A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle.

Abstract

Abstract. To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43-and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87-and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.

Role of Rapsyn Tetratricopeptide Repeat and Coiled-coil Domains in Self-association and Nicotinic Acetylcholine Receptor Clustering

“…6, was prepared by using Torpedo rapsyn as immunogen (16). Based upon epitope mapping studies it has been shown to recognize rapsyn amino acid sequence 396 -411.…”

“…The distribution of rapsyn domains fused to GFP was visualized by fluorescein isothiocyanate optics, while wild-type rapsyn was visualized by the binding of rhodamine-conjugated goat anti-mouse antibody to mAb 19F4A, a mouse monoclonal that recognizes rapsyn of many species (16). Based upon epitope mapping studies with Torpedo rapsyn, mAb194A recognizes a COOH-terminal epitope (rapsyn-(396 -411)), 3 and based upon immunofluorescence it did not recognize rapsyn-(1-360) or other smaller fragments expressed in 293T cells (data not shown).…”

Direct binding of Torpedo syntrophin to dystrophin and the 87 kDa dystrophin homologue

“…Two other dystrophin-related proteins, utrophin and the 87K protein, are products of different genes. The 87K protein, a substrate for tyrosine phosphorylation, was first discovered as a postsynaptic protein of Torpedo electric organ [13]. The 87K protein shows modest but significant homology to the CRCT region of dystrophin [14].…”

Dystrophin‐associated protein A0 is a homologue of the Torpedo 87K protein

“…The Torpedo 87K protein was first identified as a protein closely associated with the acetylcholine receptor [28], and then found to associate with Torpedo syntrophin (58K protein) [26,29]. Recently, the 87K protein was reported as a dystrophin homologue, since it was homologous to the cysteine-rich and C-terminal domains of dystrophin with an overall 27% amino acid identity over 546 amino acids [26].…”