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. 1989 Jan 1;108(1):127–139. doi: 10.1083/jcb.108.1.127

Asynchronous assembly of the acetylcholine receptor and of the 43-kD nu1 protein in the postsynaptic membrane of developing Torpedo marmorata electrocyte

PMCID: PMC2115356  PMID: 2642909

Abstract

The assembly of the nicotinic acetylcholine receptor (AchR) and the 43- kD protein (v1), the two major components of the post synaptic membrane of the electromotor synapse, was followed in Torpedo marmorata electrocyte during embryonic development by immunocytochemical methods. At the first developmental stage investigated (45-mm embryos), accumulation of AchR at the ventral pole of the newly formed electrocyte was observed within columns before innervation could be detected. No concomitant accumulation of 43-kD immunoreactivity in AchR- rich membrane domains was observed at this stage, but a transient asymmetric distribution of the extracellular protein, laminin, which paralleled that of the AchR, was noticed. At the subsequent stage studied (80-mm embryos), codistribution of the two proteins was noticed on the ventral face of the cell. Intracellular pools of AchR and 43-kD protein were followed at the EM level in 80-mm electrocytes. AchR immunoreactivity was detected within membrane compartments, which include the perinuclear cisternae of the endoplasmic reticulum and the plasma membrane. On the other hand, 43-kD immunoreactivity was not found associated with the AchR in the intracellular compartments of the cell, but codistributed with the AchR at the level of the plasma membrane. The data reported in this study suggest that AchR clustering in vivo is not initially determined by the association of the AchR with the 43-kD protein, but rather relies on AchR interaction with extracellular components, for instance from the basement membrane, laid down in the tissue before the entry of the electromotor nerve endings.

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Selected References

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  1. 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]
  2. Bloch R. J. Actin at receptor-rich domains of isolated acetylcholine receptor clusters. J Cell Biol. 1986 Apr;102(4):1447–1458. doi: 10.1083/jcb.102.4.1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bloch R. J., Geiger B. The localization of acetylcholine receptor clusters in areas of cell-substrate contact in cultures of rat myotubes. Cell. 1980 Aug;21(1):25–35. doi: 10.1016/0092-8674(80)90111-7. [DOI] [PubMed] [Google Scholar]
  5. Bridgman P. C., Carr C., Pedersen S. E., Cohen J. B. Visualization of the cytoplasmic surface of Torpedo postsynaptic membranes by freeze-etch and immunoelectron microscopy. J Cell Biol. 1987 Oct;105(4):1829–1846. doi: 10.1083/jcb.105.4.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Burden S. J., Sargent P. B., McMahan U. J. Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve. J Cell Biol. 1979 Aug;82(2):412–425. doi: 10.1083/jcb.82.2.412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Carr C., McCourt D., Cohen J. B. The 43-kilodalton protein of Torpedo nicotinic postsynaptic membranes: purification and determination of primary structure. Biochemistry. 1987 Nov 3;26(22):7090–7102. doi: 10.1021/bi00396a034. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Changeux J. P., Devillers-Thiéry A., Chemouilli P. Acetylcholine receptor: an allosteric protein. Science. 1984 Sep 21;225(4668):1335–1345. doi: 10.1126/science.6382611. [DOI] [PubMed] [Google Scholar]
  12. Claudio T., Ballivet M., Patrick J., Heinemann S. Nucleotide and deduced amino acid sequences of Torpedo californica acetylcholine receptor gamma subunit. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1111–1115. doi: 10.1073/pnas.80.4.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Devillers-Thiery A., Giraudat J., Bentaboulet M., Changeux J. P. Complete mRNA coding sequence of the acetylcholine binding alpha-subunit of Torpedo marmorata acetylcholine receptor: a model for the transmembrane organization of the polypeptide chain. Proc Natl Acad Sci U S A. 1983 Apr;80(7):2067–2071. doi: 10.1073/pnas.80.7.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Edgar D., Timpl R., Thoenen H. The heparin-binding domain of laminin is responsible for its effects on neurite outgrowth and neuronal survival. EMBO J. 1984 Jul;3(7):1463–1468. doi: 10.1002/j.1460-2075.1984.tb01997.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Fox G. Q., Richardson G. P. The developmental morphology of Torpedo marmorata: electric organ--electrogenic phase. J Comp Neurol. 1979 May 15;185(2):293–315. doi: 10.1002/cne.901850205. [DOI] [PubMed] [Google Scholar]
  17. Fox G. Q., Richardson G. P. The developmental morphology of Torpedo marmorata: electric organ--myogenic phase. J Comp Neurol. 1978 Jun 1;179(3):677–697. doi: 10.1002/cne.901790313. [DOI] [PubMed] [Google Scholar]
  18. Frail D. E., Mudd J., Shah V., Carr C., Cohen J. B., Merlie J. P. cDNAs for the postsynaptic 43-kDa protein of Torpedo electric organ encode two proteins with different carboxyl termini. Proc Natl Acad Sci U S A. 1987 Sep;84(17):6302–6306. doi: 10.1073/pnas.84.17.6302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. 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]
  23. Hucho F. The nicotinic acetylcholine receptor and its ion channel. Eur J Biochem. 1986 Jul 15;158(2):211–226. doi: 10.1111/j.1432-1033.1986.tb09740.x. [DOI] [PubMed] [Google Scholar]
  24. Kleinman H. K., Cannon F. B., Laurie G. W., Hassell J. R., Aumailley M., Terranova V. P., Martin G. R., DuBois-Dalcq M. Biological activities of laminin. J Cell Biochem. 1985;27(4):317–325. doi: 10.1002/jcb.240270402. [DOI] [PubMed] [Google Scholar]
  25. Kordeli E., Cartaud J., Nghiêm H. O., Changeux J. P. In situ localization of soluble and filamentous actin in Torpedo marmorata electrocyte. Biol Cell. 1987;59(1):61–68. doi: 10.1111/j.1768-322x.1987.tb00516.x. [DOI] [PubMed] [Google Scholar]
  26. Kordeli E., Cartaud J., Nghiêm H. O., Changeux J. P. The Torpedo electrocyte: a model system for the study of receptor-cytoskeleton interactions. J Recept Res. 1987;7(1-4):71–88. doi: 10.3109/10799898709054980. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Labat-Robert J., Saitoh T., Godeau G., Robert L., Changeux J. P. Distribution of macromolecules from the intercellular matrix in the electroplaque of Electrophorus electricus. FEBS Lett. 1980 Nov 3;120(2):259–263. doi: 10.1016/0014-5793(80)80311-5. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Louvard D., Morris C., Warren G., Stanley K., Winkler F., Reggio H. A monoclonal antibody to the heavy chain of clathrin. EMBO J. 1983;2(10):1655–1664. doi: 10.1002/j.1460-2075.1983.tb01640.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Martin G. R., Timpl R. Laminin and other basement membrane components. Annu Rev Cell Biol. 1987;3:57–85. doi: 10.1146/annurev.cb.03.110187.000421. [DOI] [PubMed] [Google Scholar]
  33. Mellinger J., Belbenoit P., Ravaille M., Szabo T. Electric organ development in Torpedo marmorata, Chondrichthyes. Dev Biol. 1978 Nov;67(1):167–188. doi: 10.1016/0012-1606(78)90307-x. [DOI] [PubMed] [Google Scholar]
  34. Merlie J. P., Smith M. M. Synthesis and assembly of acetylcholine receptor, a multisubunit membrane glycoprotein. J Membr Biol. 1986;91(1):1–10. doi: 10.1007/BF01870209. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. Noda M., Takahashi H., Tanabe T., Toyosato M., Furutani Y., Hirose T., Asai M., Inayama S., Miyata T., Numa S. Primary structure of alpha-subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence. Nature. 1982 Oct 28;299(5886):793–797. doi: 10.1038/299793a0. [DOI] [PubMed] [Google Scholar]
  38. Noda M., Takahashi H., Tanabe T., Toyosato M., Kikyotani S., Hirose T., Asai M., Takashima H., Inayama S., Miyata T. Primary structures of beta- and delta-subunit precursors of Torpedo californica acetylcholine receptor deduced from cDNA sequences. Nature. 1983 Jan 20;301(5897):251–255. doi: 10.1038/301251a0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Peng H. B., Phelan K. A. Early cytoplasmic specialization at the presumptive acetylcholine receptor cluster: a meshwork of thin filaments. J Cell Biol. 1984 Jul;99(1 Pt 1):344–349. doi: 10.1083/jcb.99.1.344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Popot J. L., Changeux J. P. Nicotinic receptor of acetylcholine: structure of an oligomeric integral membrane protein. Physiol Rev. 1984 Oct;64(4):1162–1239. doi: 10.1152/physrev.1984.64.4.1162. [DOI] [PubMed] [Google Scholar]
  43. Richardson G. P., Witzemann V. Torpedo electromotor system development: biochemical differentiation of Torpedo electrocytes in vitro. Neuroscience. 1986 Apr;17(4):1287–1296. doi: 10.1016/0306-4522(86)90095-3. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Rousselet A., Cartaud J., Devaux P. F. Importance des interactions protéine-protéine dans les maintien de la structure des fragments excitables de l'organe électrique de Torpedo marmorata. C R Seances Acad Sci D. 1979 Sep 24;289(5):461–463. [PubMed] [Google Scholar]
  46. Salpeter M. M., Loring R. H. Nicotinic acetylcholine receptors in vertebrate muscle: properties, distribution and neural control. Prog Neurobiol. 1985;25(4):297–325. doi: 10.1016/0301-0082(85)90018-8. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Sanes J. R., Marshall L. M., McMahan U. J. Reinnervation of muscle fiber basal lamina after removal of myofibers. Differentiation of regenerating axons at original synaptic sites. J Cell Biol. 1978 Jul;78(1):176–198. doi: 10.1083/jcb.78.1.176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. 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]
  51. St John P. A., Froehner S. C., Goodenough D. A., Cohen J. B. Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides. J Cell Biol. 1982 Feb;92(2):333–342. doi: 10.1083/jcb.92.2.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Vogel Z., Christian C. N., Vigny M., Bauer H. C., Sonderegger P., Daniels M. P. Laminin induces acetylcholine receptor aggregation on cultured myotubes and enhances the receptor aggregation activity of a neuronal factor. J Neurosci. 1983 May;3(5):1058–1068. doi: 10.1523/JNEUROSCI.03-05-01058.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Walker J. H., Boustead C. M., Witzemann V. The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein. EMBO J. 1984 Oct;3(10):2287–2290. doi: 10.1002/j.1460-2075.1984.tb02127.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wennogle L. P., Changeux J. P. Transmembrane orientation of proteins present in acetylcholine receptor-rich membranes from Torpedo marmorata studied by selective proteolysis. Eur J Biochem. 1980 May;106(2):381–393. doi: 10.1111/j.1432-1033.1980.tb04584.x. [DOI] [PubMed] [Google Scholar]
  55. Witzemann V., Richardson G., Boustead C. Characterization and distribution of acetylcholine receptors and acetylcholinesterase during electric organ development in Torpedo marmorata. Neuroscience. 1983;8(2):333–349. doi: 10.1016/0306-4522(83)90070-2. [DOI] [PubMed] [Google Scholar]

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