A Functional Role for Specific Spliced Variants of the α7β1 Integrin in Acetylcholine Receptor Clustering

Abstract

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 α7β1 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, α7A and α7B, and the extracellular spliced forms, α7X1 and α7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the α7β1 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-α7 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-α7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the α7A and α7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the α7X2 extracellular domain were active. These results demonstrate that the α7β1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the α7 chain, and that laminin, agrin, and the α7β1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.

Neurite Outgrowth by the Alternatively Spliced Region of Human Tenascin-C Is Mediated by Neuronal α7β1 Integrin

“…We therefore explored the hypothesis that VFDNFVLK binds to the ␣7␤1 integrin, better known as a laminin-1 receptor involved in neuromuscular (Burkin et al, 1998) and myotendinous (Crawley et al, 1997) junctions. Tenascin-C is enriched at neuromuscular and myotendinous junctions (Chiquet and Fambrough, 1984;Cifuentes-Diaz et al, 2002), colocalizing it with ␣7␤1.…”

Laminin Enriched Scaffolds for Tissue Engineering Applications

“…[35][36][37] and exogenous LM-111 supplementation promotes acetylcholine receptor (AChR) clustering and functional NMJ formation in vitro. 7,21,38 LM and agrin can act together to enhance the number, size, and rate of AChR clusters. 38,39 LM also mediates the interaction between integrin α 7 β 1 and the neural AChR clusters on the muscle membrane, potentially stabilizing them and allowing them to be more readily innervated by the incoming neurite.…”

“…7,21,38 LM and agrin can act together to enhance the number, size, and rate of AChR clusters. 38,39 LM also mediates the interaction between integrin α 7 β 1 and the neural AChR clusters on the muscle membrane, potentially stabilizing them and allowing them to be more readily innervated by the incoming neurite. 40 As a result, LM based neurogenic materials have been widely used as peripheral nerve grafts in several studies.…”

The role of synaptic laminins-α4 and -β2 in maturation and maintenance of the neuromuscular synapse

“…The loss of this integrin subunit in lamb2 -/-may have an implication on the postsynaptic maturation of the NMJ. In support of this, in-vitro studies have demonstrated the role of integrins, particularly α7β1 in aggregation of AChR clusters which are dependent on laminins (Burkin et al, 1998;Burkin et al, 2000). Burkin et al (1998) showed that integrin α7β1 were colocalised to laminininduced AChR clusters but to a lesser degree to agrin-induced AChR clusters.…”

“…These proteins include agrin, muscle specific receptor tyrosine kinase (MuSK) and rapsyn which play essential roles in the clustering of AChRs (Burden, 2002;Ghazanfari et al, 2011). Proteins that are localised at the postsynaptic membrane such as dystroglycan and integrins have also been suggested to be involved in the postsynaptic maturation of the NMJ (Burkin et al, 1998;Burkin et al, 2000;Nishimune et al, 2008). Podosomes, which are actin-rich organelles may also potentially assist in the maturation of the postsynaptic apparatus (Proszynski et al, 2009).…”