Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Feb 1;321(Pt 3):721–728. doi: 10.1042/bj3210721

Interactions of the alpha2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants.

A Wise 1, M A Watson-Koken 1, S Rees 1, M Lee 1, G Milligan 1
PMCID: PMC1218128  PMID: 9032459

Abstract

The alpha2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of 'Gi-like' pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine alpha2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1alpha, Gi2alpha and Gi3alpha or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The alpha2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5'-[gamma-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein alpha-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1alpha, Gi2alpha and Gi3alpha resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys --> Gly mutations of Gi1alpha, Gi2alpha and Gi3alpha were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1alpha was correlated highly both with the level of expression of this G-protein and with the level of expression of the alpha2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys --> Gly mutants of Gi1alpha, Gi2alpha and Gi3alpha required 10-15-fold higher concentrations of agonist than did stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the alpha2A-adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.

Full Text

The Full Text of this article is available as a PDF (476.9 KB).

Selected References

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

  1. Dell'Acqua M. L., Carroll R. C., Peralta E. G. Transfected m2 muscarinic acetylcholine receptors couple to G alpha i2 and G alpha i3 in Chinese hamster ovary cells. Activation and desensitization of the phospholipase C signaling pathway. J Biol Chem. 1993 Mar 15;268(8):5676–5685. [PubMed] [Google Scholar]
  2. Eason M. G., Kurose H., Holt B. D., Raymond J. R., Liggett S. B. Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs. J Biol Chem. 1992 Aug 5;267(22):15795–15801. [PubMed] [Google Scholar]
  3. Eason M. G., Liggett S. B. Identification of a Gs coupling domain in the amino terminus of the third intracellular loop of the alpha 2A-adrenergic receptor. Evidence for distinct structural determinants that confer Gs versus Gi coupling. J Biol Chem. 1995 Oct 20;270(42):24753–24760. doi: 10.1074/jbc.270.42.24753. [DOI] [PubMed] [Google Scholar]
  4. Federman A. D., Conklin B. R., Schrader K. A., Reed R. R., Bourne H. R. Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits. Nature. 1992 Mar 12;356(6365):159–161. doi: 10.1038/356159a0. [DOI] [PubMed] [Google Scholar]
  5. Fong H. K., Yoshimoto K. K., Eversole-Cire P., Simon M. I. Identification of a GTP-binding protein alpha subunit that lacks an apparent ADP-ribosylation site for pertussis toxin. Proc Natl Acad Sci U S A. 1988 May;85(9):3066–3070. doi: 10.1073/pnas.85.9.3066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Garcia P. D., Onrust R., Bell S. M., Sakmar T. P., Bourne H. R. Transducin-alpha C-terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells. EMBO J. 1995 Sep 15;14(18):4460–4469. doi: 10.1002/j.1460-2075.1995.tb00125.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Georgoussi Z., Milligan G., Zioudrou C. Immunoprecipitation of opioid receptor-Go-protein complexes using specific GTP-binding-protein antisera. Biochem J. 1995 Feb 15;306(Pt 1):71–75. doi: 10.1042/bj3060071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gierschik P., Sidiropoulos D., Jakobs K. H. Two distinct Gi-proteins mediate formyl peptide receptor signal transduction in human leukemia (HL-60) cells. J Biol Chem. 1989 Dec 25;264(36):21470–21473. [PubMed] [Google Scholar]
  9. Goldsmith P., Gierschik P., Milligan G., Unson C. G., Vinitsky R., Malech H. L., Spiegel A. M. Antibodies directed against synthetic peptides distinguish between GTP-binding proteins in neutrophil and brain. J Biol Chem. 1987 Oct 25;262(30):14683–14688. [PubMed] [Google Scholar]
  10. Grassie M. A., Milligan G. Analysis of the relative interactions between the alpha 2C10 adrenoceptor and the guanine-nucleotide-binding proteins G(o)1 alpha and Gi 2 alpha following co-expression of these polypeptides in rat 1 fibroblasts. Biochem J. 1995 Mar 1;306(Pt 2):525–530. doi: 10.1042/bj3060525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Green A., Johnson J. L., Milligan G. Down-regulation of Gi sub-types by prolonged incubation of adipocytes with an A1 adenosine receptor agonist. J Biol Chem. 1990 Mar 25;265(9):5206–5210. [PubMed] [Google Scholar]
  12. Hunt T. W., Carroll R. C., Peralta E. G. Heterotrimeric G proteins containing G alpha i3 regulate multiple effector enzymes in the same cell. Activation of phospholipases C and A2 and inhibition of adenylyl cyclase. J Biol Chem. 1994 Nov 25;269(47):29565–29570. [PubMed] [Google Scholar]
  13. Koski G., Klee W. A. Opiates inhibit adenylate cyclase by stimulating GTP hydrolysis. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4185–4189. doi: 10.1073/pnas.78.7.4185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Law S. F., Manning D., Reisine T. Identification of the subunits of GTP-binding proteins coupled to somatostatin receptors. J Biol Chem. 1991 Sep 25;266(27):17885–17897. [PubMed] [Google Scholar]
  15. Martin E. L., Rens-Domiano S., Schatz P. J., Hamm H. E. Potent peptide analogues of a G protein receptor-binding region obtained with a combinatorial library. J Biol Chem. 1996 Jan 5;271(1):361–366. doi: 10.1074/jbc.271.1.361. [DOI] [PubMed] [Google Scholar]
  16. Matsuoka M., Itoh H., Kozasa T., Kaziro Y. Sequence analysis of cDNA and genomic DNA for a putative pertussis toxin-insensitive guanine nucleotide-binding regulatory protein alpha subunit. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5384–5388. doi: 10.1073/pnas.85.15.5384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McClue S. J., Selzer E., Freissmuth M., Milligan G. Gi3 does not contribute to the inhibition of adenylate cyclase when stimulation of an alpha 2-adrenergic receptor causes activation of both Gi2 and Gi3. Biochem J. 1992 Jun 1;284(Pt 2):565–568. doi: 10.1042/bj2840565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McKenzie F. R., Milligan G. Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2. Biochem J. 1990 Apr 15;267(2):391–398. doi: 10.1042/bj2670391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Milligan G., Carr C., Gould G. W., Mullaney I., Lavan B. E. Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3. J Biol Chem. 1991 Apr 5;266(10):6447–6455. [PubMed] [Google Scholar]
  20. Milligan G. Techniques used in the identification and analysis of function of pertussis toxin-sensitive guanine nucleotide binding proteins. Biochem J. 1988 Oct 1;255(1):1–13. doi: 10.1042/bj2550001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Munshi R., Pang I. H., Sternweis P. C., Linden J. A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go. J Biol Chem. 1991 Nov 25;266(33):22285–22289. [PubMed] [Google Scholar]
  22. Murray-Whelan R., Schlegel W. Brain somatostatin receptor-G protein interaction. G alpha C-terminal antibodies demonstrate coupling of the soluble receptor with Gi(1-3) but not with Go. J Biol Chem. 1992 Feb 15;267(5):2960–2965. [PubMed] [Google Scholar]
  23. Osawa S., Weiss E. R. The effect of carboxyl-terminal mutagenesis of Gt alpha on rhodopsin and guanine nucleotide binding. J Biol Chem. 1995 Dec 29;270(52):31052–31058. doi: 10.1074/jbc.270.52.31052. [DOI] [PubMed] [Google Scholar]
  24. Roerig S. C., Loh H. H., Law P. Y. Identification of three separate guanine nucleotide-binding proteins that interact with the delta-opioid receptor in NG108-15 neuroblastoma x glioma hybrid cells. Mol Pharmacol. 1992 May;41(5):822–831. [PubMed] [Google Scholar]
  25. Senogles S. E. The D2 dopamine receptor isoforms signal through distinct Gi alpha proteins to inhibit adenylyl cyclase. A study with site-directed mutant Gi alpha proteins. J Biol Chem. 1994 Sep 16;269(37):23120–23127. [PubMed] [Google Scholar]
  26. Simonds W. F., Goldsmith P. K., Codina J., Unson C. G., Spiegel A. M. Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase in platelet membranes: in situ identification with G alpha C-terminal antibodies. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7809–7813. doi: 10.1073/pnas.86.20.7809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  28. Thomas D. R., Faruq S. A., Balcarek J. M., Brown A. M. Pharmacological characterisation of [35S]-GTPgammaS binding to Chinese hamster ovary cell membranes stably expressing cloned human 5-HT1D receptor subtypes. J Recept Signal Transduct Res. 1995 Jan-Mar;15(1-4):199–211. doi: 10.3109/10799899509045217. [DOI] [PubMed] [Google Scholar]
  29. Wong Y. H. Gi assays in transfected cells. Methods Enzymol. 1994;238:81–94. doi: 10.1016/0076-6879(94)38008-2. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES