Kinase domain of the muscle-specific receptor tyrosine kinase (MuSK) is sufficient for phosphorylation but not clustering of acetylcholine receptors: Required role for the MuSK ectodomain?

Abstract

Formation of the neuromuscular junction (NMJ) depends upon a nerve-derived protein, agrin, acting by means of a muscle-specific receptor tyrosine kinase, MuSK, as well as a required accessory receptor protein known as MASC. We report that MuSK does not merely play a structural role by demonstrating that MuSK kinase activity is required for inducing acetylcholine receptor (AChR) clustering. We also show that MuSK is necessary, and that MuSK kinase domain activation is sufficient, to mediate a key early event in NMJ formation-phosphorylation of the AChR. However, MuSK kinase domain activation and the resulting AChR phosphorylation are not sufficient for AChR clustering; thus we show that the MuSK ectodomain is also required. These results indicate that AChR phosphorylation is not the sole trigger of the clustering process. Moreover, our results suggest that, unlike the ectodomain of all other receptor tyrosine kinases, the MuSK ectodomain plays a required role in addition to simply mediating ligand binding and receptor dimerization, perhaps by helping to recruit NMJ components to a MuSKbased scaffold.

A Single Pulse of Agrin Triggers a Pathway That Acts To Cluster Acetylcholine Receptors

“…We showed here that continuous stimulation by agrin is not necessary. In comparison to other receptor tyrosine kinases, MuSK is very unusual and its tasks are far more complex, as previously noted by others (19). MuSK not only triggers intracellular signaling but also has to localize such signaling to the correct site of a huge syncytial cell to achieve new protein synthesis as well as relocalization of dozens of preexisting proteins at the nascent postsynapse.…”

“…In addition, MuSK is required for presynaptic specialization of the nerve terminal (20). In line with such complex roles of MuSK, experiments with chimeric receptors have shown that, in contrast to growth factor receptors, MuSK's activated intracellular domain alone is not sufficient to reproduce MuSK's biological activity (i.e., AChR clustering)-structural portions of the MuSK extracellular domain are also important (19). Our data propose that even responses as complex as those initiated by MuSK, involving structural roles as well as catalytic tasks, may be triggered by a single brief pulse of ligand.…”

The formation of neuromuscular synapses

“…Because MuSK is clustered at synapses in rapsyn mutant mice ), these results suggest a model in which matrix-bound agrin engages and clusters MuSK, MuSK clusters an unknown linker protein(s), the linker protein(s) clusters rapsyn, and rapsyn clusters AChRs. This model, however, does not take into account the kinase activity of MuSK, which is required for agrin to stimulate clustering of AChRs Glass et al 1997). Moreover, because staurosporine inhibits AChR clustering without blocking agrinstimulated tyrosine phosphorylation of MuSK (Wallace 1994;Fuhrer et al 1997), there appears to be at least one kinase in addition to MuSK that is necessary for AChR clustering (Fig.…”

Analysis of a Shc Family Adaptor Protein, ShcD/Shc4, That Associates with Muscle-Specific Kinase

“…Consistent with this expression pattern, we have found that ShcD can associate with muscle-specific kinase (MuSK), an RTK expressed at the skeletal neuromuscular junction (NMJ). On the postsynaptic muscle side of the NMJ, MuSK is stimulated by presynaptic motor neuron-derived agrin (53), which in turn leads to clustering and tyrosine phosphorylation of postsynaptic nicotinic acetylcholine receptors (AChRs) and remodeling of the neuromuscular synapse (10,11). Mice lacking either agrin or MuSK fail to develop normal neuromuscular synapses and consequently die at birth due to an inability to move or breathe (5,9).…”