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. 1987 Apr;84(8):2261–2265. doi: 10.1073/pnas.84.8.2261

Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C.

E G Lapetina, B R Reep
PMCID: PMC304629  PMID: 3470789

Abstract

We have assessed the binding of [alpha-32P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO4/PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha-32P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha-32P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S].

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Selected References

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

  1. Baenziger N. L., Majerus P. W. Isolation of human platelets and platelet surface membranes. Methods Enzymol. 1974;31:149–155. doi: 10.1016/0076-6879(74)31015-4. [DOI] [PubMed] [Google Scholar]
  2. Baldassare J. J., Fisher G. J. GTP and cytosol stimulate phosphoinositide hydrolysis in isolated platelet membranes. Biochem Biophys Res Commun. 1986 Jun 13;137(2):801–805. doi: 10.1016/0006-291x(86)91150-2. [DOI] [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. Banno Y., Nakashima S., Tohmatsu T., Nozawa Y., Lapetina E. G. GTP and GDP will stimulate platelet cytosolic phospholipase C independently of Ca2+. Biochem Biophys Res Commun. 1986 Oct 30;140(2):728–734. doi: 10.1016/0006-291x(86)90792-8. [DOI] [PubMed] [Google Scholar]
  5. Blackmore P. F., Bocckino S. B., Waynick L. E., Exton J. H. Role of a guanine nucleotide-binding regulatory protein in the hydrolysis of hepatocyte phosphatidylinositol 4,5-bisphosphate by calcium-mobilizing hormones and the control of cell calcium. Studies utilizing aluminum fluoride. J Biol Chem. 1985 Nov 25;260(27):14477–14483. [PubMed] [Google Scholar]
  6. Cockcroft S., Gomperts B. D. Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase. Nature. 1985 Apr 11;314(6011):534–536. doi: 10.1038/314534a0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Gilman A. G. G proteins and dual control of adenylate cyclase. Cell. 1984 Mar;36(3):577–579. doi: 10.1016/0092-8674(84)90336-2. [DOI] [PubMed] [Google Scholar]
  9. Gonzales R. A., Crews F. T. Guanine nucleotides stimulate production of inositol trisphosphate in rat cortical membranes. Biochem J. 1985 Dec 15;232(3):799–804. doi: 10.1042/bj2320799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Guillon G., Balestre M. N., Mouillac B., Devilliers G. Activation of membrane phospholipase C by vasopressin. A requirement for guanyl nucleotides. FEBS Lett. 1986 Feb 3;196(1):155–159. doi: 10.1016/0014-5793(86)80232-0. [DOI] [PubMed] [Google Scholar]
  11. Haslam R. J., Davidson M. M. Receptor-induced diacylglycerol formation in permeabilized platelets; possible role for a GTP-binding protein. J Recept Res. 1984;4(1-6):605–629. doi: 10.3109/10799898409042576. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Lapetina E. G. Effect of pertussis toxin on the phosphodiesteratic cleavage of the polyphosphoinositides by guanosine 5'-O-thiotriphosphate and thrombin in permeabilized human platelets. Biochim Biophys Acta. 1986 Nov 19;884(2):219–224. doi: 10.1016/0304-4165(86)90166-2. [DOI] [PubMed] [Google Scholar]
  14. Lapetina E. G., Reep B., Chang K. J. Treatment of human platelets with trypsin, thrombin, or collagen inhibits the pertussis toxin-induced ADP-ribosylation of a 41-kDa protein. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5880–5883. doi: 10.1073/pnas.83.16.5880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Litosch I., Calista C., Wallis C., Fain J. N. 5-Methyltryptamine decreases net accumulation of 32P into the polyphosphoinositides from [gamma-32P]ATP in a cell-free system from blowfly salivary glands. Activation of breakdown of the newly synthesized [32P]polyphosphoinositides. J Biol Chem. 1986 Jan 15;261(2):638–643. [PubMed] [Google Scholar]
  16. Litosch I., Fain J. N. 5-Methyltryptamine stimulates phospholipase C-mediated breakdown of exogenous phosphoinositides by blowfly salivary gland membranes. J Biol Chem. 1985 Dec 25;260(30):16052–16055. [PubMed] [Google Scholar]
  17. Litosch I., Fain J. N. Regulation of phosphoinositide breakdown by guanine nucleotides. Life Sci. 1986 Jul 21;39(3):187–194. doi: 10.1016/0024-3205(86)90529-1. [DOI] [PubMed] [Google Scholar]
  18. Litosch I., Wallis C., Fain J. N. 5-Hydroxytryptamine stimulates inositol phosphate production in a cell-free system from blowfly salivary glands. Evidence for a role of GTP in coupling receptor activation to phosphoinositide breakdown. J Biol Chem. 1985 May 10;260(9):5464–5471. [PubMed] [Google Scholar]
  19. Lucas D. O., Bajjalieh S. M., Kowalchyk J. A., Martin T. F. Direct stimulation by thyrotropin-releasing hormone (TRH) of polyphosphoinositide hydrolysis in GH3 cell membranes by a guanine nucleotide-modulated mechanism. Biochem Biophys Res Commun. 1985 Oct 30;132(2):721–728. doi: 10.1016/0006-291x(85)91192-1. [DOI] [PubMed] [Google Scholar]
  20. Martin T. F., Lucas D. O., Bajjalieh S. M., Kowalchyk J. A. Thyrotropin-releasing hormone activates a Ca2+-dependent polyphosphoinositide phosphodiesterase in permeable GH3 cells. GTP gamma S potentiation by a cholera and pertussis toxin-insensitive mechanism. J Biol Chem. 1986 Feb 25;261(6):2918–2927. [PubMed] [Google Scholar]
  21. Melin P. M., Sundler R., Jergil B. Phospholipase C in rat liver plasma membranes. Phosphoinositide specificity and regulation by guanine nucleotides and calcium. FEBS Lett. 1986 Mar 17;198(1):85–88. doi: 10.1016/0014-5793(86)81189-9. [DOI] [PubMed] [Google Scholar]
  22. Merritt J. E., Taylor C. W., Rubin R. P., Putney J. W., Jr Evidence suggesting that a novel guanine nucleotide regulatory protein couples receptors to phospholipase C in exocrine pancreas. Biochem J. 1986 Jun 1;236(2):337–343. doi: 10.1042/bj2360337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ruggiero M., Lapetina E. G. Leupeptin selectively inhibits human platelet responses induced by thrombin and trypsin; a role for proteolytic activation of phospholipase C. Biochem Biophys Res Commun. 1985 Sep 30;131(3):1198–1205. doi: 10.1016/0006-291x(85)90218-9. [DOI] [PubMed] [Google Scholar]
  24. Ruggiero M., Lapetina E. G. Protease and cyclooxygenase inhibitors synergistically prevent activation of human platelets. Proc Natl Acad Sci U S A. 1986 May;83(10):3456–3459. doi: 10.1073/pnas.83.10.3456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ruggiero M., Lapetina E. G. Sustained proteolysis is required for human platelet activation by thrombin. Thromb Res. 1986 Apr 15;42(2):247–255. doi: 10.1016/0049-3848(86)90300-2. [DOI] [PubMed] [Google Scholar]
  26. Straub R. E., Gershengorn M. C. Thyrotropin-releasing hormone and GTP activate inositol trisphosphate formation in membranes isolated from rat pituitary cells. J Biol Chem. 1986 Feb 25;261(6):2712–2717. [PubMed] [Google Scholar]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Uhing R. J., Prpic V., Jiang H., Exton J. H. Hormone-stimulated polyphosphoinositide breakdown in rat liver plasma membranes. Roles of guanine nucleotides and calcium. J Biol Chem. 1986 Feb 15;261(5):2140–2146. [PubMed] [Google Scholar]
  29. Wallace M. A., Fain J. N. Guanosine 5'-O-thiotriphosphate stimulates phospholipase C activity in plasma membranes of rat hepatocytes. J Biol Chem. 1985 Aug 15;260(17):9527–9530. [PubMed] [Google Scholar]

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