The synapse-associated protein rapsyn regulates tyrosine phosphorylation of proteins colocalized at nicotinic acetylcholine receptor clusters

Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of rapsyn with the AChR induces the tyrosine phosphorylation of the beta amd delta subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the alpha subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.