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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
. 1986 Jun;83(11):3776–3780. doi: 10.1073/pnas.83.11.3776

Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain.

H Itoh, T Kozasa, S Nagata, S Nakamura, T Katada, M Ui, S Iwai, E Ohtsuka, H Kawasaki, K Suzuki, et al.
PMCID: PMC323606  PMID: 3086867

Abstract

We have cloned cDNAs encoding alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go and determined their nucleotide sequences. Purified preparations of Gi and Go alpha subunits (Gi alpha and Go alpha) from rat brain were completely digested with trypsin, and peptides were subjected to amino acid sequence analysis. By screening of a cDNA library from rat C6 glioma cells with a synthetic probe corresponding to a 17 amino acid sequence, a clone encoding the sequence of Go alpha was obtained. Then, the library was rescreened with a Go alpha cDNA probe to isolate several strongly or weakly hybridizing clones. cDNAs encoding the complete sequences of Gi alpha and Gs alpha were thus obtained. From nucleotide sequence analysis, the amino acid sequences of Gs alpha and Gi alpha were deduced; they contain 394 and 355 amino acid residues (including the initiator methionine), respectively. The calculated molecular weights for Gs alpha and Gi alpha were 45,663 and 40,499, respectively. The Go alpha clone encoded a sequence of 310 amino acid residues that lacked the NH2 terminus. The homology of the alpha subunits of Gs, Gi, Go, transducin, and ras-encoded protein is discussed.

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Selected References

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

  1. Arai K., Clark B. F., Duffy L., Jones M. D., Kaziro Y., Laursen R. A., L'Italien J., Miller D. L., Nagarkatti S., Nakamura S. Primary structure of elongation factor Tu from Escherichia coli. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1326–1330. doi: 10.1073/pnas.77.3.1326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  3. Capon D. J., Chen E. Y., Levinson A. D., Seeburg P. H., Goeddel D. V. Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature. 1983 Mar 3;302(5903):33–37. doi: 10.1038/302033a0. [DOI] [PubMed] [Google Scholar]
  4. Cassel D., Selinger Z. Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3307–3311. doi: 10.1073/pnas.74.8.3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Codina J., Hildebrandt J. D., Sekura R. D., Birnbaumer M., Bryan J., Manclark C. R., Iyengar R., Birnbaumer L. Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases. Purification of the human erythrocyte proteins without the use of activating regulatory ligands. J Biol Chem. 1984 May 10;259(9):5871–5886. [PubMed] [Google Scholar]
  6. Gibbs J. B., Sigal I. S., Poe M., Scolnick E. M. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5704–5708. doi: 10.1073/pnas.81.18.5704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gilman A. G. G proteins and dual control of adenylate cyclase. Cell. 1984 Mar;36(3):577–579. doi: 10.1016/0092-8674(84)90336-2. [DOI] [PubMed] [Google Scholar]
  8. Gilman A. G., Nirenberg M. Effect of catecholamines on the adenosine 3':5'-cyclic monophosphate concentrations of clonal satellite cells of neurons. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2165–2168. doi: 10.1073/pnas.68.9.2165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grantham R., Gautier C., Gouy M., Mercier R., Pavé A. Codon catalog usage and the genome hypothesis. Nucleic Acids Res. 1980 Jan 11;8(1):r49–r62. doi: 10.1093/nar/8.1.197-c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Halliday K. R. Regional homology in GTP-binding proto-oncogene products and elongation factors. J Cyclic Nucleotide Protein Phosphor Res. 1983;9(6):435–448. [PubMed] [Google Scholar]
  11. Harris B. A., Robishaw J. D., Mumby S. M., Gilman A. G. Molecular cloning of complementary DNA for the alpha subunit of the G protein that stimulates adenylate cyclase. Science. 1985 Sep 20;229(4719):1274–1277. doi: 10.1126/science.3839937. [DOI] [PubMed] [Google Scholar]
  12. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  13. Hurley J. B., Fong H. K., Teplow D. B., Dreyer W. J., Simon M. I. Isolation and characterization of a cDNA clone for the gamma subunit of bovine retinal transducin. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6948–6952. doi: 10.1073/pnas.81.22.6948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jurnak F. Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. Science. 1985 Oct 4;230(4721):32–36. doi: 10.1126/science.3898365. [DOI] [PubMed] [Google Scholar]
  15. Katada T., Amano T., Ui M. Modulation by islet-activating protein of adenylate cyclase activity in C6 glioma cells. J Biol Chem. 1982 Apr 10;257(7):3739–3746. [PubMed] [Google Scholar]
  16. Katada T., Bokoch G. M., Northup J. K., Ui M., Gilman A. G. The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Properties and function of the purified protein. J Biol Chem. 1984 Mar 25;259(6):3568–3577. [PubMed] [Google Scholar]
  17. Kaziro Y. Molecular mechanism of protein biosynthesis and an approach to the mechanism of energy transduction. Mol Biol Biochem Biophys. 1980;32:333–346. doi: 10.1007/978-3-642-81503-4_26. [DOI] [PubMed] [Google Scholar]
  18. Kaziro Y. The role of guanosine 5'-triphosphate in polypeptide chain elongation. Biochim Biophys Acta. 1978 Sep 21;505(1):95–127. doi: 10.1016/0304-4173(78)90009-5. [DOI] [PubMed] [Google Scholar]
  19. Lochrie M. A., Hurley J. B., Simon M. I. Sequence of the alpha subunit of photoreceptor G protein: homologies between transducin, ras, and elongation factors. Science. 1985 Apr 5;228(4695):96–99. doi: 10.1126/science.3856323. [DOI] [PubMed] [Google Scholar]
  20. Manning D. R., Fraser B. A., Kahn R. A., Gilman A. G. ADP-ribosylation of transducin by islet-activation protein. Identification of asparagine as the site of ADP-ribosylation. J Biol Chem. 1984 Jan 25;259(2):749–756. [PubMed] [Google Scholar]
  21. Manning D. R., Gilman A. G. The regulatory components of adenylate cyclase and transducin. A family of structurally homologous guanine nucleotide-binding proteins. J Biol Chem. 1983 Jun 10;258(11):7059–7063. [PubMed] [Google Scholar]
  22. Medynski D. C., Sullivan K., Smith D., Van Dop C., Chang F. H., Fung B. K., Seeburg P. H., Bourne H. R. Amino acid sequence of the alpha subunit of transducin deduced from the cDNA sequence. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4311–4315. doi: 10.1073/pnas.82.13.4311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  24. Milligan G., Klee W. A. The inhibitory guanine nucleotide-binding protein (Ni) purified from bovine brain is a high affinity GTPase. J Biol Chem. 1985 Feb 25;260(4):2057–2063. [PubMed] [Google Scholar]
  25. Neer E. J., Lok J. M., Wolf L. G. Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase. J Biol Chem. 1984 Nov 25;259(22):14222–14229. [PubMed] [Google Scholar]
  26. Northup J. K., Sternweis P. C., Smigel M. D., Schleifer L. S., Ross E. M., Gilman A. G. Purification of the regulatory component of adenylate cyclase. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6516–6520. doi: 10.1073/pnas.77.11.6516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nukada T., Tanabe T., Takahashi H., Noda M., Hirose T., Inayama S., Numa S. Primary structure of the alpha-subunit of bovine adenylate cyclase-stimulating G-protein deduced from the cDNA sequence. FEBS Lett. 1986 Jan 20;195(1-2):220–224. doi: 10.1016/0014-5793(86)80164-8. [DOI] [PubMed] [Google Scholar]
  28. Ohta H., Okajima F., Ui M. Inhibition by islet-activating protein of a chemotactic peptide-induced early breakdown of inositol phospholipids and Ca2+ mobilization in guinea pig neutrophils. J Biol Chem. 1985 Dec 15;260(29):15771–15780. [PubMed] [Google Scholar]
  29. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pfaffinger P. J., Martin J. M., Hunter D. D., Nathanson N. M., Hille B. GTP-binding proteins couple cardiac muscarinic receptors to a K channel. Nature. 1985 Oct 10;317(6037):536–538. doi: 10.1038/317536a0. [DOI] [PubMed] [Google Scholar]
  31. Robishaw J. D., Russell D. W., Harris B. A., Smigel M. D., Gilman A. G. Deduced primary structure of the alpha subunit of the GTP-binding stimulatory protein of adenylate cyclase. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1251–1255. doi: 10.1073/pnas.83.5.1251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Seeburg P. H., Colby W. W., Capon D. J., Goeddel D. V., Levinson A. D. Biological properties of human c-Ha-ras1 genes mutated at codon 12. Nature. 1984 Nov 1;312(5989):71–75. doi: 10.1038/312071a0. [DOI] [PubMed] [Google Scholar]
  33. Sternweis P. C., Robishaw J. D. Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J Biol Chem. 1984 Nov 25;259(22):13806–13813. [PubMed] [Google Scholar]
  34. Sugimoto K., Nukada T., Tanabe T., Takahashi H., Noda M., Minamino N., Kangawa K., Matsuo H., Hirose T., Inayama S. Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence. FEBS Lett. 1985 Oct 28;191(2):235–240. doi: 10.1016/0014-5793(85)80015-6. [DOI] [PubMed] [Google Scholar]
  35. Tanabe T., Nukada T., Nishikawa Y., Sugimoto K., Suzuki H., Takahashi H., Noda M., Haga T., Ichiyama A., Kangawa K. Primary structure of the alpha-subunit of transducin and its relationship to ras proteins. Nature. 1985 May 16;315(6016):242–245. doi: 10.1038/315242a0. [DOI] [PubMed] [Google Scholar]
  36. Van Dop C., Tsubokawa M., Bourne H. R., Ramachandran J. Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin. J Biol Chem. 1984 Jan 25;259(2):696–698. [PubMed] [Google Scholar]
  37. Yatsunami K., Khorana H. G. GTPase of bovine rod outer segments: the amino acid sequence of the alpha subunit as derived from the cDNA sequence. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4316–4320. doi: 10.1073/pnas.82.13.4316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yatsunami K., Pandya B. V., Oprian D. D., Khorana H. G. cDNA-derived amino acid sequence of the gamma subunit of GTPase from bovine rod outer segments. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1936–1940. doi: 10.1073/pnas.82.7.1936. [DOI] [PMC free article] [PubMed] [Google Scholar]

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