Neuromuscular synapses form following a series of complex interactions between motor neurons, muscle fibers and Schwann cells. Signals provided by motor neurons have a key role in directing the differentiation of the muscle fiber at synaptic sites, and signals provided by muscle fibers have an important role in regulating motor axon growth and differentiation. Agrin, a 200 kD protein synthesized by motor neurons, is a critical synaptic signaling molecule that organizes postsynaptic differentiation by stimulating MuSK, a muscle-specific receptor tyrosine kinase. Mice lacking MuSK fail to form neuromuscular synapses and consequently die at birth due to their failure to move or breathe. MuSK is required for multiple aspects of presynaptic and postsynaptic differentiation, as MuSK mutant mice fail to cluster skeletal muscle-derived proteins, including acetylcholine receptors, to preferentially transcribe acetylcholine receptor genes in synaptic nuclei, and to provide retrograde signals for nerve terminal differentiation. The steps that follow MuSK activation and that lead to neuromuscular synapse formation are poorly understood. Our studies have demonstrated that the juxtamembrane domain of MuSK has a critical role in regulating MuSK signaling and directing synaptic differentiation. Experiments described here are designed to reveal how the MuSK juxtamembrane region regulates synaptic differentiation by identifying and studying the proteins that interact with this critical domain of MuSK. MuSK activation leads to tyrosine phosphorylation of the acetylcholine receptor beta and delta subunits, but the role of acetylcholine receptor tyrosine phosphorylation is not known. Molecular genetic experiments described here are designed to reveal the role that acetylcholine receptor tyrosine phosphorylation may have in synaptic differentiation.
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