Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells.

Abstract

The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of ~43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptidecoated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.An early event during the formation of the neuromuscular synapse is the aggregation of acetylcholine receptors (AChR) ~ at the postsynaptic membrane in response to innervation (1,15,50). Tissue cultures of skeletal muscle cells are an invaluable tool for the study of this process. In addition to neurally induced receptor aggregates, AChR also form clusters spontaneously (the "hot spots"; 2, 14, 48) or in response to exogenously applied stimuli in tissue culture (l 1, 23, 31, 34, 42). Although these extrasynaptic AChR clusters have proven to be a convenient system to study, the cellular and molecular mechanisms involved in the clustering process remain largely unknown. Recent studies have indicated that the cytoskeleton may be involved in the aggregation of AChR (12,28,38), but the precise nature of the AChR-cytoskeleton interaction remains elusive.Highly purified postsynaptic membranes from Torpedo Abbreviations used in this paper: AChR, acetylcholine receptor(s); FITC, fluorescein isothiocyanate; 43K protein, the major membranebound 43,000-mol-wt protein isolated from Torpedo postsynaptic membrane; mab, monoclonal antibody; R-BTX, tetramethyl rhodamine-conjugated a-bungarotoxin. 1698electric organ contain, in addition to the ACh receptor', a major peripheral membrane protein of-43,000 mol wt (43K protein) (13,19,26,36,46). Exposure to alkaline pH causes dissociation of this protein from the membranes (26) with a concomitant increase in the rotational and translational mobility of the receptor (4, 10, 25, 41). The 43K protein is distinct from actin (36) and creatine kinase (5, 16) and lies on the cytoplasmic side of the membrane (27,36,45,47,49) in close enough proximity to the receptor so that chemical crosslinking between the two proteins can be induced with a bifunctional reagent (9). Recent results indicate that the 43K protein interacts in a hydrophobic manner with th...

Acetylcholine receptor clustering and nuclear movement in muscle fibers in culture.

“…Therefore, this molecule may serve as an intermediate between the AChR and the rest of the cytoskeleton (8). Peng and Froehner (38) have seen high concentrations of this molecule beneath AChR clusters on Xenopus myocytes, and we have obtained similar results in cultures of rat muscle (44). Our results suggest that once the actin monomers have polymerized into a subcluster network, they are not as easily disrupted by treatment with cytochalasin.…”

“…T hE vertebrate neuromuscular junction is a highly specialized region of muscle characterized by high concentrations of acetylcholine receptors (AChRs), 1 acetylcholinesterase and other basal lamina components, postsynaptic folds, and a variety of postsynaptic cytoskeletal elements (6,7,26,27,33,37,38,39,47; reviewed in reference 25). Most evidence suggests that this specialized region develops after the initial interactions between the motor neuron growth cone and the muscle membrane, presumably in response to one or more factors released at the nerve terminal (13,45).…”

Expression of RNA transcripts for the postsynaptic 43 kDa protein in innervated and denervated rat skeletal muscle

“…Torpedo electrocytes ]1-4], at vertebrate neuromuscular junctions [5] and in receptor clusters on cultured muscle cells [6][7][8]. The 43 kDa protein is not required for ligand-gated ion flux through the receptor channel [9].…”

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

“…Mouse monoclonal anti-syntrophin [1351E (27)] and antirapsyn [1234 (28)] antibodies were provided by Prof. S. Froehner (University of Washington, Seattle, WA) or purchased from Abcam, UK. Monoclonal anti-transferrin receptor (H 68.4) was a gift from Prof. J. M. Trowbridge (San Diego, CA).…”