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

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.

The fibroblast growth factors (FGFs) 1 are a family of heparin-binding growth factors and oncogenes with at least 20 members (1)(2)(3). Basic fibroblast growth factor (bFGF, FGF-2) or a related heparin-binding peptide is produced by white and brown adipocytes (4,5). 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 (4). 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 (6).
The members of the fibroblast growth factor family interact with cell surface low affinity heparan sulfate that facilitates binding to their receptors (1)(2)(3)7). These consist of a family of high affinity receptor tyrosine kinases (FGF receptors 1-4) displaying overlapping affinities for the various FGFs (1)(2)(3)7). Alternative splicing generates isoforms of receptors 1-3 that exhibit unique binding characteristics (1)(2)(3). In addition, an unrelated cysteine-rich transmembrane protein of unknown function has also been identified as a high affinity receptor for FGFs (8,9).
Mature human adipocytes and 3T3 L1-preadipocytes contain a plasma membrane-bound H 2 O 2 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 (6, 10 -14).
Recent work revealed that the stimulatory effect of insulin on NADPH-dependent H 2 O 2 generation is transduced by a G protein (G␣ i2 ), whereas the inhibitory action of the ␤-adrenergic agonist, isoproterenol, which signals through a prototypical G s -coupled receptor, is mediated by G␤␥ derived from G s (13,14). In this work we examined whether the inhibitory effect of bFGF, another ligand of tyrosine kinase receptors, on NADPHdependent H 2 O 2 generation is also transmitted by a G protein.
The GST fusion protein containing a carboxyl-terminal fragment (residues 546 -670) of the ␤-adrenergic receptor kinase (␤ARK 1CT-GST) and the corresponding GST protein were a gift of Dr. R. Lefkowitz, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC (16).

Methods
Subjects, Preparation of Fat Cells, and Fat Cell Ghosts-Experimental details have been described in detail elsewhere (5, 9 -12). 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 (17) in a HEPES-buffered Krebs-Henseleit solution, pH 7.4, containing 20 mM HEPES, 10 mM NaHCO 3 , 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 mM MgCl 2 , 1 mM CaCl 2 , 5 mM KCl, and 100 mg/liter soybean trypsin inhibitor. Mechanical shaking completed cell * This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Receptor-mediated Modulation of NADPH-dependent H 2 O 2 Generation-Similar to other effector systems, such as adenylyl cyclases, it is difficult to assess the effects of inhibitory ligands on NADPH-dependent H 2 O 2 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 G␣ i2 (12).
A two-step procedure was used, as reported elsewhere (6, 10 -14). 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 CaCl 2 , 2.5 mM MgCl 2 , 10 mM NaHCO 3 , 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 MgCl 2 , 1.2 mM KH 2 PO 4 , 1 mM NaN 3, 250 M NADPH, and 10 M FAD for determination of NADPH-dependent H 2 O 2 generation. Reactions were started by addition of 250 M NADPH and terminated after 30 min at 37°C by adding HCl to yield a final concentration of 100 M (10). To assess the effects of G protein antibodies on NADPHdependent H 2 O 2 generation, membranes were first exposed to antibodies at 0°C for 45 min and were then subjected to the two-step procedure described above.
bFGF-induced Dissociation of G s -In contrast to other G proteins, G␣ s does not dissociate in the presence of GTP␥S alone under the conditions used in the present experiments (18,19). Plasma membranes (80 g) were suspended in 200 l of MOPS (30 mM, pH 7.5) containing 120 mM KCl, 2.5 mM MgCl 2 , 1.4 mM CaCl 2 , and 0.1% human serum albumin in the absence or presence of 0.3 nM bFGF for 5 min at ambient temperature. Thereafter, 50 M GTP␥S 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. G s was immunoprecipitated by an antibody directed against the COOH terminus of G␣ s (RM/1), which does not discriminate between heterotrimeric and monomeric G␣ s (18,19). 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 M leupeptin, 2 M pepstatin, and 1 unit/ml aprotinin), antibody RM/1 (at a dilution of 1:80), and 50 M GTP␥S. The samples (30 g of membrane protein/100 l 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-G␣ s (RM/1) and anti-G␤ (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 G␤ was normalized to the amount of G␣ s immunoprecipitated for each condition.
Determination of Adenylyl Cyclase Activity and Measurement of Cellular cAMP-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 MgCl 2 , 0.5 mM EDTA, 10 M GTP, 10 mM creatine phosphate, 0.1 mg creatine kinase, and 0.1% bovine serum albumin in a final volume of 100 l (20). Reactions were initiated by addition of 3-5 g 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 l 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. . 1A shows the effects of 0.1 nM bFGF on NADPH-dependent H 2 O 2 generation in the presence of increasing concentrations of GTP␥S 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, GTP␥S alone had no measurable effect under the conditions used (21). The inhibitory action of bFGF became apparent at concentrations of GTP␥S exceeding 5 M and was half-maximal at approximately 10 M GTP␥S. At a maximal concentration of GTP␥S (50 M), NADPH-dependent H 2 O 2 generation was reduced by approximately two-thirds in the presence of 0.1 nM bFGF. Fig. 1B shows a concentration-response curve for bFGF in the presence of a maximal concentration of GTP␥S (50 M). The bFGF-induced inhibition of NADPH-dependent H 2 O 2 generation was half-maximal at about 1 pM and was maximal at approximately 30 pM.

Fig
Recent work showed that the effects of another inhibitor of NADPH-dependent H 2 O 2 generation, the ␤-adrenergic agonist isoproterenol, are transduced by ␤␥ subunits derived from G s (13,14). Therefore, the possibility was explored that the effects of bFGF were also mediated by G␤␥. Indeed, the inhibitory action of bFGF was dose-dependently reversed by an antibody directed against an internal sequence of ␤ 1 /␤ 2 subunits which had no influence on basal or insulin-stimulated rates of NADPHdependent H 2 O 2 generation ( Fig. 2A). Maximal effects were observed at an antibody dilution of 1:100 in the presence of 0.3 nM bFGF (Fig. 2A).
The inhibitory effect of bFGF could also be reversed by 10 M ␤ARK-1CT, a fragment of the ␤-adrenergic receptor kinase that specifically binds free G␤␥ (16). Reversal of the bFGF-induced suppression of NADPH-dependent H 2 O 2 generation was halfmaximal at 0.5 M ␤ARK-1CT, maximal effects were observed at 10 M (Fig. 2B). 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 G s subunit composition. In contrast to other heterotrimeric G proteins, G s does not dissociate in the presence of GTP␥S alone under the conditions used (18,19). Plasma membranes were therefore treated with 50 M GTP␥S in the absence or presence of 0.3 nM bFGF, 3 nM aFGF, or 5 nM insulin. Dissociation of G s into its component subunits was monitored by an immunoblot technique utilizing antibody RM/1, which does not discriminate between heterotrimeric and monomeric G␣ s . Fig. 3 demonstrates that the amount of G␤ 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 G s . In the absence of GTP␥S, bFGF had no influence on the subunit composition of G s . Thus, the growth factor caused a guanine nucleotide triphosphate-dependent dissociation of G s 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 H 2 O 2 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. 4A). Compared with the ␤-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. 4A), whereas both compounds were of comparable efficacy in suppressing NADPH-dependent H 2 O 2 generation (Fig. 4B). 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 M isoproterenol, consistent with recent observations in NIH 3T3 cells (22). Fig. 5 shows the effects of a COOH-terminal decapeptide (peptide 385-395), a sequence comprising residues 100 -119 within the ␣-helical domain of G␣ s (peptide 100 -119), and a homologous sequence of G␣ t on the bFGF-induced stimulation of adenylyl cyclase activity and the inhibitory action of the growth factor on insulin-stimulated rates of NADPH-dependent H 2 O 2 generation. At the concentrations used, both the COOH-terminal and the ␣-helical G␣ s peptide inhibited the stimulatory effect of bFGF on cAMP formation. By contrast, the inhibitory effect of bFGF on NADPH-dependent H 2 O 2 generation was only reversed by peptide 100 -119 whereas the COOHterminal decapeptide had no effect. The effects of the peptide 100 -119 were specific in as much as a homologous peptide derived from G␣ t had no influence on the antagonistic effects of bFGF on cAMP production and H 2 O 2 generation. In addition, the peptide did not influence the stimulatory effect of insulin, which is mediated via G␣ i2 .

DISCUSSION
In this report it is shown that bFGF, a ligand of receptor protein-tyrosine kinases, stimulates adenylyl cyclase and inhibits NADPH-dependent H 2 O 2 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 G s these findings suggested that FGF receptor(s) are directly coupled to G s , 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 G␣ s and G␤ upon affinity chromatography on Sepharose coupled to antibodies directed against their carboxyl-terminal sequences and hence may both contribute to bFGF-signaling via G s . 2 Antibodies directed against the COOH termini of G␣ 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 (23)(24)(25)(26). The stimulatory effect of bFGF on adenylyl cyclase could be blocked by COOH-terminal anti-G␣ s and a peptide corresponding to its antigenic site, indicating that it was in fact transmitted by G␣ s.
The bFGF-induced inhibition of NADPH-dependent H 2 O 2 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 G␤␥ subunits, however, based on observations that this latter effect was specifically reversed by anti-G␤ or a COOH-terminal ␤-adrenergic receptor kinase fragment (␤ARK-CT) that sequesters free G␤␥, and was mimicked by exogenously supplied G␤␥ subunits (14,15).
In contrast to the stimulatory effect of bFGF on adenylyl cyclase, its inhibitory action on NADPH-dependent H 2 O 2 generation was not influenced by COOH-terminal anti-G␣ s . A possible explanation for this unexpected finding would be that activation of G s by bFGF receptors may not yield enough, or inappropriately composed, ␤␥ subunits to account for inhibition of NADPH-dependent H 2 O 2 generation, as has been proposed for the regulation of type II adenylyl cyclase (27,28). However, the effects of COOH-terminal G␣ antibodies on G␤␥-mediated responses are variable and may depend on receptors, G proteins, and cell types studied (13,14,(23)(24)(25)(26). Therefore, the lacking effects of COOH-terminal anti-G␣ s do not permit conclusions to be made about the origin of the G␤␥ complexes transmitting the inhibitory action of bFGF. Indeed, we have recently shown that G s is capable of providing enough appropriately composed G␤␥ subunits to account for the inhibition of NADPH-dependent H 2 O 2 generation seen in the presence of isoproterenol (14). However, bFGF was considerably less efficacious than isoproterenol in activating adenylyl cyclase, although both compounds were of comparable efficacy in inhibiting of NADPHdependent H 2 O 2 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 GTP␥S.
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 ac- tivated ␤-adrenergic receptors recruit more G s for signaling than the receptor(s) for bFGF. It therefore appeared possible that ␤␥ complexes derived from G proteins other than G s 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 (29,30).
To obtain definitive information about the origin of the G␤␥ complexes transmitting the inhibitory effects of bFGF, we utilized a peptide comprising residues 100 -119 of G␣ s (K-20). Recent work showed that this peptide antagonized the antagonistic effects of the ␤-adrenergic agonist isoproterenol on adenylyl cyclase and NADPH oxidase that are transmitted by G␣ s and G␤␥ s , respectively (13,14). The present studies revealed that peptide 100 -119 reversed the effects of bFGF on NADPHdependent H 2 O 2 generation and cAMP production as well. The peptide did not influence the stimulatory effect of insulin, which is mediated by G␣ i2 . In addition, a homologous peptide derived from G␣ t had no influence on the antagonistic effects of bFGF on cAMP production and H 2 O 2 generation, indicating that peptide 100 -119 is specific for G␣ s , and does not block the interactions of receptors with other G proteins. Thus, the latter finding strongly supported the view that the ␤␥ subunits mediating the bFGF-induced inhibition of NADPH-dependent H 2 O 2 generation were exclusively derived from G s . 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 H 2 O 2 generation, which is mediated by G␣ i2 (12), these include activation of cardiac adenylyl cyclase by epidermal growth factor through G␣ s and induction of fibroblast transformation by TGF␤1 via G␣ i1 , respectively (31,32). The receptors of some growth factors, such as insulin and epidermal growth factor, may couple to multiple G proteins (12,33,34). Intriguingly, at least one ligand of a receptor protein-tyrosine kinase, insulin-like growth factor-1, appears to be capable of utilizing G␣ as well as G␤␥ subunits for signaling, albeit in different cells (35)(36)(37). However, bFGF is the first example of a ligand of receptor proteintyrosine 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 (14), the mechanisms by which G␣ i2 (which mediates the stimulatory effects of insulin) and G␤␥ s 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 ␣-subunit, and this is thought to faciltate dissociation into ␣ and ␤␥ subunits (38,39). G protein-sensitive effectors are then directly regulated by GTPliganded ␣-subunits, ␤␥ subunits, or both (38,39). Consistently, the mechanisms by which insulin and bFGF modulated NADPH-dependent H 2 O 2 generation were confined to the plasma membrane and were independent of soluble second messengers, making it likely that activated G␣ i2 and G␤␥ s acted upon NADPH oxidase directly. However, indirect mechanisms of action involving intermediate membrane-associated effectors or association of G␣ i2 and G␤␥ s to yield the inactive G i2 heterotrimer cannot definitively be ruled out (40 -42).
The generation of H 2 O 2 and other reactive oxygen species seems to be a common signaling event for hormones and cytokines that regulate cell growth and differentiation (40 -44). Consistently, previous work from this laboratory revealed that growth factors that stimulated NADPH-dependent H 2 O 2 generation promoted adipogenesis in 3T3 L1-preadipocytes, whereas inhibitory ligands, such as bFGF, were antiadipogenic (6). Intriguingly, it has been reported that inhibition of adipogenesis may be mediated via a G s pathway that does not involve cAMP (45). The finding that the signal of bFGF, an inhibitor of adipogenesis, is transduced by ␤␥ subunits that are derived from G s is consistent with these latter observations and provides further evidence in support of the concept that the H 2 O 2 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 G s (46).
In conclusion, the present findings show that bFGF, a ligand of receptor protein-tyrosine kinases, is capable of utilizing G␣ s as well as G␤␥ s 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 G s . These results confirm and extend recent observations demonstrating that the human fat cell oxidase is under antagonistic control by G␣ and G␤␥ 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.