Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1989 Sep 1;262(2):403–408. doi: 10.1042/bj2620403

Guanine-nucleotide-binding proteins expressed in rat white adipose tissue. Identification of both mRNAs and proteins corresponding to Gi1, Gi2 and Gi3.

F M Mitchell 1, S L Griffiths 1, E D Saggerson 1, M D Houslay 1, J T Knowler 1, G Milligan 1
PMCID: PMC1133282  PMID: 2508627

Abstract

Considerable debate has focused on the molecular identity of the guanine-nucleotide-binding proteins (G-proteins) in adipose tissue which can be detected following pertussis-toxin-catalysed ADP-ribosylation [Rapiejko, Northup, Evans, Brown & Malbon (1986) Biochem. J. 240, 35-40; Hinsch, Rosenthal, Spicher, Binder, Gausepohl, Frank, Schultz & Joost (1988) FEBS Lett. 238, 191-196]. We have used a panel of selective anti-peptide antisera which are able to discriminate between the different pertussis-toxin-sensitive G-proteins to assess which of these are expressed in rat adipose tissue. We demonstrate that plasma membranes of rat white adipocytes contain alpha subunits corresponding to each of Gi1, Gi2 and Gi3. Furthermore, using synthetic oligonucleotides complimentary to unique regions of each of the three polypeptides, we demonstrate that the mRNAs for the three G-protein alpha subunits can also be detected in adipose tissue.

Full text

PDF
403

Images in this article

Selected References

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

  1. Backlund P. S., Jr, Aksamit R. R., Unson C. G., Goldsmith P., Spiegel A. M., Milligan G. Immunochemical and electrophoretic characterization of the major pertussis toxin substrate of the RAW264 macrophage cell line. Biochemistry. 1988 Mar 22;27(6):2040–2046. doi: 10.1021/bi00406a034. [DOI] [PubMed] [Google Scholar]
  2. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Codina J., Olate J., Abramowitz J., Mattera R., Cook R. G., Birnbaumer L. Alpha i-3 cDNA encodes the alpha subunit of Gk, the stimulatory G protein of receptor-regulated K+ channels. J Biol Chem. 1988 May 15;263(14):6746–6750. [PubMed] [Google Scholar]
  5. Gierschik P., Falloon J., Milligan G., Pines M., Gallin J. I., Spiegel A. Immunochemical evidence for a novel pertussis toxin substrate in human neutrophils. J Biol Chem. 1986 Jun 15;261(17):8058–8062. [PubMed] [Google Scholar]
  6. 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]
  7. Goldsmith P., Backlund P. S., Jr, Rossiter K., Carter A., Milligan G., Unson C. G., Spiegel A. Purification of heterotrimeric GTP-binding proteins from brain: identification of a novel form of Go. Biochemistry. 1988 Sep 6;27(18):7085–7090. doi: 10.1021/bi00418a062. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Hinsch K. D., Rosenthal W., Spicher K., Binder T., Gausepohl H., Frank R., Schultz G., Joost H. G. Adipocyte plasma membranes contain two Gi subtypes but are devoid of Go. FEBS Lett. 1988 Sep 26;238(1):191–196. doi: 10.1016/0014-5793(88)80254-0. [DOI] [PubMed] [Google Scholar]
  10. Huff R. M., Axton J. M., Neer E. J. Physical and immunological characterization of a guanine nucleotide-binding protein purified from bovine cerebral cortex. J Biol Chem. 1985 Sep 5;260(19):10864–10871. [PubMed] [Google Scholar]
  11. 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]
  12. Katada T., Ui M. Islet-activating protein. A modifier of receptor-mediated regulation of rat islet adenylate cyclase. J Biol Chem. 1981 Aug 25;256(16):8310–8317. [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. Milligan G., Mullaney I., Unson C. G., Marshall L., Spiegel A. M., McArdle H. GTP analogues promote release of the alpha subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells. Biochem J. 1988 Sep 1;254(2):391–396. doi: 10.1042/bj2540391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Milligan G., Spiegel A. M., Unson C. G., Saggerson E. D. Chemically induced hypothyroidism produces elevated amounts of the alpha subunit of the inhibitory guanine nucleotide binding protein (Gi) and the beta subunit common to all G-proteins. Biochem J. 1987 Oct 1;247(1):223–227. doi: 10.1042/bj2470223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Milligan G., Tanfin Z., Goureau O., Unson C., Harbon S. Identification of both Gi2 and a novel, immunologically distinct, form of Go in rat myometrial membranes. FEBS Lett. 1989 Feb 27;244(2):411–416. doi: 10.1016/0014-5793(89)80574-5. [DOI] [PubMed] [Google Scholar]
  21. Mullaney I., Magee A. I., Unson C. G., Milligan G. Differential regulation of amounts of the guanine-nucleotide-binding proteins Gi and Go in neuroblastoma x glioma hybrid cells in response to dibutyryl cyclic AMP. Biochem J. 1988 Dec 1;256(2):649–656. doi: 10.1042/bj2560649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mumby S. M., Kahn R. A., Manning D. R., Gilman A. G. Antisera of designed specificity for subunits of guanine nucleotide-binding regulatory proteins. Proc Natl Acad Sci U S A. 1986 Jan;83(2):265–269. doi: 10.1073/pnas.83.2.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Pines M., Gierschik P., Milligan G., Klee W., Spiegel A. Antibodies against the carboxyl-terminal 5-kDa peptide of the alpha subunit of transducin crossreact with the 40-kDa but not the 39-kDa guanine nucleotide binding protein from brain. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4095–4099. doi: 10.1073/pnas.82.12.4095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rapiejko P. J., Northup J. K., Evans T., Brown J. E., Malbon C. C. G-proteins of fat-cells. Role in hormonal regulation of intracellular inositol 1,4,5-trisphosphate. Biochem J. 1986 Nov 15;240(1):35–40. doi: 10.1042/bj2400035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ros M., Northup J. K., Malbon C. C. Steady-state levels of G-proteins and beta-adrenergic receptors in rat fat cells. Permissive effects of thyroid hormones. J Biol Chem. 1988 Mar 25;263(9):4362–4368. [PubMed] [Google Scholar]
  29. 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]
  30. 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]

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

RESOURCES