Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Jan 2;116(2):385–393. doi: 10.1083/jcb.116.2.385

Expression of fusion proteins of the nicotinic acetylcholine receptor from mammalian muscle identifies the membrane-spanning regions in the alpha and delta subunits

PMCID: PMC2289298  PMID: 1730761

Abstract

We have investigated the topology of the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) from mammalian muscle synthesized in an in vitro translation system supplemented with dog pancreatic microsomes. Fusion proteins were expressed in which a carboxy-terminal fragment of bovine prolactin was attached downstream of each of the major putative transmembrane domains, M1-M4 and MA, in the AChR subunits. The orientation of the prolactin domain relative to the microsomal membrane was then determined for each protein by a proteolysis protection assay. Since the prolactin domain contains no information which either directs or prevents its translocation, its transmembrane orientation depends solely on sequences within the AChR subunit portion of the fusion protein. When subunit-prolactin fusion proteins with the prolactin domain fused after either M2 or M4 were tested, prolactin-immunoreactive peptides that were larger than the prolactin domain itself were recovered. No prolactin-immunoreactive peptides were recovered after proteolysis of fusion proteins containing prolactin fused after M1, M3, or MA. These results support a model of AChR subunit topology in which M1-M4, but not MA, are transmembrane domains and the carboxy terminus is extracellular.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

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

  1. Anderson D. J., Blobel G. In vitro synthesis, glycosylation, and membrane insertion of the four subunits of Torpedo acetylcholine receptor. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5598–5602. doi: 10.1073/pnas.78.9.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson D. J., Blobel G. Molecular events in the synthesis and assembly of a nicotinic acetylcholine receptor. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 1):125–134. doi: 10.1101/sqb.1983.048.01.015. [DOI] [PubMed] [Google Scholar]
  3. Anderson D. J., Blobel G., Tzartos S., Gullick W., Lindstrom J. Transmembrane orientation of an early biosynthetic form of acetylcholine receptor delta subunit determined by proteolytic dissection in conjunction with monoclonal antibodies. J Neurosci. 1983 Sep;3(9):1773–1784. doi: 10.1523/JNEUROSCI.03-09-01773.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson D. J., Walter P., Blobel G. Signal recognition protein is required for the integration of acetylcholine receptor delta subunit, a transmembrane glycoprotein, into the endoplasmic reticulum membrane. J Cell Biol. 1982 May;93(2):501–506. doi: 10.1083/jcb.93.2.501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boquet P. L., Manoil C., Beckwith J. Use of TnphoA to detect genes for exported proteins in Escherichia coli: identification of the plasmid-encoded gene for a periplasmic acid phosphatase. J Bacteriol. 1987 Apr;169(4):1663–1669. doi: 10.1128/jb.169.4.1663-1669.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boyd D., Manoil C., Beckwith J. Determinants of membrane protein topology. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8525–8529. doi: 10.1073/pnas.84.23.8525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brodsky M. H., Warton M., Myers R. M., Littman D. R. Analysis of the site in CD4 that binds to the HIV envelope glycoprotein. J Immunol. 1990 Apr 15;144(8):3078–3086. [PubMed] [Google Scholar]
  8. Chavez R. A., Hall Z. W. The transmembrane topology of the amino terminus of the alpha subunit of the nicotinic acetylcholine receptor. J Biol Chem. 1991 Aug 15;266(23):15532–15538. [PubMed] [Google Scholar]
  9. Chepuri V., Gennis R. B. The use of gene fusions to determine the topology of all of the subunits of the cytochrome o terminal oxidase complex of Escherichia coli. J Biol Chem. 1990 Aug 5;265(22):12978–12986. [PubMed] [Google Scholar]
  10. 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]
  11. Criado M., Hochschwender S., Sarin V., Fox J. L., Lindstrom J. Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2004–2008. doi: 10.1073/pnas.82.7.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Criado M., Sarin V., Fox J. L., Lindstrom J. Structural localization of the sequence alpha 235-242 of the nicotinic acetylcholine receptor. Biochem Biophys Res Commun. 1985 Apr 30;128(2):864–871. doi: 10.1016/0006-291x(85)90126-3. [DOI] [PubMed] [Google Scholar]
  13. Das M. K., Lindstrom J. The main immunogenic region of the nicotinic acetylcholine receptor: interaction of monoclonal antibodies with synthetic peptides. Biochem Biophys Res Commun. 1989 Dec 15;165(2):865–871. doi: 10.1016/s0006-291x(89)80046-4. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. DiPaola M., Czajkowski C., Karlin A. The sidedness of the COOH terminus of the acetylcholine receptor delta subunit. J Biol Chem. 1989 Sep 15;264(26):15457–15463. [PubMed] [Google Scholar]
  16. Finer-Moore J., Stroud R. M. Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor. Proc Natl Acad Sci U S A. 1984 Jan;81(1):155–159. doi: 10.1073/pnas.81.1.155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Grenningloh G., Rienitz A., Schmitt B., Methfessel C., Zensen M., Beyreuther K., Gundelfinger E. D., Betz H. The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Nature. 1987 Jul 16;328(6127):215–220. doi: 10.1038/328215a0. [DOI] [PubMed] [Google Scholar]
  19. Gutierrez C., Barondess J., Manoil C., Beckwith J. The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. Analysis of osmotically regulated genes in Escherichia coli. J Mol Biol. 1987 May 20;195(2):289–297. doi: 10.1016/0022-2836(87)90650-4. [DOI] [PubMed] [Google Scholar]
  20. Guy H. R. A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations. Biophys J. 1984 Jan;45(1):249–261. doi: 10.1016/S0006-3495(84)84152-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hamilton S. L., McLaughlin M., Karlin A. Formation of disulfide-linked oligomers of acetylcholine receptor in membrane from torpedo electric tissue. Biochemistry. 1979 Jan 9;18(1):155–163. doi: 10.1021/bi00568a024. [DOI] [PubMed] [Google Scholar]
  22. Imoto K., Busch C., Sakmann B., Mishina M., Konno T., Nakai J., Bujo H., Mori Y., Fukuda K., Numa S. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature. 1988 Oct 13;335(6191):645–648. doi: 10.1038/335645a0. [DOI] [PubMed] [Google Scholar]
  23. Imoto K., Methfessel C., Sakmann B., Mishina M., Mori Y., Konno T., Fukuda K., Kurasaki M., Bujo H., Fujita Y. Location of a delta-subunit region determining ion transport through the acetylcholine receptor channel. Nature. 1986 Dec 18;324(6098):670–674. doi: 10.1038/324670a0. [DOI] [PubMed] [Google Scholar]
  24. Isenberg K. E., Mudd J., Shah V., Merlie J. P. Nucleotide sequence of the mouse muscle nicotinic acetylcholine receptor alpha subunit. Nucleic Acids Res. 1986 Jun 25;14(12):5111–5111. doi: 10.1093/nar/14.12.5111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jackson R. J., Hunt T. Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol. 1983;96:50–74. doi: 10.1016/s0076-6879(83)96008-1. [DOI] [PubMed] [Google Scholar]
  26. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. LaPolla R. J., Mayne K. M., Davidson N. Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7970–7974. doi: 10.1073/pnas.81.24.7970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. LaRochelle W. J., Wray B. E., Sealock R., Froehner S. C. Immunochemical demonstration that amino acids 360-377 of the acetylcholine receptor gamma-subunit are cytoplasmic. J Cell Biol. 1985 Mar;100(3):684–691. doi: 10.1083/jcb.100.3.684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Lindstrom J., Criado M., Hochschwender S., Fox J. L., Sarin V. Immunochemical tests of acetylcholine receptor subunit models. Nature. 1984 Oct 11;311(5986):573–575. doi: 10.1038/311573a0. [DOI] [PubMed] [Google Scholar]
  31. Lindstrom J., Criado M., Ratnam M., Whiting P., Ralston S., Rivier J., Sarin V., Sargent P. Using monoclonal antibodies to determine the structures of acetylcholine receptors from electric organs, muscles, and neurons. Ann N Y Acad Sci. 1987;505:208–225. doi: 10.1111/j.1749-6632.1987.tb51293.x. [DOI] [PubMed] [Google Scholar]
  32. Lindstrom J., Hochschwender S., Wan K., Ratnam M., Criado M. Use of monoclonal antibodies in exploring the structure of the acetylcholine receptor. Biochem Soc Trans. 1985 Feb;13(1):14–16. doi: 10.1042/bst0130014. [DOI] [PubMed] [Google Scholar]
  33. Lingappa V. R., Devillers-Thiery A., Blobel G. Nascent prehormones are intermediates in the biosynthesis of authentic bovine pituitary growth hormone and prolactin. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2432–2436. doi: 10.1073/pnas.74.6.2432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. McCrea P. D., Popot J. L., Engelman D. M. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit. EMBO J. 1987 Dec 1;6(12):3619–3626. doi: 10.1002/j.1460-2075.1987.tb02693.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mishina M., Tobimatsu T., Imoto K., Tanaka K., Fujita Y., Fukuda K., Kurasaki M., Takahashi H., Morimoto Y., Hirose T. Location of functional regions of acetylcholine receptor alpha-subunit by site-directed mutagenesis. 1985 Jan 31-Feb 6Nature. 313(6001):364–369. doi: 10.1038/313364a0. [DOI] [PubMed] [Google Scholar]
  38. Mitra A. K., McCarthy M. P., Stroud R. M. Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at 22 A by low dose electron microscopy and x-ray diffraction to 12.5 A. J Cell Biol. 1989 Aug;109(2):755–774. doi: 10.1083/jcb.109.2.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Oiki S., Danho W., Madison V., Montal M. M2 delta, a candidate for the structure lining the ionic channel of the nicotinic cholinergic receptor. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8703–8707. doi: 10.1073/pnas.85.22.8703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Patrick J., Boulter J., Goldman D., Gardner P., Heinemann S. Molecular biology of nicotinic acetylcholine receptors. Ann N Y Acad Sci. 1987;505:194–207. doi: 10.1111/j.1749-6632.1987.tb51292.x. [DOI] [PubMed] [Google Scholar]
  42. Pedersen S. E., Bridgman P. C., Sharp S. D., Cohen J. B. Identification of a cytoplasmic region of the Torpedo nicotinic acetylcholine receptor alpha-subunit by epitope mapping. J Biol Chem. 1990 Jan 5;265(1):569–581. [PubMed] [Google Scholar]
  43. Perara E., Lingappa V. R. A former amino terminal signal sequence engineered to an internal location directs translocation of both flanking protein domains. J Cell Biol. 1985 Dec;101(6):2292–2301. doi: 10.1083/jcb.101.6.2292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ratnam M., Lindstrom J. Structural features of the nicotinic acetylcholine receptor revealed by antibodies to synthetic peptides. Biochem Biophys Res Commun. 1984 Aug 16;122(3):1225–1233. doi: 10.1016/0006-291x(84)91223-3. [DOI] [PubMed] [Google Scholar]
  45. Ratnam M., Nguyen D. L., Rivier J., Sargent P. B., Lindstrom J. Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles. Biochemistry. 1986 May 6;25(9):2633–2643. doi: 10.1021/bi00357a052. [DOI] [PubMed] [Google Scholar]
  46. Ratnam M., Sargent P. B., Sarin V., Fox J. L., Nguyen D. L., Rivier J., Criado M., Lindstrom J. Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping. Biochemistry. 1986 May 6;25(9):2621–2632. doi: 10.1021/bi00357a051. [DOI] [PubMed] [Google Scholar]
  47. Rothman R. E., Andrews D. W., Calayag M. C., Lingappa V. R. Construction of defined polytopic integral transmembrane proteins. The role of signal and stop transfer sequence permutations. J Biol Chem. 1988 Jul 25;263(21):10470–10480. [PubMed] [Google Scholar]
  48. Sargent P. B., Hedges B. E., Tsavaler L., Clemmons L., Tzartos S., Lindstrom J. M. Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies. J Cell Biol. 1984 Feb;98(2):609–618. doi: 10.1083/jcb.98.2.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schofield P. R., Darlison M. G., Fujita N., Burt D. R., Stephenson F. A., Rodriguez H., Rhee L. M., Ramachandran J., Reale V., Glencorse T. A. Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super-family. Nature. 1987 Jul 16;328(6127):221–227. doi: 10.1038/328221a0. [DOI] [PubMed] [Google Scholar]
  50. Takai T., Noda M., Mishina M., Shimizu S., Furutani Y., Kayano T., Ikeda T., Kubo T., Takahashi H., Takahashi T. Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle. 1985 Jun 27-Jul 3Nature. 315(6022):761–764. doi: 10.1038/315761a0. [DOI] [PubMed] [Google Scholar]
  51. Toyoshima C., Unwin N. Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes. Nature. 1988 Nov 17;336(6196):247–250. doi: 10.1038/336247a0. [DOI] [PubMed] [Google Scholar]
  52. Walter P., Blobel G. Preparation of microsomal membranes for cotranslational protein translocation. Methods Enzymol. 1983;96:84–93. doi: 10.1016/s0076-6879(83)96010-x. [DOI] [PubMed] [Google Scholar]
  53. Yost C. S., Hedgpeth J., Lingappa V. R. A stop transfer sequence confers predictable transmembrane orientation to a previously secreted protein in cell-free systems. Cell. 1983 Oct;34(3):759–766. doi: 10.1016/0092-8674(83)90532-9. [DOI] [PubMed] [Google Scholar]
  54. Young E. F., Ralston E., Blake J., Ramachandran J., Hall Z. W., Stroud R. M. Topological mapping of acetylcholine receptor: evidence for a model with five transmembrane segments and a cytoplasmic COOH-terminal peptide. Proc Natl Acad Sci U S A. 1985 Jan;82(2):626–630. doi: 10.1073/pnas.82.2.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yun C. H., Van Doren S. R., Crofts A. R., Gennis R. B. The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides. J Biol Chem. 1991 Jun 15;266(17):10967–10973. [PubMed] [Google Scholar]

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

RESOURCES