The function of cortactin in the clustering of acetylcholine receptors at the vertebrate neuromuscular junction.
BACKGROUND
Postsynaptic enrichment of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (NMJ) depends on the activation of the muscle receptor tyrosine MuSK by neural agrin. Agrin-stimulation of MuSK is known to initiate an intracellular signaling cascade that leads to the clustering of AChRs in an actin polymerization-dependent manner, but the molecular steps which link MuSK activation to AChR aggregation remain incompletely defined.


RESULTS
In this study we used biochemical, cell biological and molecular assays to investigate a possible role in AChR clustering of cortactin, a protein which is a tyrosine kinase substrate and a regulator of F-actin assembly and which has also been previously localized at AChR clustering sites. We report that cortactin was co-enriched at AChR clusters in situ with its target the Arp2/3 complex, which is a key stimulator of actin polymerization in cells. Cortactin was further preferentially tyrosine phosphorylated at AChR clustering sites and treatment of myotubes with agrin significantly enhanced the tyrosine phosphorylation of cortactin. Importantly, forced expression in myotubes of a tyrosine phosphorylation-defective cortactin mutant (but not wild-type cortactin) suppressed agrin-dependent AChR clustering, as did the reduction of endogenous cortactin levels using RNA interference, and introduction of the mutant cortactin into muscle cells potently inhibited synaptic AChR aggregation in response to innervation.


CONCLUSIONS
Our results suggest a novel function of phosphorylation-dependent cortactin signaling downstream from agrin/MuSK in facilitating AChR clustering at the developing NMJ.
Madhavan
Raghavan
R
Department of Biology, The Hong Kong University of Science and Technology, Kowloon, Hong Kong, China.
Gong
Zhuolin L
ZL
Ma
Jin Jin
JJ
Chan
Ariel W S
AW
Peng
H Benjamin
HB
D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D018171:Agrin / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D051356:Cortactin / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008954:Models, Biological; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009469:Neuromuscular Junction / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014714:Vertebrates / Q000378:metabolism*; D014981:Xenopus
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D018171:Agrin; D051356:Cortactin; D011950:Receptors, Cholinergic; D019000:Phosphotyrosine