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Myasthenia gravis (MG) is a severely debilitating autoimmune disease that is due to a decrease in the efficiency of synaptic transmission at neuromuscular synapses. MG is caused by antibodies against postsynaptic proteins, including (i) acetylcholine receptors, the neurotransmitter receptor, (ii) muscle-specific kinase (MuSK), a receptor tyrosine kinase essential for the formation and maintenance of neuromuscular synapses, and (iii) low-density lipoprotein receptor-related protein 4 (Lrp4), which responds to neural Agrin by binding and stimulating MuSK. Passive transfer studies in mice have shown that IgG4 antibodies from MuSK MG patients cause disease without requiring complement or other immune components, suggesting that these MuSK antibodies cause disease by directly interfering with MuSK function. Here we show that pathogenic IgG4 antibodies to MuSK bind to a structural epitope in the first Ig-like domain of MuSK, prevent binding between MuSK and Lrp4, and inhibit Agrin-stimulated MuSK phosphorylation. In contrast, these IgG4 antibodies have no direct effect on MuSK dimerization or MuSK internalization. These results provide insight into the unique pathogenesis of MuSK MG and provide clues toward development of specific treatment options. [ABSTRACT FROM AUTHOR]

At the neuromuscular junction, the acetylcholine receptor (AChR) is specifically clustered in the postsynaptic membrane via interactions with rapsyn and other scaffolding proteins. However, it remains unclear where these proteins bind on the AChR and how the interactions are regulated. Here, we define a phosphorylation-dependent binding site on the receptor that mediates agrin-induced clustering. Using chimeric proteins in which CD4 is fused to the large intracellular loop of each of theAChRsubunits we found that agrin induced clustering of only chimeras containing the β subunit loop. By making deletions in the β loop we defined a 20 amino-acid sequence that is sufficient for clustering. The sequence contains a conserved tyrosine (β390) whose phosphorylation is induced by agrin and whose mutation abolished clustering of β loop chimeras and their ability to inhibit agrin-induced clustering of the endogenous AChR. Phosphorylation of the AChRβ subunit is correlated with increased rapsyn/AChR binding, suggesting that the effect ofβ390 phosphorylation on clustering is mediated by rapsyn. Indeed, we found that rapsyn associated with CD4-β loop chimeras in a phosphorylation-dependent manner, and that agrin increased the ratio of rapsyn binding to wild type AChR but not to AChR-β3F/3F, which lacks β loop tyrosine phosphorylation sites. Together, these findings suggest that agrin-induced phosphorylation of the β subunit motif increases the stoichiometry of rapsyn binding to the AChR, thereby helping to stably cluster the receptor and anchor it at high density in the postsynaptic membrane. [ABSTRACT FROM AUTHOR]

Agrin activates MuSK, a receptor tyrosine kinase expressed in skeletal muscle, leading to tyrosine phosphorylation of the acetylcholine receptor (AChR) β-subunit and clustering of AChRs. The importance of AChR β-subunit tyrosine phosphorylation in clustering AChRs and regulating synaptic differentiation is poorly understood. We generated mice with targeted mutations in the three intracellular tyrosines of the AChR β-subunit (AChR-β3F/3F). Mice lacking AChR β-subunit tyrosine phosphorylation thrive postnatally and have no overt behavioral defects, indicating that AChR β-subunit tyrosine phosphorylation is not essential for the formation of neuromuscular synapses. Nonetheless, the size of synapses and the density of synaptic AChRs are reduced in AChR-β3F/3F mutant mice. Moreover, synapses are structurally simplified and the organization of postjunctional folds is aberrant in mice lacking tyrosine phosphorylation of the AChR β-subunit. Furthermore, mutant AChRs cluster poorly in response to agrin and are readily extracted from the cell surface of cultured myotubes by non-ionic detergent. These data indicate that tyrosine phosphorylation of the AChR β-subunit has an important role in organizing AChRs and regulating synaptic differentiation. [ABSTRACT FROM AUTHOR]