The location, organization and lateral mobility of nicotinic acetylcholine receptors at the neuromuscular junction are believed to be controlled by a transmembrane complex of cytoskeletal and basal lamina elements. Thus far, only a few proteins which presumably form part of this complex have been identified. To advance the hypothesis further, we need to identify more of these proteins and to see how they interact together. This project aims at these questions through identification and characterization of proteins in the corresponding complex in electric tissue of electric rays, a muscle-derived tissue. In one part, the project focuses on 2 proteins already identified, namely, a 58kD protein and the electric tissue homolog of dystrophin, the protein of normal muscle which is absent or defective in Duchenne and Becker muscular dystrophy. Whether these proteins occur in a complex with actin and other proteins on the postsynaptic membrane will be determined. In the second part, new proteins will be identified by monoclonal antibody methods. The immunogen will be material extracted from a more intact preparation of electric tissue postsynaptic membranes than has been used heretofore. The antibodies so generated, as well as antibodies already available against dystrophin and the 58kD protein, will be used to localize identified proteins relative to accumulations of receptor and to membrane-associated structural elements in electric tissue and muscle. The methods will be immunofluorescence and immunoelectron microscopy (thin section and freeze-etch methods). The identified proteins will be purified by immunoaffinity methods for determination of their molecular morphologies by electron microscopy and for identification of copurifying proteins with which they may interact. In this way, we hope to identify cytoplasmic and transmembrane components of the transmembrane complex and their shapes. Equally important, we can expect to identify additional proteins which, like the 58kD protein and dystrophin, are components of the non-synaptic sarcolemma as well as the neuromuscular junction. Any of these proteins could be potentially involved in the action of dystrophin to protect the sarcolemma against mechanical damage during contraction. This action is also probably mediated through a transmembrane complex between dystrophin and extracellular materials. The proposed project thus may have direct bearing on two problems at once, one of these being relevant to a major disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS015293-11A1
Application #
3396089
Study Section
Neurological Sciences Subcommittee 1 (NLS)
Project Start
1979-04-01
Project End
1995-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
11
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Type
Schools of Medicine
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Peters, M F; Kramarcy, N R; Sealock, R et al. (1994) beta 2-Syntrophin: localization at the neuromuscular junction in skeletal muscle. Neuroreport 5:1577-80
Butler, M H; Douville, K; Murnane, A A et al. (1992) Association of the Mr 58,000 postsynaptic protein of electric tissue with Torpedo dystrophin and the Mr 87,000 postsynaptic protein. J Biol Chem 267:6213-8
Hamilton, E H; Sealock, R; Wallace, N R et al. (1992) Trichohyalin: purification from porcine tongue epithelium and characterization of the native protein. J Invest Dermatol 98:881-9
Lai, F A; Liu, Q Y; Xu, L et al. (1992) Amphibian ryanodine receptor isoforms are related to those of mammalian skeletal or cardiac muscle. Am J Physiol 263:C365-72
Turner, C E; Kramarcy, N; Sealock, R et al. (1991) Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle. Exp Cell Res 192:651-5
Sealock, R; Butler, M H; Kramarcy, N R et al. (1991) Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle. J Cell Biol 113:1133-44
Kramarcy, N R; Sealock, R (1991) Commercial preparations of colloidal gold-antibody complexes frequently contain free active antibody. J Histochem Cytochem 39:37-9
Kramarcy, N R; Sealock, R (1990) Dystrophin as a focal adhesion protein. Collocalization with talin and the Mr 48,000 sarcolemmal protein in cultured Xenopus muscle. FEBS Lett 274:171-4
Chen, Q; Sealock, R; Peng, H B (1990) A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction. J Cell Biol 110:2061-71

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