Abstract
Agrin, released by motor neurons, promotes neuromuscular synapse formation by stimulating MuSK, a receptor tyrosine kinase expressed in skeletal muscle. Phosphorylated MuSK recruits docking protein-7 (Dok-7), an adaptor protein that is expressed selectively in muscle. In the absence of Dok-7, neuromuscular synapses fail to form, and mutations that impair Dok-7 are a major cause of congenital myasthenia in humans. How Dok-7 stimulates synaptic differentiation is poorly understood. Once recruited to MuSK, Dok-7 directly stimulates MuSK kinase activity. This unusual activity of an adapter protein is mediated by the N-terminal region of Dok-7, whereas most mutations that cause congenital myasthenia truncate the C-terminal domain. Here, we demonstrate that Dok-7 also functions downstream from MuSK, and we identify the proteins that are recruited to the C-terminal domain of Dok-7. We show that Agrin stimulates phosphorylation of two tyrosine residues in the C-terminal domain of Dok-7, which leads to recruitment of two adapter proteins: Crk and Crk-L. Furthermore, we show that selective inactivation of Crk and Crk-L in skeletal muscle leads to severe defects in neuromuscular synapses in vivo, revealing a critical role for Crk and Crk-L downstream from Dok-7 in presynaptic and postsynaptic differentiation.
Publication types
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Adaptor Proteins, Signal Transducing / genetics
-
Adaptor Proteins, Signal Transducing / metabolism*
-
Agrin / pharmacology
-
Animals
-
Blotting, Western
-
Cell Line
-
Green Fluorescent Proteins / genetics
-
Green Fluorescent Proteins / metabolism
-
HEK293 Cells
-
Humans
-
Mice
-
Mice, Knockout
-
Microscopy, Confocal
-
Muscle Fibers, Skeletal / cytology
-
Muscle Fibers, Skeletal / drug effects
-
Muscle Fibers, Skeletal / metabolism
-
Muscle Proteins / genetics
-
Muscle Proteins / metabolism*
-
Muscle, Skeletal / embryology
-
Muscle, Skeletal / metabolism
-
Mutation
-
Neuromuscular Junction / metabolism*
-
Nuclear Proteins / genetics
-
Nuclear Proteins / metabolism*
-
Phosphorylation / drug effects
-
Proto-Oncogene Proteins c-crk / genetics
-
Proto-Oncogene Proteins c-crk / metabolism*
-
Receptor Protein-Tyrosine Kinases / genetics
-
Receptor Protein-Tyrosine Kinases / metabolism
-
Receptors, Cholinergic / genetics
-
Receptors, Cholinergic / metabolism
-
Synapses / metabolism*
-
Time Factors
-
Tyrosine / genetics
-
Tyrosine / metabolism
Substances
-
Adaptor Proteins, Signal Transducing
-
Agrin
-
CRKL protein
-
Crk protein, mouse
-
Dok-7 protein, mouse
-
Muscle Proteins
-
Nuclear Proteins
-
Proto-Oncogene Proteins c-crk
-
Receptors, Cholinergic
-
Green Fluorescent Proteins
-
Tyrosine
-
MuSK protein, mouse
-
Receptor Protein-Tyrosine Kinases