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 clustering 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 KORAP myotubes expressed rapsyn at physiological levels. These formed large AChR-rapsyn clusters in response to agrin, just like wild-type myotubes. KORAP myotubes that overexpressed rapsyn formed only scattered AChR-rapsyn microaggregates, irrespective of agrin treatment. KORAP cells were then transfected 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 phosphorylation of this site is unlikely to mediate agrin-induced clustering. The results indicate that rapsyn expression is essential for agrin-induced AChR clustering but that its overexpression inhibits this pathway. The approach of using rapsyn-deficient muscle cells opens the way for defining the role of rapsyn in agrin-induced AChR clustering.
Han
H
H
Institute for Biomedical Research, Department of Physiology, University of Sydney, Sydney NSW, Australia.
Noakes
P G
PG
Phillips
W D
WD
D018171:Agrin / Q000378:metabolism*; D000818:Animals; D002461:Cell Line, Transformed; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000378:metabolism; D005260:Female; D017353:Gene Deletion; D015870:Gene Expression / Q000502:physiology; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D051379:Mice; D008817:Mice, Mutant Strains; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000378:metabolism*; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000294:innervation; D016296:Mutagenesis / Q000502:physiology; D010446:Peptide Fragments / Q000235:genetics; D010766:Phosphorylation; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D014162:Transfection; D049411:Utrophin
D018171:Agrin; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; C483600:Utrn protein, mouse; D049411:Utrophin; C051609:peripheral membrane protein 43K; D012694:Serine