Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Jan 1;120(1):185–195. doi: 10.1083/jcb.120.1.185

Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells

PMCID: PMC2119477  PMID: 7678011

Abstract

Aggregation of the nicotinic acetylcholine receptor (AChR) at sites of nerve-muscle contact is one of the earliest events to occur during the development of the neuromuscular junction. The stimulus presented to the muscle by nerve and the mechanisms underlying postsynaptic differentiation are not known. The purpose of this study was to examine the distribution of phosphotyrosine (PY)-containing proteins in cultured Xenopus muscle cells in response to AChR clustering stimuli. Results demonstrated a distinct accumulation of PY at AChR clusters induced by several stimuli, including nerve, the culture substratum, and polystyrene microbeads. AChR microclusters formed by external cross- linking did not show PY colocalization, implying that the accumulation of PY in response to clustering stimuli was not due to the aggregation of basally phosphorylated AChRs. A semi-quantitative determination of the time course for development of PY labeling at bead contacts revealed early PY accumulation within 15 min of contact before significant AChR aggregation. At later stages (within 15 h), the AChR signal came to approximate the PY signal. We have reported the inhibition of bead-induced AChR clustering in response to beads by a tyrphostin tyrosine kinase inhibitor (RG50864) (Peng, H. B., L. P. Baker, and Q. Chen. 1991. Neuron. 6:237-246). RG50864 also inhibited PY accumulation at bead contacts, providing evidence for tyrosine kinase activation in response to the bead stimulus. These results suggest that tyrosine phosphorylation may play an important role in the generative stages of cluster formation, and may involve protein(s) other than or in addition to AChRs.

Full Text

The Full Text of this article is available as a PDF (2.5 MB).

Selected References

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

  1. Anderson M. J., Champaneria S., Swenarchuk L. E. Synaptic differentiation can be evoked by polymer microbeads that mimic localized pericellular proteolysis by removing proteins from adjacent surfaces. Dev Biol. 1991 Oct;147(2):464–479. doi: 10.1016/0012-1606(91)90305-m. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Anderson M. J., Cohen M. W., Zorychta E. Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):731–756. doi: 10.1113/jphysiol.1977.sp011879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Axelrod D. Crosslinkage and visualization of acetylcholine receptors on myotubes with biotinylated alpha-bungarotoxin and fluorescent avidin. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4823–4827. doi: 10.1073/pnas.77.8.4823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baker L. P., Chen Q., Peng H. B. Induction of acetylcholine receptor clustering by native polystyrene beads. Implication of an endogenous muscle-derived signalling system. J Cell Sci. 1992 Jul;102(Pt 3):543–555. doi: 10.1242/jcs.102.3.543. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Bloch R. J., Pumplin D. W. Molecular events in synaptogenesis: nerve-muscle adhesion and postsynaptic differentiation. Am J Physiol. 1988 Mar;254(3 Pt 1):C345–C364. doi: 10.1152/ajpcell.1988.254.3.C345. [DOI] [PubMed] [Google Scholar]
  10. Bozyczko D., Decker C., Muschler J., Horwitz A. F. Integrin on developing and adult skeletal muscle. Exp Cell Res. 1989 Jul;183(1):72–91. doi: 10.1016/0014-4827(89)90419-9. [DOI] [PubMed] [Google Scholar]
  11. Bridgman P. C., Nakajima Y. Distribution of filipin-sterol complexes on cultured muscle cells: cell-substratum contact areas associated with acetylcholine receptor clusters. J Cell Biol. 1983 Feb;96(2):363–372. doi: 10.1083/jcb.96.2.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Burridge K., Fath K., Kelly T., Nuckolls G., Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol. 1988;4:487–525. doi: 10.1146/annurev.cb.04.110188.002415. [DOI] [PubMed] [Google Scholar]
  13. Carlson S. S., Wight T. N. Nerve terminal anchorage protein 1 (TAP-1) is a chondroitin sulfate proteoglycan: biochemical and electron microscopic characterization. J Cell Biol. 1987 Dec;105(6 Pt 2):3075–3086. doi: 10.1083/jcb.105.6.3075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Connolly J. A. Role of the cytoskeleton in the formation, stabilization, and removal of acetylcholine receptor clusters in cultured muscle cells. J Cell Biol. 1984 Jul;99(1 Pt 1):148–154. doi: 10.1083/jcb.99.1.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Davis S., Lu M. L., Lo S. H., Lin S., Butler J. A., Druker B. J., Roberts T. M., An Q., Chen L. B. Presence of an SH2 domain in the actin-binding protein tensin. Science. 1991 May 3;252(5006):712–715. doi: 10.1126/science.1708917. [DOI] [PubMed] [Google Scholar]
  17. DeClue J. E., Martin G. S. Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells. Mol Cell Biol. 1987 Jan;7(1):371–378. doi: 10.1128/mcb.7.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ferns M. J., Hall Z. W. How many agrins does it take to make a synapse? Cell. 1992 Jul 10;70(1):1–3. doi: 10.1016/0092-8674(92)90525-h. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Froehner S. C. The submembrane machinery for nicotinic acetylcholine receptor clustering. J Cell Biol. 1991 Jul;114(1):1–7. doi: 10.1083/jcb.114.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Glenney J. R., Jr, Zokas L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol. 1989 Jun;108(6):2401–2408. doi: 10.1083/jcb.108.6.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Hirst R., Horwitz A., Buck C., Rohrschneider L. Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6470–6474. doi: 10.1073/pnas.83.17.6470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Huganir R. L., Greengard P. Regulation of neurotransmitter receptor desensitization by protein phosphorylation. Neuron. 1990 Nov;5(5):555–567. doi: 10.1016/0896-6273(90)90211-w. [DOI] [PubMed] [Google Scholar]
  25. Huganir R. L., Greengard P. cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1130–1134. doi: 10.1073/pnas.80.4.1130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Ito S., Werth D. K., Richert N. D., Pastan I. Vinculin phosphorylation by the src kinase. Interaction of vinculin with phospholipid vesicles. J Biol Chem. 1983 Dec 10;258(23):14626–14631. [PubMed] [Google Scholar]
  28. Luther P. W., Bloch R. J. Formaldehyde-amine fixatives for immunocytochemistry of cultured Xenopus myocytes. J Histochem Cytochem. 1989 Jan;37(1):75–82. doi: 10.1177/37.1.2491754. [DOI] [PubMed] [Google Scholar]
  29. Luther P. W., Peng H. B. Membrane-related specializations associated with acetylcholine receptor aggregates induced by electric fields. J Cell Biol. 1985 Jan;100(1):235–244. doi: 10.1083/jcb.100.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Maher P. A., Pasquale E. B., Wang J. Y., Singer S. J. Phosphotyrosine-containing proteins are concentrated in focal adhesions and intercellular junctions in normal cells. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6576–6580. doi: 10.1073/pnas.82.19.6576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McGuire P. G., Seeds N. W. Degradation of underlying extracellular matrix by sensory neurons during neurite outgrowth. Neuron. 1990 Apr;4(4):633–642. doi: 10.1016/0896-6273(90)90121-u. [DOI] [PubMed] [Google Scholar]
  32. Miles K., Greengard P., Huganir R. L. Calcitonin gene-related peptide regulates phosphorylation of the nicotinic acetylcholine receptor in rat myotubes. Neuron. 1989 May;2(5):1517–1524. doi: 10.1016/0896-6273(89)90198-0. [DOI] [PubMed] [Google Scholar]
  33. Nastuk M. A., Lieth E., Ma J. Y., Cardasis C. A., Moynihan E. B., McKechnie B. A., Fallon J. R. The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering. Neuron. 1991 Nov;7(5):807–818. doi: 10.1016/0896-6273(91)90283-6. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Orida N., Poo M. M. Electrophoretic movement and localisation of acetylcholine receptors in the embryonic muscle cell membrane. Nature. 1978 Sep 7;275(5675):31–35. doi: 10.1038/275031a0. [DOI] [PubMed] [Google Scholar]
  36. Pasquale E. B., Maher P. A., Singer S. J. Talin is phosphorylated on tyrosine in chicken embryo fibroblasts transformed by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5507–5511. doi: 10.1073/pnas.83.15.5507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Peng H. B., Baker L. P., Chen Q. Induction of synaptic development in cultured muscle cells by basic fibroblast growth factor. Neuron. 1991 Feb;6(2):237–246. doi: 10.1016/0896-6273(91)90359-8. [DOI] [PubMed] [Google Scholar]
  38. Peng H. B., Baker L. P., Chen Q. Tissue culture of Xenopus neurons and muscle cells as a model for studying synaptic induction. Methods Cell Biol. 1991;36:511–526. doi: 10.1016/s0091-679x(08)60294-0. [DOI] [PubMed] [Google Scholar]
  39. Peng H. B., Baker L. P., Dai Z. A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells. J Cell Biol. 1993 Jan;120(1):197–204. doi: 10.1083/jcb.120.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  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. 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]
  43. Qu Z. C., Moritz E., Huganir R. L. Regulation of tyrosine phosphorylation of the nicotinic acetylcholine receptor at the rat neuromuscular junction. Neuron. 1990 Mar;4(3):367–378. doi: 10.1016/0896-6273(90)90049-l. [DOI] [PubMed] [Google Scholar]
  44. Safran A., Sagi-Eisenberg R., Neumann D., Fuchs S. Phosphorylation of the acetylcholine receptor by protein kinase C and identification of the phosphorylation site within the receptor delta subunit. J Biol Chem. 1987 Aug 5;262(22):10506–10510. [PubMed] [Google Scholar]
  45. Schroeder W., Meyer H. E., Buchner K., Bayer H., Hucho F. Phosphorylation sites of the nicotinic acetylcholine receptor. A novel site detected in position delta S362. Biochemistry. 1991 Apr 9;30(14):3583–3588. doi: 10.1021/bi00228a032. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Sefton B. M., Hunter T., Ball E. H., Singer S. J. Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell. 1981 Apr;24(1):165–174. doi: 10.1016/0092-8674(81)90512-2. [DOI] [PubMed] [Google Scholar]
  48. Stadler H., Kiene M. L. Synaptic vesicles in electromotoneurones. II. Heterogeneity of populations is expressed in uptake properties; exocytosis and insertion of a core proteoglycan into the extracellular matrix. EMBO J. 1987 Aug;6(8):2217–2221. doi: 10.1002/j.1460-2075.1987.tb02493.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stollberg J., Fraser S. E. Acetylcholine receptors and concanavalin A-binding sites on cultured Xenopus muscle cells: electrophoresis, diffusion, and aggregation. J Cell Biol. 1988 Oct;107(4):1397–1408. doi: 10.1083/jcb.107.4.1397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Tank D. W., Wu E. S., Webb W. W. Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints. J Cell Biol. 1982 Jan;92(1):207–212. doi: 10.1083/jcb.92.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Turner C. E., Kramarcy N., Sealock R., Burridge K. Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle. Exp Cell Res. 1991 Feb;192(2):651–655. doi: 10.1016/0014-4827(91)90090-h. [DOI] [PubMed] [Google Scholar]
  52. Turner C. E. Paxillin is a major phosphotyrosine-containing protein during embryonic development. J Cell Biol. 1991 Oct;115(1):201–207. doi: 10.1083/jcb.115.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Usdin T. B., Fischbach G. D. Purification and characterization of a polypeptide from chick brain that promotes the accumulation of acetylcholine receptors in chick myotubes. J Cell Biol. 1986 Aug;103(2):493–507. doi: 10.1083/jcb.103.2.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wallace B. G., Qu Z., Huganir R. L. Agrin induces phosphorylation of the nicotinic acetylcholine receptor. Neuron. 1991 Jun;6(6):869–878. doi: 10.1016/0896-6273(91)90227-q. [DOI] [PubMed] [Google Scholar]
  55. Yarden Y., Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443–478. doi: 10.1146/annurev.bi.57.070188.002303. [DOI] [PubMed] [Google Scholar]

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

RESOURCES