The role for proteoglycans in acetylcholine receptor clustering on cultured muscle.

Abstract

The present dissertation investigated the possible role of proteoglycans (PGs) in acetycholine receptor (AChR) clustering on cultured C2 myotubes. Analysis of variant muscle cell lines and their hybrid products supported the hypothesis that PGs are required for the clustering of AChRs. Three PG-defective genetic variants derived from the C2 cell line form myotubes but fail to spontaneously cluster AChRs. The three variants show different broad-spectrum defects in glycosaminoglycan (GAG) biosynthesis and are especially deficient in the synthesis of chondroitin sulfate (CS) chain. Formation of heterokaryon myotubes containing nuclei from two different variants spontaneously clustered AChRs and recovered synthesis of GAGs, especially of CS. It strongly suggests that there is a requirement for proper GAG biosynthesis in AChR clustering. Chlorate was found to inhibit both GAG synthesis and the clustering of AChRs in a dose-dependent manner. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild type C2 myotubes, both the frequency of spontaneous AChR clusters and the level of CS were increased. Culture of wild type C2 myotubes in the presence of chondroitinase ABC eliminated CS and prevented the formation of AChR clusters. Treatment with chondroitinase ABC only prevented AChR clustering if begun prior to the formation of spontaneous clusters. This suggests that CS is required in the initiation but not the maintenance of AChR clusters. Agrin-induced AChR clustering was dramatically reduced by digestion of CS. Unlike calcium, however, agrin action on AChR clustering did not affect levels of CS. Also, agrin-induced AChR clustering was restored on S27 myotubes by adding calcium-treated C2 conditioned medium. The IIH6 monoclonal antibody against α-dystroglycan, laminin, and agrin all bound to a specific fraction of C2 cell extracts separated on ion exchange chromatography. Also IIH6, laminin, and agrin affinity-precipitations showed a smeared sulfate labelled band above 120kD which is close in molecular weight to that of $\alpha$-dystroglycan. The band disappeared after chondroitinase ABC treatment. Protease-digested IIH6 immunoprecipitate eluted corresponding to CS. This result strongly suggests that CS is required for agrin activity on AChR clustering.