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. 1995 Feb 15;306(Pt 1):71–75. doi: 10.1042/bj3060071

Immunoprecipitation of opioid receptor-Go-protein complexes using specific GTP-binding-protein antisera.

Z Georgoussi 1, G Milligan 1, C Zioudrou 1
PMCID: PMC1136483  PMID: 7864831

Abstract

Solubilization of opioid receptors from rat cortical membranes that retained high-affinity guanine nucleotide-sensitive agonist binding was achieved using 10 mM CHAPS. We report the nature of the interactions of mu and delta opioid receptors with the guanine nucleotide-binding protein G(o) by immunoprecipitation of CHAPS extracts with selective G(o)alpha-subunit protein antisera. Antiserum IM1 raised against amino acids 22-35 of G(o)alpha selectively co-immunoprecipitated G(o)alpha-mu and G(o)alpha-delta opioid receptor complexes detected in the immunoprecipitates by specific [3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin and [3H][D-Ser2,Leu5,Thr6]enkephalin binding respectively. By contrast, antisera directed against the C-terminal decapeptide (OC2) and the N-terminal hexadecapeptide (ON1) of isoforms of G(o)alpha were unable to immunoprecipitate solubilized opioid receptor-G(o) complexes, although both were able to immunoprecipitate solubilized G(o)alpha and have been shown to reduce the affinity of [D-Ala2,D-Leu5]enkephalin for opioid receptors in rat cortical membranes [Georgoussi, Carr and Milligan (1993) Mol. Pharmacol. 44, 62-69]. These findings demonstrate that CHAPS-solubilized mu and delta opioid receptors from rat cortical membranes form stable complexes with one or more variants of G(o).

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Selected References

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

  1. Carter B. D., Medzihradsky F. Go mediates the coupling of the mu opioid receptor to adenylyl cyclase in cloned neural cells and brain. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4062–4066. doi: 10.1073/pnas.90.9.4062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Childers S. R. Opioid receptor-coupled second messenger systems. Life Sci. 1991;48(21):1991–2003. doi: 10.1016/0024-3205(91)90154-4. [DOI] [PubMed] [Google Scholar]
  3. Cote T. E., Gosse M. E., Weems H. B. Solubilization of high-affinity, guanine nucleotide-sensitive mu-opioid receptors from 7315c cell membranes. J Neurochem. 1993 Sep;61(3):973–978. doi: 10.1111/j.1471-4159.1993.tb03610.x. [DOI] [PubMed] [Google Scholar]
  4. Evans C. J., Keith D. E., Jr, Morrison H., Magendzo K., Edwards R. H. Cloning of a delta opioid receptor by functional expression. Science. 1992 Dec 18;258(5090):1952–1955. doi: 10.1126/science.1335167. [DOI] [PubMed] [Google Scholar]
  5. Fitzgerald T. J., Uhing R. J., Exton J. H. Solubilization of the vasopressin receptor from rat liver plasma membranes. Evidence for a receptor X GTP-binding protein complex. J Biol Chem. 1986 Dec 25;261(36):16871–16877. [PubMed] [Google Scholar]
  6. Georgoussi Z., Carr C., Milligan G. Direct measurements of in situ interactions of rat brain opioid receptors with the guanine nucleotide-binding protein Go. Mol Pharmacol. 1993 Jul;44(1):62–69. [PubMed] [Google Scholar]
  7. 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]
  8. Kieffer B. L., Befort K., Gaveriaux-Ruff C., Hirth C. G. The delta-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):12048–12052. doi: 10.1073/pnas.89.24.12048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Laugwitz K. L., Offermanns S., Spicher K., Schultz G. mu and delta opioid receptors differentially couple to G protein subtypes in membranes of human neuroblastoma SH-SY5Y cells. Neuron. 1993 Feb;10(2):233–242. doi: 10.1016/0896-6273(93)90314-h. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Masters S. B., Sullivan K. A., Miller R. T., Beiderman B., Lopez N. G., Ramachandran J., Bourne H. R. Carboxyl terminal domain of Gs alpha specifies coupling of receptors to stimulation of adenylyl cyclase. Science. 1988 Jul 22;241(4864):448–451. doi: 10.1126/science.2899356. [DOI] [PubMed] [Google Scholar]
  12. Matesic D. F., Manning D. R., Wolfe B. B., Luthin G. R. Pharmacological and biochemical characterization of complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein. J Biol Chem. 1989 Dec 25;264(36):21638–21645. [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. McPherson G. A. A practical computer-based approach to the analysis of radioligand binding experiments. Comput Programs Biomed. 1983 Aug-Oct;17(1-2):107–113. doi: 10.1016/0010-468x(83)90031-4. [DOI] [PubMed] [Google Scholar]
  16. Mullaney I., Milligan G. Identification of two distinct isoforms of the guanine nucleotide binding protein G0 in neuroblastoma X glioma hybrid cells: independent regulation during cyclic AMP-induced differentiation. J Neurochem. 1990 Dec;55(6):1890–1898. doi: 10.1111/j.1471-4159.1990.tb05773.x. [DOI] [PubMed] [Google Scholar]
  17. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  18. Offermanns S., Schultz G., Rosenthal W. Evidence for opioid receptor-mediated activation of the G-proteins, Go and Gi2, in membranes of neuroblastoma x glioma (NG108-15) hybrid cells. J Biol Chem. 1991 Feb 25;266(6):3365–3368. [PubMed] [Google Scholar]
  19. Ofri D., Ritter A. M., Liu Y. F., Gioannini T. L., Hiller J. M., Simon E. J. Characterization of solubilized opioid receptors: reconstitution and uncoupling of guanine nucleotide-sensitive agonist binding. J Neurochem. 1992 Feb;58(2):628–635. doi: 10.1111/j.1471-4159.1992.tb09764.x. [DOI] [PubMed] [Google Scholar]
  20. Okuma Y., Reisine T. Immunoprecipitation of alpha 2a-adrenergic receptor-GTP-binding protein complexes using GTP-binding protein selective antisera. Changes in receptor/GTP-binding protein interaction following agonist binding. J Biol Chem. 1992 Jul 25;267(21):14826–14831. [PubMed] [Google Scholar]
  21. Reisine T., Bell G. I. Molecular biology of opioid receptors. Trends Neurosci. 1993 Dec;16(12):506–510. doi: 10.1016/0166-2236(93)90194-q. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Sadée W., Yu V. C., Richards M. L., Preis P. N., Schwab M. R., Brodsky F. M., Biedler J. L. Expression of neurotransmitter receptors and myc protooncogenes in subclones of a human neuroblastoma cell line. Cancer Res. 1987 Oct 1;47(19):5207–5212. [PubMed] [Google Scholar]
  24. Schmidt C. J., Neer E. J. In vitro synthesis of G protein beta gamma dimers. J Biol Chem. 1991 Mar 5;266(7):4538–4544. [PubMed] [Google Scholar]
  25. Senogles S. E., Spiegel A. M., Padrell E., Iyengar R., Caron M. G. Specificity of receptor-G protein interactions. Discrimination of Gi subtypes by the D2 dopamine receptor in a reconstituted system. J Biol Chem. 1990 Mar 15;265(8):4507–4514. [PubMed] [Google Scholar]
  26. Seward E., Hammond C., Henderson G. Mu-opioid-receptor-mediated inhibition of the N-type calcium-channel current. Proc Biol Sci. 1991 May 22;244(1310):129–135. doi: 10.1098/rspb.1991.0061. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Simonds W. F., Goldsmith P. K., Woodard C. J., Unson C. G., Spiegel A. M. Receptor and effector interactions of Gs. Functional studies with antibodies to the alpha s carboxyl-terminal decapeptide. FEBS Lett. 1989 Jun 5;249(2):189–194. doi: 10.1016/0014-5793(89)80622-2. [DOI] [PubMed] [Google Scholar]
  29. Simonds W. F., Koski G., Streaty R. A., Hjelmeland L. M., Klee W. A. Solubilization of active opiate receptors. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4623–4627. doi: 10.1073/pnas.77.8.4623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Surprenant A., Shen K. Z., North R. A., Tatsumi H. Inhibition of calcium currents by noradrenaline, somatostatin and opioids in guinea-pig submucosal neurones. J Physiol. 1990 Dec;431:585–608. doi: 10.1113/jphysiol.1990.sp018349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ueda H., Harada H., Nozaki M., Katada T., Ui M., Satoh M., Takagi H. Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go. Proc Natl Acad Sci U S A. 1988 Sep;85(18):7013–7017. doi: 10.1073/pnas.85.18.7013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wong Y. H., Demoliou-Mason C. D., Barnard E. A. Opioid receptors in magnesium-digitonin-solubilized rat brain membranes are tightly coupled to a pertussis toxin-sensitive guanine nucleotide-binding protein. J Neurochem. 1989 Apr;52(4):999–1009. doi: 10.1111/j.1471-4159.1989.tb01840.x. [DOI] [PubMed] [Google Scholar]

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