IMMOBILIZATION OF NICOTINIC ACETYLCHOLINE-RECEPTORS IN MOUSE C2 MYOTUBES BY AGRIN-INDUCED PROTEIN-TYROSINE PHOSPHORYLATION

Agrin induces the formation of highly localized specializations on myo tubes at which nicotinic acetylcholine receptors (AChRs) and many othe r components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine ph osphorylation. Treatments that inhibit tyrosine phosphorylation preven t AChR aggregation. To examine further the relationship between tyrosi ne phosphorylation and receptor aggregation, we have used the techniqu e of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes trea ted with agrin or with pervanadate, a protein tyrosine phosphatase inh ibitor. Agrin induced the formation of patches in C2 myotubes that sta ined intensely with anti-phosphotyrosine antibodies and within which A ChRs were relatively immobile, Pervanadate, on the other hand, increas ed protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without af fecting the mobility of other membrane proteins. Pervanadate, like agr in, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscul ar junctions in embryonic rat muscles also labeled with anti-phosphoty rosine antibodies, suggesting that tyrosine phosphorylation could medi ate AChR aggregation in vivo as well. Thus, agrin appears to induce AC hR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.