Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Apr 15;267(2):391–398. doi: 10.1042/bj2670391

Delta-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2.

F R McKenzie 1, G Milligan 1
PMCID: PMC1131301  PMID: 2159280

Abstract

Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.

Full text

PDF
391

Images in this article

393Fig. 1.
on p.393
394Fig. 2.
on p.394
394Fig. 3.
on p.394
394Fig. 4.
on p.394

Selected References

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

  1. Chohan P., Carpenter C., Saggerson E. D. Changes in the anti-lipolytic action and binding to plasma membranes of N6-L-phenylisopropyladenosine in adipocytes from starved and hypothyroid rats. Biochem J. 1984 Oct 1;223(1):53–59. doi: 10.1042/bj2230053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Florio V. A., Sternweis P. C. Reconstitution of resolved muscarinic cholinergic receptors with purified GTP-binding proteins. J Biol Chem. 1985 Mar 25;260(6):3477–3483. [PubMed] [Google Scholar]
  3. 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]
  4. Hamprecht B., Glaser T., Reiser G., Bayer E., Propst F. Culture and characteristics of hormone-responsive neuroblastoma X glioma hybrid cells. Methods Enzymol. 1985;109:316–341. doi: 10.1016/0076-6879(85)09096-6. [DOI] [PubMed] [Google Scholar]
  5. Hescheler J., Rosenthal W., Trautwein W., Schultz G. The GTP-binding protein, Go, regulates neuronal calcium channels. 1987 Jan 29-Feb 4Nature. 325(6103):445–447. doi: 10.1038/325445a0. [DOI] [PubMed] [Google Scholar]
  6. Houslay M. D., Metcalfe J. C., Warren G. B., Hesketh T. R., Smith G. A. The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it. Biochim Biophys Acta. 1976 Jun 17;436(2):489–494. doi: 10.1016/0005-2736(76)90210-8. [DOI] [PubMed] [Google Scholar]
  7. Jones D. T., Reed R. R. Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J Biol Chem. 1987 Oct 15;262(29):14241–14249. [PubMed] [Google Scholar]
  8. Katada T., Ui M. ADP ribosylation of the specific membrane protein of C6 cells by islet-activating protein associated with modification of adenylate cyclase activity. J Biol Chem. 1982 Jun 25;257(12):7210–7216. [PubMed] [Google Scholar]
  9. 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]
  10. McFadzean I., Mullaney I., Brown D. A., Milligan G. Antibodies to the GTP binding protein, Go, antagonize noradrenaline-induced calcium current inhibition in NG108-15 hybrid cells. Neuron. 1989 Aug;3(2):177–182. doi: 10.1016/0896-6273(89)90030-5. [DOI] [PubMed] [Google Scholar]
  11. McKenzie F. R., Kelly E. C., Unson C. G., Spiegel A. M., Milligan G. Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates. Biochem J. 1988 Feb 1;249(3):653–659. doi: 10.1042/bj2490653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Milligan G. Foetal-calf serum stimulates a pertussis-toxin-sensitive high-affinity GTPase activity in rat glioma C6 BU1 cells. Biochem J. 1987 Jul 15;245(2):501–505. doi: 10.1042/bj2450501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Milligan G., Simonds W. F., Streaty R. A., Tocque B., Klee W. A. Functional control of the delta-opiate receptor by the inhibitory guanine nucleotide-binding protein. Biochem Soc Trans. 1985 Dec;13(6):1110–1113. doi: 10.1042/bst0131110. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Milligan G. Tissue distribution and subcellular location of guanine nucleotide binding proteins: implications for cellular signalling. Cell Signal. 1989;1(5):411–419. doi: 10.1016/0898-6568(89)90027-2. [DOI] [PubMed] [Google Scholar]
  16. Milligan G., Unson C. G. Persistent activation of the alpha subunit of Gs promotes its removal from the plasma membrane. Biochem J. 1989 Jun 15;260(3):837–841. doi: 10.1042/bj2600837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mitchell F. M., Griffiths S. L., Saggerson E. D., Houslay M. D., Knowler J. T., Milligan G. Guanine-nucleotide-binding proteins expressed in rat white adipose tissue. Identification of both mRNAs and proteins corresponding to Gi1, Gi2 and Gi3. Biochem J. 1989 Sep 1;262(2):403–408. doi: 10.1042/bj2620403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Rall T., Harris B. A. Identification of the lesion in the stimulatory GTP-binding protein of the uncoupled S49 lymphoma. FEBS Lett. 1987 Nov 30;224(2):365–371. doi: 10.1016/0014-5793(87)80486-6. [DOI] [PubMed] [Google Scholar]
  20. Sabol S. L., Nirenberg M. Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. I. Inhibition of adenylate cyclase mediated by alpha receptors. J Biol Chem. 1979 Mar 25;254(6):1913–1920. [PubMed] [Google Scholar]
  21. 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]
  22. Suki W. N., Abramowitz J., Mattera R., Codina J., Birnbaumer L. The human genome encodes at least three non-allellic G proteins with alpha i-type subunits. FEBS Lett. 1987 Aug 10;220(1):187–192. doi: 10.1016/0014-5793(87)80900-6. [DOI] [PubMed] [Google Scholar]
  23. Sullivan K. A., Miller R. T., Masters S. B., Beiderman B., Heideman W., Bourne H. R. Identification of receptor contact site involved in receptor-G protein coupling. Nature. 1987 Dec 24;330(6150):758–760. doi: 10.1038/330758a0. [DOI] [PubMed] [Google Scholar]
  24. Yatani A., Mattera R., Codina J., Graf R., Okabe K., Padrell E., Iyengar R., Brown A. M., Birnbaumer L. The G protein-gated atrial K+ channel is stimulated by three distinct Gi alpha-subunits. Nature. 1988 Dec 15;336(6200):680–682. doi: 10.1038/336680a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES