Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Jun 1;104(6):1633–1646. doi: 10.1083/jcb.104.6.1633

A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle

PMCID: PMC2114519  PMID: 3294859

Abstract

In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.

Full Text

The Full Text of this article is available as a PDF (5.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson M. J., Fambrough D. M. Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan sulfate proteoglycan on the surface of skeletal muscle fibers. J Cell Biol. 1983 Nov;97(5 Pt 1):1396–1411. doi: 10.1083/jcb.97.5.1396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson M. J., Klier F. G., Tanguay K. E. Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction. J Cell Biol. 1984 Nov;99(5):1769–1784. doi: 10.1083/jcb.99.5.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson M. J. Nerve-induced remodeling of muscle basal lamina during synaptogenesis. J Cell Biol. 1986 Mar;102(3):863–877. doi: 10.1083/jcb.102.3.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Angelides K. J. Fluorescently labelled Na+ channels are localized and immobilized to synapses of innervated muscle fibres. Nature. 1986 May 1;321(6065):63–66. doi: 10.1038/321063a0. [DOI] [PubMed] [Google Scholar]
  5. Atsumi S. Development of neuromuscular junctions of fast and slow muscles in the chick embryo: a light and electron microscopic study. J Neurocytol. 1977 Dec;6(6):691–709. doi: 10.1007/BF01176380. [DOI] [PubMed] [Google Scholar]
  6. Barrantes F. J., Neugebauer D. C., Zingsheim H. P. Peptide extraction by alkaline treatment is accompanied by rearrangement of the membrane-bound acetylcholine receptor from Torpedo marmorata. FEBS Lett. 1980 Mar 24;112(1):73–78. doi: 10.1016/0014-5793(80)80131-1. [DOI] [PubMed] [Google Scholar]
  7. Beam K. G., Caldwell J. H., Campbell D. T. Na channels in skeletal muscle concentrated near the neuromuscular junction. Nature. 1985 Feb 14;313(6003):588–590. doi: 10.1038/313588a0. [DOI] [PubMed] [Google Scholar]
  8. Berg D. K., Kelly R. B., Sargent P. B., Williamson P., Hall Z. W. Binding of -bungarotoxin to acetylcholine receptors in mammalian muscle (snake venom-denervated muscle-neonatal muscle-rat diaphragm-SDS-polyacrylamide gel electrophoresis). Proc Natl Acad Sci U S A. 1972 Jan;69(1):147–151. doi: 10.1073/pnas.69.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bloch R. J., Froehner S. C. The relationship of the postsynaptic 43K protein to acetylcholine receptors in receptor clusters isolated from cultured rat myotubes. J Cell Biol. 1987 Mar;104(3):645–654. doi: 10.1083/jcb.104.3.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bloch R. J., Hall Z. W. Cytoskeletal components of the vertebrate neuromuscular junction: vinculin, alpha-actinin, and filamin. J Cell Biol. 1983 Jul;97(1):217–223. doi: 10.1083/jcb.97.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  12. Burden S. J., DePalma R. L., Gottesman G. S. Crosslinking of proteins in acetylcholine receptor-rich membranes: association between the beta-subunit and the 43 kd subsynaptic protein. Cell. 1983 Dec;35(3 Pt 2):687–692. doi: 10.1016/0092-8674(83)90101-0. [DOI] [PubMed] [Google Scholar]
  13. Burden S. J. The subsynaptic 43-kDa protein is concentrated at developing nerve-muscle synapses in vitro. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8270–8273. doi: 10.1073/pnas.82.23.8270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Burden S. Identification of an intracellular postsynaptic antigen at the frog neuromuscular junction. J Cell Biol. 1982 Sep;94(3):521–530. doi: 10.1083/jcb.94.3.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cartaud J., Sobel A., Rousselet A., Devaux P. F., Changeux J. P. Consequences of alkaline treatment for the ultrastructure of the acetylcholine-receptor-rich membranes from Torpedo marmorata electric organ. J Cell Biol. 1981 Aug;90(2):418–426. doi: 10.1083/jcb.90.2.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Elliott J., Blanchard S. G., Wu W., Miller J., Strader C. D., Hartig P., Moore H. P., Racs J., Raftery M. A. Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins. Biochem J. 1980 Mar 1;185(3):667–677. doi: 10.1042/bj1850667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fallon J. R., Nitkin R. M., Reist N. E., Wallace B. G., McMahan U. J. Acetylcholine receptor-aggregating factor is similar to molecules concentrated at neuromuscular junctions. Nature. 1985 Jun 13;315(6020):571–574. doi: 10.1038/315571a0. [DOI] [PubMed] [Google Scholar]
  18. Fertuck H. C., Salpeter M. M. Localization of acetylcholine receptor by 125I-labeled alpha-bungarotoxin binding at mouse motor endplates. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1376–1378. doi: 10.1073/pnas.71.4.1376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Froehner S. C., Douville K., Klink S., Culp W. J. Monoclonal antibodies to cytoplasmic domains of the acetylcholine receptor. J Biol Chem. 1983 Jun 10;258(11):7112–7120. [PubMed] [Google Scholar]
  20. Froehner S. C., Gulbrandsen V., Hyman C., Jeng A. Y., Neubig R. R., Cohen J. B. Immunofluorescence localization at the mammalian neuromuscular junction of the Mr 43,000 protein of Torpedo postsynaptic membranes. Proc Natl Acad Sci U S A. 1981 Aug;78(8):5230–5234. doi: 10.1073/pnas.78.8.5230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Froehner S. C. Peripheral proteins of postsynaptic membranes from Torpedo electric organ identified with monoclonal antibodies. J Cell Biol. 1984 Jul;99(1 Pt 1):88–96. doi: 10.1083/jcb.99.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Giloh H., Sedat J. W. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science. 1982 Sep 24;217(4566):1252–1255. doi: 10.1126/science.7112126. [DOI] [PubMed] [Google Scholar]
  23. Godfrey E. W., Nitkin R. M., Wallace B. G., Rubin L. L., McMahan U. J. Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells. J Cell Biol. 1984 Aug;99(2):615–627. doi: 10.1083/jcb.99.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gysin R., Wirth M., Flanagan S. D. Structural heterogeneity and subcellular distribution of nicotinic synapse-associated proteins. J Biol Chem. 1981 Nov 25;256(22):11373–11376. [PubMed] [Google Scholar]
  25. Hall Z. W., Lubit B. W., Schwartz J. H. Cytoplasmic actin in postsynaptic structures at the neuromuscular junction. J Cell Biol. 1981 Sep;90(3):789–792. doi: 10.1083/jcb.90.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hall Z. W. Multiple forms of acetylcholinesterase and their distribution in endplate and non-endplate regions of rat diaphragm muscle. J Neurobiol. 1973;4(4):343–361. doi: 10.1002/neu.480040404. [DOI] [PubMed] [Google Scholar]
  27. Heuser J. E., Salpeter S. R. Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane. J Cell Biol. 1979 Jul;82(1):150–173. doi: 10.1083/jcb.82.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kelly A. M., Zacks S. I. The fine structure of motor endplate morphogenesis. J Cell Biol. 1969 Jul;42(1):154–169. doi: 10.1083/jcb.42.1.154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kordeli E., Cartaud J., Nghiêm H. O., Pradel L. A., Dubreuil C., Paulin D., Changeux J. P. Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes. J Cell Biol. 1986 Mar;102(3):748–761. doi: 10.1083/jcb.102.3.748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. LaRochelle W. J., Froehner S. C. Determination of the tissue distributions and relative concentrations of the postsynaptic 43-kDa protein and the acetylcholine receptor in Torpedo. J Biol Chem. 1986 Apr 25;261(12):5270–5274. [PubMed] [Google Scholar]
  31. Lo M. M., Garland P. B., Lamprecht J., Barnard E. A. Rotational mobility of the membrane-bound acetylcholine receptor of Torpedo electric organ measured by phosphorescence depolarisation. FEBS Lett. 1980 Mar 10;111(2):407–412. doi: 10.1016/0014-5793(80)80838-6. [DOI] [PubMed] [Google Scholar]
  32. Loring R. H., Salpeter M. M. Denervation increases turnover rate of junctional acetylcholine receptors. Proc Natl Acad Sci U S A. 1980 Apr;77(4):2293–2297. doi: 10.1073/pnas.77.4.2293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matthews-Bellinger J., Salpeter M. M. Distribution of acetylcholine receptors at frog neuromuscular junctions with a discussion of some physiological implications. J Physiol. 1978 Jun;279:197–213. doi: 10.1113/jphysiol.1978.sp012340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  35. McMahan U. J., Sanes J. R., Marshall L. M. Cholinesterase is associated with the basal lamina at the neuromuscular junction. Nature. 1978 Jan 12;271(5641):172–174. doi: 10.1038/271172a0. [DOI] [PubMed] [Google Scholar]
  36. Miledi R., Slater C. R. On the degeneration of rat neuromuscular junctions after nerve section. J Physiol. 1970 Apr;207(2):507–528. doi: 10.1113/jphysiol.1970.sp009076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Neubig R. R., Krodel E. K., Boyd N. D., Cohen J. B. Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides. Proc Natl Acad Sci U S A. 1979 Feb;76(2):690–694. doi: 10.1073/pnas.76.2.690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nghiêm H. O., Cartaud J., Dubreuil C., Kordeli C., Buttin G., Changeux J. P. Production and characterization of a monoclonal antibody directed against the 43,000-dalton v1 polypeptide from Torpedo marmorata electric organ. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6403–6407. doi: 10.1073/pnas.80.20.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nitkin R. M., Wallace B. G., Spira M. E., Godfrey E. W., McMahan U. J. Molecular components of the synaptic basal lamina that direct differentiation of regenerating neuromuscular junctions. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 2):653–665. doi: 10.1101/sqb.1983.048.01.069. [DOI] [PubMed] [Google Scholar]
  40. Patrick J., McMillan J., Wolfson H., O'Brien J. C. Acetylcholine receptor metabolism in a nonfusing muscle cell line. J Biol Chem. 1977 Mar 25;252(6):2143–2153. [PubMed] [Google Scholar]
  41. Peng H. B., Cheng P. C. Formation of postsynaptic specializations induced by latex beads in cultured muscle cells. J Neurosci. 1982 Dec;2(12):1760–1774. doi: 10.1523/JNEUROSCI.02-12-01760.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Peng H. B. Cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor cluster in cell cultures. J Cell Biol. 1983 Aug;97(2):489–498. doi: 10.1083/jcb.97.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Peng H. B., Froehner S. C. Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells. J Cell Biol. 1985 May;100(5):1698–1705. doi: 10.1083/jcb.100.5.1698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Peng H. B., Nakajima Y. Membrane particle aggregates in innervated and noninnervated cultures of Xenopus embryonic muscle cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):500–504. doi: 10.1073/pnas.75.1.500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Porter S., Froehner S. C. Characterization and localization of the Mr = 43,000 proteins associated with acetylcholine receptor-rich membranes. J Biol Chem. 1983 Aug 25;258(16):10034–10040. [PubMed] [Google Scholar]
  46. Pruss R. M., Mirsky R., Raff M. C., Thorpe R., Dowding A. J., Anderton B. H. All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody. Cell. 1981 Dec;27(3 Pt 2):419–428. doi: 10.1016/0092-8674(81)90383-4. [DOI] [PubMed] [Google Scholar]
  47. Ravdin P., Axelrod D. Fluorescent tetramethyl rhodamine derivatives of alpha-bungarotoxin: preparation, separation, and characterization. Anal Biochem. 1977 Jun;80(2):585–592. doi: 10.1016/0003-2697(77)90682-0. [DOI] [PubMed] [Google Scholar]
  48. Rotundo R. L. Purification and properties of the membrane-bound form of acetylcholinesterase from chicken brain. Evidence for two distinct polypeptide chains. J Biol Chem. 1984 Nov 10;259(21):13186–13194. [PubMed] [Google Scholar]
  49. Rousselet A., Cartaud J., Devaux P. F., Changeux J. P. The rotational diffusion of the acetylcholine receptor in Torpeda marmorata membrane fragments studied with a spin-labelled alpha-toxin: importance of the 43 000 protein(s). EMBO J. 1982;1(4):439–445. doi: 10.1002/j.1460-2075.1982.tb01188.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sanes J. R., Chiu A. Y. The basal lamina of the neuromuscular junction. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 2):667–678. doi: 10.1101/sqb.1983.048.01.070. [DOI] [PubMed] [Google Scholar]
  51. Sanes J. R., Hall Z. W. Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina. J Cell Biol. 1979 Nov;83(2 Pt 1):357–370. doi: 10.1083/jcb.83.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sanes J. R. Laminin, fibronectin, and collagen in synaptic and extrasynaptic portions of muscle fiber basement membrane. J Cell Biol. 1982 May;93(2):442–451. doi: 10.1083/jcb.93.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sealock R., Kavookjian A. Postsynaptic distribution of acetylcholine receptors in electroplax of the torpedine ray, Narcine brasiliensis. Brain Res. 1980 May 19;190(1):81–93. doi: 10.1016/0006-8993(80)91161-0. [DOI] [PubMed] [Google Scholar]
  54. Sealock R., Paschal B., Beckerle M., Burridge K. Talin is a post-synaptic component of the rat neuromuscular junction. Exp Cell Res. 1986 Mar;163(1):143–150. doi: 10.1016/0014-4827(86)90566-5. [DOI] [PubMed] [Google Scholar]
  55. Sealock R., Wray B. E., Froehner S. C. Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies. J Cell Biol. 1984 Jun;98(6):2239–2244. doi: 10.1083/jcb.98.6.2239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Silberstein L., Inestrosa N. C., Hall Z. W. Aneural muscle cell cultures make synaptic basal lamina components. Nature. 1982 Jan 14;295(5845):143–145. doi: 10.1038/295143a0. [DOI] [PubMed] [Google Scholar]
  57. Sobel A., Weber M., Changeux J. P. Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ. Eur J Biochem. 1977 Oct 17;80(1):215–224. doi: 10.1111/j.1432-1033.1977.tb11874.x. [DOI] [PubMed] [Google Scholar]
  58. Tokuyasu K. T., Singer S. J. Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol. 1976 Dec;71(3):894–906. doi: 10.1083/jcb.71.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Vigny M., Koenig J., Rieger F. The motor end-plate specific form of acetylcholinesterase: appearance during embryogenesis and re-innervation of rat muscle. J Neurochem. 1976 Dec;27(6):1347–1353. doi: 10.1111/j.1471-4159.1976.tb02614.x. [DOI] [PubMed] [Google Scholar]
  60. Wallace B. G., Nitkin R. M., Reist N. E., Fallon J. R., Moayeri N. N., McMahan U. J. Aggregates of acetylcholinesterase induced by acetylcholine receptor-aggregating factor. Nature. 1985 Jun 13;315(6020):574–577. doi: 10.1038/315574a0. [DOI] [PubMed] [Google Scholar]
  61. Wray B. E., Sealock R. Ultrastructural immunocytochemistry of particulate fractions using polyvinyl chloride microculture wells. J Histochem Cytochem. 1984 Oct;32(10):1117–1120. doi: 10.1177/32.10.6481151. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES