Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Feb 15;330(Pt 1):489–495. doi: 10.1042/bj3300489

Rapid activation of sodium-proton exchange and extracellular signal-regulated protein kinase in fibroblasts by G protein-coupled 5-HT1A receptor involves distinct signalling cascades.

M N Garnovskaya 1, Y Mukhin 1, J R Raymond 1
PMCID: PMC1219164  PMID: 9461547

Abstract

These experiments tested the hypothesis that signalling elements involved in the activation of the extracellular signal-regulated protein kinase (ERK) mediate rapid activation of sodium-proton exchange (NHE) in fibroblasts when both signals are initiated by a single G protein-coupled receptor, the 5-HT1A receptor. Similarities between the two processes were comparable concentration-response curves and time-courses, and overlapping sensitivity to some pharmacological inhibitors of tyrosine kinases (staurosporine and genistein), and phosphoinositide 3'-kinase (wortmannin and LY204002). Activation of NHE was much more sensitive to the phosphatidylcholine-specific phospholipase inhibitor (D609) than was ERK. Neither pathway was sensitive to manoeuvres designed to block PKC. In contrast, Src or related kinases appear to be required to activate ERK, but not NHE. Transfection of cDNA constructs encoding inactive mutant phosphoinositide 3'-kinase, Grb2, Sos, Ras, and Raf molecules were successful in attenuating ERK, but had essentially no effect upon NHE activation. Finally, PD98059, an inhibitor of mitogen activated/extracellular signal regulated kinase kinase, blocked ERK but not NHE activation. Thus, in CHO fibroblast cells, activation by the 5-HT1A receptor of ERK and NHE share a number of overlapping features. However, our studies do not support a major role for ERK, when activated by the 5-HT1A receptor, as a short-term upstream regulator of NHE activity.

Full Text

The Full Text of this article is available as a PDF (355.0 KB).

Selected References

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

  1. Aharonovitz O., Granot Y. Stimulation of mitogen-activated protein kinase and Na+/H+ exchanger in human platelets. Differential effect of phorbol ester and vasopressin. J Biol Chem. 1996 Jul 12;271(28):16494–16499. doi: 10.1074/jbc.271.28.16494. [DOI] [PubMed] [Google Scholar]
  2. Barber D. L. Mechanisms of receptor-mediated regulation of Na-H exchange. Cell Signal. 1991;3(5):387–397. doi: 10.1016/0898-6568(91)90069-7. [DOI] [PubMed] [Google Scholar]
  3. Bertin B., Freissmuth M., Breyer R. M., Schütz W., Strosberg A. D., Marullo S. Functional expression of the human serotonin 5-HT1A receptor in Escherichia coli. Ligand binding properties and interaction with recombinant G protein alpha-subunits. J Biol Chem. 1992 Apr 25;267(12):8200–8206. [PubMed] [Google Scholar]
  4. Bianchini L., L'Allemain G., Pouysségur J. The p42/p44 mitogen-activated protein kinase cascade is determinant in mediating activation of the Na+/H+ exchanger (NHE1 isoform) in response to growth factors. J Biol Chem. 1997 Jan 3;272(1):271–279. doi: 10.1074/jbc.272.1.271. [DOI] [PubMed] [Google Scholar]
  5. Blumer K. J., Johnson G. L. Diversity in function and regulation of MAP kinase pathways. Trends Biochem Sci. 1994 Jun;19(6):236–240. doi: 10.1016/0968-0004(94)90147-3. [DOI] [PubMed] [Google Scholar]
  6. Brunn G. J., Williams J., Sabers C., Wiederrecht G., Lawrence J. C., Jr, Abraham R. T. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 1996 Oct 1;15(19):5256–5267. [PMC free article] [PubMed] [Google Scholar]
  7. Butkerait P., Zheng Y., Hallak H., Graham T. E., Miller H. A., Burris K. D., Molinoff P. B., Manning D. R. Expression of the human 5-hydroxytryptamine1A receptor in Sf9 cells. Reconstitution of a coupled phenotype by co-expression of mammalian G protein subunits. J Biol Chem. 1995 Aug 4;270(31):18691–18699. doi: 10.1074/jbc.270.31.18691. [DOI] [PubMed] [Google Scholar]
  8. Cai H., Erhardt P., Troppmair J., Diaz-Meco M. T., Sithanandam G., Rapp U. R., Moscat J., Cooper G. M. Hydrolysis of phosphatidylcholine couples Ras to activation of Raf protein kinase during mitogenic signal transduction. Mol Cell Biol. 1993 Dec;13(12):7645–7651. doi: 10.1128/mcb.13.12.7645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cartwright C. A., Eckhart W., Simon S., Kaplan P. L. Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain. Cell. 1987 Apr 10;49(1):83–91. doi: 10.1016/0092-8674(87)90758-6. [DOI] [PubMed] [Google Scholar]
  10. Clark J. D., Limbird L. E. Na(+)-H+ exchanger subtypes: a predictive review. Am J Physiol. 1991 Dec;261(6 Pt 1):C945–C953. doi: 10.1152/ajpcell.1991.261.6.C945. [DOI] [PubMed] [Google Scholar]
  11. Cowen D. S., Sowers R. S., Manning D. R. Activation of a mitogen-activated protein kinase (ERK2) by the 5-hydroxytryptamine1A receptor is sensitive not only to inhibitors of phosphatidylinositol 3-kinase, but to an inhibitor of phosphatidylcholine hydrolysis. J Biol Chem. 1996 Sep 13;271(37):22297–22300. doi: 10.1074/jbc.271.37.22297. [DOI] [PubMed] [Google Scholar]
  12. Dhanasekaran N., Prasad M. V., Wadsworth S. J., Dermott J. M., van Rossum G. Protein kinase C-dependent and -independent activation of Na+/H+ exchanger by G alpha 12 class of G proteins. J Biol Chem. 1994 Apr 22;269(16):11802–11806. [PubMed] [Google Scholar]
  13. Donowitz M., Montgomery J. L., Walker M. S., Cohen M. E. Brush-border tyrosine phosphorylation stimulates ileal neutral NaCl absorption and brush-border Na(+)-H+ exchange. Am J Physiol. 1994 Apr;266(4 Pt 1):G647–G656. doi: 10.1152/ajpgi.1994.266.4.G647. [DOI] [PubMed] [Google Scholar]
  14. Dudley D. T., Pang L., Decker S. J., Bridges A. J., Saltiel A. R. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7686–7689. doi: 10.1073/pnas.92.17.7686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Feig L. A., Cooper G. M. Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol Cell Biol. 1988 Aug;8(8):3235–3243. doi: 10.1128/mcb.8.8.3235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fukushima T., Waddell T. K., Grinstein S., Goss G. G., Orlowski J., Downey G. P. Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases. J Cell Biol. 1996 Mar;132(6):1037–1052. doi: 10.1083/jcb.132.6.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Garnovskaya M. N., Gettys T. W., van Biesen T., Prpic V., Chuprun J. K., Raymond J. R. 5-HT1A receptor activates Na+/H+ exchange in CHO-K1 cells through Gialpha2 and Gialpha3. J Biol Chem. 1997 Mar 21;272(12):7770–7776. doi: 10.1074/jbc.272.12.7770. [DOI] [PubMed] [Google Scholar]
  18. Garnovskaya M. N., van Biesen T., Hawe B., Casañas Ramos S., Lefkowitz R. J., Raymond J. R. Ras-dependent activation of fibroblast mitogen-activated protein kinase by 5-HT1A receptor via a G protein beta gamma-subunit-initiated pathway. Biochemistry. 1996 Oct 29;35(43):13716–13722. doi: 10.1021/bi961764n. [DOI] [PubMed] [Google Scholar]
  19. Gishizky M. L., Cortez D., Pendergast A. M. Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10889–10893. doi: 10.1073/pnas.92.24.10889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Good D. W. Hyperosmolality inhibits bicarbonate absorption in rat medullary thick ascending limb via a protein-tyrosine kinase-dependent pathway. J Biol Chem. 1995 Apr 28;270(17):9883–9889. doi: 10.1074/jbc.270.17.9883. [DOI] [PubMed] [Google Scholar]
  21. Hagag N., Lacal J. C., Graber M., Aaronson S., Viola M. V. Microinjection of ras p21 induces a rapid rise in intracellular pH. Mol Cell Biol. 1987 May;7(5):1984–1988. doi: 10.1128/mcb.7.5.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hara K., Yonezawa K., Sakaue H., Ando A., Kotani K., Kitamura T., Kitamura Y., Ueda H., Stephens L., Jackson T. R. 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7415–7419. doi: 10.1073/pnas.91.16.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hausdorff S. F., Bennett A. M., Neel B. G., Birnbaum M. J. Different signaling roles of SHPTP2 in insulin-induced GLUT1 expression and GLUT4 translocation. J Biol Chem. 1995 Jun 2;270(22):12965–12968. doi: 10.1074/jbc.270.22.12965. [DOI] [PubMed] [Google Scholar]
  24. Hu Q., Klippel A., Muslin A. J., Fantl W. J., Williams L. T. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science. 1995 Apr 7;268(5207):100–102. doi: 10.1126/science.7701328. [DOI] [PubMed] [Google Scholar]
  25. Huang C. L., Takenawa T., Ives H. E. Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors. J Biol Chem. 1991 Mar 5;266(7):4045–4048. [PubMed] [Google Scholar]
  26. Inglese J., Koch W. J., Touhara K., Lefkowitz R. J. G beta gamma interactions with PH domains and Ras-MAPK signaling pathways. Trends Biochem Sci. 1995 Apr;20(4):151–156. doi: 10.1016/s0968-0004(00)88992-6. [DOI] [PubMed] [Google Scholar]
  27. Kaplan D. L., Boron W. F. Long-term expression of c-H-ras stimulates Na-H and Na(+)-dependent Cl-HCO3 exchange in NIH-3T3 fibroblasts. J Biol Chem. 1994 Feb 11;269(6):4116–4124. [PubMed] [Google Scholar]
  28. Kapus A., Grinstein S., Wasan S., Kandasamy R., Orlowski J. Functional characterization of three isoforms of the Na+/H+ exchanger stably expressed in Chinese hamster ovary cells. ATP dependence, osmotic sensitivity, and role in cell proliferation. J Biol Chem. 1994 Sep 23;269(38):23544–23552. [PubMed] [Google Scholar]
  29. Kawamoto S., Hidaka H. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets. Biochem Biophys Res Commun. 1984 Nov 30;125(1):258–264. doi: 10.1016/s0006-291x(84)80362-9. [DOI] [PubMed] [Google Scholar]
  30. Klippel A., Escobedo J. A., Hirano M., Williams L. T. The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity. Mol Cell Biol. 1994 Apr;14(4):2675–2685. doi: 10.1128/mcb.14.4.2675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. L'Allemain G., Paris S., Pouysségur J. Growth factor action and intracellular pH regulation in fibroblasts. Evidence for a major role of the Na+/H+ antiport. J Biol Chem. 1984 May 10;259(9):5809–5815. [PubMed] [Google Scholar]
  32. Levine S. A., Montrose M. H., Tse C. M., Donowitz M. Kinetics and regulation of three cloned mammalian Na+/H+ exchangers stably expressed in a fibroblast cell line. J Biol Chem. 1993 Dec 5;268(34):25527–25535. [PubMed] [Google Scholar]
  33. Lin X., Voyno-Yasenetskaya T. A., Hooley R., Lin C. Y., Orlowski J., Barber D. L. Galpha12 differentially regulates Na+-H+ exchanger isoforms. J Biol Chem. 1996 Sep 13;271(37):22604–22610. doi: 10.1074/jbc.271.37.22604. [DOI] [PubMed] [Google Scholar]
  34. Lorenz U., Ravichandran K. S., Pei D., Walsh C. T., Burakoff S. J., Neel B. G. Lck-dependent tyrosyl phosphorylation of the phosphotyrosine phosphatase SH-PTP1 in murine T cells. Mol Cell Biol. 1994 Mar;14(3):1824–1834. doi: 10.1128/mcb.14.3.1824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lowe J. H., Huang C. L., Ives H. E. Sphingosine differentially inhibits activation of the Na+/H+ exchanger by phorbol esters and growth factors. J Biol Chem. 1990 May 5;265(13):7188–7194. [PubMed] [Google Scholar]
  36. Luttrell L. M., Hawes B. E., van Biesen T., Luttrell D. K., Lansing T. J., Lefkowitz R. J. Role of c-Src tyrosine kinase in G protein-coupled receptor- and Gbetagamma subunit-mediated activation of mitogen-activated protein kinases. J Biol Chem. 1996 Aug 9;271(32):19443–19450. doi: 10.1074/jbc.271.32.19443. [DOI] [PubMed] [Google Scholar]
  37. Ma Y. H., Reusch H. P., Wilson E., Escobedo J. A., Fantl W. J., Williams L. T., Ives H. E. Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma. J Biol Chem. 1994 Dec 2;269(48):30734–30739. [PubMed] [Google Scholar]
  38. Ma Y. H., Reusch H. P., Wilson E., Escobedo J. A., Fantl W. J., Williams L. T., Ives H. E. Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma. J Biol Chem. 1994 Dec 2;269(48):30734–30739. [PubMed] [Google Scholar]
  39. Maly K., Uberall F., Loferer H., Doppler W., Oberhuber H., Groner B., Grunicke H. H. Ha-ras activates the Na+/H+ antiporter by a protein kinase C-independent mechanism. J Biol Chem. 1989 Jul 15;264(20):11839–11842. [PubMed] [Google Scholar]
  40. Meyers R., Cantley L. C. Cloning and characterization of a wortmannin-sensitive human phosphatidylinositol 4-kinase. J Biol Chem. 1997 Feb 14;272(7):4384–4390. doi: 10.1074/jbc.272.7.4384. [DOI] [PubMed] [Google Scholar]
  41. Milarski K. L., Saltiel A. R. Expression of catalytically inactive Syp phosphatase in 3T3 cells blocks stimulation of mitogen-activated protein kinase by insulin. J Biol Chem. 1994 Aug 19;269(33):21239–21243. [PubMed] [Google Scholar]
  42. Nada S., Okada M., MacAuley A., Cooper J. A., Nakagawa H. Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. Nature. 1991 May 2;351(6321):69–72. doi: 10.1038/351069a0. [DOI] [PubMed] [Google Scholar]
  43. Nakanishi S., Kakita S., Takahashi I., Kawahara K., Tsukuda E., Sano T., Yamada K., Yoshida M., Kase H., Matsuda Y. Wortmannin, a microbial product inhibitor of myosin light chain kinase. J Biol Chem. 1992 Feb 5;267(4):2157–2163. [PubMed] [Google Scholar]
  44. Navé B. T., Haigh R. J., Hayward A. C., Siddle K., Shepherd P. R. Compartment-specific regulation of phosphoinositide 3-kinase by platelet-derived growth factor and insulin in 3T3-L1 adipocytes. Biochem J. 1996 Aug 15;318(Pt 1):55–60. doi: 10.1042/bj3180055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Noël J., Pouysségur J. Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms. Am J Physiol. 1995 Feb;268(2 Pt 1):C283–C296. doi: 10.1152/ajpcell.1995.268.2.C283. [DOI] [PubMed] [Google Scholar]
  46. Powis G., Bonjouklian R., Berggren M. M., Gallegos A., Abraham R., Ashendel C., Zalkow L., Matter W. F., Dodge J., Grindey G. Wortmannin, a potent and selective inhibitor of phosphatidylinositol-3-kinase. Cancer Res. 1994 May 1;54(9):2419–2423. [PubMed] [Google Scholar]
  47. Rivard N., McKenzie F. R., Brondello J. M., Pouysségur J. The phosphotyrosine phosphatase PTP1D, but not PTP1C, is an essential mediator of fibroblast proliferation induced by tyrosine kinase and G protein-coupled receptors. J Biol Chem. 1995 May 5;270(18):11017–11024. doi: 10.1074/jbc.270.18.11017. [DOI] [PubMed] [Google Scholar]
  48. Rozengurt E. Early signals in the mitogenic response. Science. 1986 Oct 10;234(4773):161–166. doi: 10.1126/science.3018928. [DOI] [PubMed] [Google Scholar]
  49. Sabe H., Knudsen B., Okada M., Nada S., Nakagawa H., Hanafusa H. Molecular cloning and expression of chicken C-terminal Src kinase: lack of stable association with c-Src protein. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2190–2194. doi: 10.1073/pnas.89.6.2190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sakaue M., Bowtell D., Kasuga M. A dominant-negative mutant of mSOS1 inhibits insulin-induced Ras activation and reveals Ras-dependent and -independent insulin signaling pathways. Mol Cell Biol. 1995 Jan;15(1):379–388. doi: 10.1128/mcb.15.1.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schaap D., van der Wal J., Howe L. R., Marshall C. J., van Blitterswijk W. J. A dominant-negative mutant of raf blocks mitogen-activated protein kinase activation by growth factors and oncogenic p21ras. J Biol Chem. 1993 Sep 25;268(27):20232–20236. [PubMed] [Google Scholar]
  52. Snyder M. A., Bishop J. M., McGrath J. P., Levinson A. D. A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity. Mol Cell Biol. 1985 Jul;5(7):1772–1779. doi: 10.1128/mcb.5.7.1772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Tang T. L., Freeman R. M., Jr, O'Reilly A. M., Neel B. G., Sokol S. Y. The SH2-containing protein-tyrosine phosphatase SH-PTP2 is required upstream of MAP kinase for early Xenopus development. Cell. 1995 Feb 10;80(3):473–483. doi: 10.1016/0092-8674(95)90498-0. [DOI] [PubMed] [Google Scholar]
  55. Thomason P. A., James S. R., Casey P. J., Downes C. P. A G-protein beta gamma-subunit-responsive phosphoinositide 3-kinase activity in human platelet cytosol. J Biol Chem. 1994 Jun 17;269(24):16525–16528. [PubMed] [Google Scholar]
  56. Toullec D., Pianetti P., Coste H., Bellevergue P., Grand-Perret T., Ajakane M., Baudet V., Boissin P., Boursier E., Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991 Aug 25;266(24):15771–15781. [PubMed] [Google Scholar]
  57. Vlahos C. J., Matter W. F., Hui K. Y., Brown R. F. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994 Feb 18;269(7):5241–5248. [PubMed] [Google Scholar]
  58. Voyno-Yasenetskaya T., Conklin B. R., Gilbert R. L., Hooley R., Bourne H. R., Barber D. L. G alpha 13 stimulates Na-H exchange. J Biol Chem. 1994 Feb 18;269(7):4721–4724. [PubMed] [Google Scholar]
  59. Xiao S., Rose D. W., Sasaoka T., Maegawa H., Burke T. R., Jr, Roller P. P., Shoelson S. E., Olefsky J. M. Syp (SH-PTP2) is a positive mediator of growth factor-stimulated mitogenic signal transduction. J Biol Chem. 1994 Aug 19;269(33):21244–21248. [PubMed] [Google Scholar]
  60. Yamaji Y., Amemiya M., Cano A., Preisig P. A., Miller R. T., Moe O. W., Alpern R. J. Overexpression of csk inhibits acid-induced activation of NHE-3. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6274–6278. doi: 10.1073/pnas.92.14.6274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zhang J., Zhang J., Benovic J. L., Sugai M., Wetzker R., Gout I., Rittenhouse S. E. Sequestration of a G-protein beta gamma subunit or ADP-ribosylation of Rho can inhibit thrombin-induced activation of platelet phosphoinositide 3-kinases. J Biol Chem. 1995 Mar 24;270(12):6589–6594. doi: 10.1074/jbc.270.12.6589. [DOI] [PubMed] [Google Scholar]
  62. van Biesen T., Hawes B. E., Luttrell D. K., Krueger K. M., Touhara K., Porfiri E., Sakaue M., Luttrell L. M., Lefkowitz R. J. Receptor-tyrosine-kinase- and G beta gamma-mediated MAP kinase activation by a common signalling pathway. Nature. 1995 Aug 31;376(6543):781–784. doi: 10.1038/376781a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES