING-MS domain self-associates and clusters dystroglycan, but not nAChR s, These results emphasize the modular character of the rapsyn structural d omains.