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Extracellular synaptic factors induce clustering of acetylcholine receptors stably expressed in fibroblasts

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Abstract

The clustering of nicotinic acetylcholine receptors (AChRs) is one of the first events observed during formation of the neuromuscular junction. To determine the mechanism involved in AChR clustering, we established a nonmuscle cell line (mouse fibroblast L cells) that stably expresses just one muscle-specific gene product, the AChR. We have shown that when Torpedo californica AChRs are expressed in fibroblasts, their immunological, biochemical, and electrophysiological properties all indicate that fully functional cell surface AChRs are produced. In the present study, the cell surface distribution and stability of Torpedo AChRs expressed in fibroblasts (AChR-fibroblasts) were analyzed and shown to be similar to nonclustered AChRs expressed in muscle cells. AChR-fibroblasts incubated with antibodies directed against the AChR induced the formation of small AChR microclusters (less than 0.5 micron 2) and caused an increase in the internalization rate and degradation of surface AChRs (antigenic modulation) in a manner similar to that observed in muscle cells. Two disparate sources of AChR clustering factors, extracellular matrix isolated from Torpedo electric organ and conditioned media from a rodent neuroblastoma-glioma hybrid cell line, each induced large (1-3 microns 2), stable AChR clusters with no change in the level of surface AChR expression. By exploiting the temperature-sensitive nature of Torpedo AChR assembly, we were able to demonstrate that factor-induced clusters were produced by mobilization of preexisting surface AChRs, not by directed insertion of newly synthesized AChRs. AChR clusters were never observed in the absence of extracellular synaptic factors. Our results suggest that these factors can interact directly with the AChR

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Publisher: The Rockefeller University Press
OAI identifier: oai:pubmedcentral.nih.gov:2289913
Provided by: PubMed Central
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    1. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
    2. (1977). Accelerated degradation of acetylcholine receptor from cultured rat myotubes with myasthenia gravis sera and globulins .
    3. (1979). Acetylcholine and local anesthetic binding to Torpedo nicotinic post-synaptic membranes after removal ofnon-receptor peptides.
    4. (1985). Acetylcholine receptor aggregating factor is similar to molecules concentrated at neuromuscular junctions.
    5. (1988). Acetylcholine receptor-aggregating proteins are associated with the extraeellular matrix of many tissues in Torpedo.
    6. (1990). Activation of Torpedoacetylcholine receptors expressed in mouse flbroblasts.
    7. (1987). Agrin-like molecules at 176synaptic sites in normal, denervated, and damaged muscles.
    8. (1989). Anunusualbeta-spectrin associatedwith clustered acetylcholine receptors.
    9. (1989). Asynchronous assembly ofthe acetylcholine receptorand ofthe 43-kD v, protein in the postsynaptic membrane ofdeveloping Torpedo marmorata electrocyte.
    10. (1982). Brain extract causes acetylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions.
    11. (1983). Characterization and localization of the M, = 43,000 proteins associated with acetylcholine receptor-rich membranes.
    12. (1987). Comparison ofthe postsynaptic 43 kDa protein frommuscle cellsthatdiffer inacetylcholine receptor clustering activity.
    13. (1991). Components of Torpedo electric organ and muscle that cause aggregation of acetylcholinereceptors on culturedmuscle cells.
    14. (1983). Construction ofa retrovirus packaging mutantand itsuse to produce helper-free defective rretroviras.
    15. (1983). Crosslinkingofproteins in acetylcholine receptor-rich membranes: Association between the beta-subunit and the 43 kd subsynaptic protein.
    16. (1986). Determinationofthetissuedistributions and relative concentrations of the postsynaptic 43-kDa protein and the acetylcholine receptor in Torpedo.
    17. (1987). Development and neural control ofthe neuromuscular junction andofthejunctional acetylcholinereceptor.
    18. (1983). Development ofneuromuscular synapses.
    19. (1987). Developmental regulation ofnicotinic acetylcholine receptors.
    20. (1990). Developmentalexpressionofthe 43K and 58Kpostsynaptic membrane proteins and nicotinic acetylcholine receptors in Torpedo electrocytes.
    21. (1983). Diffusely distributed acetylcholine receptors can participate in cluster formation on cultured rat myotubes.
    22. (1983). Direct observation of the rapid aggregation of acetylcholine receptors on identified cultured myotubes after exposure to embryonic brain extract.
    23. (1978). Distinct protein components of Torpedomembranes carrythe acetylcholinereceptorsite and the binding site forlocal anesthetics and histrionicotoxin.
    24. (1989). Distribution of Na' channels and ankyrin in neuromuscularjunctionsis complementary tothatofacetylcholine receptors and the 43 kd protein.
    25. (1990). Domains of human c-myc protein required for autosuppression and cooperation with ras oncogenes are overlapping.
    26. (1991). Efficiency of acetylcholine receptor subunit assembly and its regulation by cAMP.
    27. (1989). Expression ofRNA transcripts for the postsynaptic 43 kDa protein in innervated and denervated rat skeletal muscle.
    28. (1989). Fibroblasts transfected with Torpedo acetylcholine receptor 0-, y-, and b-subunit cDNAs express functional receptors when infected with a retroviral a recombinant.
    29. (1985). Formation of acetylcholine receptor clusters at neuromuscular junction in Xenopus cultures.
    30. (1989). Formation of acetylcholine receptor clusters in chick myoubes: Migration or new insertion?
    31. (1979). Formation ofdisulfidelinked oligomers of acetylcholine receptor in membrane from Torpedo electric tissue.
    32. (1987). Genetic reconstitution of functional acetylcholine receptor channels in mouse fibroblasts. Science (Wash.
    33. (1987). Identification of agrin, a synaptic organizing protein from Torpedo electric organ.
    34. (1988). Identification ofthe mouse muscle 43,000-dalton acetylcholinereceptor-associatedprotein (RAPsyn) by cDNA cloning.
    35. (1981). Immunofluorescenc e localization at the mammalian neuromuscular junction of the M, 43,000 protein of Torpedo postsynaptic membrane.
    36. (1978). Inununofluorescence analysis of surface acetylcholine receptors on muscle: modulation by auto-antibodies.
    37. (1988). Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes.
    38. (1984). Isolation and characterisation of a cDNA clone for the complete coding region of the b subunit of the mouse acetylcholine receptor.
    39. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases.
    40. (1979). Ligand induced changes in stability and distribution of acetylcholine receptors on surface membranes of muscle cells. Life Sci.
    41. (1977). Modulationofacetylcholine receptor by antibody against the receptor.
    42. (1988). Molecular events in synaptogenesis: nerve-muscle adhesion and postsynaptic differentiation.
    43. (1980). Monoclonal antibodies used to probe acetylcholine receptor structure: localization of the main immunogenic region and detection of similarities between subunits.
    44. (1990). Multipleconductanceclasses ofmousenicotinic acetylcholine receptorsexpressed in Xenopusoocytes.
    45. (1978). Myasthenic antibodies cross-link acetylcholine receptors to accelerate degradation.
    46. (1989). Myristic acid is the NHZterminal blocking group of the 43-kDa protein from Torpedo nicotinic postsynaptic membranes.
    47. (1977). Nerve-induced and spontaneous redistribution ofacetylcholinereceptors on cultured musclecells
    48. (1983). Neuromuscular development in tissue culture.
    49. (1985). On the mechanism of ACh receptor accumulation at newly formed synapses on chick myoubes.
    50. (1974). Periodate-lysine-paraformaldehyde fixative-a new fixative for immunoelectron microscopy.
    51. (1980). Purification of Torpedocalifornicapost-synaptic membranesand fractionation oftheirconstituent proteins.
    52. (1984). Redistribution of acetylcholine receptors on developing rat myotubes.
    53. (1988). Regulation of transcript encoding the 43K subsynaptic protein during development and after denervation.
    54. (1988). Regulation ofagrin-inducedacetylcholine receptoraggregation by Ca+* and phorbol ester.
    55. (1985). Specificityofneuronal factors whichaggregateacetylcholine receptors on cultured myotubes.
    56. (1989). Stable expression ofmultisubunit protein complexes in mammalian cells.
    57. (1987). Stable expression oftransfected Torpedo acetylcholine receptor a subunit in mouse fibroblast L cells.
    58. (1981). Structural heterogeneity and subcellular distribution of nicotinic synapse-associated proteins.
    59. (1990). Synaptic contact between embryonic neurons and acetylcholine receptorfibroblasts.
    60. (1990). Temperature-sensitive expressionofallTorpedo and Torpedo-rat hybrid AChR in mammalian muscle cells.
    61. (1984). The 43K protein, vr, associated with acetylcholine receptor containing membrane fragments is an actin-bindingprotein.
    62. (1988). The biology and enzymology of eukaryotic protein acylation.
    63. (1977). The distribution of alpha-bungarotoxin binding sites on mammalian skeletal muscle developing in vivo.
    64. (1987). The extracellular matrix and subsynaptic sarcoplasm at nerve-muscle synapses.
    65. (1989). The mammalian 43-kD acetylcholine receptor-associatedprotein (RAPsyn) is expressed in some nonmusele cells.
    66. (1990). The postsynaptic 43K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes.
    67. (1987). The relationship ofthe postsynaptic 43K protein toacetylcholine receptors in receptorclusters isolated from cultured rat myoubes.
    68. (1986). The role ofthe postsynaptic cytoskeleton in AChR organization. Trends Neurosci.
    69. (1985). Thesubsynaptic43-kDaprotein isconcentrated at developing nerve-muscle synapses in vitro.
    70. (1989). Three-dimensional structure ofthe nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at22A by low doseelectron microscopy and X-ray diffraction to 12.5A.
    71. (1984). Ultrastructural localization of the M, 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies.
    72. (1987). Visualization ofthe cytoplasmic surface ofTorpedo postsynaptic membraneby freeze-etch and immunoelectron microscopy.

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