Dispersing Clusters with Calpain

Abstract

A reciprocal relationship exists between agrin and acetylcholine (ACh) in controlling acetylcholine receptor (AChR) clustering at the neuromuscular junction: Agrin promotes clustering, and ACh disperses clusters. Chen et al . provide insight into how these opposing signals may allow stabilization of AChR at the neuromuscular junction by differential activity of the calcium-activated proteases calpains. Exposure of C2C12 myotubes in culture to the cholinergic agonist carbachol triggered a calcium transient, activation of calpain, and stimulation of cyclin-dependent kinase 5 (CDK5) through production of the activator p25 by cleavage of the CDK5 regulatory partner p35. (CDK5 has been previously implicated in AChR dispersal, and p35 is a known substrate for calpain.) Activation of CDK5 by carbachol was blocked by pharmacological inhibition of calpain with calpeptin, and the dispersal of AChR clusters induced by agrin was also prevented if calpain activity was blocked (pharmacologically or by RNAi). In vivo, pharmacologic inhibition of calpain (by injection of pregnant mice) in mice deficient for agrin rescued the formation of AChR clusters, although not all clusters were apposed to the nerve terminals. In transgenic mice overexpressing a calpain-inhibitory peptide calpastatin in skeletal muscle, p25 abundance was lower in the skeletal muscles from the transgenic mice, and AChR clustering was increased compared with control mice. A yeast two-hybrid screen revealed that rapsyn interacted with the large subunit of m-calpain, and mutagenesis analysis identified that the two proteins interacted through the tetratricopeptide repeat (TPR) domain of rapsyn and the regulatory domain (domain III) of calpain. Rapsyn is a protein that interacts with the AChR and is necessary for cluster stabilization. Calpain activity in vitro or in transfected cells was inhibited by rapsyn, and RNAi experiments to knock down rapsyn in C2C12 myotubes showed that p25 was more abundant in control and carbachol-treated cells. Rapsyn, calpain, and the AChR coimmunoprecipitated from C2C12 cells, and agrin stimulated this interaction. Analysis of the synaptic and nonsynaptic regions of diaphragm suggested that m-calpain was relatively evenly distributed between these two regions, but the activity of calpain was substantially less in the synaptic region (based on the abundance of p25). Thus, the authors propose that agrin promotes the interaction of calpain with the inhibitor rapsyn at the synapse, allowing AChR to cluster, and in nonsynaptic regions, calpain is activated in response to the calcium signals induced by ACh and disperses nonsynaptic ACh clusters. F. Chen, L. Qian, Z.-H. Yang, Y. Huang, S. T. Ngo, N.-J. Ruan, J. Wang, C. Schneider, P. G. Noakes, Y.-Q. Ding, L. Mei, Z.-G. Luo, Rapsyn interaction with calpain stabilizes AChR clusters at the neuromuscular junction. Neuron 55 , 247-260 (2007). [PubMed]