Abstract
The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of approximately 43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptide-coated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.
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- 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]
- 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]
- Barrantes F. J., Mieskes G., Wallimann T. Creatine kinase activity in the Torpedo electrocyte and in the nonreceptor, peripheral v proteins from acetylcholine receptor-rich membranes. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5440–5444. doi: 10.1073/pnas.80.17.5440. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- Frank E., Fischbach G. D. Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses. J Cell Biol. 1979 Oct;83(1):143–158. doi: 10.1083/jcb.83.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- Kuromi H., Kidokoro Y. Nerve disperses preexisting acetylcholine receptor clusters prior to induction of receptor accumulation in Xenopus muscle cultures. Dev Biol. 1984 May;103(1):53–61. doi: 10.1016/0012-1606(84)90006-x. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Peng H. B., Cheng P. C., Luther P. W. Formation of ACh receptor clusters induced by positively charged latex beads. Nature. 1981 Aug 27;292(5826):831–834. doi: 10.1038/292831a0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Peng H. B. Participation of calcium and calmodulin in the formation of acetylcholine receptor clusters. J Cell Biol. 1984 Feb;98(2):550–557. doi: 10.1083/jcb.98.2.550. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Porter S., Froehner S. C. Interaction of the 43K protein with components of Torpedo postsynaptic membranes. Biochemistry. 1985 Jan 15;24(2):425–432. doi: 10.1021/bi00323a028. [DOI] [PubMed] [Google Scholar]
- 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]
- RODRIGUEZ J., DEINHARDT F. Preparation of a semipermanent mounting medium for fluorescent antibody studies. Virology. 1960 Oct;12:316–317. doi: 10.1016/0042-6822(60)90205-1. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Schaffner A. E., Daniels M. P. Conditioned medium from cultures of embryonic neurons contains a high molecular weight factor which induces acetylcholine receptor aggregation on cultured myotubes. J Neurosci. 1982 May;2(5):623–632. doi: 10.1523/JNEUROSCI.02-05-00623.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sealock R. Visualization at the mouse neuromuscular junction of a submembrane structure in common with Torpedo postsynaptic membranes. J Neurosci. 1982 Jul;2(7):918–923. doi: 10.1523/JNEUROSCI.02-07-00918.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]