Molecular signaling mechanisms regulating the development and maintenance of the neuromuscular junction.

摘要

One of the key questions in synaptic development is how the appropriate neurotransmitter receptors are trafficked to the synapse. At the neuromuscular junction (NMJ), a receptor tyrosine kinase, MuSK, propagates a tyrosine phosphorylation cascade that leads to the synaptic clustering of nicotinic acetylcholine receptors (nAChRs), and this cascade is also dependent on the nAChR-binding protein rapsyn. Here we describe two sets of experiments that further elucidate this signaling cascade. First, through immunocytochemistry and immunoprecipitation, we identify Disabled-1 as a phosphotyrosine binding (PTB) domain-containing protein that interacts with MuSK at the adult synapse. This interaction, determined by co-immunoprecipitation in heterologous cells, is dependent both on the PTB domain of Disabled-1 and on the nonphosphorylated state of MuSK. Overexpression of Dab1 in cultured myotubes inhibits MuSK-mediated nAChR clustering in a PTB-dependent manner. These results suggest that Dab1 is a negative regulator of MuSK signaling and that it may play a role in the maintenance and homeostasis of the mature neuromuscular junction. The second set of experiments describes our efforts to determine the function of rapsyn in MuSK-mediated nAChR clustering. We show that rapsyn is a ubiquitin ligase and that this ubiquitin ligase activity is required for agrin-induced clustering and for the rapsyn-mediated internalization of nAChRs. We propose that rapsyn directly ubiquitinates the beta1 subunit of the nAChR, and show by immunoprecipitation that the beta1 subunits of surface nAChRs are highly ubiquitinated in C2C12 myotubes, but less so in rapsyn-/- myotubes. Specifically, a mutant beta1 subunit in which the one conserved lysine of the major cytoplasmic loop has been mutated to arginine is able to reach the cell surface, but fails to be incorporated into agrin-induced clusters and simultaneously inhibits the clustering of wild-type receptors. Hence, we propose a novel model in which rapsyn ubiquitinates the conserved lysine of the cytoplasmic loop of the nAChR beta1 subunit, thereby causing the entire receptor to be internalized and redirected to the synapse.