Overexpression of rapsyn inhibits agrin-induced acetylcholine receptor clustering in muscle cells

Rapsyn is a protein on the cytoplasmic face of the postsynaptic membrane of skeletal muscle that is essential for clustering acetylcholine receptors ( AChR). Here we show that transfection of rapsyn cDNA can restore AChR clust ering function to muscle cells cultured from rapsyn deficient (KORAP) mice. KORAP myotubes displayed no AChR aggregates before or after treatment with neural agrin. After transfection with rapsyn expression plasmid, some KORA P myotubes expressed rapsyn at physiological levels. These formed large ACh R-rapsyn clusters in response to agrin, just like wild-type myotubes. KORAP myotubes that overexpressed rapsyn formed only scattered AChR-rapsyn micro aggregates, irrespective of agrin treatment. KORAP cells were then transfec ted with mutant forms of rapsyn. A deletion mutant lacking residues 16-254 formed rapsyn microaggregates, but failed to aggregate AChRs. Substitution mutation to the C-terminal serine phosphorylation site of rapsyn (M43(D405, D406)) did not impair the response to agrin, showing that differential phos phorylation of this site is unlikely to mediate agrin-induced clustering. T he results indicate that rapsyn expression is essential for agrin-induced A ChR clustering but that its overexpression inhibits this pathway. The appro ach of using rapsyn-deficient muscle cells opens the way for defining the r ole of rapsyn in agrin-induced AChR clustering.