Identification of a Motif in the Acetylcholine Receptor β Subunit Whose Phosphorylation Regulates Rapsyn Association and Postsynaptic Receptor Localization

Abstract

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.

Determinants in the β and δ Subunit Cytoplasmic Loop Regulate Golgi Trafficking and Surface Expression of the Muscle Acetylcholine Receptor

“…To identify molecular determinants for AChR trafficking we performed an unbiased screen using chimeric constructs consisting of mouse CD4 extracellular and transmembrane domains fused with the major cytoplasmic loop of each mouse muscle nAChR subunit (19) (Fig. 1A).…”

“…1A). The CD4-loop chimeras do not assemble with endogenous AChR subunits in muscle cells (19) and lack a previously identified ER retention motif in the first transmembrane domain of each subunit (10). Thus, they allow us to test each subunit cytoplasmic loop for molecular signals that act in later, post-ER trafficking steps.…”

“…CD4-Subunit Loop Constructs-Chimeric constructs consisting of mouse CD4 extracellular and transmembrane domains fused with the major cytoplasmic loop of each mouse nAChR subunit were generated as described previously (19). Briefly, a BglII site was introduced by site-directed mutagenesis at the end of the transmembrane domain of mouse CD4.…”

“…For immunostaining experiments, myoblasts were grown on 8-well chamber slides (Nalgene Nunc, Inc.) and transfected at ϳ60 -70% confluence using FuGENE (Roche Applied Science). Upon reaching confluence the cells were incubated with fusion medium (DMEM supplemented with 5% horse serum and 2 mM L-glutamine) and allowed to differentiate into myotubes for 3-4 days prior to analysis (19).…”

ColQ Controls Postsynaptic Differentiation at the Neuromuscular Junction

“…6C, black bars). We also analyzed cell surface ␤-AChR subunit phosphorylation on Y390 residue, a process known to increase AChR-rapsyn interactions and AChR clustering in presence of agrin (Borges et al, 2008). The p␤-AChR-to-␤-AChR ratio was two to threefold less important in ColQ Ϫ/Ϫ cells treated with agrin compared to wt cells (Fig.…”