At the developing neuromuscular junction, a motoneuron-derived factor called agrin signals through the muscle-specific kinase receptor to induce postsynaptic aggregation of the acetylcholine receptor (AChR). The agrin signaling pathway involves tyrosine phosphorylation of the AChR β subunit, and we have tested its role in receptor localization by expressing tagged, tyrosine-minus forms of the β subunit in mouse Sol8 myotubes. We find that agrin-induced phosphorylation of the β subunit occurs only on cell surface AChR, and that AChR-containing tyrosine-minus β subunit is targeted normally to the plasma membrane. Surface AChR that is tyrosine phosphorylated is less detergent extractable than nonphosphorylated AChR, indicating that it is preferentially linked to the cytoskeleton. Consistent with this, we find that agrin treatment reduces the detergent extractability of AChR that contains tagged wild-type β subunit but not tyrosine-minus β subunit. In addition, agrin-induced clustering of AChR containing tyrosine-minus β subunit is reduced in comparison to wild-type receptor. Thus, we find that agrin-induced phosphorylation of AChR β subunit regulates cytoskeletal anchoring and contributes to the clustering of the AChR, and this is likely to play an important role in the postsynaptic localization of the receptor at the developing synapse.

The acetylcholine receptor (AChR)-associated protein rapsyn is essential for neuromuscular synapse formation and clustering of AChRs, but its mode of action remains unclear. We have investigated whether agrin, a key nerve-derived synaptogenic factor, influences rapsyn-AChR interactions and how this affects clustering and cytoskeletal linkage of AChRs. By precipitating AChRs and probing for associated rapsyn, we found that in denervated diaphragm rapsyn associates with synaptic as well as with extrasynaptic AChRs showing that rapsyn interacts with unclustered AChRs in vivo. Interestingly, synaptic AChRs are associated with more rapsyn suggesting that clustering of AChRs may require increased interaction with rapsyn. In similar experiments in cultured myotubes, rapsyn interacted with intracellular AChRs and with unclustered AChRs at the cell surface, although surface interactions are much more prominent. Remarkably, agrin induces recruitment of additional rapsyn to surface AChRs and clustering of AChRs independently of the secretory pathway. This agrin-induced increase in rapsyn-AChR interaction strongly correlates with clustering, because staurosporine and herbimycin blocked both the increase and clustering. Conversely, laminin and calcium induced both increased rapsynAChR interaction and AChR clustering. Finally, time course experiments revealed that the agrin-induced increase occurs with AChRs that become cytoskeletally linked, and that this precedes receptor clustering. Thus, we propose that neural agrin controls postsynaptic aggregation of the AChR by enhancing rapsyn interaction with surface AChRs and inducing cytoskeletal anchoring and that this is an important precursor step for AChR clustering.

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.

Agrin is a motoneuron-derived factor that initiates neuromuscular synapse formation; however, the signaling pathway underlying postsynaptic differentiation is not yet understood. We have investigated the role of calcium in agrin signaling through the MuSK receptor tyrosine kinase and in the intracellular signaling cascade that leads to AChR phosphorylation and clustering. We find that agrin- and neuramindase-induced MuSK activation in cultured myotubes is completely blocked by removal of extracellular calcium, but only slightly reduced by clamping of intracellular calcium transients with BAPTA. Following agrin's activation of MuSK, we find that the downstream tyrosine phosphorylation of the AChR beta-subunit was inhibited by BAPTA but not by a slower acting chelator, EGTA. Similarly, agrin-induced clustering of the AChR was blocked by BAPTA but not EGTA. These findings indicate that extracellular calcium is required for the formation of a MuSK signaling complex, and that intracellular calcium regulates phosphorylation and clustering of the AChR in the postsynaptic membrane.