A functional role for specific spliced variants of the α7β1 integrin in acetylcholine receptor clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha 7 beta 1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha 7A and alpha 7B, and the extracellular spliced forms, alpha 7X1 and alpha 7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha 7 beta 1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha 7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha 7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha 7A and alpha 7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha 7X2 extracellular domain were active. These results demonstrate that the alpha 7 beta 1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha 7 chain, and that laminin, agrin, and the alpha 7 beta 1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.