Identification of a motif in the acetylcholine receptor β subunit whose phosphorylation regulates rapsyn association and postsynaptic receptor localization

Lucia S. Borges, Sergey Yechikhov, Young I. Lee, John B. Rudell, Matthew B. Friese, Steven J. Burden, Michael J Ferns

Research output: Contribution to journalArticle

43 Scopus citations

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 β subunit loop. By making deletions in the β 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 β loop chimeras and their ability to inhibit agrin-induced clustering of the endogenous AChR. Phosphorylation of the AChRβ subunit is correlated with increased rapsyn/AChR binding, suggesting that the effect of βY390 phosphorylation on clustering is mediated by rapsyn. Indeed, we found that rapsyn associated with CD4-β loop chimeras in a phosphorylation- dependent manner, and that agrin increased the ratio of rapsyn binding to wild type AChR but not to AChR-β3F/3F, which lacks β loop tyrosine phosphorylation sites. Together, these findings suggest that agrin-induced phosphorylation of the β 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.

Original languageEnglish (US)
Pages (from-to)11468-11476
Number of pages9
JournalJournal of Neuroscience
Volume28
Issue number45
DOIs
StatePublished - Nov 5 2008

Keywords

  • Agrin
  • Neuromuscular junction
  • Nicotinic acetylcholine receptor
  • Phosphorylation
  • Postsynaptic membrane
  • Synaptogenesis

ASJC Scopus subject areas

  • Neuroscience(all)

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