title: Rapsyn carboxyl terminal domains mediate muscle specific kinase-induced phosphorylation of the muscle acetylcholine receptor.
abstract: At the developing vertebrate neuromuscular junction, postsynaptic localization of the acetylcholine receptor (AChR) is regulated by agrin signaling via the muscle specific kinase (MuSK) and requires an intracellular scaffolding protein called rapsyn. In addition to its structural role, rapsyn is also necessary for agrin-induced tyrosine phosphorylation of the AChR, which regulates some aspects of receptor localization. Here, we have investigated the molecular mechanism by which rapsyn mediates AChR phosphorylation at the rodent neuromuscular junction. In a heterologous COS cell system, we show that MuSK and rapsyn induced phosphorylation of beta subunit tyrosine 390 (Y390) and delta subunit Y393, as in muscle cells. Mutation of beta Y390 or delta Y393 did not inhibit MuSK/rapsyn-induced phosphorylation of the other subunit in COS cells, and mutation of beta Y390 did not inhibit agrin-induced phosphorylation of the delta subunit in Sol8 muscle cells; thus, their phosphorylation occurs independently, downstream of MuSK activation. In COS cells, we further show that MuSK-induced phosphorylation of the beta subunit was mediated by rapsyn, as MuSK plus rapsyn increased beta Y390 phosphorylation more than rapsyn alone and MuSK alone had no effect. Intriguingly, MuSK also induced tyrosine phosphorylation of rapsyn itself. We then used deletion mutants to map the rapsyn domains responsible for activation of cytoplasmic tyrosine kinases that phosphorylate the AChR subunits. We found that rapsyn C-terminal domains (amino acids 212-412) are both necessary and sufficient for activation of tyrosine kinases and induction of cellular tyrosine phosphorylation. Moreover, deletion of the rapsyn RING domain (365-412) abolished MuSK-induced tyrosine phosphorylation of the AChR beta subunit. Together, these findings suggest that rapsyn facilitates AChR phosphorylation by activating or localizing tyrosine kinases via its C-terminal domains.
authors.affiliation: Departments of Anesthesiology and Physiology and Membrane Biology, One Shields Avenue, University of California-Davis, Davis, CA 95616, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Lee
authors.forename: J
authors.initials: J
authors.lastname: Rudell
authors.forename: S
authors.initials: S
authors.lastname: Yechikhov
authors.forename: R
authors.initials: R
authors.lastname: Taylor
authors.forename: S
authors.initials: S
authors.lastname: Swope
authors.forename: M
authors.initials: M
authors.lastname: Ferns
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002038:Bungarotoxins / Q000494:pharmacology; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D051379:Mice; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism*; D009154:Mutation; D009469:Neuromuscular Junction / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000145:classification / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D014162:Transfection / Q000379:methods
chemical_list: D000906:Antibodies; D002038:Bungarotoxins; D009124:Muscle Proteins; D021122:Protein Subunits; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; C051609:peripheral membrane protein 43K; C475048:MuSK protein, rat; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases