MAB1645 Sigma-AldrichAnti-Dystrophin Antibody, clone 1808

Syntrophins are cytoplasmic peripheral membrane proteins of the dystrophin-associated protein complex (DAPC). Three syntrophin isoforms, alpha1, beta1, and beta2, are encoded by distinct genes. Each contains two pleckstrin homology (PH) domains, a syntrophin-unique (SU) domain, and a PDZ domain. The name PDZ comes from the first three proteins found to contain repeats of this domain (PSD-95, Drosophila discs large protein, and the zona occludens protein 1). PDZ domains in other proteins bind to the C termini of ion channels and neurotransmitter receptors containing the consensus sequence (S/T)XV-COOH and mediate the clustering or synaptic localization of these proteins. Two voltage-gated sodium channels (NaChs), SkM1 and SkM2, of skeletal and cardiac muscle, respectively, have this consensus sequence. Because NaChs are sarcolemmal components like syntrophins, we have investigated possible interactions between these proteins. NaChs copurify with syntrophin and dystrophin from extracts of skeletal and cardiac muscle. Peptides corresponding to the C-terminal 10 amino acids of SkM1 and SkM2 are sufficient to bind detergent-solubilized muscle syntrophins, to inhibit the binding of native NaChs to syntrophin PDZ domain fusion proteins, and to bind specifically to PDZ domains from alpha1-, beta1-, and beta2-syntrophin. These peptides also inhibit binding of the syntrophin PDZ domain to the PDZ domain of neuronal nitric oxide synthase, an interaction that is not mediated by C-terminal sequences. Brain NaChs, which lack the (S/T)XV consensus sequence, also copurify with syntrophin and dystrophin, an interaction that does not appear to be mediated by the PDZ domain of syntrophin. Collectively, our data suggest that syntrophins link NaChs to the actin cytoskeleton and the extracellular matrix via dystrophin and the DAPC.

We studied the location, relative abundance, and stability of dystrophin in clusters of ACh receptors (AChRs) isolated from primary cultures of neonatal rat myotubes. Although variable amounts of dystrophin were found at receptor clusters, dystrophin was always associated with organized, receptor-rich domains (AChR domains). Dystrophin was occasionally seen in focal contact domains, but never in clathrin-coated domains. Dystrophin was also present in a diffuse, punctate distribution in regions of myotube membrane that did not contain AChR clusters. Immunogold labeling at the ultrastructural level localized dystrophin in a spectrin-rich filamentous network closely applied to the cytoplasmic surface of the cell membrane at AChR domains. Dystrophin was not associated with overlying actin filaments. Semiquantitative immunofluorescence studies indicated that dystrophin was present in relatively small amounts in these preparations, with only one molecule of dystrophin for every approximately 5 AChR, 43 kDa and 58 kDa molecules, and for every approximately 20-35 beta-spectrin molecules. Clusters were disrupted, but the total amount of dystrophin was not significantly reduced, when myotubes were incubated with sodium azide or in Ca(2+)-free medium, and when isolated AChR clusters were extracted at low ionic strength, at high pH, or in 6 M urea. These treatments extract other peripheral membrane proteins from AChR clusters. Labeling for dystrophin was completely eliminated when clusters were incubated with chymotrypsin, however. Thus, dystrophin forms part of a membrane skeleton at AChR clusters, but it is more difficult to remove than other proteins in the network. This suggests that dystrophin attaches to cluster membrane in a unique way.

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.

Highly purified postsynaptic membranes from Torpedo electric organ contain the acetylcholine receptor as well as other proteins. To identify synapse-specific components, we prepared monoclonal antibodies (mabs) to proteins extracted from the membranes with either lithium diiodosalicylate or alkaline treatment. 10 mabs specific for three different proteins were obtained. Seven mabs reacted with a major 43,000-mol-wt protein (43K protein). This protein is composed of isoelectric variants (pl = 7.2-7.8) and each of the mabs reacted with all of the variants. Analysis of these mabs by competition for binding to 43K protein and by reaction with proteolytic fragments of 43K protein in immunoblots showed that they recognize at least five different epitopes. Two mabs reacted with a protein of 90,000 mol wt (90K protein) and one with a protein of 58,000 mol wt composed of isoelectric variants (pl = 6.4-6.7) (58K protein). The 43K and 58K proteins appeared to co-purify with the receptor-containing membranes while the 90K protein did not. Immunofluorescence experiments indicated that the anti-43K mabs bind to the innervated face of Torpedo electrocytes and that a component related to the 43K protein is found at the rat neuromuscular junction. The anti-58K mab stained the innervated face, although rather weakly, while the anti-90K mabs reacted intensely with the non-innervated membrane. Thus, the 43K protein and possibly also the 58K protein are synaptic components while the 90K protein is predominantly nonsynaptic.