Dual role for calcium in agrin signaling and acetylcholine receptor clustering.
Agrin is a motoneuron-derived factor that initiates neuromuscular synapse formation; however, the signaling pathway underlying postsynaptic differentiation is not yet understood. We have investigated the role of calcium in agrin signaling through the MuSK receptor tyrosine kinase and in the intracellular signaling cascade that leads to AChR phosphorylation and clustering. We find that agrin- and neuramindase-induced MuSK activation in cultured myotubes is completely blocked by removal of extracellular calcium, but only slightly reduced by clamping of intracellular calcium transients with BAPTA. Following agrin's activation of MuSK, we find that the downstream tyrosine phosphorylation of the AChR beta-subunit was inhibited by BAPTA but not by a slower acting chelator, EGTA. Similarly, agrin-induced clustering of the AChR was blocked by BAPTA but not EGTA. These findings indicate that extracellular calcium is required for the formation of a MuSK signaling complex, and that intracellular calcium regulates phosphorylation and clustering of the AChR in the postsynaptic membrane.
Borges
Lucia S
LS
Department of Neurology and Neurosurgery, McGill University, and Centre for Research in Neuroscience, Montreal General Hospital Research Institute, 1650 Cedar Ave, Montreal, Quebec, H3G 1A4, Canada.
Lee
Young
Y
Ferns
Michael
M
D018171:Agrin / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002478:Cells, Cultured; D019281:Dimerization; D004789:Enzyme Activation; D005110:Extracellular Space / Q000502:physiology; D051379:Mice; D009046:Motor Neurons / Q000502:physiology; D009439:Neuraminidase / Q000494:pharmacology; D009469:Neuromuscular Junction / Q000502:physiology; D010766:Phosphorylation; D020794:Receptor Protein-Tyrosine Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D013569:Synapses / Q000502:physiology; D014162:Transfection
D018171:Agrin; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; C095714:MUSK protein, human; D020794:Receptor Protein-Tyrosine Kinases; D009439:Neuraminidase; D002118:Calcium