Wnt proteins regulate acetylcholine receptor clustering in muscle cells

Abstract

Background: The neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish.

Results: In the study we systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR) cluster formation. We identified five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16) that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, we demonstrated that Wnt induction of AChR clusters was dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. We provide evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4.

Conclusions: These results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4.

Figure 1

Wnt proteins induce AChR clusters in muscle cells. A, C2 myotubes were stimulated with conditioned media containing Wnt proteins or control medium for 16 h. AChR clusters were visualized by R-BTX staining and indicated by arrows. B. Quantification data of A. AChR clusters greater than 4 μm in length were quantified. *, p < 0.01, Student's t test. C, Wnt mRNAs are expressed in muscle. Total RNAs were extracted from skeletal muscles of P0, P15, P30 and adult mice and used for qRT-PCR. Relative expression was shown in histograms.

Figure 2

Characterization of Wnt effect on AChR clustering. Myotubes were stimulated with increasing concentrations of agrin alone (A) or together with 1 nM Wnt9a or Wnt11 (B). AChR clusters were assayed as in Figure 1.

Figure 3

Wnt effect on AChR clustering requires interaction with MuSK. A, Wnt9a and Wnt11, but not Wnt7a, bind MuSK in vitro. Flag-Wnts immobilized beads were incubated with MuSKect-Myc. Bound proteins were isolated, resolved by SDS-PAGE and blotted with antibodies against Myc and Flag. B, Wnt11 binds CRD of MuSK. Flag-Wnt11 immobilized beads were incubated with MuSKect, MuSKectΔCRD or MuSKCRD. Interactions were assayed as in A. *, p < 0.05, n = 3. C, Wnt-induced AChR clustering is rescued by MuSK, but not MuSKΔCRD, in MuSK-/- cells. MuSK-/- cells were transfected without or with respective constructs and resulting myotubes (identified by GFP encoded by the expression construct) were assayed for AChR cluster formation in response to agrin or Wnts as in Figure 1. Arrows, AChR clusters. *, p < 0.01, Student's t test. D, Wnt9a and Wnt11 induce MuSK tyrosine phosphorylation. C2C12 myotubes were treated by agrin or Wnts for 1 h. MuSK was purified by immunoprecipitation and probed with the 4G10 antibody. E, Wnt9a and Wnt11 induce MuSK dimerization. Flag-MuSK and MuSK-Myc were transfected into C2C12 myoblasts and resulting myotubes were treated by Wnts, agrin or control media for 1 h. Flag-MuSK was precipitated by Flag antibody and associated MuSK-Myc was determined by anti-Myc antibody. Band intensity of immunoblot was analyzed by ImageJ software. *, p < 0.01, one-way ANOVA with Student's t test.

Figure 4

LRP4 is required for Wnt-induced AChR clusters. A, Wnt9a and Wnt11 fail to stimulate AChR clustering in LRP4^mitt muscle cells. Primary muscle cells were cultured from wt and mt mice and assayed for AChR clusters as in Figure 1. Histogram shows quantitative analysis of AChR clusters. B, Wnts bind to ectodomain of LRP4. Flag-tagged Wnt7a, Wnt9a or Wnt11 were immobilized with beads and incubated with LRP4N-Myc. Interactions were determined by precipitation and Western blot using anti-Myc antibody.