Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Oct 1;271(1):223–229. doi: 10.1042/bj2710223

GTP analogues cause preferential translocation of an 18 kDa cytosolic G-protein to the membrane fraction in the ZR-75-1 human breast-cancer cell line.

J Levy 1, R J King 1
PMCID: PMC1149536  PMID: 2121131

Abstract

Several G-proteins (GTP-binding proteins) were identified by SDS/PAGE in the cytosol (105,000 g supernatant) and membrane fractions of the oestrogen-dependent human mammary-tumour cell line ZR-75-1. These proteins, with molecular masses in the range 18-29 kDa, specifically bind [alpha-32P]GTP, which can be displaced by unlabelled GTP, GDP and their non-hydrolysable analogues guanosine 5'-[delta-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]), but not by GMP, ATP, ADP, AMP and other unrelated nucleotides. The apparent dissociation constant for GTP was approx. 2 x 10(-8)M. Homogenization of ZR-75-1 cells in high-salt buffer (1 M-KCl), and successive washing of the membrane fraction, suggested that, among the major G-proteins found, the 18 kDa protein is predominantly soluble, whereas the 27-29 kDa complex is primarily bound to the membrane fraction under the experimental conditions employed. Possible translocation of these G-proteins between membrane and cytosol was analysed. No redistribution of the 27-29 kDa complex was observed, whereas GTP[S] in the presence of Mg2+ caused apparent translocation of the 18 kDa protein to the membrane fraction. This effect was specific for GTP and stable GTP analogues, whereas GDP, GMP, ATP, ADP, AMP and other unrelated nucleotides were ineffective. GTP[S] and guanosine 5'-[beta gamma-imido]-triphosphate (p[NH]ppG) were equally potent (apparent Kd approximately 5 x 10(-6)M), whereas GTP was rather weak. The nucleotide effect is temperature-, time- and concentration-dependent. The translocation process was reversible, slow, and reached its maximum between 30 and 60 min at 37 degrees C. The apparent translocation of this small G-protein from the cytosol to the membrane fraction, and the specific effect of GTP analogues, suggest that this process may have functional significance in mammary-tumour cells.

Full text

PDF
223

Images in this article

224Fig. 1.
on p.224
226Fig. 3.
on p.226

Selected References

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

  1. Adari H., Lowy D. R., Willumsen B. M., Der C. J., McCormick F. Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. Science. 1988 Apr 22;240(4851):518–521. doi: 10.1126/science.2833817. [DOI] [PubMed] [Google Scholar]
  2. Audigier Y., Nigam S. K., Blobel G. Identification of a G protein in rough endoplasmic reticulum of canine pancreas. J Biol Chem. 1988 Nov 5;263(31):16352–16357. [PubMed] [Google Scholar]
  3. Baldassare J. J., Fisher G. J. Regulation of membrane-associated and cytosolic phospholipase C activities in human platelets by guanosine triphosphate. J Biol Chem. 1986 Sep 15;261(26):11942–11944. [PubMed] [Google Scholar]
  4. Baldassare J. J., Knipp M. A., Henderson P. A., Fisher G. J. GTP gamma S-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate soluble phospholipase C from human platelets requires soluble GTP-binding protein. Biochem Biophys Res Commun. 1988 Jul 15;154(1):351–357. doi: 10.1016/0006-291x(88)90692-4. [DOI] [PubMed] [Google Scholar]
  5. Bhullar R. P., Haslam R. J. Detection of 23-27 kDa GTP-binding proteins in platelets and other cells. Biochem J. 1987 Jul 15;245(2):617–620. doi: 10.1042/bj2450617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bourne H. R. GTP-binding proteins. One molecular machine can transduce diverse signals. 1986 Jun 26-Jul 2Nature. 321(6073):814–816. doi: 10.1038/321814a0. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  8. Buss J. E., Mumby S. M., Casey P. J., Gilman A. G., Sefton B. M. Myristoylated alpha subunits of guanine nucleotide-binding regulatory proteins. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7493–7497. doi: 10.1073/pnas.84.21.7493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Coffer A. I., King R. J. Characterization of p29, an estrogen-receptor associated tumor marker. J Steroid Biochem. 1988 Nov;31(5):745–750. doi: 10.1016/0022-4731(88)90281-6. [DOI] [PubMed] [Google Scholar]
  10. Darbre P., Yates J., Curtis S., King R. J. Effect of estradiol on human breast cancer cells in culture. Cancer Res. 1983 Jan;43(1):349–354. [PubMed] [Google Scholar]
  11. Deckmyn H., Tu S. M., Majerus P. W. Guanine nucleotides stimulate soluble phosphoinositide-specific phospholipase C in the absence of membranes. J Biol Chem. 1986 Dec 15;261(35):16553–16558. [PubMed] [Google Scholar]
  12. Drobak B. K., Allan E. F., Comerford J. G., Roberts K., Dawson A. P. Presence of guanine nucleotide-binding proteins in a plant hypocotyl microsomal fraction. Biochem Biophys Res Commun. 1988 Feb 15;150(3):899–903. doi: 10.1016/0006-291x(88)90713-9. [DOI] [PubMed] [Google Scholar]
  13. Evans T., Brown M. L., Fraser E. D., Northup J. K. Purification of the major GTP-binding proteins from human placental membranes. J Biol Chem. 1986 May 25;261(15):7052–7059. [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Ikeda K., Kikuchi A., Takai Y. Small molecular weight GTP-binding proteins in human erythrocyte ghosts. Biochem Biophys Res Commun. 1988 Oct 31;156(2):889–897. doi: 10.1016/s0006-291x(88)80927-6. [DOI] [PubMed] [Google Scholar]
  17. Jacobs M., Thelen M. P., Farndale R. W., Astle M. C., Rubery P. H. Specific guanine nucleotide binding by membranes from Cucurbita pepo seedlings. Biochem Biophys Res Commun. 1988 Sep 30;155(3):1478–1484. doi: 10.1016/s0006-291x(88)81308-1. [DOI] [PubMed] [Google Scholar]
  18. Kahn R. A., Gilman A. G. Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin. J Biol Chem. 1984 May 25;259(10):6228–6234. [PubMed] [Google Scholar]
  19. Kahn R. A., Gilman A. G. The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein. J Biol Chem. 1986 Jun 15;261(17):7906–7911. [PubMed] [Google Scholar]
  20. Kikuchi A., Yamashita T., Kawata M., Yamamoto K., Ikeda K., Tanimoto T., Takai Y. Purification and characterization of a novel GTP-binding protein with a molecular weight of 24,000 from bovine brain membranes. J Biol Chem. 1988 Feb 25;263(6):2897–2904. [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Lapetina E. G., Lacal J. C., Reep B. R., Molina y Vedia L. A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets. Proc Natl Acad Sci U S A. 1989 May;86(9):3131–3134. doi: 10.1073/pnas.86.9.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lapetina E. G., Reep B. R. Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2261–2265. doi: 10.1073/pnas.84.8.2261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McArdle H., Mullaney I., Magee A., Unson C., Milligan G. GTP analogues cause release of the alpha subunit of the GTP binding protein, GO, from the plasma membrane of NG108-15 cells. Biochem Biophys Res Commun. 1988 Apr 15;152(1):243–251. doi: 10.1016/s0006-291x(88)80706-x. [DOI] [PubMed] [Google Scholar]
  25. Melançon P., Glick B. S., Malhotra V., Weidman P. J., Serafini T., Gleason M. L., Orci L., Rothman J. E. Involvement of GTP-binding "G" proteins in transport through the Golgi stack. Cell. 1987 Dec 24;51(6):1053–1062. doi: 10.1016/0092-8674(87)90591-5. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Pfaffinger P. J., Martin J. M., Hunter D. D., Nathanson N. M., Hille B. GTP-binding proteins couple cardiac muscarinic receptors to a K channel. Nature. 1985 Oct 10;317(6037):536–538. doi: 10.1038/317536a0. [DOI] [PubMed] [Google Scholar]
  29. Segal T., Levy J., Sharoni Y. GnRH analogs stimulate phospholipase C activity in mammary tumor membranes: modulation by GTP. Mol Cell Endocrinol. 1987 Oct;53(3):239–243. doi: 10.1016/0303-7207(87)90179-1. [DOI] [PubMed] [Google Scholar]
  30. Stryer L. Cyclic GMP cascade of vision. Annu Rev Neurosci. 1986;9:87–119. doi: 10.1146/annurev.ne.09.030186.000511. [DOI] [PubMed] [Google Scholar]
  31. Toki C., Oda K., Ikehara Y. Demonstration of GTP-binding proteins and ADP-ribosylated proteins in rat liver Golgi fraction. Biochem Biophys Res Commun. 1989 Oct 16;164(1):333–338. doi: 10.1016/0006-291x(89)91722-1. [DOI] [PubMed] [Google Scholar]
  32. Wang P., Toyoshima S., Osawa T. Concanavalin A-induced translocation of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes. J Biochem. 1988 Aug;104(2):169–172. doi: 10.1093/oxfordjournals.jbchem.a122435. [DOI] [PubMed] [Google Scholar]
  33. Yamamoto K., Kondo J., Hishida T., Teranishi Y., Takai Y. Purification and characterization of a GTP-binding protein with a molecular weight of 20,000 in bovine brain membranes. Identification as the rho gene product. J Biol Chem. 1988 Jul 15;263(20):9926–9932. [PubMed] [Google Scholar]

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

RESOURCES