Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Mar 14;92(6):2283–2287. doi: 10.1073/pnas.92.6.2283

Activation-dependent carboxyl methylation of neutrophil G-protein gamma subunit.

M R Philips 1, R Staud 1, M Pillinger 1, A Feoktistov 1, C Volker 1, J B Stock 1, G Weissmann 1
PMCID: PMC42468  PMID: 7892262

Abstract

The gamma subunits of heterotrimeric guanine nucleotide-binding regulatory (G) proteins (G gamma) are post-translationally processed at their C termini by prenylation, proteolysis, and carboxyl methylation. Whereas prenylation of G gamma is required for membrane association of G proteins, the role of carboxyl methylation is unknown. Here we show that human neutrophils express G gamma 2 but not G gamma 3 or G gamma 7 and that carboxyl methylation of G gamma 2 is associated with signal transduction. In a reconstituted cell-free system, neutrophil G gamma 2 was labeled by the methyl donor S-[methyl-3H]adenosyl-L-methionine. Carboxyl methylation was confirmed by alkaline hydrolysis and quantitation of volatile [3H]methanol. Neutrophil G gamma 2 methylation was stimulated by activation of G protein with guanosine 5'-[beta, gamma-thio]triphosphate. We estimate that after 1 hr of G-protein activation at least 6% of the total pool of G gamma 2 was carboxyl-methylated. The inflammatory agonist fMet-Leu-Phe stimulated guanosine 5'-[beta,gamma-thio]triphosphate-dependent carboxyl methylation. Methylation of G gamma 2 was inhibited by the carboxyl methyltransferase inhibitor N-acetyl-S-trans,trans-farnesylcysteine at concentrations that affected signal transduction in neutrophils. These results demonstrate that activation of neutrophil Gi is associated with alpha-carboxyl methyl esterification of G gamma 2 and suggest that carboxyl methylation of G gamma may play a role in signal transduction.

Full text

PDF
2283

Images in this article

2284Fig. 1
on p.2284
2284Fig. 2
on p.2284
2285Fig. 3
on p.2285
2285Fig. 4
on p.2285
2286Fig. 5
on p.2286
2286Fig. 6
on p.2286

Selected References

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

  1. Backlund P. S., Jr, Simonds W. F., Spiegel A. M. Carboxyl methylation and COOH-terminal processing of the brain G-protein gamma-subunit. J Biol Chem. 1990 Sep 15;265(26):15572–15576. [PubMed] [Google Scholar]
  2. Bokoch G. M., Bickford K., Bohl B. P. Subcellular localization and quantitation of the major neutrophil pertussis toxin substrate, Gn. J Cell Biol. 1988 Jun;106(6):1927–1936. doi: 10.1083/jcb.106.6.1927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bommakanti R. K., Bokoch G. M., Tolley J. O., Schreiber R. E., Siemsen D. W., Klotz K. N., Jesaitis A. J. Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation. J Biol Chem. 1992 Apr 15;267(11):7576–7581. [PubMed] [Google Scholar]
  4. Cali J. J., Balcueva E. A., Rybalkin I., Robishaw J. D. Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning. J Biol Chem. 1992 Nov 25;267(33):24023–24027. [PubMed] [Google Scholar]
  5. Camps M., Carozzi A., Schnabel P., Scheer A., Parker P. J., Gierschik P. Isozyme-selective stimulation of phospholipase C-beta 2 by G protein beta gamma-subunits. Nature. 1992 Dec 17;360(6405):684–686. doi: 10.1038/360684a0. [DOI] [PubMed] [Google Scholar]
  6. Camps M., Hou C., Sidiropoulos D., Stock J. B., Jakobs K. H., Gierschik P. Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits. Eur J Biochem. 1992 Jun 15;206(3):821–831. doi: 10.1111/j.1432-1033.1992.tb16990.x. [DOI] [PubMed] [Google Scholar]
  7. Carlson K. E., Brass L. F., Manning D. R. Thrombin and phorbol esters cause the selective phosphorylation of a G protein other than Gi in human platelets. J Biol Chem. 1989 Aug 5;264(22):13298–13305. [PubMed] [Google Scholar]
  8. Chelsky D., Ruskin B., Koshland D. E., Jr Methyl-esterified proteins in a mammalian cell line. Biochemistry. 1985 Nov 5;24(23):6651–6658. doi: 10.1021/bi00344a053. [DOI] [PubMed] [Google Scholar]
  9. Clapham D. E., Neer E. J. New roles for G-protein beta gamma-dimers in transmembrane signalling. Nature. 1993 Sep 30;365(6445):403–406. doi: 10.1038/365403a0. [DOI] [PubMed] [Google Scholar]
  10. Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem. 1992;61:355–386. doi: 10.1146/annurev.bi.61.070192.002035. [DOI] [PubMed] [Google Scholar]
  11. Del Buono B. J., Luscinskas F. W., Simons E. R. Preparation and characterization of plasma membrane vesicles from human polymorphonuclear leukocytes. J Cell Physiol. 1989 Dec;141(3):636–644. doi: 10.1002/jcp.1041410323. [DOI] [PubMed] [Google Scholar]
  12. Fukada Y., Matsuda T., Kokame K., Takao T., Shimonishi Y., Akino T., Yoshizawa T. Effects of carboxyl methylation of photoreceptor G protein gamma-subunit in visual transduction. J Biol Chem. 1994 Feb 18;269(7):5163–5170. [PubMed] [Google Scholar]
  13. Fukada Y., Takao T., Ohguro H., Yoshizawa T., Akino T., Shimonishi Y. Farnesylated gamma-subunit of photoreceptor G protein indispensable for GTP-binding. Nature. 1990 Aug 16;346(6285):658–660. doi: 10.1038/346658a0. [DOI] [PubMed] [Google Scholar]
  14. Gierschik P., Sidiropoulos D., Spiegel A., Jakobs K. H. Purification and immunochemical characterization of the major pertussis-toxin-sensitive guanine-nucleotide-binding protein of bovine-neutrophil membranes. Eur J Biochem. 1987 May 15;165(1):185–194. doi: 10.1111/j.1432-1033.1987.tb11210.x. [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. Hancock J. F., Cadwallader K., Marshall C. J. Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras(B). EMBO J. 1991 Mar;10(3):641–646. doi: 10.1002/j.1460-2075.1991.tb07992.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hancock J. F., Paterson H., Marshall C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990 Oct 5;63(1):133–139. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  18. Huzoor-Akbar, Wang W., Kornhauser R., Volker C., Stock J. B. Protein prenylcysteine analog inhibits agonist-receptor-mediated signal transduction in human platelets. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):868–872. doi: 10.1073/pnas.90.3.868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Inglese J., Koch W. J., Caron M. G., Lefkowitz R. J. Isoprenylation in regulation of signal transduction by G-protein-coupled receptor kinases. Nature. 1992 Sep 10;359(6391):147–150. doi: 10.1038/359147a0. [DOI] [PubMed] [Google Scholar]
  20. Iñiguez-Lluhi J. A., Simon M. I., Robishaw J. D., Gilman A. G. G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma. J Biol Chem. 1992 Nov 15;267(32):23409–23417. [PubMed] [Google Scholar]
  21. Katz A., Wu D., Simon M. I. Subunits beta gamma of heterotrimeric G protein activate beta 2 isoform of phospholipase C. Nature. 1992 Dec 17;360(6405):686–689. doi: 10.1038/360686a0. [DOI] [PubMed] [Google Scholar]
  22. Kisselev O. G., Ermolaeva M. V., Gautam N. A farnesylated domain in the G protein gamma subunit is a specific determinant of receptor coupling. J Biol Chem. 1994 Aug 26;269(34):21399–21402. [PubMed] [Google Scholar]
  23. Lee J., Stock J. Protein phosphatase 2A catalytic subunit is methyl-esterified at its carboxyl terminus by a novel methyltransferase. J Biol Chem. 1993 Sep 15;268(26):19192–19195. [PubMed] [Google Scholar]
  24. Logothetis D. E., Kurachi Y., Galper J., Neer E. J., Clapham D. E. The beta gamma subunits of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature. 1987 Jan 22;325(6102):321–326. doi: 10.1038/325321a0. [DOI] [PubMed] [Google Scholar]
  25. Muntz K. H., Sternweis P. C., Gilman A. G., Mumby S. M. Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes. Mol Biol Cell. 1992 Jan;3(1):49–61. doi: 10.1091/mbc.3.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Northup J. K., Smigel M. D., Gilman A. G. The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding. J Biol Chem. 1982 Oct 10;257(19):11416–11423. [PubMed] [Google Scholar]
  27. Parish C. A., Rando R. R. Functional significance of G protein carboxymethylation. Biochemistry. 1994 Aug 23;33(33):9986–9991. doi: 10.1021/bi00199a023. [DOI] [PubMed] [Google Scholar]
  28. Philips M. R., Abramson S. B., Kolasinski S. L., Haines K. A., Weissmann G., Rosenfeld M. G. Low molecular weight GTP-binding proteins in human neutrophil granule membranes. J Biol Chem. 1991 Jan 15;266(2):1289–1298. [PubMed] [Google Scholar]
  29. Philips M. R., Buyon J. P., Winchester R., Weissmann G., Abramson S. B. Up-regulation of the iC3b receptor (CR3) is neither necessary nor sufficient to promote neutrophil aggregation. J Clin Invest. 1988 Aug;82(2):495–501. doi: 10.1172/JCI113623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Philips M. R., Pillinger M. H., Staud R., Volker C., Rosenfeld M. G., Weissmann G., Stock J. B. Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils. Science. 1993 Feb 12;259(5097):977–980. doi: 10.1126/science.8438158. [DOI] [PubMed] [Google Scholar]
  31. Pillinger M. H., Volker C., Stock J. B., Weissmann G., Philips M. R. Characterization of a plasma membrane-associated prenylcysteine-directed alpha carboxyl methyltransferase in human neutrophils. J Biol Chem. 1994 Jan 14;269(2):1486–1492. [PubMed] [Google Scholar]
  32. Pérez-Sala D., Tan E. W., Cañada F. J., Rando R. R. Methylation and demethylation reactions of guanine nucleotide-binding proteins of retinal rod outer segments. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3043–3046. doi: 10.1073/pnas.88.8.3043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rotrosen D., Gallin J. I., Spiegel A. M., Malech H. L. Subcellular localization of Gi alpha in human neutrophils. J Biol Chem. 1988 Aug 5;263(22):10958–10964. [PubMed] [Google Scholar]
  34. Scheer A., Gierschik P. Farnesylcysteine analogues inhibit chemotactic peptide receptor-mediated G-protein activation in human HL-60 granulocyte membranes. FEBS Lett. 1993 Mar 15;319(1-2):110–114. doi: 10.1016/0014-5793(93)80047-x. [DOI] [PubMed] [Google Scholar]
  35. Silvius J. R., l'Heureux F. Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers. Biochemistry. 1994 Mar 15;33(10):3014–3022. doi: 10.1021/bi00176a034. [DOI] [PubMed] [Google Scholar]
  36. Simonds W. F., Butrynski J. E., Gautam N., Unson C. G., Spiegel A. M. G-protein beta gamma dimers. Membrane targeting requires subunit coexpression and intact gamma C-A-A-X domain. J Biol Chem. 1991 Mar 25;266(9):5363–5366. [PubMed] [Google Scholar]
  37. Stephens L., Smrcka A., Cooke F. T., Jackson T. R., Sternweis P. C., Hawkins P. T. A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits. Cell. 1994 Apr 8;77(1):83–93. doi: 10.1016/0092-8674(94)90237-2. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Stock J. B., Clarke S., Koshland D. E., Jr The protein carboxylmethyltransferase involved in Escherichia coli and Salmonella typhimurium chemotaxis. Methods Enzymol. 1984;106:310–321. doi: 10.1016/0076-6879(84)06031-6. [DOI] [PubMed] [Google Scholar]
  40. Tang W. J., Gilman A. G. Type-specific regulation of adenylyl cyclase by G protein beta gamma subunits. Science. 1991 Dec 6;254(5037):1500–1503. doi: 10.1126/science.1962211. [DOI] [PubMed] [Google Scholar]
  41. Volker C., Miller R. A., McCleary W. R., Rao A., Poenie M., Backer J. M., Stock J. B. Effects of farnesylcysteine analogs on protein carboxyl methylation and signal transduction. J Biol Chem. 1991 Nov 15;266(32):21515–21522. [PubMed] [Google Scholar]
  42. Volpp B. D., Nauseef W. M., Clark R. A. Subcellular distribution and membrane association of human neutrophil substrates for ADP-ribosylation by pertussis toxin and cholera toxin. J Immunol. 1989 May 1;142(9):3206–3212. [PubMed] [Google Scholar]
  43. Whiteway M., Hougan L., Dignard D., Thomas D. Y., Bell L., Saari G. C., Grant F. J., O'Hara P., MacKay V. L. The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein. Cell. 1989 Feb 10;56(3):467–477. doi: 10.1016/0092-8674(89)90249-3. [DOI] [PubMed] [Google Scholar]
  44. Yamane H. K., Farnsworth C. C., Xie H. Y., Howald W., Fung B. K., Clarke S., Gelb M. H., Glomset J. A. Brain G protein gamma subunits contain an all-trans-geranylgeranylcysteine methyl ester at their carboxyl termini. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5868–5872. doi: 10.1073/pnas.87.15.5868. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES