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. 1988 Jun 1;252(2):369–373. doi: 10.1042/bj2520369

Opioid peptides promote cholera-toxin-catalysed ADP-ribosylation of the inhibitory guanine-nucleotide-binding protein (Gi) in membranes of neuroblastoma x glioma hybrid cells.

G Milligan 1, F R McKenzie 1
PMCID: PMC1149154  PMID: 3137927

Abstract

NG108-15 neuroblastoma x glioma hybrid cells express a major 45 kDa substrate for cholera toxin and a 40 kDa substrate(s) for pertussis toxin when ADP-ribosylation is performed in the presence of GTP. In the absence of exogenous GTP, however, cholera toxin was shown to catalyse incorporation of radioactivity into a 40 kDa protein as well as into the 45 kDa polypeptide. In membranes of cells which had been pretreated in vivo with pertussis toxin, the 40 kDa band was no longer a substrate for either pertussis or cholera toxin in vitro, whereas in membranes from cholera-toxin-pretreated cells the 40 kDa band was still a substrate for fresh cholera toxin in vitro and for pertussis toxin. In this cell line, opioid peptides have been shown to inhibit adenylate cyclase exclusively by interacting with Gi (inhibitory G-protein) and with no other pertussis-toxin-sensitive G-protein. Opioid agonists, but not antagonists, promoted the cholera-toxin-catalysed ADP-ribosylation of the 40 kDa polypeptide, hence demonstrating that this cholera-toxin substrate was indeed the alpha-subunit of Gi. These results demonstrate that Gi can be a substrate for either cholera or pertussis toxin under appropriate conditions.

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Selected References

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

  1. Aksamit R. R., Backlund P. S., Jr, Cantoni G. L. Cholera toxin inhibits chemotaxis by a cAMP-independent mechanism. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7475–7479. doi: 10.1073/pnas.82.22.7475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Gawler D., Milligan G., Spiegel A. M., Unson C. G., Houslay M. D. Abolition of the expression of inhibitory guanine nucleotide regulatory protein Gi activity in diabetes. Nature. 1987 May 21;327(6119):229–232. doi: 10.1038/327229a0. [DOI] [PubMed] [Google Scholar]
  3. Gierschik P., Milligan G., Pines M., Goldsmith P., Codina J., Klee W., Spiegel A. Use of specific antibodies to quantitate the guanine nucleotide-binding protein Go in brain. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2258–2262. doi: 10.1073/pnas.83.7.2258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  5. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  6. Masters S. B., Stroud R. M., Bourne H. R. Family of G protein alpha chains: amphipathic analysis and predicted structure of functional domains. Protein Eng. 1986 Oct-Nov;1(1):47–54. [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Milligan G., Gierschik P., Spiegel A. M., Klee W. A. The GTP-binding regulatory proteins of neuroblastoma x glioma, NG108-15, and glioma, C6, cells. Immunochemical evidence of a pertussis toxin substrate that is neither Ni nor No. FEBS Lett. 1986 Jan 20;195(1-2):225–230. doi: 10.1016/0014-5793(86)80165-x. [DOI] [PubMed] [Google Scholar]
  10. Milligan G. Guanine nucleotide regulation of the pertussis and cholera toxin substrates of rat glioma C6 BU1 cells. Biochim Biophys Acta. 1987 Jul 6;929(2):197–202. doi: 10.1016/0167-4889(87)90176-5. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Milligan G., Streaty R. A., Gierschik P., Spiegel A. M., Klee W. A. Development of opiate receptors and GTP-binding regulatory proteins in neonatal rat brain. J Biol Chem. 1987 Jun 25;262(18):8626–8630. [PubMed] [Google Scholar]
  13. 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]
  14. O'Brien R. M., Houslay M. D., Milligan G., Siddle K. The insulin receptor tyrosyl kinase phosphorylates holomeric forms of the guanine nucleotide regulatory proteins Gi and Go. FEBS Lett. 1987 Feb 23;212(2):281–288. doi: 10.1016/0014-5793(87)81361-3. [DOI] [PubMed] [Google Scholar]
  15. Owens J. R., Frame L. T., Ui M., Cooper D. M. Cholera toxin ADP-ribosylates the islet-activating protein substrate in adipocyte membranes and alters its function. J Biol Chem. 1985 Dec 15;260(29):15946–15952. [PubMed] [Google Scholar]
  16. Sharma S. K., Nirenberg M., Klee W. A. Morphine receptors as regulators of adenylate cyclase activity. Proc Natl Acad Sci U S A. 1975 Feb;72(2):590–594. doi: 10.1073/pnas.72.2.590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Spiegel A. M. Signal transduction by guanine nucleotide binding proteins. Mol Cell Endocrinol. 1987 Jan;49(1):1–16. doi: 10.1016/0303-7207(87)90058-x. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Verghese M., Uhing R. J., Snyderman R. A pertussis/choleratoxin-sensitive N protein may mediate chemoattractant receptor signal transduction. Biochem Biophys Res Commun. 1986 Jul 31;138(2):887–894. doi: 10.1016/s0006-291x(86)80579-4. [DOI] [PubMed] [Google Scholar]

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