Inhibitory Effect of Isoproterenol on NADPH-dependent H2O2 Generation in Human Adipocyte Plasma Membranes Is Mediated by βγ-Subunits Derived from Gs *

Previous studies revealed that human fat cell plasma membranes contain a multireceptor-linked H2O2-generating system that is under antagonistic control by hormones and cytokines and is stimulated by insulin via Gαi2. In this report, it is shown that the inhibitory action of the β-adrenergic agonist isoproterenol is mediated by G protein βγ-subunits, based on observations that its action was specifically reversed by anti-Gβ antibodies or a C-terminal β-adrenergic receptor kinase-1 fragment containing the Gβγ-binding site of the enzyme, and was mimicked by exogenously supplied G protein βγ-subunits. Isoproterenol signals through a prototypical Gs-coupled receptor. Consistent with these results, direct activation of Gs by cholera toxin or by an anti-Gαs antibody exhibiting β-adrenergic receptor-mimetic properties (K-20) resulted in an isoproterenol-like inhibition of NADPH-dependent H2O2generation. In addition, a peptide corresponding to the target sequence of K-20 blocked the action of the catecholamine, apparently by competition between the peptide and Gs for activated β-adrenergic receptors, indicating that the G protein βγ-subunits mediating the inhibitory effects of the catecholamine were in fact derived from Gs.

Previous studies revealed that human fat cell plasma membranes contain a multireceptor-linked H 2 O 2 -generating system that is under antagonistic control by hormones and cytokines and is stimulated by insulin via G␣ i2 . In this report, it is shown that the inhibitory action of the ␤-adrenergic agonist isoproterenol is mediated by G protein ␤␥-subunits, based on observations that its action was specifically reversed by anti-G␤ antibodies or a C-terminal ␤-adrenergic receptor kinase-1 fragment containing the G␤␥-binding site of the enzyme, and was mimicked by exogenously supplied G protein ␤␥-subunits. Isoproterenol signals through a prototypical G scoupled receptor. Consistent with these results, direct activation of G s by cholera toxin or by an anti-G␣ s antibody exhibiting ␤-adrenergic receptor-mimetic properties (K-20) resulted in an isoproterenol-like inhibition of NADPH-dependent H 2 O 2 generation. In addition, a peptide corresponding to the target sequence of K-20 blocked the action of the catecholamine, apparently by competition between the peptide and G s for activated ␤-adrenergic receptors, indicating that the G protein ␤␥-subunits mediating the inhibitory effects of the catecholamine were in fact derived from G s .
There is growing evidence that redox-active biomolecules play important roles in signaling by hormones and cytokines (1)(2)(3). Ligand-induced changes in cellular redox status modulate tyrosine phosphorylation-dependent pathways of signal transduction; alter DNA binding and transactivation activities of many transcriptional activators; and may influence key steps in the synthesis, degradation, and action of cAMP and cGMP as well (2,3). Previous work showed that human fat cells possess a plasma membrane-bound H 2 O 2 -generating system that is under antagonistic control by a large and diverse group of hormones and cytokines, including insulin, the ␤-adrenergic agonist isoproterenol, and different isoforms of fibroblast and platelet-derived growth factors (4 -7). The mechanisms by which hormones and cytokines regulated NADPH-dependent H 2 O 2 generation were confined to the plasma membrane, operated in the absence of ATP, and were independent of soluble second messengers, indicating that established pathways of signal transduction were not involved. These findings placed human fat cell oxidase in a position comparable with adenylyl cyclase and suggested a physical interaction between receptors and NADPH oxidase or receptor-effector coupling via signaltransducing protein(s). Indeed, recent work revealed that the stimulatory effect of insulin on NADPH-dependent H 2 O 2 generation is transduced via G␣ i2 (7).
In this study, we have investigated whether the effects of the ␤-adrenergic agonist isoproterenol are also mediated by heterotrimeric G protein(s). Using specific antibodies against the ␣and ␤-subunits of heterotrimeric G proteins and a peptide that specifically binds G␤␥, it is shown that the inhibitory effects of isoproterenol on NADPH-dependent H 2 O 2 generation are mediated by ␤␥-subunits. Taking advantage of the unique properties of a commercially available antibody directed against residues 100 -119 within the ␣-helical domain of G␣ s (K-20) and of the peptide corresponding to its target sequence, which are summarized in a recent publication (8), it is shown that the ␤␥-subunits mediating the inhibitory action of isoproterenol were derived from G s .

Materials
The characteristics and sources of the antibodies and G s -derived peptides used in these experiments are listed in Table I. Cholera toxin subunit A, recombinant G␣ s , and protein A-agarose were from Calbiochem. Hybond polyvinylidene difluoride membranes were obtained from Amersham Pharmacia Biotech (Braunschweig, Germany). Insulin, GTP␥S, 1 GDP␤S, and GDP were from Roche Molecular Biochemicals (Mannheim, Germany). Isoproterenol was from Sigma (Mü nchen, Germany). Human albumin and luminol were obtained from Behring Werke (Marburg, Germany).

Subjects and Preparation of Fat Cells and Fat Cell
Ghosts-Experimental details have been described in detail elsewhere (4 -7). 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 (11) 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 resuspended 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. Cell lysis was completed by mechan-* This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Federal Republic of 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.
‡ To whom correspondence should be addressed.
ical shaking, and fat cell ghosts were collected by low speed centrifugation (1000 ϫ g, 4°C, 20 min) and washed with lysing medium. These crude fat cell plasma membranes (fat cell ghosts) contained Ͻ10% of total cellular NADPH:cytochrome P430 reductase and Ͻ2% of NADPHdependent glutamate dehydrogenase (4).
Receptor-mediated Modulation of NADPH-dependent H 2 O 2 Generation-A two-step procedure was used, as reported elsewhere (4 -7). 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 NaCl, 1.4 mM 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. After 20 min, ghosts were collected by centrifugation; washed; and then resuspended 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 stopped with 0.1 N HCl. H 2 O 2 was assayed using oxidation of luminol in the presence of peroxidase as the indicator reaction (5).
To assess the effects of anti-G protein antibodies and peptides corresponding to their target sequences on NADPH-dependent H 2 O 2 generation, membranes were exposed to both types of agents at 0°C for 45 min, as indicated in the figure and table legends, and were then subjected to the two-step procedure described above. For cholera toxin labeling, membranes (200 g) were preincubated for 45 min at 37°C in 0.5 ml of 30 mM MOPS, pH 7.5, containing 2.5 mM MgCl 2 , 1.4 mM CaCl 2 , 120 mM NaCl, 10 mM thymidine, 10 mM arginine, 10 M GTP␥S, 10 M NAD or 10 M [ 32 P]NAD (12.5 Ci/assay), 0.1% bovine serum albumin, and various concentrations of cholera toxin subunit A, as indicated (7).
Determination of Adenylyl Cyclase Activity-Adenylyl cyclase activity of human fat cell plasma membranes was determined in 30 mM Tris-HCl, pH 7.5, containing 1 mM ATP, 2.5 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM 3-isobutyl-1-methylxanthine, 10 M GTP, 10 mM creatine phosphate, 0.1 mg of creatine kinase, and 0.1% bovine serum albumin in a final volume of 100 l (12). Reactions were initiated by addition of 3-5 g of membrane protein and were continued for 15 min at 37°C in the absence or presence of isoproterenol, as indicated. Reactions were terminated by 100 l of ice-cold perchloric acid (5%). cAMP was determined by radioimmunoassay (Amersham Pharmacia Biotech) after neutralization. To assess the effect of anti-G␣ s C terminus antibody or a peptide corresponding to its antigenic site on adenylyl cyclase activity, membranes were pretreated with the antibody (RM/1) or peptide as described above.

Isoproterenol Acts as an Inhibitor of NADPH-dependent H 2 O 2 Generation in the Presence of GTP␥S-Previous work has
shown that isoproterenol causes an inhibition of NADPH-dependent H 2 O 2 generation in human fat cell plasma membranes that can be overcome by the ␤-adrenergic receptor antagonist propranolol, indicating that it is mediated by a ␤-adrenergic receptor (5). Initial studies suggested that the regulatory effects of hormones and cytokines on NADPH-dependent H 2 O 2 generation may be critically dependent on supraphysiological concentrations of manganese (4 -6). However, as pointed out previously (7), divalent cations such as Mn 2ϩ may obscure an involvement of G proteins in receptor signaling. Indeed, manganese, which is active at millimolar concentrations, can be replaced by micromolar concentrations of GTP␥S. Fig. 1 illustrates the mutual interrelationship of the effects of isoproterenol and GTP␥S. In the absence of exogenously supplied GTP␥S, isoproterenol caused a small decrease in NADPH-dependent H 2 O 2 production of ϳ3 nmol ϫ (mg pro-tein ϫ min) Ϫ1 , which probably reflected the presence of trace amounts of endogenous GTP (data not shown). The synergistic effect of GTP␥S became apparent at ϳ0.3 M and was halfmaximal at 3 M (Fig. 1A). At a maximal concentration of GTP␥S (50 M), insulin-stimulated rates of NADPH-dependent H 2 O 2 generation were suppressed by two-thirds in the presence of 1 M isoproterenol.   terenol on NADPH-dependent H 2 O 2 generation may be transduced by a G protein.
Inhibitory Effects of Isoproterenol Are Mediated by ␤␥-Subunits-The inhibitory action of isoproterenol was dose-dependently reversed by an antibody directed against an internal sequence of ␤ 1 /␤ 2 -subunits that had no influence on basal or insulin-stimulated rates of NADPH-dependent H 2 O 2 generation ( Fig. 2A). Maximal effects were observed at an antibody dilution of 1:100 in the presence of 10 M isoproterenol.
The inhibitory effects of isoproterenol could also be reversed by ␤ARK1-CT (10), a ␤-adrenergic receptor kinase-1 fragment that includes the G␤␥-binding region of this kinase (Fig. 2B). At a concentration of 10 M, the peptide reversed the inhibition seen in the presence of 0.5 M isoproterenol completely, whereas the GST protein alone had no effect.
Consistent with the results summarized above, addition of G␤␥ purified from bovine brain caused a concentration-dependent inhibition of insulin-stimulated NADPH-dependent H 2 O 2 generation that was maximal at 200 nM G␤␥ (Fig. 3A). Addition of 300 nM GDP-liganded ␣-subunits from bovine brain or re-combinant G␣ s resulted in complete reversal of the inhibition induced by G␤␥ (Fig. 3B). Boiled G␤␥ (200 nM) had no effect on NADPH-dependent H 2 O 2 generation. Thus, three independent lines of evidence indicated that the inhibitory effects of isoproterenol on NADPH-dependent H 2 O 2 generation were transmitted by ␤␥-subunits.
G Protein ␤␥-Subunits Mediating the ␤-Adrenergic Inhibition of H 2 O 2 Generation Are Derived from G s -Mammalian ␤-adrenergic receptors are prototypical examples of G protein-coupled receptors that are highly selective for G s , the G protein stimulatory with respect to adenylyl cyclase (13), suggesting that the ␤␥-subunits mediating the inhibitory effects of isoproterenol on NADPH-dependent H 2 O 2 generation might have been derived from G s . Antibodies directed against the C termini of G␣ subunits and peptides corresponding to their target sequences are thought to block receptor-G protein recognition and activation and are therefore widely used in functional studies for assessing receptor-G protein coupling (14 -17). Anti-G␣ s C terminus antibody and the peptide corresponding to its antigenic site were highly efficient in suppressing the stimulatory effect of isoproterenol on adenylyl cyclase, which is transduced through G␣ s (8). However, we previously showed that both compounds failed to reverse the suppression of NADPH-dependent H 2 O 2 generation seen in the presence of isoproterenol over the whole range of concentrations tested (8). One possibility to explain this unexpected finding would be that activation of G s may not yield enough, or inappropriately composed, ␤␥-subunits to inhibit NADPH-dependent H 2 O 2 generation, as has been proposed for the regulation of type II adenylyl cyclase (18, 19). The latter contention implies that isoproterenol may be able to activate multiple G proteins, which seems in fact to be the case under certain conditions (20).
However, as pointed out elsewhere (8), the effects of anti-G␣ C terminus antibodies on G␤␥-mediated responses are variable and may depend on receptors, G proteins, and cell types studied (14 -17). Therefore, the lack of effects of anti-G␣ s antibody (and of the peptide corresponding to its target sequence) does not necessarily indicate that the ␤␥-subunits transmitting the inhibitory effect of isoproterenol were derived from G proteins other than G s .
Considering the variability in antibody action, the effects of cholera toxin, which directly activates G s (21), were explored next. Indeed, a direct activation of G s by cholera toxin resulted in a concentration-dependent inhibition of NADPH-dependent H 2 O 2 generation. At the concentration employed in previous studies (50 g/ml), the bacterial toxin caused a rightward shift of the dose-response curve for insulin by 2 orders of magnitude, but did not suppress the maximal response seen at 10 nM insulin (data not shown). At higher concentrations, the toxin also inhibited the response seen in the presence of 10 nM insulin (Fig. 4A). The latter effect was dose-dependent and was paralleled by a corresponding increase in G s labeling (Fig. 4B). At the highest toxin concentration used (200 g/ml), insulinstimulated H 2 O 2 production was suppressed by approximately two-thirds, suggesting that trimeric G s contained enough appropriately composed G protein ␤␥-subunits to transduce a pronounced inhibition of human fat cell oxidase.
To provide definitive proof that the isoproterenol-induced inhibition of NADPH-dependent H 2 O 2 generation was mediated by functionally specific G␤␥ derived from G s , we used a novel approach circumventing the shortcomings of anti-G␣ C terminus antibodies. As shown recently (8), a commercially available antibody recognizing residues 100 -119 within the ␣-helical domain of G␣ s (K-20) stimulates G s in a manner similar to activated ␤-adrenergic receptors. This receptor-mimetic antibody was as efficacious as isoproterenol in suppressing NADPH-dependent H 2 O 2 generation, indicating that G s can in fact provide sufficient amounts of appropriately composed ␤␥-subunits to account for the inhibition of NADPH oxidase activity seen in the presence of isoproterenol (8). More important, a peptide corresponding to the target sequence of K-20 effectively antagonized the ␤-adrenergic inhibition of NADPH-dependent H 2 O 2 generation, apparently through competition between the peptide and G s for binding to activated ␤-adrenergic receptors (8). At a concentration of 2.5 M, the peptide reduced the inhibitory action of a maximally effective concentration of isoproterenol by ϳ80%, whereas the homologous sequence of G t had no effect at all (data not shown). Considering the specificity of K-20 and its target sequence, the latter finding provided definitive proof that the ␤␥-subunits mediating the ␤-adrenergic inhibition of NADPH-dependent H 2 O 2 generation were in fact mainly, if not completely, derived from G s . Consistent with these results, activation of G s -coupled receptors has been shown to produce sufficient amounts of ␤␥-subunits to provide membrane anchorage of G protein-coupled receptor kinase (22), to activate mitogen-activated protein kinases (23,24), or to inhibit type I adenylyl cyclase (25).
Conclusions-The mechanisms by which H 2 O 2 and other reactive oxygen species such as superoxide are generated has been extensively studied in neutrophils and macrophages (26 -28). The phagocyte system, which is critical to the inflammatory response, is a multicomponent enzyme that is activated by assembling four cytosolic components (p40 phox , p47 phox , p67 phox , and the small GTPase Rac) with the transmembrane electron carrier flavocytochrome b 558 (26 -28). Human fat cell oxidase differs from neutrophil oxidase in that all components required for H 2 O 2 generation are membrane-bound. In addition, the system produces nontoxic amounts of H 2 O 2 , consistent with its potential role in signaling by hormones, growth factors, and cytokines. Despite these differences, human fat cell plasma membranes contain a spectrally detectable cytochrome b 558 (4), suggesting that some of the components of human fat cell oxidase may be similar or identical to those of phagocyte oxidase, as has also been proposed for stimulus-sensitive redox activities of other non-phagocytic cells such as endothelia (29), fibroblasts (30,31), mesangial cells (32), chondrocytes (33), vascular smooth muscle cells (34 -36), and adventitial cells (37).
As pointed out previously, there is precedent for an involvement of heterotrimeric G proteins in the regulation of membrane-bound redox activities (7). The NADPH oxidases of endothelia and professional phagocytes can be triggered by peptides binding to G protein-coupled receptors (26 -29). In parietal cells, the opening-closing behavior of a housekeeping Cl Ϫ channel is controlled by superoxide production mediated by a pertussis toxin-sensitive GTP-binding protein (38). Finally, intestinal smooth muscle cells appear to possess a hormonesensitive NO synthetase that is localized to the plasma membrane and that is coupled to G i1/2 (39). However, human fat cell oxidase is the first example of a ligand-regulated redox system that is under antagonistic control by ␣and ␤␥-subunits derived from different G proteins, as has also been reported for other important effector systems, including certain types of adenylyl cyclase (18,19,25), phospholipase C (40), and inwardly rectifying K ϩ channels (41).
The mechanisms by which G␣ i2 and G␤␥ s modulate the activity of 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 GDP for GTP on the ␣-subunit, and this is thought to facilitate dissociation into ␣and ␤␥-subunits (42,43). G protein-sensitive effectors are then directly regulated by GTP-liganded ␣-subunits, ␤␥-subunits, or both. Consistent with these results, the mechanisms by which insulin and isoproterenol modulate NADPH-dependent H 2 O 2 generation were confined to the plasma membrane and are independent of soluble second messengers, making it likely that activated G␣ i2 and G␤␥ s act upon NADPH oxidase directly, although indirect mechanisms of action involving intermediate membrane-associated effectors such as Rac cannot be ruled out (26,31).
In conclusion, the present findings show that the ␤-adrenergic agonist isoproterenol utilizes ␤␥-subunits to inhibit human fat cell oxidase and illustrate that antibodies directed against the C termini of G protein ␣-subunits, which are often used in functional studies, are of limited value for determining their origin. The latter observation has also been made by others, but is rarely mentioned (44). Taking advantage of the serendipitous discovery that an antibody directed against residues 100 -119 within the ␣-helical domain of G␣ s (K-20) has ␤-adrenergic receptor-mimetic properties, whereas the peptide corresponding to its target sequence acts as a ␤-adrenergic receptor antagonist, it could be shown that the G protein ␤␥-subunits utilized by isoproterenol are derived from G s . Along with previous observations demonstrating that the stimulatory effect of insulin on NADPH-dependent H 2 O 2 generation is transduced via G␣ i2 , the present findings thus reveal that the stimulussensitive NADPH oxidase present in human fat cells is the first example of a ligand-regulated redox system that is under antagonistic control by both types of component subunits of heterotrimeric G proteins. The complex regulation of human fat cell oxidase provides further evidence in support of the concept that this membrane-bound redox activity meets all the criteria of a universal effector system that translates binding of hormones, growth factors, and cytokines to cell-surface receptors into changes in intracellular redox equilibrium and may be important in the regulation of fat cell differentiation and maintenance of the differentiated state.