Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Jul;82(14):4852–4856. doi: 10.1073/pnas.82.14.4852

Mouse-Torpedo hybrid acetylcholine receptors: functional homology does not equal sequence homology.

M M White, K M Mayne, H A Lester, N Davidson
PMCID: PMC391003  PMID: 3860826

Abstract

The nicotinic acetylcholine (AcCho) receptor (AcChoR) is a multisubunit protein complex of stoichiometry alpha 2 beta gamma delta. The several subunits show homology with each other within a given species; in addition, homology is found between analogous subunits between species. We have used the phage SP6 RNA polymerase transcription system to produce single-species RNA in vitro for various AcChoR subunits from cDNAs. Injection of an equimolar mixture of RNA for the alpha, beta, gamma, and delta subunits of Torpedo californica AcChoR into Xenopus oocytes results in the appearance of functional receptors in the oocyte membrane. No response to AcCho is detected when the beta or gamma subunit RNA is omitted, and a small response is seen when the delta subunit RNA is omitted. Replacement of Torpedo delta subunit RNA by the mouse BC3H-1 cell line AcChoR delta subunit RNA leads to the formation of functional receptors that show a 3-4-fold greater response to AcCho than does the full Torpedo complex. No response is seen when the mouse delta RNA replaces Torpedo gamma RNA. By amino acid homology profile comparisons, the mouse delta subunit appears to be moderately but not highly similar to the Torpedo delta subunit; the apparent similarity to the Torpedo gamma subunit is only slightly less. Therefore, the features of the primary sequence that determine the functional delta character of the mouse polypeptide are not revealed by simple homology comparisons.

Full text

PDF
4853

Images in this article

Selected References

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

  1. Barnard E. A., Miledi R., Sumikawa K. Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes. Proc R Soc Lond B Biol Sci. 1982 May 22;215(1199):241–246. doi: 10.1098/rspb.1982.0040. [DOI] [PubMed] [Google Scholar]
  2. Barrantes F. J. Recent developments in the structure and function of the acetylcholine receptor. Int Rev Neurobiol. 1983;24:259–341. doi: 10.1016/s0074-7742(08)60224-x. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Conti-Tronconi B. M., Raftery M. A. The nicotinic cholinergic receptor: correlation of molecular structure with functional properties. Annu Rev Biochem. 1982;51:491–530. doi: 10.1146/annurev.bi.51.070182.002423. [DOI] [PubMed] [Google Scholar]
  5. Contreras R., Cheroutre H., Degrave W., Fiers W. Simple, efficient in vitro synthesis of capped RNA useful for direct expression of cloned eukaryotic genes. Nucleic Acids Res. 1982 Oct 25;10(20):6353–6362. doi: 10.1093/nar/10.20.6353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Contreras R., Cheroutre H., Fiers W. A simple apparatus for injection of nanoliter quantities into Xenopus laevis oocytes. Anal Biochem. 1981 May 1;113(1):185–187. doi: 10.1016/0003-2697(81)90063-4. [DOI] [PubMed] [Google Scholar]
  7. Dascal N., Landau E. M., Lass Y. Xenopus oocyte resting potential, muscarinic responses and the role of calcium and guanosine 3',5'-cyclic monophosphate. J Physiol. 1984 Jul;352:551–574. doi: 10.1113/jphysiol.1984.sp015310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Furuichi Y., LaFiandra A., Shatkin A. J. 5'-Terminal structure and mRNA stability. Nature. 1977 Mar 17;266(5599):235–239. doi: 10.1038/266235a0. [DOI] [PubMed] [Google Scholar]
  10. Green M. R., Maniatis T., Melton D. A. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell. 1983 Mar;32(3):681–694. doi: 10.1016/0092-8674(83)90054-5. [DOI] [PubMed] [Google Scholar]
  11. Gurdon J. B., Melton D. A. Gene transfer in amphibian eggs and oocytes. Annu Rev Genet. 1981;15:189–218. doi: 10.1146/annurev.ge.15.120181.001201. [DOI] [PubMed] [Google Scholar]
  12. Konarska M. M., Padgett R. A., Sharp P. A. Recognition of cap structure in splicing in vitro of mRNA precursors. Cell. 1984 Oct;38(3):731–736. doi: 10.1016/0092-8674(84)90268-x. [DOI] [PubMed] [Google Scholar]
  13. Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. LEIBOVITZ A. THE GROWTH AND MAINTENANCE OF TISSUE-CELL CULTURES IN FREE GAS EXCHANGE WITH THE ATMOSPHERE. Am J Hyg. 1963 Sep;78:173–180. doi: 10.1093/oxfordjournals.aje.a120336. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Lane C. D. The fate of foreign proteins introduced in Xenopus oocytes. Cell. 1981 May;24(2):281–282. doi: 10.1016/0092-8674(81)90315-9. [DOI] [PubMed] [Google Scholar]
  17. Lester H. A., Changeux J. P., Sheridan R. E. Conductance increases produced by bath application of cholinergic agonists to Electrophorus electroplaques. J Gen Physiol. 1975 Jun;65(6):797–816. doi: 10.1085/jgp.65.6.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Manthey A. A. The effect of calcium on the desensitization of membrane receptors at the neuromuscular junction. J Gen Physiol. 1966 May;49(5):963–976. doi: 10.1085/jgp.49.5.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Mishina M., Kurosaki T., Tobimatsu T., Morimoto Y., Noda M., Yamamoto T., Terao M., Lindstrom J., Takahashi T., Kuno M. Expression of functional acetylcholine receptor from cloned cDNAs. Nature. 1984 Feb 16;307(5952):604–608. doi: 10.1038/307604a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Neubig R. R., Cohen J. B. Permeability control by cholinergic receptors in Torpedo postsynaptic membranes: agonist dose-response relations measured at second and millisecond times. Biochemistry. 1980 Jun 10;19(12):2770–2779. doi: 10.1021/bi00553a036. [DOI] [PubMed] [Google Scholar]
  25. Noda M., Furutani Y., Takahashi H., Toyosato M., Tanabe T., Shimizu S., Kikyotani S., Kayano T., Hirose T., Inayama S. Cloning and sequence analysis of calf cDNA and human genomic DNA encoding alpha-subunit precursor of muscle acetylcholine receptor. 1983 Oct 27-Nov 2Nature. 305(5937):818–823. doi: 10.1038/305818a0. [DOI] [PubMed] [Google Scholar]
  26. Noda M., Takahashi H., Tanabe T., Toyosato M., Kikyotani S., Furutani Y., Hirose T., Takashima H., Inayama S., Miyata T. Structural homology of Torpedo californica acetylcholine receptor subunits. Nature. 1983 Apr 7;302(5908):528–532. doi: 10.1038/302528a0. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Penefsky H. S. Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1977 May 10;252(9):2891–2899. [PubMed] [Google Scholar]
  29. 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]
  30. Sumikawa K., Houghton M., Emtage J. S., Richards B. M., Barnard E. A. Active multi-subunit ACh receptor assembled by translation of heterologous mRNA in Xenopus oocytes. Nature. 1981 Aug 27;292(5826):862–864. doi: 10.1038/292862a0. [DOI] [PubMed] [Google Scholar]
  31. Sumikawa K., Houghton M., Smith J. C., Bell L., Richards B. M., Barnard E. A. The molecular cloning and characterisation of cDNA coding for the alpha subunit of the acetylcholine receptor. Nucleic Acids Res. 1982 Oct 11;10(19):5809–5822. doi: 10.1093/nar/10.19.5809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Takai T., Noda M., Furutani Y., Takahashi H., Notake M., Shimizu S., Kayano T., Tanabe T., Tanaka K., Hirose T. Primary structure of gamma subunit precursor of calf-muscle acetylcholine receptor deduced from the cDNA sequence. Eur J Biochem. 1984 Aug 15;143(1):109–115. doi: 10.1111/j.1432-1033.1984.tb08348.x. [DOI] [PubMed] [Google Scholar]
  33. Tanabe T., Noda M., Furutani Y., Takai T., Takahashi H., Tanaka K., Hirose T., Inayama S., Numa S. Primary structure of beta subunit precursor of calf muscle acetylcholine receptor deduced from cDNA sequence. Eur J Biochem. 1984 Oct 1;144(1):11–17. doi: 10.1111/j.1432-1033.1984.tb08424.x. [DOI] [PubMed] [Google Scholar]
  34. Wallace R. A., Jared D. W., Dumont J. N., Sega M. W. Protein incorporation by isolated amphibian oocytes. 3. Optimum incubation conditions. J Exp Zool. 1973 Jun;184(3):321–333. doi: 10.1002/jez.1401840305. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES