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. 1988 Sep 1;107(3):1139–1145. doi: 10.1083/jcb.107.3.1139

Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells

PMCID: PMC2115270  PMID: 3417778

Abstract

We have investigated the mechanisms regulating the clustering of nicotinic acetylcholine receptor (AChR) on the surface of cultured embryonic chick muscle cells. Treatment of these cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, was found to cause a rapid dispersal of AChR clusters, as monitored by fluorescence microscopy of cells labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin. The loss of AChR clusters was not accompanied by an appreciable change in the amount of AChR on the surface of these cells, as measured by the specific binding of [125I]Bgt. Analysis of the phosphorylation pattern of immunoprecipitable AChR subunits showed that the gamma- and delta- subunits are phosphorylated by endogenous protein kinase activity in the intact muscle cells, and that the delta-subunit displays increased phosphorylation in response to TPA. Structural analogues of TPA which do not stimulate protein kinase C have no effect on AChR surface topography or phosphorylation. Exposure of chick myotubes to the cholinergic agonist carbamylcholine was found to cause a dispersal of AChR clusters with a time course similar to that of TPA. Like TPA, carbamylcholine enhances the phosphorylation of the delta-subunit of AChR. The carbamylcholine-induced redistribution and phosphorylation of AChR is blocked by the nicotinic AChR antagonist d-tubocurarine. TPA and carbamylcholine have no effect on cell morphology during the time- course of these experiments. These findings indicate that cell surface topography of AChR may be regulated by phosphorylation of its subunits and suggest a mechanism for dispersal of AChR clusters by agonist activation.

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Selected References

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  1. Adamo S., Zani B. M., Nervi C., Senni M. I., Molinaro M., Eusebi F. Acetylcholine stimulates phosphatidylinositol turnover at nicotinic receptors of cultured myotubes. FEBS Lett. 1985 Oct 7;190(1):161–164. doi: 10.1016/0014-5793(85)80449-x. [DOI] [PubMed] [Google Scholar]
  2. Albuquerque E. X., Deshpande S. S., Aracava Y., Alkondon M., Daly J. W. A possible involvement of cyclic AMP in the expression of desensitization of the nicotinic acetylcholine receptor. A study with forskolin and its analogs. FEBS Lett. 1986 Apr 7;199(1):113–120. doi: 10.1016/0014-5793(86)81235-2. [DOI] [PubMed] [Google Scholar]
  3. Anderson M. J., Cohen M. W. Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):757–773. doi: 10.1113/jphysiol.1977.sp011880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Axelrod D., Ravdin P., Koppel D. E., Schlessinger J., Webb W. W., Elson E. L., Podleski T. R. Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4594–4598. doi: 10.1073/pnas.73.12.4594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bloch R. J. Acetylcholine receptor clustering in rat myotubes: requirement for CA2+ and effects of drugs which depolymerize microtubules. J Neurosci. 1983 Dec;3(12):2670–2680. doi: 10.1523/JNEUROSCI.03-12-02670.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Bloch R. J. Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism. J Cell Biol. 1979 Sep;82(3):626–643. doi: 10.1083/jcb.82.3.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Bloch R. J. Loss of acetylcholine receptor clusters induced by treatment of cultured rat myotubes with carbachol. J Neurosci. 1986 Mar;6(3):691–700. doi: 10.1523/JNEUROSCI.06-03-00691.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bloch R. J., Steinbach J. H. Reversible loss of acetylcholine receptor clusters at the developing rat neuromuscular junction. Dev Biol. 1981 Jan 30;81(2):386–391. doi: 10.1016/0012-1606(81)90305-5. [DOI] [PubMed] [Google Scholar]
  12. Browning M. D., Huganir R., Greengard P. Protein phosphorylation and neuronal function. J Neurochem. 1985 Jul;45(1):11–23. doi: 10.1111/j.1471-4159.1985.tb05468.x. [DOI] [PubMed] [Google Scholar]
  13. Bursztajn S., McManaman J. L., Appel S. H. Organization of acetylcholine receptor clusters in cultured rat myotubes is calcium dependent. J Cell Biol. 1984 Feb;98(2):507–517. doi: 10.1083/jcb.98.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U., Nishizuka Y. Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem. 1982 Jul 10;257(13):7847–7851. [PubMed] [Google Scholar]
  15. Christian C. N., Daniels M. P., Sugiyama H., Vogel Z., Jacques L., Nelson P. G. A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells. Proc Natl Acad Sci U S A. 1978 Aug;75(8):4011–4015. doi: 10.1073/pnas.75.8.4011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cohen S. A., Fischbach G. D. Clusters of acetylcholine receptors located at identified nerve-muscle synapses in vitro. Dev Biol. 1977 Aug;59(1):24–38. doi: 10.1016/0012-1606(77)90237-8. [DOI] [PubMed] [Google Scholar]
  17. Davis R. J., Ganong B. R., Bell R. M., Czech M. P. sn-1,2-Dioctanoylglycerol. A cell-permeable diacylglycerol that mimics phorbol diester action on the epidermal growth factor receptor and mitogenesis. J Biol Chem. 1985 Feb 10;260(3):1562–1566. [PubMed] [Google Scholar]
  18. Dennis M. J. Development of the neuromuscular junction: inductive interactions between cells. Annu Rev Neurosci. 1981;4:43–68. doi: 10.1146/annurev.ne.04.030181.000355. [DOI] [PubMed] [Google Scholar]
  19. Devreotes P. N., Fambrough D. M. Acetylcholine receptor turnover in membranes of developing muscle fibers. J Cell Biol. 1975 May;65(2):335–358. doi: 10.1083/jcb.65.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Eusebi F., Molinaro M., Zani B. M. Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes. J Cell Biol. 1985 Apr;100(4):1339–1342. doi: 10.1083/jcb.100.4.1339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fertuck H. C., Salpeter M. M. Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. J Cell Biol. 1976 Apr;69(1):144–158. doi: 10.1083/jcb.69.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fischbach G. D., Cohen S. A. The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture. Dev Biol. 1973 Mar;31(1):147–162. doi: 10.1016/0012-1606(73)90326-6. [DOI] [PubMed] [Google Scholar]
  23. Gardner J. M., Fambrough D. M. Acetylcholine receptor degradation measured by density labeling: effects of cholinergic ligands and evidence against recycling. Cell. 1979 Mar;16(3):661–674. doi: 10.1016/0092-8674(79)90039-4. [DOI] [PubMed] [Google Scholar]
  24. Geiger B. Membrane-cytoskeleton interaction. Biochim Biophys Acta. 1983 Aug 11;737(3-4):305–341. doi: 10.1016/0304-4157(83)90005-9. [DOI] [PubMed] [Google Scholar]
  25. Geiger B., Volk T., Volberg T. Molecular heterogeneity of adherens junctions. J Cell Biol. 1985 Oct;101(4):1523–1531. doi: 10.1083/jcb.101.4.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Hirokawa N., Heuser J. E. Internal and external differentiations of the postsynaptic membrane at the neuromuscular junction. J Neurocytol. 1982 Jun;11(3):487–510. doi: 10.1007/BF01257990. [DOI] [PubMed] [Google Scholar]
  28. Huganir R. L., Delcour A. H., Greengard P., Hess G. P. Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization. Nature. 1986 Jun 19;321(6072):774–776. doi: 10.1038/321774a0. [DOI] [PubMed] [Google Scholar]
  29. Huganir R. L., Miles K., Greengard P. Phosphorylation of the nicotinic acetylcholine receptor by an endogenous tyrosine-specific protein kinase. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6968–6972. doi: 10.1073/pnas.81.22.6968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jessell T. M., Siegel R. E., Fischbach G. D. Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5397–5401. doi: 10.1073/pnas.76.10.5397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lee S. A., Holz R. W. Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol. J Biol Chem. 1986 Dec 25;261(36):17089–17098. [PubMed] [Google Scholar]
  32. Meigs J. B., Wang Y. L. Reorganization of alpha-actinin and vinculin induced by a phorbol ester in living cells. J Cell Biol. 1986 Apr;102(4):1430–1438. doi: 10.1083/jcb.102.4.1430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Middleton P., Jaramillo F., Schuetze S. M. Forskolin increases the rate of acetylcholine receptor desensitization at rat soleus endplates. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4967–4971. doi: 10.1073/pnas.83.13.4967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nef P., Mauron A., Stalder R., Alliod C., Ballivet M. Structure linkage, and sequence of the two genes encoding the delta and gamma subunits of the nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7975–7979. doi: 10.1073/pnas.81.24.7975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nishikawa M., Hidaka H., Adelstein R. S. Phosphorylation of smooth muscle heavy meromyosin by calcium-activated, phospholipid-dependent protein kinase. The effect on actin-activated MgATPase activity. J Biol Chem. 1983 Dec 10;258(23):14069–14072. [PubMed] [Google Scholar]
  36. Nishizuka Y. Turnover of inositol phospholipids and signal transduction. Science. 1984 Sep 21;225(4668):1365–1370. doi: 10.1126/science.6147898. [DOI] [PubMed] [Google Scholar]
  37. Noble M. D., Brown T. H., Peacock J. H. Regulation of acetylcholine receptor levels by a cholinergic agonist in mouse muscle cell cultures. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3488–3492. doi: 10.1073/pnas.75.7.3488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Olek A. J., Krikorian J. G., Daniels M. P. Early stages in the formation and stabilization of acetylcholine receptor aggregates on cultured myotubes: sensitivity to temperature and azide. Dev Biol. 1986 Sep;117(1):24–34. doi: 10.1016/0012-1606(86)90344-1. [DOI] [PubMed] [Google Scholar]
  39. Olek A. J., Ling A., Daniels M. P. Development of ultrastructural specializations during the formation of acetylcholine receptor aggregates on cultured myotubes. J Neurosci. 1986 Feb;6(2):487–497. doi: 10.1523/JNEUROSCI.06-02-00487.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Olek A. J., Pudimat P. A., Daniels M. P. Direct observation of the rapid aggregation of acetylcholine receptors on identified cultured myotubes after exposure to embryonic brain extract. Cell. 1983 Aug;34(1):255–264. doi: 10.1016/0092-8674(83)90156-3. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Podleski T. R., Axelrod D., Ravdin P., Greenberg I., Johnson M. M., Salpeter M. M. Nerve extract induces increase and redistribution of acetylcholine receptors on cloned muscle cells. Proc Natl Acad Sci U S A. 1978 Apr;75(4):2035–2039. doi: 10.1073/pnas.75.4.2035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Prives J., Bar-Sagi D. Effect of tunicamycin, an inhibitor of protein glycosylation, on the biological properties of acetylcholine receptor in cultured muscle cells. J Biol Chem. 1983 Feb 10;258(3):1775–1780. [PubMed] [Google Scholar]
  45. Prives J., Fulton A. B., Penman S., Daniels M. P., Christian C. N. Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture. J Cell Biol. 1982 Jan;92(1):231–236. doi: 10.1083/jcb.92.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Prives J., Silman I., Amsterdam A. Appearance and disappearance of acetycholine receptor during differentiation of chick skeletal muscle in vitro. Cell. 1976 Apr;7(4):543–550. doi: 10.1016/0092-8674(76)90204-x. [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. Ross A. F., Rapuano M., Schmidt J. H., Prives J. M. Phosphorylation and assembly of nicotinic acetylcholine receptor subunits in cultured chick muscle cells. J Biol Chem. 1987 Oct 25;262(30):14640–14647. [PubMed] [Google Scholar]
  49. 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]
  50. Salpeter M. M., Spanton S., Holley K., Podleski T. R. Brain extract causes acetylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions. J Cell Biol. 1982 May;93(2):417–425. doi: 10.1083/jcb.93.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schliwa M., Nakamura T., Porter K. R., Euteneuer U. A tumor promoter induces rapid and coordinated reorganization of actin and vinculin in cultured cells. J Cell Biol. 1984 Sep;99(3):1045–1059. doi: 10.1083/jcb.99.3.1045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stya M., Axelrod D. Mobility and detergent extractability of acetylcholine receptors on cultured rat myotubes: a correlation. J Cell Biol. 1983 Jul;97(1):48–51. doi: 10.1083/jcb.97.1.48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stya M., Axelrod D. Mobility of extrajunctional acetylcholine receptors on denervated adult muscle fibers. J Neurosci. 1984 Jan;4(1):70–74. doi: 10.1523/JNEUROSCI.04-01-00070.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sytkowski A. J., Vogel Z., Nirenberg M. W. Development of acetylcholine receptor clusters on cultured muscle cells. Proc Natl Acad Sci U S A. 1973 Jan;70(1):270–274. doi: 10.1073/pnas.70.1.270. [DOI] [PMC free article] [PubMed] [Google Scholar]

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