The formation of complex acetylcholine receptor clusters requires MuSK kinase activity and structural information from the MuSK extracellular domain

Abstract

Efficient synaptic transmission at the neuromuscular junction (NMJ) requires the topological maturation of the postsynaptic apparatus from an oval acetylcholine receptor (AChR)-rich plaque into a complex pretzel-shaped array of branches. However, compared to NMJ formation very little is known about the mechanisms that regulate NMJ maturation. Recently the process of in vivo transformation from plaque into pretzel has been reproduced in vitro by culturing myotubes aneurally on laminin-coated substrate. It was proposed that the formation of complex AChR clusters is regulated by a MuSK-dependent muscle intrinsic program. To elucidate the structure–function role of MuSK in the aneural maturation of AChR pretzels, we used muscle cell lines expressing MuSK mutant and chimeric proteins. Here we report, that besides its role during agrin-induced AChR clustering, MuSK kinase activity is also necessary for substrate-dependent cluster formation. Constitutive-active MuSK induces larger AChR clusters, a faster cluster maturation on laminin and increases the anchorage of AChRs to the cytoskeleton compared to MuSK wild-type. In addition, we find that the juxtamembrane region of MuSK, which has previously been shown to regulate agrin-induced AChR clustering, is unable to induce complex AChR clusters on laminin substrate. Most interestingly, MuSK kinase activity is not sufficient for laminin-dependent AChR cluster formation since the MuSK ectodomain is also required suggesting a so far undiscovered instructive role for the extracellular domain of MuSK.

Identification of a Dutch founder mutation in MUSK causing fetal akinesia deformation sequence

“…The important role of both domains in tyrosine kinase activation and Dok-7 signaling suggests that the variant may affect MUSK signaling and AChR clustering. 14 , 15 To investigate this, local tyrosine kinase activity and AChR clusters were visualized at nascent motor endplates in muscle biopsies from an affected fetus (case 8, 23 weeks of gestation) and a control fetus aborted at 22 weeks due to cervical insufficiency ( Figure 3d–k ). Ingrowing axons from motor neurons were visualized with the synaptic vesicle marker Synaptotagmin-1.…”

Induced Formation and Maturation of Acetylcholine Receptor Clusters in a Defined 3D Bio-Artificial Muscle

“…Functional NMJ requires the topological maturation of the postsynaptic apparatus from an oval plaque of AchR clusters into a complex postnatal topology characterized by a pretzel-shaped and branched morphology [27]. Although the artificial muscle lacks mature AchR clusters that are essential for innervation, the BAM is capable of responding to agrin, a nerve-derived protein, markedly and effectively forming a large number of AchR clusters.…”

Global Analysis of Muscle-specific Kinase Signaling by Quantitative Phosphoproteomics

“…The next day protein A agarose (Pierce) was added for 1 to 3 h, the beads were washed three times with lysis buffer, and precipitated protein complexes were subjected to SDS-PAGE. AChRs were isolated from cleared lysates as described previously (15). Following SDS-PAGE, proteins were transferred to PVDF membrane (Millipore, Darmstadt, Germany).…”