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Basic Fibroblast Growth Factor Utilizes Both Types of Component Subunits of Gs for Dual Signaling in Human Adipocytes

2000; Elsevier BV; Volume: 275; Issue: 46 Linguagem: Inglês

10.1074/jbc.m002490200

ISSN

1083-351X

Autores

Heidemarie I. Krieger‐Brauer, Pankaj Medda, H Kather,

Tópico(s)

Growth Hormone and Insulin-like Growth Factors

Resumo

Basic fibroblast growth factor (bFGF), a ligand of receptor protein-tyrosine kinases, promoted the dissociation of Gs and had antagonistic stimulatory and inhibitory effects on adenylyl cyclase and NADPH oxidase in human fat cell plasma membranes. The bFGF-induced activation of adenylyl cyclase was blocked by COOH-terminal anti-Gαs, indicating that it was mediated by Gαs. The inhibitory action of bFGF was mimicked by exogenously supplied Gβγ-subunits and was reversed by anti-Gβ1/2, or βARK-CT, a COOH-terminal β-adrenergic receptor kinase fragment that specifically binds free Gβγ, indicating that it was transduced by Gβγ complexes. The bFGF-induced inhibition of NADPH-dependent H2O2 generation was also reversed by peptide 100–119, an inhibitor of Gs activation by ligand-occupied β-adrenergic receptors, indicating that the Gβγ complexes mediating the inhibitory action of the growth factor are derived from Gs. The findings suggest a direct, non-kinase-dependent, coupling of bFGF receptor(s) to Gs and provide the first example of a ligand of receptor protein-tyrosine kinases that is capable of utilizing both types of component subunits of a single heterotrimeric G protein for dual signaling in a single cell type. Basic fibroblast growth factor (bFGF), a ligand of receptor protein-tyrosine kinases, promoted the dissociation of Gs and had antagonistic stimulatory and inhibitory effects on adenylyl cyclase and NADPH oxidase in human fat cell plasma membranes. The bFGF-induced activation of adenylyl cyclase was blocked by COOH-terminal anti-Gαs, indicating that it was mediated by Gαs. The inhibitory action of bFGF was mimicked by exogenously supplied Gβγ-subunits and was reversed by anti-Gβ1/2, or βARK-CT, a COOH-terminal β-adrenergic receptor kinase fragment that specifically binds free Gβγ, indicating that it was transduced by Gβγ complexes. The bFGF-induced inhibition of NADPH-dependent H2O2 generation was also reversed by peptide 100–119, an inhibitor of Gs activation by ligand-occupied β-adrenergic receptors, indicating that the Gβγ complexes mediating the inhibitory action of the growth factor are derived from Gs. The findings suggest a direct, non-kinase-dependent, coupling of bFGF receptor(s) to Gs and provide the first example of a ligand of receptor protein-tyrosine kinases that is capable of utilizing both types of component subunits of a single heterotrimeric G protein for dual signaling in a single cell type. fibroblast growth factor basic fibroblast growth factor guanosine 5′-O-(3-thiotriphosphate) guanyl 5′-(β,γ-imino)triphosphate 3-(N-morpholino)propanesulfonic acid 2-(N-morpholino)ethanesulfonic acid glutathioneS-transferase β-adrenergic receptor kinase guanosine 5′-O-2-(thio)diphosphate The fibroblast growth factors (FGFs)1 are a family of heparin-binding growth factors and oncogenes with at least 20 members (1Fernig D.G. Gallagher J.T. Prog. Growth Factor Res. 1994; 5: 353-377Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 2Burke D. Wilkes D. Blundell T.L. Malcolm S. Trends Biochem. Sci. 1998; 23: 59-62Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 3Szebenyi G. Fallon J.S. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar). Basic fibroblast growth factor (bFGF, FGF-2) or a related heparin-binding peptide is produced by white and brown adipocytes (4Teichert-Kuliszewska K. Hamilton B.S. Deitel M. Roncari D.A.K. J. Clin. Invest. 1992; 90: 1226-1231Crossref PubMed Scopus (36) Google Scholar,5Yamashita H. Sato N. Kizaki T. Oh-ishi S. Segawa M. Saitoh D. Ohira Y. Ohno H. Cell Growth Differ. 1995; 6: 1457-1462PubMed Google Scholar). The growth factor stimulates the proliferation of preadipocytes and inhibits their conversion to adipocytes. The expression of bFGF is decreased appreciably during adipose differentiation and is increased in obesity (4Teichert-Kuliszewska K. Hamilton B.S. Deitel M. Roncari D.A.K. J. Clin. Invest. 1992; 90: 1226-1231Crossref PubMed Scopus (36) Google Scholar). Interestingly, another member of the FGF family, aFGF (FGF-1), accelerated the conversion of 3T3 L1-preadipocytes to adipocytes in the presence of insulin and was adipogenic in itself (6Krieger-Brauer H.I. Kather H. Biochem. J. 1995; 307: 549-556Crossref PubMed Scopus (119) Google Scholar). The members of the fibroblast growth factor family interact with cell surface low affinity heparan sulfate that facilitates binding to their receptors (1Fernig D.G. Gallagher J.T. Prog. Growth Factor Res. 1994; 5: 353-377Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 2Burke D. Wilkes D. Blundell T.L. Malcolm S. Trends Biochem. Sci. 1998; 23: 59-62Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 3Szebenyi G. Fallon J.S. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar, 7Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2075) Google Scholar). These consist of a family of high affinity receptor tyrosine kinases (FGF receptors 1–4) displaying overlapping affinities for the various FGFs (1Fernig D.G. Gallagher J.T. Prog. Growth Factor Res. 1994; 5: 353-377Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 2Burke D. Wilkes D. Blundell T.L. Malcolm S. Trends Biochem. Sci. 1998; 23: 59-62Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 3Szebenyi G. Fallon J.S. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar, 7Yayon A. Klagsbrun M. Esko J.D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2075) Google Scholar). Alternative splicing generates isoforms of receptors 1–3 that exhibit unique binding characteristics (1Fernig D.G. Gallagher J.T. Prog. Growth Factor Res. 1994; 5: 353-377Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 2Burke D. Wilkes D. Blundell T.L. Malcolm S. Trends Biochem. Sci. 1998; 23: 59-62Abstract Full Text PDF PubMed Scopus (153) Google Scholar, 3Szebenyi G. Fallon J.S. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar). In addition, an unrelated cysteine-rich transmembrane protein of unknown function has also been identified as a high affinity receptor for FGFs (8Burrus L.W. Zuber M.E. Luedecke B.A. Olwin B.B. Mol. Cell. Biol. 1992; 12: 5600-5609Crossref PubMed Scopus (104) Google Scholar, 9Mason I.J. Cell. 1994; 78: 547-552Abstract Full Text PDF PubMed Scopus (525) Google Scholar). Mature human adipocytes and 3T3 L1-preadipocytes contain a plasma membrane-bound H2O2 generating system that is under antagonistic control by various hormones, growth factors, and cytokines, including ligands of receptor protein-tyrosine kinases, such as insulin and various isoforms of PDGF and FGF (6Krieger-Brauer H.I. Kather H. Biochem. J. 1995; 307: 549-556Crossref PubMed Scopus (119) Google Scholar, 10Krieger-Brauer H.I. Kather H. J. Clin. Invest. 1992; 89: 1006-1013Crossref PubMed Scopus (184) Google Scholar, 11Krieger-Brauer H.I. Kather H. Biochem. J. 1995; 307: 543-548Crossref PubMed Scopus (67) Google Scholar, 12Krieger-Brauer H.I. Medda P.K. Kather H. J. Biol. Chem. 1997; 272: 10135-10143Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 13Krieger-Brauer H.I. Medda P.K. Hebling U. Kather H. J. Biol. Chem. 1999; 274: 28308-28313Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 14Krieger-Brauer H.I. Medda P.K. Sattel B. Kather H. J. Biol. Chem. 2000; 275: 2486-2490Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Recent work revealed that the stimulatory effect of insulin on NADPH-dependent H2O2 generation is transduced by a G protein (Gαi2), whereas the inhibitory action of the β-adrenergic agonist, isoproterenol, which signals through a prototypical Gs-coupled receptor, is mediated by Gβγ derived from Gs (13Krieger-Brauer H.I. Medda P.K. Hebling U. Kather H. J. Biol. Chem. 1999; 274: 28308-28313Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 14Krieger-Brauer H.I. Medda P.K. Sattel B. Kather H. J. Biol. Chem. 2000; 275: 2486-2490Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In this work we examined whether the inhibitory effect of bFGF, another ligand of tyrosine kinase receptors, on NADPH-dependent H2O2 generation is also transmitted by a G protein. Antibodies directed against the C terminus of Gαs(RM/1), common anti-Gβ (SW/1), and anti-Gβ1/2 were from PerkinElmer Life Sciences, Bad Homburg, Germany (RM/1 and SW/1), and Upstate Biotechnology Inc., Lake Placid, NY, respectively. Recombinant Gαi2, Gαs, and protein A-agarose were from Calbiochem. Hybond polyvinylidene difluoride membranes were obtained from Amersham Pharmacia Biotech-Buchler, Braunschweig, Germany. Insulin, GTPγS, GDPβS, and GTP were from Roche Molecular Biochemicals, Mannheim, Germany. Isoproterenol and rbFGF were from Sigma AG, München, Germany. Human albumin and luminol were obtained from Behring Werke, Marburg, Germany. The GST fusion protein containing a carboxyl-terminal fragment (residues 546–670) of the β-adrenergic receptor kinase (βARK 1CT-GST) and the corresponding GST protein were a gift of Dr. R. Lefkowitz, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC (16Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). Experimental details have been described in detail elsewhere (5Yamashita H. Sato N. Kizaki T. Oh-ishi S. Segawa M. Saitoh D. Ohira Y. Ohno H. Cell Growth Differ. 1995; 6: 1457-1462PubMed Google Scholar, 9Mason I.J. Cell. 1994; 78: 547-552Abstract Full Text PDF PubMed Scopus (525) Google Scholar, 10Krieger-Brauer H.I. Kather H. J. Clin. Invest. 1992; 89: 1006-1013Crossref PubMed Scopus (184) Google Scholar, 11Krieger-Brauer H.I. Kather H. Biochem. J. 1995; 307: 543-548Crossref PubMed Scopus (67) Google Scholar, 12Krieger-Brauer H.I. Medda P.K. Kather H. J. Biol. Chem. 1997; 272: 10135-10143Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Briefly, adipose tissue was from nondiabetic subjects undergoing elective abdominal or cosmetic breast surgery. The specimens were cut into small pieces, and fat cells were isolated by the method of Rodbell (17Rodbell M. J. Biol. Chem. 1964; 239: 375-384Abstract Full Text PDF PubMed Google Scholar) in a HEPES-buffered Krebs-Henseleit solution, pH 7.4, containing 20 mm HEPES, 10 mmNaHCO3, 5 mm glucose, 20 g/liter albumin, and 1 mg/ml collagenase (CLS, Worthington). After 30 min, fat cells were washed and re-suspended in 10 volumes of an ice-cold lysing medium containing 20 mm MES, pH 6.0, 2 mmMgCl2, 1 mm CaCl2, 5 mmKCl, and 100 mg/liter soybean trypsin inhibitor. Mechanical shaking completed cell lysis, and fat cell ghosts were collected by low speed centrifugation (1,000 × g, 4 °C, 20 min). Similar to other effector systems, such as adenylyl cyclases, it is difficult to assess the effects of inhibitory ligands on NADPH-dependent H2O2 generation under basal conditions, where enzyme activity is low. Since direct activators of NADPH oxidase are presently unknown, enzyme activity was increased by insulin, which stimulates NADPH oxidase via Gαi2 (12Krieger-Brauer H.I. Medda P.K. Kather H. J. Biol. Chem. 1997; 272: 10135-10143Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). A two-step procedure was used, as reported elsewhere (6Krieger-Brauer H.I. Kather H. Biochem. J. 1995; 307: 549-556Crossref PubMed Scopus (119) Google Scholar, 10Krieger-Brauer H.I. Kather H. J. Clin. Invest. 1992; 89: 1006-1013Crossref PubMed Scopus (184) Google Scholar, 11Krieger-Brauer H.I. Kather H. Biochem. J. 1995; 307: 543-548Crossref PubMed Scopus (67) Google Scholar, 12Krieger-Brauer H.I. Medda P.K. Kather H. J. Biol. Chem. 1997; 272: 10135-10143Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 13Krieger-Brauer H.I. Medda P.K. Hebling U. Kather H. J. Biol. Chem. 1999; 274: 28308-28313Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 14Krieger-Brauer H.I. Medda P.K. Sattel B. Kather H. J. Biol. Chem. 2000; 275: 2486-2490Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Plasma membranes from untreated or insulin-treated cells were first exposed to hormones and growth factors and were then assayed for NADPH oxidase activity. The activation step was carried out in 30 mm MOPS, pH 7.5, containing 120 mm KCl, 1.4 CaCl2, 2.5 mm MgCl2, 10 mm NaHCO3, and 0.1% human albumin. Membranes were first incubated with various concentrations of hormones and growth factors for 5 min to allow receptor occupation. Thereafter, guanine nucleotides were added, as indicated. After 20 min, ghosts were collected by centrifugation, washed, and then re-suspended in 30 mm MES, pH 5.8, containing 120 mm NaCl, 4 mm MgCl2, 1.2 mmKH2PO4, 1 mm NaN3, 250 μm NADPH, and 10 μm FAD for determination of NADPH-dependent H2O2 generation. Reactions were started by addition of 250 μm NADPH and terminated after 30 min at 37 °C by adding HCl to yield a final concentration of 100 μm (10Krieger-Brauer H.I. Kather H. J. Clin. Invest. 1992; 89: 1006-1013Crossref PubMed Scopus (184) Google Scholar). To assess the effects of G protein antibodies on NADPH-dependent H2O2 generation, membranes were first exposed to antibodies at 0 °C for 45 min and were then subjected to the two-step procedure described above. In contrast to other G proteins, Gαs does not dissociate in the presence of GTPγS alone under the conditions used in the present experiments (18Toyoshige M. Okuya S. Rebois R.V. Biochemistry. 1994; 33: 4865-4871Crossref PubMed Scopus (26) Google Scholar, 19Toyoshige M. Basi N.S. Rebois R.V. J. Biol. Chem. 1996; 271: 8791-8795Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Plasma membranes (80 μg) were suspended in 200 μl of MOPS (30 mm, pH 7.5) containing 120 mm KCl, 2.5 mm MgCl2, 1.4 mm CaCl2, and 0.1% human serum albumin in the absence or presence of 0.3 nm bFGF for 5 min at ambient temperature. Thereafter, 50 μm GTPγS was added and incubations were continued for another 25 min. The membranes were recovered by centrifugation and solubilized in 1% Triton X-100 for 30 min at 0 °C. Gswas immunoprecipitated by an antibody directed against the COOH terminus of Gαs (RM/1), which does not discriminate between heterotrimeric and monomeric Gαs (18Toyoshige M. Okuya S. Rebois R.V. Biochemistry. 1994; 33: 4865-4871Crossref PubMed Scopus (26) Google Scholar, 19Toyoshige M. Basi N.S. Rebois R.V. J. Biol. Chem. 1996; 271: 8791-8795Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Immunoprecipitations were carried out in 10 mm Tris-HCl, pH 7.4, containing 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.5% Nonidet P-40, proteinase inhibitors (0.4 mm phenylmethylsulfonyl fluoride, 2 μmleupeptin, 2 μm pepstatin, and 1 unit/ml aprotinin), antibody RM/1 (at a dilution of 1:80), and 50 μm GTPγS. The samples (30 μg of membrane protein/100 μl of precipitation buffer) were incubated overnight at 4 °C. The immune complexes were captured with protein A-agarose beads and washed three times with 1 ml of washing buffer (10 mm Tris-HCl, pH 7.4, containing 1% Triton X-100, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, and proteinase inhibitors). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Western blotting was performed using anti-Gαs (RM/1) and anti-Gβ (SW/1) antibodies (PerkinElmer Life Sciences). Bands were visualized by chemiluminescence using the ECL kit from Amersham Pharmacia Biotech and quantified by densitometric analysis. The amount of Gβ was normalized to the amount of Gαs immunoprecipitated for each condition. Adenylyl cyclase activity of human fat cell plasma membranes was determined in 50 mm Tris-HCl, pH 7.5, containing 1 mm ATP, 2.5 mm MgCl2, 0.5 mm EDTA, 10 μm GTP, 10 mmcreatine phosphate, 0.1 mg creatine kinase, and 0.1% bovine serum albumin in a final volume of 100 μl (20Katada T. Amano T. Ui M. J. Biol. Chem. 1982; 257: 3739-3746Abstract Full Text PDF PubMed Google Scholar). Reactions were initiated by addition of 3–5 μg of membrane protein and incubated for 15 min at 37 °C in the absence or presence of isoproterenol or bFGF, as indicated. Reactions were terminated by 100 μl of ice-cold perchloric acid (5%). cAMP was determined by radioimmunoassay after neutralization. For determination of cellular cAMP, isolated adipocytes (50,000 cells) were incubated in HEPES-buffered Krebs-Henseleit solution, pH 7.4, containing 0.5 mm isobutylmethylxanthine, 2% human serum albumin, and isoproterenol or bFGF, as indicated, for 15 min at 37 °C in a total volume of 0.5 ml. Incubations were terminated by an equal volume of 5% ice-cold perchloric acid, and cAMP was determined as described above. Fig. 1 A shows the effects of 0.1 nm bFGF on NADPH-dependent H2O2 generation in the presence of increasing concentrations of GTPγS in membranes from cells that had been pretreated with 10 nm insulin. Similar to early observations with the turkey erythrocyte adenylyl cyclase, where activation by GMP(PNP) appeared to be totally dependent on hormone because the hormone-independent rate of activation was slow, GTPγS alone had no measurable effect under the conditions used (21Orly J. Schramm M. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3433-3437Crossref PubMed Scopus (168) Google Scholar). The inhibitory action of bFGF became apparent at concentrations of GTPγS exceeding 5 μm and was half-maximal at approximately 10 μm GTPγS. At a maximal concentration of GTPγS (50 μm), NADPH-dependent H2O2 generation was reduced by approximately two-thirds in the presence of 0.1 nm bFGF. Fig. 1 B shows a concentration-response curve for bFGF in the presence of a maximal concentration of GTPγS (50 μm). The bFGF-induced inhibition of NADPH-dependent H2O2 generation was half-maximal at about 1 pm and was maximal at approximately 30 pm. Recent work showed that the effects of another inhibitor of NADPH-dependent H2O2 generation, the β-adrenergic agonist isoproterenol, are transduced by βγ subunits derived from Gs (13Krieger-Brauer H.I. Medda P.K. Hebling U. Kather H. J. Biol. Chem. 1999; 274: 28308-28313Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 14Krieger-Brauer H.I. Medda P.K. Sattel B. Kather H. J. Biol. Chem. 2000; 275: 2486-2490Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Therefore, the possibility was explored that the effects of bFGF were also mediated by Gβγ. Indeed, the inhibitory action of bFGF was dose-dependently reversed by an antibody directed against an internal sequence of β1/β2 subunits which had no influence on basal or insulin-stimulated rates of NADPH-dependent H2O2 generation (Fig. 2 A). Maximal effects were observed at an antibody dilution of 1:100 in the presence of 0.3 nm bFGF (Fig. 2 A). The inhibitory effect of bFGF could also be reversed by 10 μm βARK-1CT, a fragment of the β-adrenergic receptor kinase that specifically binds free Gβγ (16Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). Reversal of the bFGF-induced suppression of NADPH-dependent H2O2 generation was half-maximal at 0.5 μm βARK-1CT, maximal effects were observed at 10 μm (Fig. 2 B). The GST protein alone failed to reverse the inhibitory effects of isoproterenol and bFGF (not shown). Fig. 3 shows the effects of aFGF, bFGF, and insulin on Gs subunit composition. In contrast to other heterotrimeric G proteins, Gs does not dissociate in the presence of GTPγS alone under the conditions used (18Toyoshige M. Okuya S. Rebois R.V. Biochemistry. 1994; 33: 4865-4871Crossref PubMed Scopus (26) Google Scholar, 19Toyoshige M. Basi N.S. Rebois R.V. J. Biol. Chem. 1996; 271: 8791-8795Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Plasma membranes were therefore treated with 50 μm GTPγS in the absence or presence of 0.3 nm bFGF, 3 nmaFGF, or 5 nm insulin. Dissociation of Gs into its component subunits was monitored by an immunoblot technique utilizing antibody RM/1, which does not discriminate between heterotrimeric and monomeric Gαs. Fig. 3 demonstrates that the amount of Gβ recovered in RM/1 immunoprecipitates was selectively reduced in membranes that had been pretreated with bFGF, while aFGF and insulin failed to influence the subunit composition of immunoprecipitated Gs. In the absence of GTPγS, bFGF had no influence on the subunit composition of Gs. Thus, the growth factor caused a guanine nucleotide triphosphate-dependent dissociation of Gs which is characteristic for a receptor-mediated activation. The latter finding suggested that bFGF may also stimulate adenylyl cyclase, which was in fact the case. Fig.4 shows the effects of bFGF and isoproterenol on cAMP production and NADPH-dependent H2O2 generation in human fat cell plasma membranes. bFGF caused a concentration-dependent increase of cAMP accumulation that became apparent at picomolar concentrations, suggesting that it was transmitted by discrete ligand-specific receptors (Fig. 4 A). Compared with the β-adrenergic agonist isoproterenol, which elicited a 6-fold increase in cAMP production, bFGF was able to induce only a modest (3-fold) activation of adenylyl cyclase in fat cell plasma membranes (Fig. 4 A), whereas both compounds were of comparable efficacy in suppressing NADPH-dependent H2O2 generation (Fig. 4 B). The differential ability of isoproterenol and bFGF to increase cAMP became even more apparent in intact cells, where a bFGF-induced increase in cAMP was only detectable in the presence of a phosphodiesterase inhibitor (isobutylmethylxanthine). On the average, cellular cAMP levels were doubled by 10 nm bFGF under these conditions, a figure that contrasts to a more than 100-fold elevation seen in the presence of 1 μm isoproterenol, consistent with recent observations in NIH 3T3 cells (22Pursiheimo J.P. Jalkanen M. Taskén K. Jaakola P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 168-173Crossref PubMed Scopus (29) Google Scholar). Fig. 5 shows the effects of a COOH-terminal decapeptide (peptide 385–395), a sequence comprising residues 100–119 within the α-helical domain of Gαs(peptide 100–119), and a homologous sequence of Gαt on the bFGF-induced stimulation of adenylyl cyclase activity and the inhibitory action of the growth factor on insulin-stimulated rates of NADPH-dependent H2O2 generation. At the concentrations used, both the COOH-terminal and the α-helical Gαs peptide inhibited the stimulatory effect of bFGF on cAMP formation. By contrast, the inhibitory effect of bFGF on NADPH-dependent H2O2 generation was only reversed by peptide 100–119 whereas the COOH-terminal decapeptide had no effect. The effects of the peptide 100–119 were specific in as much as a homologous peptide derived from Gαt had no influence on the antagonistic effects of bFGF on cAMP production and H2O2 generation. In addition, the peptide did not influence the stimulatory effect of insulin, which is mediated via Gαi2. In this report it is shown that bFGF, a ligand of receptor protein-tyrosine kinases, stimulates adenylyl cyclase and inhibits NADPH-dependent H2O2 generation in human fat cell plasma membranes. The mechanisms by which bFGF elicited its antagonistic stimulatory and inhibitory effects on adenylyl cyclase and NADPH oxidase were confined to the plasma membrane, independent of second messengers, and operated in the absence of ATP, indicating that established pathways of signal transduction, including the tyrosine kinase activity of bFGF receptor(s) were not involved. Along with the observation that bFGF promoted dissociation of Gs these findings suggested that FGF receptor(s) are directly coupled to Gs, via a non-tyrosine kinase-dependent mechanism. Indeed, human adipocytes express at least two members of the FGF receptor family, e.g. FGFR1 and FGFR2. Both isoforms co-eluted with Gαs and Gβ upon affinity chromatography on Sepharose coupled to antibodies directed against their carboxyl-terminal sequences and hence may both contribute to bFGF-signaling via Gs. 2H. I. Krieger-Brauer, unpublished results. Antibodies directed against the COOH termini of Gα subunits and peptides corresponding to their target sequences are thought to block receptor recognition and activation, and are therefore widely used in functional studies aimed at assessing receptor/G protein coupling (23Stehno-Bittel L. Krapivinsky G. Krapivinsky L. Perez-Terzic C. Clapham D.E. J. Biol. Chem. 1995; 270: 30068-30074Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24Macrez N. Morel J.L. Kalkbrenner K. Viard P. Schultz G. Mironneau J. J. Biol. Chem. 1997; 272: 23180-23185Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 25Bomsel M. Mostov K.E. J. Biol. Chem. 1993; 268: 25824-25835Abstract Full Text PDF PubMed Google Scholar, 26Murthy K.S. Coy D.H. Makhlouf G.M. J. Biol. Chem. 1996; 271: 23458-23463Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The stimulatory effect of bFGF on adenylyl cyclase could be blocked by COOH-terminal anti-Gαs and a peptide corresponding to its antigenic site, indicating that it was in fact transmitted by Gαs. The bFGF-induced inhibition of NADPH-dependent H2O2 generation also required the participation of a G protein. In contrast to its stimulatory effects on adenylyl cyclase, the inhibitory action of bFGF appeared to be mediated via Gβγ subunits, however, based on observations that this latter effect was specifically reversed by anti-Gβ or a COOH-terminal β-adrenergic receptor kinase fragment (βARK-CT) that sequesters free Gβγ, and was mimicked by exogenously supplied Gβγ subunits (14Krieger-Brauer H.I. Medda P.K. Sattel B. Kather H. J. Biol. Chem. 2000; 275: 2486-2490Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 15Degtiar V.E. Harhammer R. Nürnberg B. J. Physiol. 1997; 502: 321-333Crossref PubMed Scopus (41) Google Scholar). In contrast to the stimulatory effect of bFGF on adenylyl cyclase, its inhibitory action on NADPH-dependent H2O2 generation was not influenced by COOH-terminal anti-Gαs. A possible explanation for this unexpected finding would be that activation of Gs by bFGF receptors may not yield enough, or inappropriately composed, βγ subunits to account for inhibition of NADPH-dependent H2O2 generation, as has been proposed for the regulation of type II adenylyl cyclase (27Taussig R. Tang W.J. Hepler J.R. Gilman A.G. J. Biol. Chem. 1994; 269: 6093-6100Abstract Full Text PDF PubMed Google Scholar, 28Bayewitch M.L. Avidor-Reiss T. Levy R. Pfeuffer T. Nevo I. Simonds W.F. Vogel Z. J. Biol. Chem. 1998; 273: 2273-2276Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). However, the effects of COOH-terminal Gα antibodies on Gβγ-mediated responses are variable and may depend on receptors, G proteins, and cell types studied (13Krieger-Brauer H.I. Medda P.K. Hebling U. Kather H. J. Biol. Chem. 1999; 274: 28308-28313Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 14Krieger-Brauer H.I. Medda P.K. Sattel B. Kather H. J. Biol. Chem. 2000; 275: 2486-2490Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 23Stehno-Bittel L. Krapivinsky G. Krapivinsky L. Perez-Terzic C. Clapham D.E. J. Biol. Chem. 1995; 270: 30068-30074Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 24Macrez N. Morel J.L. Kalkbrenner K. Viard P. Schultz G. Mironneau J. J. Biol. Chem. 1997; 272: 23180-23185Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 25Bomsel M. Mostov K.E. J. Biol. Chem. 1993; 268: 25824-25835Abstract Full Text PDF PubMed Google Scholar, 26Murthy K.S. Coy D.H. Makhlouf G.M. J. Biol. Chem. 1996; 271: 23458-23463Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Therefore, the lacking effects of COOH-terminal anti-Gαs do not permit conclusions to be made about the origin of the Gβγ complexes transmitting the inhibitory action of bFGF. Indeed, we have recently shown that Gs is capable of providing enough appropriately composed Gβγ subunits to account for the inhibition of NADPH-dependent H2O2 generation seen in the presence of isoproterenol (14Krieger-Brauer H.I. Medda P.K. Sattel B. Kather H. J. Biol. Chem. 2000; 275: 2486-2490Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). However, bFGF was considerably less efficacious than isoproterenol in activating adenylyl cyclase, although both compounds were of comparable efficacy in inhibiting of NADPH-dependent H2O2 generation. It is possible that the differential effect of bFGF on both systems is merely an apparent one, because the adenylyl cyclase assays contained GTP, whereas NADPH oxidase activity was determined in the presence of GTPγS. However, the differential ability of bFGF and isoproterenol to act as stimulators of adenylyl cyclase occurred under identical conditions and was even more pronounced in intact cells, indicating that it was physiologically relevant. A possible explanation, among others, for this difference would be that activated β-adrenergic receptors recruit more Gs for signaling than the receptor(s) for bFGF. It therefore appeared possible that βγ complexes derived from G proteins other than Gs participated in bFGF receptor signaling. Indeed, it has been reported that bFGF receptor(s) may also couple to pertussis toxin-sensitive G proteins in certain cell types (29Sa G. Fox P.L. J. Biol. Chem. 1994; 269: 3219-3225Abstract Full Text PDF PubMed Google Scholar, 30Fedorov Y.V. Jones N.C. Olwin B.B. Mol. Cell. Biol. 1998; 18: 5780-5788Crossref PubMed Scopus (49) Google Scholar). To obtain definitive information about the origin of the Gβγ complexes transmitting the inhibitory effects of bFGF, we utilized a peptide comprising residues 100–119 of Gαs (K-20). Recent work showed that this peptide antagonized the antagonistic effects of the β-adrenergic agonist isoproterenol on adenylyl cyclase and NADPH oxidase that are transmitted by Gαs and Gβγs, respectively (13Krieger-Brauer H.I. Medda P.K. Hebling U. Kather H. J. Biol. Chem. 1999; 274: 28308-28313Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 14Krieger-Brauer H.I. Medda P.K. Sattel B. Kather H. J. Biol. Chem. 2000; 275: 2486-2490Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The present studies revealed that peptide 100–119 reversed the effects of bFGF on NADPH-dependent H2O2 generation and cAMP production as well. The peptide did not influence the stimulatory effect of insulin, which is mediated by Gαi2. In addition, a homologous peptide derived from Gαt had no influence on the antagonistic effects of bFGF on cAMP production and H2O2 generation, indicating that peptide 100–119 is specific for Gαs, and does not block the interactions of receptors with other G proteins. Thus, the latter finding strongly supported the view that the βγ subunits mediating the bFGF-induced inhibition of NADPH-dependent H2O2 generation were exclusively derived from Gs. G proteins are typically coupled to heptahelical receptors. However, it is becoming increasingly clear that G proteins may be responsible for transmitting signals of other types of receptors as well, including receptor kinases. Aside from the stimulatory effect of insulin on NADPH-dependent H2O2 generation, which is mediated by Gαi2 (12Krieger-Brauer H.I. Medda P.K. Kather H. J. Biol. Chem. 1997; 272: 10135-10143Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), these include activation of cardiac adenylyl cyclase by epidermal growth factor through Gαs and induction of fibroblast transformation by TGFβ1 via Gαi1, respectively (31Nair B.G. Parikh B. Milligan G. Patel T.B. J. Biol. Chem. 1990; 265: 21317-21322Abstract Full Text PDF PubMed Google Scholar, 32Kataoka R. Sherlock J. Lanier S.M. J. Biol. Chem. 1993; 268: 19851-19857Abstract Full Text PDF PubMed Google Scholar). The receptors of some growth factors, such as insulin and epidermal growth factor, may couple to multiple G proteins (12Krieger-Brauer H.I. Medda P.K. Kather H. J. Biol. Chem. 1997; 272: 10135-10143Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 33Jo H. Cha B.Y. Davis H.W. Mc Donald J.M. Endocrinology. 1992; 131: 2855-2862Crossref PubMed Scopus (28) Google Scholar, 34Stryjek-Kaminska D. Piiper A. Zeuzem S. Biochem. 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However, bFGF is the first example of a ligand of receptor protein-tyrosine kinases that has been shown to be capable of signaling through both types of G protein subunits of a single class of heterotrimeric G proteins in a single cell type. As pointed out recently (14Krieger-Brauer H.I. Medda P.K. Sattel B. Kather H. J. Biol. Chem. 2000; 275: 2486-2490Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), the mechanisms by which Gαi2 (which mediates the stimulatory effects of insulin) and Gβγs modulate the activity of the human fat cell oxidase remain to be defined. Current knowledge suggests that activation of heterotrimeric G proteins by ligand-receptor complexes is achieved by exchange of GTP for GDP on the α-subunit, and this is thought to faciltate dissociation into α and βγ subunits (38Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (934) Google Scholar,39Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1283) Google Scholar). G protein-sensitive effectors are then directly regulated by GTP-liganded α-subunits, βγ subunits, or both (38Hamm H.E. J. Biol. Chem. 1998; 273: 669-672Abstract Full Text Full Text PDF PubMed Scopus (934) Google Scholar, 39Neer E.J. Cell. 1995; 80: 249-257Abstract Full Text PDF PubMed Scopus (1283) Google Scholar). Consistently, the mechanisms by which insulin and bFGF modulated NADPH-dependent H2O2 generation were confined to the plasma membrane and were independent of soluble second messengers, making it likely that activated Gαi2and Gβγs acted upon NADPH oxidase directly. However, indirect mechanisms of action involving intermediate membrane-associated effectors or association of Gαi2 and Gβγs to yield the inactive Gi2 heterotrimer cannot definitively be ruled out (40Knaus U.G. Morris S. Dong H.J. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Crossref PubMed Scopus (357) Google Scholar, 41Irani K. Xia Y. Zweier J.L. Sollot S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1424) Google Scholar, 42Katada T. Bokoch G.M. Smigel M.D. Ui M. Gilman A.G. J. Biol. Chem. 1984; 259: 3586-3595Abstract Full Text PDF PubMed Google Scholar). The generation of H2O2 and other reactive oxygen species seems to be a common signaling event for hormones and cytokines that regulate cell growth and differentiation (40Knaus U.G. Morris S. Dong H.J. Chernoff J. Bokoch G.M. Science. 1995; 269: 221-223Crossref PubMed Scopus (357) Google Scholar, 41Irani K. Xia Y. Zweier J.L. Sollot S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1424) Google Scholar, 42Katada T. Bokoch G.M. Smigel M.D. Ui M. Gilman A.G. J. Biol. 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The finding that the signal of bFGF, an inhibitor of adipogenesis, is transduced by βγ subunits that are derived from Gs is consistent with these latter observations and provides further evidence in support of the concept that the H2O2 produced in response to hormones, growth factors, and cytokines may be important in the regulation of adipocyte differentiation and maintenance of the differentiated state. The physiological role of the bFGF-induced stimulation of cAMP production, if any, is currently unknown. In intact cells, a measurable elevation of cAMP levels by bFGF was only observed in the simultaneous presence of a phosphodiesterase inhibitor. In addition, preliminary evidence suggests that bFGF has no lipolytic activity in human adipocytes.2 An interesting possibility that will be addressed in future studies is raised by observations in hamster fibroblasts showing that bFGF alone has no effects on cAMP levels but may be capable of potentiating the effects of other stimulators of adenylyl cyclase via activation of Gs (46Magnaldo I. Pouyssegur J. Paris S. Cell. Signal. 1989; 1: 507-517Crossref PubMed Scopus (8) Google Scholar). In conclusion, the present findings show that bFGF, a ligand of receptor protein-tyrosine kinases, is capable of utilizing Gαs as well as Gβγs for dual signaling in a single cell type via a non-kinase-dependent mechanism, and suggest a physical interaction of one or more members of the FGF receptor family with Gs. These results confirm and extend recent observations demonstrating that the human fat cell oxidase is under antagonistic control by Gα and Gβγ subunits derived from different G proteins and provide further evidence in support of the concept that this membrane-bound redox system represents an universal effector system for hormones, growth factors, and cytokines linking ligand binding to cell surface receptors to changes in the intracellular redox equilibrium. We are indebted to Brigitte Sattel and Ulrike Hebling for expert technical assistance, and thank Dr. G. Schultz, Freie Universität Berlin, Germany, for supplying purified G protein subunits, and Dr. R. Lefkowitz, Duke University Medical Center, Durham, NC, for supplying the GST fusion protein containing a carboxyl-terminal fragment of the β-adrenergic receptor kinase (βARK 1CT-GST) and the corresponding GST protein.

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