At the neuromuscular junction, the acetylcholine receptor (AChR) is specifically clustered in the postsynaptic membrane via interactions with rapsyn and other scaffolding proteins. However, it remains unclear where these proteins bind on the AChR and how the interactions are regulated. Here, we define a phosphorylation-dependent binding site on the receptor that mediates agrin-induced clustering. Using chimeric proteins in which CD4 is fused to the large intracellular loop of each of the AChR subunits we found that agrin induced clustering of only chimeras containing the  subunit loop. By making deletions in the  loop we defined a 20 amino-acid sequence that is sufficient for clustering. The sequence contains a conserved tyrosine (Y390) whose phosphorylation is induced by agrin and whose mutation abolished clustering of  loop chimeras and their ability to inhibit agrin-induced clustering of the endogenous AChR. Phosphorylation of the AChR  subunit is correlated with increased rapsyn/AChR binding, suggesting that the effect of Y390 phosphorylation on clustering is mediated by rapsyn. Indeed, we found that rapsyn associated with CD4- loop chimeras in a phosphorylation-dependent manner, and that agrin increased the ratio of rapsyn binding to wild type AChR but not to AChR- 3F/3F , which lacks  loop tyrosine phosphorylation sites. Together, these findings suggest that agrin-induced phosphorylation of the  subunit motif increases the stoichiometry of rapsyn binding to the AChR, thereby helping to stably cluster the receptor and anchor it at high density in the postsynaptic membrane.
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