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 importa nt role in the clustering of the nicotinic acetylcholine receptor (ACh R) 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 exami ned whether tyrosine phosphorylation is involved in this rapsyn-induce d AChR clustering, Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with an ti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rap syn clusters is seen in the absence of AChR expression, indicating tha t the AChR is required for the appearance of phosphotyrosine at cluste rs. In addition, coexpression of rapsyn with the AChR induces the tyro sine phosphorylation of the beta and delta subunits of the AChR. surpr isingly, mutation of the tyrosine phosphorylation sites in the AChR di d not inhibit rapsyn-induced clustering of the AChR and clusters of th e mutant AChRs still contained high levels of phosphotyrosine. Experim ents with single AChR subunits demonstrate that the alpha subunit of t he AChR appears to be necessary and sufficient for codistribution of p hosphotyrosine 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 th erefore regulate clustering at least in part by regulating the tyrosin e phosphorylation of cellular proteins.