A Mechanism for Acetylcholine Receptor Clustering Distinct from Agrin Signaling

Abstract

Acetylcholine receptors (AChRs) and other postsynaptic molecules cluster spontaneously on cultured C2 myotubes. The frequency of clustering is enhanced by neural agrin, neuraminidase, or calcium through a signaling pathway which includes tyrosine phosphorylation of a muscle-specific kinase (MuSK) and the AChR β-subunit. Vicia villosa agglutinin (VVA) lectin, previously shown to potentiate agrin-induced clustering on C2 myotubes, is shown here to also potentiate neuraminidase- and calcium-induced clustering of AChRs, while having no effect on the level of tyrosine phosphorylation of MuSK or the AChR β-subunit. We propose that VVA lectin increases the frequency of AChR clustering through a mechanism that is distinct from agrin signaling, and that may involve α-dystroglycan.

Differential Vicia villosa Agglutinin Reactivity Identifies Three Distinct Dystroglycan Complexes in Skeletal Muscle

“…VVA was shown to modestly induce AChR clustering by itself and to potentiate agrin-induced AChR clustering, whereas ␤-N-acetylhexosaminidase digestion dramatically inhibited agrin-induced AChR clustering in C2C12 myotubes (8). Like laminin-1-induced AChR clustering (33,34), VVA-induced clustering was shown to occur in MuSK Ϫ/Ϫ myotubes (35) and in the absence of MuSK or AChR ␤-subunit phosphorylation (31). Furthermore, ␣-dystroglycan in C2C12 myotubes displayed both patent VVA reactivity and also latent reactivity after neuraminidase treatment (31).…”

“…Like laminin-1-induced AChR clustering (33,34), VVA-induced clustering was shown to occur in MuSK Ϫ/Ϫ myotubes (35) and in the absence of MuSK or AChR ␤-subunit phosphorylation (31). Furthermore, ␣-dystroglycan in C2C12 myotubes displayed both patent VVA reactivity and also latent reactivity after neuraminidase treatment (31). Finally, VVA-induced AChR clustering was nearly abolished in myotubes from mice in which the dystrophin/utrophin-glycoprotein complex protein ␣-dystrobrevin was ablated (4).…”

“…Neuraminidase Stimulates AChR Clustering and ␣-Dystroglycan VVA Reactivity in C2C12 but Not L6 Myotubes-Treatment of C2C12 myotubes with neuraminidase has been shown to dramatically increase cell surface labeling by VVA (8), stimulate AChR clustering in the absence of agrin (8,36), and increase the binding of C2C12 ␣-dystroglycan to VVA-agarose (31). Furthermore, we previously demonstrated that purified skeletal muscle ␣-dystroglycan becomes VVA-reactive after treatment with neuraminidase (6).…”

“…Distinct ␣-Dystroglycan Glycoforms in Cultured Myotubes-␣-Dystroglycan in C2C12 myotubes was recently shown to bind to VVA-agarose (31). However, it remained to be determined what fraction of total C2C12 ␣-dystroglycan was patently reactive with VVA and whether VVA-reactive ␣-dystroglycan may also be expressed in other clonal myotube cell lines.…”

Reduced Glycosaminoglycan Sulfation Diminishes the Agrin Signal Transduction Pathway

“…Neuraminidase and sialic acid have complementary effects on the clustering process in C2C12 myotube culture, with neuraminidase increasing [Grow et al, 1999a] and sialic acid decreasing [Grow and Gordon, 2000b] agrin-induced AChR clustering and agrin-induced tyrosine phosphorylation of the AChR ß-subunit. Other experimental manipulations that increase the frequency of AChR clustering in muscle cell culture include laminin [Vogel et al, 1983;Sugiyama et al, 1997;Montanaro et al, 1998] or VVA (Vicia villosa agglutinin) lectin [Martin and Sanes, 1995;Grow et al, 1999b]. VVA lectin binds ·-dystroglycan [Ervasti et al, 1997] and may stabilize AChR clusters in a manner similar to laminin [Grow et al, 1999b].…”