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

Abstract

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 ␤␥-sub- There is growing evidence that redox-active biomolecules play important roles in signaling by hormones and cytokines (1-3). 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 .

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

“…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␤␥.…”

“…Alternative splicing generates isoforms of receptors 1-3 that exhibit unique binding characteristics (1-3). 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.…”

“…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.…”

Mouse β3a‐ and β3b‐adrenoceptors expressed in Chinese hamster ovary cells display identical pharmacology but utilize distinct signalling pathways

“…So while a PI3K pathway is present, the mechanism of activation remains to be elucidated. Recent reports show that Gbg subunits from G s can couple to other intracellular signalling events such as isoprenaline-mediated b 2 -AR inhibition of NADPH-dependent H 2 O 2 generation in human adipocytes (Krieger-Brauer et al, 2000). Thus mechanisms involving G s may not universally require elevation of cyclic AMP and activation of PKA.…”

R-α-Lipoic Acid Action on Cell Redox Status, the Insulin Receptor, and Glucose Uptake in 3T3-L1 Adipocytes

“…Indeed, hydrogen peroxide was hypothesized to act as a second messenger for the observed effects of insulin (41). Recently, human fat cells were shown to possess a multireceptor-linked, plasma membrane-bound H 2 O 2 generating system that is under antagonistic control by a large and diverse group of hormones, including insulin, the ␤-adrenergic agonist isoproterenol, and different isoforms of fibroblast and platelet-derived growth factors, providing further evidence in support of a second messenger function for oxidants such as hydrogen peroxide (42)(43)(44)(45)(46).…”