Insulin-induced Activation of NADPH-dependent H2O2 Generation in Human Adipocyte Plasma Membranes Is Mediated by Galpha i2*

(Received for publication, August 5, 1996, and in revised form, January 15, 1997)

Heidemarie I. Krieger-Brauer , Pankaj K. Medda and Horst Kather

From the Klinisches Institut für Herzinfarktforschung an der Medizinischen Universitätsklinik Heidelberg, Bergheimerstraße 58, Heidelberg 69115, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDPbeta S (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Galpha i1-2 or the corresponding peptide.

Consistently, manganese could be replaced by micromolar concentrations of GTPgamma S (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPgamma S to the left (>10-fold) and increased the maximal response. In the presence of 10 µM GTPgamma S, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor.

The insulin receptor and Gi were co-adsorbed on anti-Galpha i and anti-insulin receptor beta -subunit (anti-IRbeta ) affinity columns. Partially purified insulin receptor preparations contained Galpha s, Galpha i2, and Gbeta gamma (but no Galpha i1 or Galpha i3). The functional nature of the insulin receptor-Gi2 complex was made evident by insulin's ability to modulate labeling of Gi by bacterial toxins. Insulin action was mimicked by activated Galpha i, but not by Galpha o or Gbeta gamma , indicating that insulin's signal was transduced via Galpha i2. Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.


INTRODUCTION

The insulin receptor is a heterotetrameric transmembrane protein consisting of two alpha - and beta -subunits (1). Insulin binding produces a conformational change leading to activation of its intrinsic tyrosine kinase activity. This tyrosine kinase activity is one of the earliest steps in insulin action and may be essential for many of insulin's biological effects. Recently, we demonstrated that insulin activates a H2O2-generating system in human fat cell plasma membranes via a mechanism bypassing the receptor kinase (2-4). Together with the results of others (5-9), these findings indicated that tyrosine phosphorylation may not be essential in all cases for insulin receptor signaling.

An alternative pathway of insulin receptor signaling for which tyrosine phosphorylation may not be essential could be via G proteins1 (6-11). In intact cells, pertussis toxin, which ADP-ribosylates the alpha -subunits of members of the Gi/Go family and uncouples them from receptors, inhibited a number of insulin-stimulated cellular events, such as glucose transport and its metabolism (9-11), whereas the effects of the bacterial toxin on insulin's antilipolytic action have remained controversial (11-13).

Plasma membranes from BC3 H-1 myocytes, adipocytes, and hepatocytes have been used to show that insulin promotes GTP binding or GDP release (7, 14, 15). It is also reported that insulin attenuates the pertussis toxin sensitivity of a 40-kDa Gi-like protein (6, 8). Finally, GTPgamma S, a nonhydrolyzable GTP analog, inhibited insulin binding to its receptor and modulated autophosphorylation of the receptor and its phosphotransferase activity (16, 17). Consistently, the insulin receptor has at least two G protein binding sites that coincide with autophosphorylation sites and appears to be associated with several distinct GTP-binding proteins, including a 40-kDa pertussis toxin substrate and a 60-67-kDa protein (18-22).

Several lines of evidence indicated that the insulin receptor kinase has no role in the communication between the insulin receptor and G proteins: (i) no phosphotyrosine was detectable in anti-Gi immunoprecipitates from lysates of 32P-labeled insulin-stimulated hepatocytes (6); (ii) ATP and ATP analogs had no effect on the insulin-induced acceleration of binding of GTPgamma S to BC3 H-1 membranes (7); and (iii) a monoclonal antibody to the insulin receptor (Ma-20) inhibited pertussis toxin (PTX)-catalyzed ADP-ribosylation of Gi without activating insulin receptor kinase (8).

Thus, there is evidence to suggest that a non-kinase-dependent pathway of insulin receptor signaling involving G proteins exists. However, an effector system that couples to a G protein associated with the insulin receptor has not yet been identified. Thus, despite many efforts, a role of G proteins in insulin receptor signaling remains to be established.


EXPERIMENTAL PROCEDURES

Materials

Synthetic peptides corresponding to residues 345-354 of Galpha i1-2, 345-354 of Galpha i3, and 385-394 of Galpha s and the corresponding COOH-terminal antibodies, recombinant alpha -subunits of Gi1, Gi2, Gi3, and Go, as well as the A-protomers of cholera and pertussis toxin were from Calbiochem-Novabiochem GmbH (Bad Soden, Germany). Purified beta gamma subunits and alpha i/alpha o-subunits from brain were kindly provided by Dr. G. Schultz (Institut für Pharmakologie, Freie Universität Berlin, Germany).

Peroxidase-conjugated anti-rabbit IgG was from Dianova (Hamburg, Germany), and rabbit polyclonal anti-Gbeta and protein A-agarose were from Upstate Biotechnology Inc. (Lake Placid, NY). Rabbit polyclonal anti-Galpha i (I-20) was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit insulin receptor antibody (beta -subunit) was from Transduction Laboratories (Lexington, KY). Antisera raised against internal decapeptide sequences of Galpha i1 (MIKKI), Galpha i2 (IINES), or against the COOH terminus of Galpha i3 (VELMU) were kindly donated by Dr. Y. J. Ohisalo (Department of Medical Chemistry, University of Helsinki, Finland). [32P]NAD (800 Ci/mmol) was purchased from NEN, Du Pont, Bad Homburg, Germany, Hybond PVDF membranes were from Amersham (Braunschweig, Germany). Insulin, GTPgamma S, GDPbeta S, and GDP were from Boehringer Mannheim (Mannheim, Germany). Human serum albumin and luminol were from Behring Werke (Marburg, Germany) and Fluka AG (Basel, Switzerland), respectively.

Methods

Subjects, Preparation of Fat Cells, and Fat Cell Ghosts

Experimental procedures have been described in detail elsewhere (2-4). Briefly, adipose tissue was from nondiabetic subjects undergoing elective abdominal or cosmetic breast surgery. The tissue specimens were cut into small pieces, and fat cells were isolated by the method of Rodbell (23) in a HEPES-buffered Krebs-Henseleit solution, pH 7.4, containing 20 mM HEPES, 10 mM NaHCO3, 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 MgCl2, 1 mM CaCl2, 5 mM KCl, and 100 mg/liter soybean trypsin inhibitor. Cell lysis was completed by mechanical shaking, and fat cell ghosts were collected by low speed centrifugation (1000 × g, 4 °C, 20 min).

Receptor-mediated Modulation of NADPH-dependent H2O2 Generation in Fat Cell Ghosts

A two-step procedure was used as reported elsewhere (2-4). Plasma membranes were first exposed to insulin and various cofactors (activation step) 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 CaCl2, 2.5 mM MgCl2, 10 mM NaHCO3, and 0.1% human albumin. Membranes were first incubated with various concentrations of insulin for 5 min to allow receptor occupation. Thereafter, guanine nucleotides or Mn2+ were added as indicated in the legends to figures and tables. 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 MgCl2, 1.2 mM KH2PO4, 1 mM NaN3, 250 µM NADPH, and 10 µM FAD for determination of NADPH-dependent H2O2 generation (2-4).

Treatment of Membranes with Bacterial Toxins

For toxin labeling, membranes (100 µg) were incubated for 45 min at 37 °C in 0.1 ml of 30 mM MOPS, pH 7.5, containing 2.5 mM MgCl2, 1.4 mM CaCl2, 120 mM NaCl, 10 mM thymidine, 10 mM arginine, 100 µM ATP, and 10 µM 32P-NAD (10 µCi/assay), 0.01% bovine serum albumin, and 0.2 µg of PTX A protomer, or 5 µg of cholera toxin (CTX) subunit A. In the absence of insulin, 100 µM GDP (pertussis toxin) or 10 µM GTPgamma S (cholera toxin) were routinely included in the reaction media.

For assessing the effects of insulin on the action of both toxins as well as modulation of PTX labeling by Mn2+ in plasma membranes, GTPgamma S or GDP was omitted (6, 24). The ligand-induced ADP-ribosylation of Gi by cholera toxin was initiated by the simultaneous addition of 100 nM insulin and CTX (5 µg/sample). By contrast, for determining the inhibition of PTX labeling, membranes were first exposed to 10 nM insulin or 3 mM MnCl2, respectively. After 15 min, PTX A-protomer (0.2 µg/sample) was added, and incubations were continued for another 30 min. Reactions were terminated by centrifugation; the pellets were solubilized with 150 µl of Laemmli buffer (25). Proteins were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 11% gel), electrophoretically transferred to Hybond PVDF, and visualized by autoradiography. Autoradiographic data were quantified by laser densitometry.

The conditions used for assessing the effects of bacterial toxins on NADPH-dependent H2O2 generation were identical with those described above, except that 32P-NAD was omitted and albumin concentration was increased to 0.1% (w/v). Control values containing all reagents except bacterial toxins were run in parallel for each condition. After 45 min., membranes were pelleted, resuspended in 30 mM MOPS, pH 7.5, containing 120 mM NaCl, 1.4 mM CaCl2, 2.5 mM MgCl2, 10 mM NaHCO3, and 0.1% human serum albumin, and were then subjected to the two-step procedure for determining NADPH-dependent H2O2 generation described above.

Partial Purification of Insulin Receptors

The purification of insulin receptors was performed as described by Katota (26). Plasma membranes were solubilized in 25 mM HEPES, pH 7.6, containing 4 mM EDTA, 4 mM EGTA, 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, 1 unit/ml aprotinin, 2 µM leupeptin, and 2 µM pepstatin. After centrifugation, the supernatant was applied to a column of wheat germ agglutinin (WGA) coupled to agarose. Bound material was eluted with 0.3 M acetyl-D-glucosamine in 25 mM HEPES pH 7.6, containing 0.1% Triton X-100 and the protease inhibitors used for solubilization of insulin receptors. Fractions containing insulin receptors were identified by immunoblotting.

Western Blot Analyses

Proteins resolved by 11% SDS-PAGE were transferred to Hybond PVDF membranes. The membranes were blocked for 2 h with 5% polyvinylpyrrolidone and were then exposed for 12-14 h at 4 °C to polyclonal antibodies raised against decapeptide sequences of Galpha s and different forms of Galpha i, Gbeta , or IRbeta , as indicated in the legends to figures and tables. The blots were washed with TBS containing 0.1% Tween 20 (TBS-T buffer) and were incubated for 1 h at room temperature with peroxidase-conjugated anti-rabbit IgG (Dianova; 1:20,000) in TBS-T buffer with 1% polyvinylpyrrolidone. Visualization was accomplished by enhanced chemiluminescence (Amersham).

Immunoaffinity Chromatography

Immunoaffinity columns were prepared using CNBr-activated Sepharose (Pharmacia Biotech Inc.) and polyclonal antibodies (I-20) raised against a peptide corresponding to amino acids 93-112 of Galpha i or directed against the beta -subunit of insulin receptor, respectively. Antibodies were dialyzed against coupling buffer, pH 8.9 (0.1 M NaHCO3 containing 0.5 M NaCl), added to the gel, and incubated for 3 h at room temperature. An excess of antibodies was removed with coupling buffer; the remaining active groups were blocked with 0.75 M ethanolamine, pH 8.0, for 3 h; and the gels were washed with 0.1 M acetate buffer, pH 4.0, containing 0.5 M NaCl. Solubilized membrane proteins were applied to 1.6 ml of the immunoaffinity matrix in 20 mM Tris, pH 7.4, containing 300 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.4 mM phenylmethylsulfonyl fluoride, 10 µg/ml Aprotinin, 1% Nonidet-40 and 2% Triton X-100. After 12-14 h, columns were washed with 10 column volumes of the same buffer. Galpha or IRbeta and associated proteins were eluted with 0.1 M glycin buffer, pH 2.75, containing 0.1 M NaCl.

Reconstitution with Galpha i/o Subunits

Purified or recombinant Galpha i/o was activated by incubation with 30 mM MOPS, pH 7.5, containing 120 mM NaCl, 1.4 mM CaCl2, 2.5 mM MgCl2, 0.1% human serum albumin, and 500 µM GTPgamma S at room temperature for 60 min (27). GDP-ligated Galpha i/o was prepared under identical conditions except that GTPgamma S was replaced by 500 µM GDP.

To determine the effects of activated Galpha i/o-subunits on NADPH-dependent H2O2 generation, 60-70 µg of membrane protein was incubated with 0.5 µM nucleotide-ligated G protein alpha -subunits for 25 min at 25 °C in a total volume of 400 µl without prior removal of free guanine nucleotides. Controls contained 25 µM GTPgamma S or GDP, respectively, corresponding to the concentrations of free nucleotides carried over by the addition of Galpha i/o-GTPgamma S or Galpha i/o-GDP. Incubations were terminated by washing followed by determination of NADPH-dependent H2O2 generation as described above.


RESULTS

Effects of Stable Guanine Nucleotide Analogues

As reported previously and shown in Table I (2, 4), the stimulatory effect of insulin on NADPH-dependent H2O2 generation was critically dependent on supraphysiological concentrations of manganese. In the absence of Mn2+, insulin increased NADPH-dependent H2O2 generation by approximately 10%, whereas a 2-fold increase was seen in the presence of the divalent cation. As pointed out previously and discussed below, divalent cations, such as manganese, have a variety of biological effects involving changes in G protein function (28-30). To explore the possibility that a G protein regulated NADPH-dependent H2O2 generation, we evaluated whether the stimulatory effect produced by insulin in the presence of 3 mM Mn2+ could be inhibited by GDPbeta S. At a concentration of 100 µM, the GDP-analogue suppressed the basal rate of H2O2 generation by approximately one-third. Concomitantly, insulin's stimulatory action was almost completely reversed, suggesting that receptor-mediated activation of NADPH-dependent H2O2 generation was in fact mediated by a G protein (Table I). Consistently, manganese, which is active at millimolar concentrations only, could be replaced by micromolar concentrations of GTPgamma S (Table I, Fig. 1).

Table I.

Effects of manganese and stable guanine nucleotide analogs on basal and insulin-stimulated rates of H2O2 generation

Plasma membranes were first incubated in the absence or presence of 5 nM insulin for 5 min. Thereafter, 3 mM MnCl2, 10 µM GTPgamma S and 100 µM GDPbeta S were added, as indicated. After 25 min, membranes were washed and assayed for NADPH-dependent H2O2 generation. Values are means ± S.D. of six experiments for each condition.


Additions H2O2 generation
Basal Insulin

nmol min-1 mg-1
None 28  ± 3 35  ± 5
Mn2+ 29  ± 2 58  ± 6
Mn2+ + GDPbeta S 24  ± 5 33  ± 7a
GDPbeta S 20  ± 6 23  ± 5
Mn2+ + GTPgamma S 31  ± 2 62  ± 10b
GTPgamma S 35  ± 3 60  ± 9b

a Significantly different from insulin-stimulated H2O2 generation (p <=  0.001); Student's t test).
b Significantly different from basal rates (p <=  0.001; Student's t test).


Fig. 1. Concentration-response curves for GTPgamma S in the absence and presence of insulin. Plasma membranes (100-150 µg) were first incubated in the absence (open circle ------open circle ) or presence (bullet ------bullet ) of 5 nM insulin for 5 min. Thereafter, increasing concentrations of GTPgamma S were added, and incubations were continued for another 20 min. Incubations were terminated by washing, and membranes were assayed for NADPH-dependent H2O2 generation, as described under "Methods." Values are means ± S.D. of three separate experiments.
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Fig. 1 shows dose-response curves for GTPgamma S in the absence and presence of a maximal concentration of insulin (5 nM). GTPgamma S caused a concentration-dependent enhancement of NADPH-dependent H2O2 generation, which at maximum concentrations amounted to an increase of about 40% above basal levels. Half-maximal effects were observed at approximately 10 µM GTPgamma S. Insulin stimulated NADPH-dependent H2O2 generation by increasing the sensitivity and maximal responsiveness to activation by the nonhydrolyzable GTP analogue. This resulted in a more than 10-fold shift in the apparent affinity; the maximal response to the GTP analogue was markedly increased.

Fig. 2 shows activity profiles produced by increasing concentrations of insulin in the simultaneous presence of 3 mM Mn2+ or 10 µM GTPgamma S, respectively. The dose-response curves were superimposable over the whole range of insulin concentrations tested, suggesting that identical mechanisms of signal transmission were involved under both conditions. As noted in a previous publication (3), insulin was extremely potent in stimulating NADPH-dependent H2O2 generation. Significant increases in NADPH-dependent H2O2 generation were observed at 1 pM, and the maximal effect occurred with about 100 pM insulin under both conditions, indicating that insulin acted via its cognate receptor.


Fig. 2. Activation of NADPH-dependent H2O2 generation by increasing concentrations of insulin in the presence of MnCl2 or GTPgamma S, respectively. Membranes (100-150 µg/ml) were incubated with increasing concentrations of insulin for 5 min. Thereafter, 3 mM MnCl2 (triangle ) or 10 µM GTPgamma S (open circle ) was added, and samples were processed further, as described in the legend to Fig. 1. Values are means ± S.D. of five separate experiments.
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Effects of Bacterial Toxins

To characterize further the G proteins that transduce insulin's stimulatory effect on NADPH-dependent H2O2 generation, the effects of pretreating membranes with the A protomers of PTX and CTX were tested. A pretreatment of membranes with 1 µg/ml of the A protomer of PTX for 30 min had little effect on nonstimulated rates of NADPH-dependent H2O2 generation. However, the stimulatory effect of insulin was impaired, regardless of whether GTPgamma S or Mn2+ was used as a cofactor (Fig. 3).


Fig. 3. Effects of pertussis toxin on insulin-stimulated rates of NADPH-dependent H2O2 generation. Membranes were preincubated in the absence or presence of 2 µg/ml pertussis toxin A-protomer for 45 min. After washing, membranes were incubated for 25 min with 3 mM MnCl2 or 10 µg/ml GTPgamma S either alone or in combination with 5 nM insulin, as indicated. Insulin-induced activation of NADPH-dependent H2O2 generation was determined after washing as described under "Methods." Values are means ± S.D. of 6-10 paired experiments carried out with different membrane preparations. The stimulatory effect of insulin is abolished by PTX regardless of whether GTPgamma S or Mn2+ is used as a cofactor. * and **, significantly different from corresponding controls (*, p <=  0.001; **, p <=  0.01; Student's t test for paired observations).
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It has long been known that ADP-ribosylation of Gi/Go by pertussis toxin is inhibited by divalent cations, with Mn2+ being more potent than magnesium (6, 8, 28). Indeed, ADP-ribosylation of Gi by pertussis toxin was severely impaired in the presence of 3 mM Mn2+ (Fig. 4A). Accordingly, the PTX-induced impairment of insulin action could be prevented if the pretreatment with pertussis toxin was carried out in the presence of 3 mM Mn2+ (Fig. 4B).


Fig. 4. Pertussis toxin-catalyzed ADP-ribosylation of Gi and the resulting suppression of insulin action are prevented by MnCl2. Membranes were incubated in the presence and absence of 3 mM MnCl2, as indicated. After 15 min, pertussis toxin A-protomer (2 µg/ml) was added, and membranes were incubated for another 30 min at 37 °C. Panel A shows the effect of MnCl2 on ADP-ribosylation of human fat cell membrane proteins by pertussis toxin. The samples were subjected to SDS-PAGE, and autoradiography was performed as described under "Methods." Panel B shows that the PTX-induced suppression of insulin's stimulatory action was prevented by MnCl2. Membranes were activated by 5 nM insulin in the presence of 10 µM GTPgamma S, followed by washing and determination of NADPH-dependent H2O2 generation as described under "Methods" and in the legend to Fig. 1. Values are means ± S.D. of three separate experiments. *, significantly different from values obtained in membranes treated with PTX alone (p <=  0.05; Student's t test for paired observations).
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As has been reported by others (6, 8), ADP-ribosylation of Gi by pertussis toxin was also inhibited by insulin. Inclusion of 10 nM insulin during pertussis toxin treatment resulted in a decrease of PTX labeling of Galpha i2 by approximately one-third (Fig. 5).


Fig. 5. Inhibition of pertussis toxin-induced ADP-ribosylation by insulin. Fat cell plasma membranes were exposed to pertussis toxin in the absence or presence of 100 nM insulin and subjected to SDS-PAGE and autoradiography as described under "Methods." A, representative autoradiograph of pertussis toxin-catalyzed ADP-ribosylation in the presence and absence of insulin. B, densitometric analyses of PTX labeling of Gi in the absence (control) or presence of insulin (10-7 M). Values are means ± S.D. of three separate experiments. *, significantly different from values obtained in membranes treated with PTX alone (p <=  0.05; Student's t test for paired observations).
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Cholera toxin catalyzes the ADP-ribosylation of Galpha s leading to its constitutive activation (31). The members of the Gi/Go family can also, under some conditions, be ADP-ribosylated by CTX (32-34). Regarding the requirements for CTX-catalyzed ADP-ribosylation of G proteins, two situations have emerged. Gs can be ADP-ribosylated in the absence of ligands, whereas proteins of the Gi/Go class are only sensitive to CTX in the presence of their corresponding agonist-activated receptors. Indeed, when cholera toxin-catalyzed ADP-ribosylation was performed in the presence of 0.1 mM GTP without insulin, radioactivity was incorporated into two proteins of ~ 43 and 45 kDa, which could be shown by immunoblotting to represent the short and long form of Gs. In the absence of exogenously added guanine nucleotides, radiolabel was also incorporated into a polypeptide of 40 kDa, resembling Galpha i, on Western blots (Fig. 6). The addition of insulin enhanced the cholera toxin-catalyzed ADP-ribosylation of the 40-kDa polypeptide in a concentration-dependent manner but did not affect the incorporation of radioactivity into either the 43- or 45-kDa bands. (Fig. 6).


Fig. 6. Enhancement of cholera toxin labeling of Gi by insulin. Membranes were incubated with 50 µg/ml of cholera toxin A-subunit and 3H-NAD (37 °C, 45 min) either alone (control) or in combination with various concentrations of insulin in the absence of exogenously supplied guanine nucleotides Subsequently, membranes were solubilized and subjected to SDS-PAGE and autoradiography as described under "Methods." A, insulin 100 nM caused a selective increase in cholera toxin labeling of Gi without affecting ADP-ribosylation of Gs in the absence of GTP. B, densitometric analysis of cholera toxin labeling of Gi in the absence and presence of two different concentrations of insulin. Values are means ± S.D. of three separate experiments. *, significantly different from values obtained in membranes treated with CTX alone (p <=  0.05; Student's t test for paired observations).
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Concomitantly, a pretreatment of membranes with CTX alone caused a small decrease in basal activity but had no influence on insulin-stimulated rates of NADPH-dependent H2O2 generation (Fig. 7). The stimulatory effect of insulin on NADPH-dependent H2O2 generation was not affected when membranes were pretreated for 45 min with 10 nM insulin alone. However, when insulin was applied together with CTX in the absence of exogenous GTP, the subsequent response to a maximal concentration of insulin was reduced from 100% above basal levels to approximately 15% (Fig. 7).


Fig. 7. Insulin action is impaired upon exposure of membranes to 100 nM insulin and 50 µg/ml cholera toxin but remains unaffected by a pretreatment with either agent alone. Membranes were first exposed to 50 µg/ml of cholera toxin A-subunit (37 °C, 45 min) in the absence or presence of 100 nM insulin, washed, and activated by 5 nM insulin and 10 µM GTPgamma S for 25 min. Activated membranes were washed again and assayed for NADPH-dependent H2O2 generation as described under "Methods." Values are means ± S.D. of six experiments. *, significantly different from controls (p <=  0.01; Student's t test for paired observations).
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Effects of COOH-terminal Antibodies and the Corresponding Peptides

The identity of the G protein(s) involved in insulin receptor-NADPH oxidase coupling was further probed with antibodies against the carboxyl-terminal region of G protein alpha -subunits and synthetic peptides corresponding to specific regions of the carboxyl termini of Galpha . These antibodies and peptides have been shown to prevent G protein interaction with various receptors (35, 36). Membranes were preincubated on ice for 1 h with various dilutions of anti-Galpha s, anti-Galpha i1-2, or anti-Galpha i3, respectively. At the concentrations used, none of the antisera or peptides had a significant effect on basal rates of NADPH-dependent H2O2 generation. The stimulatory effect of insulin was selectively blocked by antibodies to Galpha i1-2, regardless of whether insulin action was assessed in the presence of 10 µM GTPgamma S or 3 mM Mn2+. Antibodies directed toward Galpha i3 or Galpha s were without effect (Table II). Consistently, the COOH-terminal peptide from Galpha i3, at concentrations up to 20 µM, failed to affect the stimulatory effect of insulin on NADPH-dependent H2O2 generation. In contrast, the Galpha i2 peptide, at a concentration of 20 µM, inhibited insulin's stimulatory effect by approximately 80% (Table III).

Table II.

Selective inhibition of insulin-stimulated H2O2 generation by COOH-terminal anti-Galpha i 1-2

Membranes were preincubated with antibodies (dilution 1:1000) directed against the carboxyl termini of G protein alpha -subunits for 1 h on ice and subsequently activated by 5 nM insulin in the simultaneous presence of 10 µM GTPgamma S or 3 mM MnCl2, respectively. Incubations were terminated by washing, and membranes were assayed for NADPH-dependent H2O2 generation, as described under "Methods." Values are means ± S.D. of three separate experiments.


Antibody H2O2 generation
Basal Insulin, GTPgamma S Insulin, Mn2+

nmol min-1 mg-1
None 27  ± 5 57  ± 10 54  ± 3
Anti-Galpha 1 1-2 28  ± 4 30  ± 5a 35  ± 5a
Anti-Galpha i 3 33  ± 6 58  ± 7
Anti-Galpha s 30  ± 4 65  ± 11

a Significantly different from corresponding controls (p <=  0.01; Student's t test).

Table III.

Effects of decapeptides corresponding to the COOH-terminal sequences of G protein alpha -subunits on insulin-stimulated H2O2 generation

Membranes were preincubated for 30 min at 22 °C with 20 µM concentrations of different COOH-terminal peptides and were then activated by insulin and GTPgamma S and assayed for NADPH-dependent H2O2 generation, as described under "Methods" and in the legends to Tables I and II. Values are means ± S.D. of four separate experiments.


Agent H2O2 generation

nmol min-1 mg-1
None 63  ± 6
Galpha i 1-2 peptide (345-354) 34  ± 5a
Galpha i 3 peptide (345-354) 61  ± 6
Galpha s peptide (385-394) 65  ± 10

a Significantly different from control (p <=  0.01; Student's t test).

Collectively, the experiments using bacterial toxins as well as antibodies against the COOH-terminal sequences of Galpha i or the corresponding peptides demonstrated that insulin's stimulatory action on NADPH-dependent H2O2 generation was transduced via Gi1-2 and strongly supported the view that the insulin receptor interacted with Gi1, Gi2, or both of these proteins directly.

Physical Association of Galpha i2 and the Insulin Receptor

Since the insulin receptor and the Gi protein transducing the insulin signal to NADPH oxidase seemed to interact directly, the association of G proteins with the insulin receptor was investigated. Fig. 8 shows the relative distribution of Gs and Gi proteins in fat cell plasma membranes and partially purified insulin receptor preparations using antibodies directed toward internal sequences of Galpha i1 and Galpha i2, as well as COOH-terminal anti-Galpha i3 and anti-Gs. Human fat cell membranes contained Gs and the three isoforms of Gi, a finding that is in general agreement with a previous report showing that human adipocytes contain Gi1, Gi2, and low amounts of Gi3 (37). Interestingly, Galpha i2 was selectively enriched in partially purified insulin receptor preparations. Only Galpha s, Galpha i2, and Gbeta gamma were recovered after WGA chromatography, whereas neither Galpha i1 nor Galpha i3 could be detected. Essentially the same results were obtained by immunoaffinity chromatography on Sepharose coupled to antibodies directed against the beta  subunit of the insulin receptor (Fig. 9). Together these findings suggested that the insulin receptor may be associated with heterotrimeric Gi2 (and Gs). Consistently, the Gi2 present in insulin receptor fractions could be labeled by pertussis toxin, which ribosylates heterotrimeric Gi only (Fig. 10). A treatment of lectin-purified insulin receptors with 100 nM insulin for 15 min resulted in a dramatic decrease in PTX labeling (>90%), indicating that virtually all Gi2 recovered after WGA chromatography was functionally associated with the insulin receptor (Fig. 10).


Fig. 8. Immunodetection of Galpha - and Gbeta -subunits in adipocyte plasma membranes and partially purified insulin receptor preparations. Recombinant Galpha -subunits and brain Gbeta gamma (S), membrane proteins (M), and partially purified insulin receptors recovered in acetyl-D-glucosamine eluates after adsorption to WGA were subjected to SDS-PAGE, and Western blotting was performed as described under "Methods." Antisera used for immunodetection are indicated to the left.
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Fig. 9. Co-immunoadsorption of IRbeta and Galpha i by anti-IRbeta Sepharose. Plasma membranes were solubilized, and the lysates were subjected to immunoaffinity chromatography on Sepharose coupled to anti-IRbeta as described under "Methods." Eluate fractions containing insulin receptors were identified by immunoblotting and subjected to SDS-PAGE and Western blotting. The blots were probed with anti-IRbeta (aIRbeta , lane a) or with anti-Galpha i (aGalpha i, lane b). The blot shown in lane b was stripped and reprobed with anti-Gbeta (aGbeta , lane c). The experiment was reproduced three times using different membrane preparations.
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Fig. 10. Inhibition of pertussis toxin-induced ADP-ribosylation by insulin in partially purified insulin receptor preparations. Partially purified insulin receptors recovered in acetyl-D-glucosamine eluates after adsorption to WGA were exposed to pertussis toxin and 10 µM GDP in the absence or presence of 100 nM insulin for 30 min and subjected to SDS-PAGE and autoradiography as described under "Methods." A representative experiment out of three carried out with different membrane preparations is shown.
[View Larger Version of this Image (51K GIF file)]


Conversely, when human fat cell plasma membranes were solubilized and immunoprecipitated with antibodies against a common N-terminal sequence of Galpha i or anti-Gbeta , the immunoprecipitates contained a ~95-kDa species that reacted with an antibody directed toward the beta -subunit of the insulin receptor (data not shown). Accordingly, the insulin receptor co-eluted with Galpha i and Gbeta upon affinity chromatography on anti Galpha i-Sepharose (Fig. 11). Overall, it thus appeared that insulin's stimulatory effect on NADPH-dependent H2O2 generation was transduced via a direct interaction of the insulin receptor with Gi2. However, bacterial toxins and immunological approaches alone cannot distinguish which G protein subunit (Galpha i2 or Gbeta gamma ) transduces the signal to NADPH oxidase.


Fig. 11. Co-immunoadsorption of IRbeta and Galpha i by anti-Galpha i-Sepharose. Plasma membranes were solubilized, and the lysates were subjected to immunoaffinity chromatography on Sepharose coupled to anti-Galpha i as described under "Methods." Insulin receptor-Galpha i complexes were subjected to SDS-PAGE and transferred to PVDF membranes. The blots were probed with anti-IRbeta (aIR, lane a) or with anti-Galpha i (aGalpha i, lane b). The blot shown in lane b was stripped and reprobed with anti-Gbeta (aGbeta , lane c). The experiment was reproduced three times using different membrane preparations.
[View Larger Version of this Image (48K GIF file)]


The Stimulatory Action of Insulin Is Mimicked by Activated Galpha i2

To determine which G protein subunit transduces the insulin signal to NADPH oxidase, we examined the ability of purified and recombinant G protein subunits to activate NADPH-dependent H2O2 generation (Table IV). To assure that the Galpha subunits were intact in functional terms, they were assayed for their capacity to be ADP-ribosylated by pertussis toxin in the presence of purified Gbeta gamma from brain. This is a sensitive assay for detection of functional Gi/Go heterotrimers (38). Controls contained 25 µM GDP or GTPgamma S, respectively, corresponding to the concentrations of free nucleotides carried over by the addition of nucleotide-ligated G protein alpha -subunits. In the presence of GTPgamma S, nonstimulated rates of NADPH-dependent H2O2 generation were approximately 20% higher than with GDP (Table IV). The addition of brain Galpha -GTPgamma S, but not brain Galpha -GDP, caused an increase in NADPH-dependent H2O2 generation that was comparable with that observed in the presence of a maximal concentration of insulin. The addition of activated recombinant Galpha i1, Galpha i2, and Galpha i3 also stimulated NADPH-dependent H2O2 generation. Galpha i2 caused a greater activation than Galpha i1 or Galpha i3, whereas Galpha o was without effect. In contrast to Galpha i-GDP, the addition of brain Gbeta gamma subunits resulted in inhibition of insulin-stimulated H2O2 generation in the presence of 25 µM GTPgamma S, which causes a dissociation of heterotrimeric G proteins and hence prevents a complexation of free beta gamma -subunits.

Table IV.

Stimulation of NADPH-dependent H2O2 generation by recombinant and purified G protein subunits

Membranes (60-80 µg) were incubated with nucleotide-liganded purified (0.3 µM) or recombinant (0.5 µM) Galpha -subunits (rGalpha ) as well as Gbeta gamma (0.2 µM) in the absence or presence of 5 nM insulin (Ins), as indicated. Controls contained 25 µM free GTPgamma S or GDP corresponding to the concentrations of free nucleotides carried over by the addition of Galpha i/o-GTPgamma S or Galpha i/o-GDP. Incubations were terminated by washing. Therefore, neither free guanine nucleotides, buffer components, nor detergent was carried over into the assay for determination of NADPH-dependent H2O2 generation. Values are means ± S.E. of 3-6 experiments, as indicated in the right-hand column.


Additions H2O2 generation n

nmol min-1 mg-1
GDP 30  ± 7 6
GTPgamma S 36  ± 6 6
Ins, GTPgamma S 65  ± 8 6
Brain Galpha -GTPgamma S 73  ± 8 3
Brain Galpha -GDP 33  ± 5 3
rGalpha i 1-GTPgamma S 60  ± 8a 3
rGalpha i 2-GTPgamma S 77  ± 9a 6
rGalpha i 3-GTPgamma S 53  ± 5a 3
rGalpha o-GTPgamma S 32  ± 7 3
rGalpha i 2-GDP 33  ± 7 6
Ins, GTPgamma S; Gbeta gamma 29  ± 5 4
Ins, GTPgamma S; rGalpha i 2-GDP 60  ± 7 3

a Significantly different from controls (p <=  0.01; Student's t test).


DISCUSSION

The Stimulatory Effect of Insulin on NADPH-dependent H2O2 Generation Is Mediated by a Member of the Gi/Go Class of Heterotrimeric G Proteins

In previous studies the regulatory effects of insulin and other ligands on NADPH-dependent H2O2 generation were critically dependent on millimolar concentrations of manganese but occurred in the absence of exogenously supplied GTP (2-4). Interestingly, supraphysiological concentrations of Mn2+ are also essential for a ligand-induced activation of receptor protein-tyrosine kinases in cell-free preparations (29). Thus, the Mn2+ requirement seemed to provide further evidence in support of the widely held view that the receptor kinase activity is essential for most, if not all, of insulin's biological effects (1). However, the mechanism(s) by which insulin activated NADPH-dependent H2O2 generation were independent of ATP, indicating that an alternative pathway bypassing the receptor kinase had been activated (2). As pointed out in the introduction, one potential pathway could be via G proteins. Indeed, divalent cations, such as Mn2+, are known to potentiate the effects of GTP in some systems (28-30), and this could explain why no exogenous GTP was required in the presence of Mn2+. Accordingly, several independent lines of evidence indicated that the stimulatory effects of insulin on NADPH-dependent H2O2 generation were in fact mediated via a heterotrimeric G protein. First, the increase in NADPH-dependent H2O2 generation induced by insulin in the presence of Mn2+ was inhibited by GDPbeta S, a specific antagonist of GTP at G proteins. Second, the stimulatory effect of insulin seen in the presence of Mn2+ was prevented by pretreating the membranes with pertussis toxin, consistent with several reports suggesting that a pertussis toxin substrate may contribute to insulin receptor signaling (6-8). Conversely, a prior treatment with 3 mM Mn2+ prevented the PTX-induced ADP-ribosylation of Gi. Concomitantly, the bacterial toxin lost its ability to block insulin's stimulatory effect on NADPH-dependent H2O2 generation. The latter observations confirm early reports, demonstrating that divalent cations, such as Mn2+, can convert heterotrimeric G proteins to forms that cannot be ADP-ribosylated by bacterial toxins (28) and provide additional evidence in support of the view that Mn2+ action involved a change in G protein function. Third, studies using antibodies directed against the carboxyl terminus of the alpha -subunits of different heterotrimeric G proteins and synthetic peptides corresponding to specific regions of the carboxyl termini of Galpha i showed that the insulin-induced increase of NADPH-dependent H2O2 generation was mediated via Galpha i1-2 or a related protein. Finally, manganese, which is active at millimolar concentrations, could be replaced by micromolar concentrations of GTPgamma S. The synergistic activation of NADPH-dependent H2O2 generation by insulin and GTPgamma S was also blocked by GDPbeta S, pertussis toxin, and anti-Galpha i1-2 or the corresponding peptide, indicating that insulin activated the same G protein, regardless of whether Mn2+ or GTPgamma S was used as a cofactor.

Overall, it thus appeared that the involvement of Galpha i1-2 in insulin receptor signaling had been masked by Mn2+ in previous studies (2-4), possibly because the divalent cation potentiated the effect of endogenous GTP. This observation has important practical implications. As pointed out above, investigations into the functions of receptor protein-tyrosine kinases are routinely carried out in the presence of millimolar concentrations of manganese, because this cation is thought to be essential for a ligand-induced activation of their catalytic activity in cell-free preparations. Manganese is not the sole compound routinely present in receptor kinase assays that tends to obscure an involvement of G proteins in insulin receptor signaling. Another example of this type is the phosphatase inhibitor, vanadate, which dissociates heterotrimeric G proteins and prevents pertussis toxin labeling of Gi/o (6). Thus, one of the main obstacles in elucidating the role of G proteins in signaling by receptor kinases may reside in the fact that the conditions thought to be optimal for assessing phosphorylation-dependent signaling events are inappropriate for investigating G protein-dependent pathways of signal transduction in cell-free preparations.

Specificity of Insulin Action

Insulin, insulin-like growth factor (IGF)-1, and IGF-2 have distinct cell surface receptors, each of which can bind insulin, IGF-1, and IGF-2 with varying affinity (39). The receptors for insulin and IGF-1 are both alpha 2beta 2-heterotetramers and have approximately 60% sequence identity; the receptor for IGF-2 consists of a single membrane-spanning polypeptide that lacks intrinsic tyrosine kinase activity. Type I IGF receptors are not detectable in rat adipocytes (40). However, there is indirect evidence that they may be present in human adipocytes (41). Indeed, using antibodies against the beta -subunit of the type I IGF receptor, we were able to show that this protein is in fact present in human fat cell membranes.2 However, it is unlikely that IGF-1 receptors mediated insulin's stimulatory effect on NADPH-dependent H2O2 generation for two reasons. First, the stimulatory effect of insulin was observed at picomolar concentrations, which would cause negligible cross-reactivity with IGF-1 receptors. Second, although IGF-1 is another stimulator of NADPH-dependent H2O2 generation (3), its effect was not inhibited by pertussis toxin, indicating that the receptors for insulin and IGF-1 utilized different pathways of receptor signaling.2

The Insulin Receptor Interacts with Gi2 Directly

Heterotrimeric G proteins are typically coupled to seven-helix receptors. However, it is becoming increasingly clear that G proteins may be responsible for transducing signals of other types of receptors as well, including receptor protein-tyrosine kinases (42, 44-46). The present findings confirm and extend previous observations suggesting that the insulin receptor is an example of a non-seven-transmembrane receptor that appears to be associated with multiple G proteins, including Gi (20).

The association with Gi was detectable after immunoprecipitation with antibodies directed against Galpha i or IRbeta and survived wheat germ agglutinin chromatography as well as immunoaffinity chromatography on Sepharose coupled to anti-Galpha i or anti-IRbeta , respectively. Partially purified insulin receptor preparations contained only Galpha i2 and Gbeta gamma (but not Galpha i1 or Galpha i3), indicating that the insulin receptor was associated with the Gi2 heterotrimer.

The functional nature of the insulin receptor-Gi2 complex is made evident by the GTP-dependence of insulin's stimulatory effect on NADPH-dependent H2O2 generation and by its ability to modulate the ADP-ribosylation of Galpha i/Galpha o by bacterial toxins. Insulin reduced the PTX-induced ADP-ribosylation of total Gi/Go present in crude plasma membranes by approximately one-third, indicating that the insulin receptor caused a conformational change of a major fraction of plasma membrane-bound Gi/Go upon ligand binding (38). Accordingly, pertussis toxin labeling of Gi2 was virtually abolished in WGA extracts of insulin-treated membranes. The latter finding demonstrated that nearly all Gi2 recovered in WGA extracts is functionally associated with the insulin receptor. It has been reported that Gi2 comprises approximately half of total Gi/Go present in human adipocyte plasma membranes (37). Given that insulin inhibited PTX labeling of total plasma membrane-bound Gi/Go by one-third and considering that the insulin receptor selectively interacts with Gi2, it thus appears that the ligand-occupied insulin receptor communicates with at least two-thirds of the total Gi2 present in human fat cell membranes, suggesting that this alternative pathway may be as important for insulin receptor signaling as its intrinsic tyrosine kinase activity.

Another strategy for confirming the functional nature of the insulin receptor-Gi2 complex was based on the ability of cholera toxin to ADP-ribosylate Gi activated by a ligand-occupied receptor in the absence of GTP (32-34). As yet, a ligand-induced ADP-ribosylation of Gi has only been demonstrated for ligands of members of the superfamily of heptahelical receptors. In this paper we show that the addition of insulin, a ligand of a tyrosine kinase receptor, to membranes that are maintained in the absence of GTP stimulated the CTX-dependent ADP-ribosylation of Gi. The observation that the ability of mediating a ligand-induced ADP-ribosylation of Gi by cholera toxin is not restricted to heptahelical receptors but is shared by at least one receptor protein-tyrosine kinase strongly suggested that both types of receptors activated Gi via similar mechanisms. Indeed, as pointed out previously (2-4) and shown herein, the stimulatory effect of insulin occurred in the absence of ATP, indicating that the interaction between Gi1-2 and the insulin receptor took place via a noncovalent mechanism that may be similar to that used by heptahelical receptors.

NADPH Oxidase Is an Effector for Galpha i2

Current knowledge suggests that activation of heterotrimeric G proteins by ligand-receptor complexes is achieved by exchange of GDP for GTP on the alpha -subunit, and this is thought to facilitate dissociation into alpha -and beta gamma -subunits (47). G protein-sensitive effectors are then directly regulated by GTP-liganded alpha -subunits, beta gamma -subunits, or both (48). Accordingly, the mechanism(s) by which insulin stimulated NADPH-dependent H2O2 generation was membrane-delimited and independent of soluble second messengers, making it likely that activated Gi2 acted upon NADPH oxidase directly, although indirect mechanisms of action involving intermediate membrane-associated effectors cannot entirely be ruled out.

Recently, evidence has been presented to suggest that stimulation of the MAP kinase pathway by IGF-1 receptor requires the participation of beta gamma subunits derived from PTX-sensitive G proteins (49). Because the receptors for insulin and IGF-1 are closely related, it appeared possible that the insulin-induced increase in NADPH-dependent H2O2 generation was mediated by beta gamma -subunits derived from Gi2. However, insulin's stimulatory effect was mimicked by activated Galpha subunits, whereas beta gamma -subunits suppressed insulin-activated rates of NADPH-dependent H2O2 generation. Galpha i2 appeared to cause a greater activation than Galpha i1 or Galpha i3, and Galpha o was without effect, suggesting specificity in the interaction with the effector NADPH oxidase, but detailed titration studies will be required to determine the relative potency and efficacy of various PTX-sensitive Galpha subunits. Identification of unambiguous effectors for Galpha i subunits has been difficult (50). The present findings implicate NADPH oxidase as an effector system for activated PTX-sensitive Galpha i proteins, fulfilling most of the criteria utilized to establish adenylate cyclase as an effector for Galpha i.

There is precedent for an involvement of G proteins in regulating plasma membrane-bound redox activities. The activation of NADPH oxidases of endothelia and professional phagocytes can be triggered by peptides binding to G protein-coupled receptors (51). In parietal cells, the opening-closing behavior of a housekeeping Cl- channel is controlled by superoxide production mediated by a PTX-sensitive GTP-binding protein (52). Finally, intestinal smooth muscle cells appear to possess a hormone-sensitive NO synthetase that is localized to the plasma membrane and is coupled to Gi1-2 (53). Thus, a scheme emerges in which G protein-coupled redox systems activated by hormones or cytokines and sensitive to pertussis toxin are responsible for the generation of reactive oxygen species at the plasma membrane.

NADPH Oxidase Is the First Example of an Effector System Coupled to the Insulin Receptor via a Heterotrimeric G Protein

As pointed out in the introduction, an apparent association of insulin receptors with G proteins has been supported by several indirect studies, and most recent evidence indicated that Galpha i2 is a critical regulator of insulin action in vivo (54). However, despite intensive efforts, an effector system that is regulated by the ligand-occupied insulin receptor via the intermediacy of a G protein has not yet been identified. It has been proposed that adenylate cyclase or glycosyl-phosphatidylinositol-specific phospholipase C may be coupled to the insulin receptor via Gi or a related protein (55, 56). However, recent work has indicated that insulin's regulatory effects on these latter systems are a consequence of its stimulatory action on protein phosphatase activity and glucose metabolism, respectively (57, 58). Thus, the stimulus-sensitive NADPH oxidase of human fat cells is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.

Previous work demonstrated that the stimulus-sensitive H2O2-generating system that is present in human fat cell plasma membranes meets important criteria of a universal effector system for hormones and cytokines (2-4). By demonstrating that the stimulatory effect of insulin on NADPH-dependent H2O2 generation is transmitted via Galpha i2, the present findings provide further evidence in support of the idea that H2O2 has a second messenger function. Most recent work using transgenic mice harboring RNA antisense to the gene for Galpha i2 has suggested that Galpha i2 may be critical for insulin action (54), and evidence has been presented to suggest that the levels and functions of Gi proteins may be altered in diabetes (8, 43). Together with these latter observations, the current findings argue for a central role of NADPH-dependent H2O2 generation in insulin receptor signaling.

In conclusion, previous studies showed that the mechanism(s) by which insulin stimulated NADPH-dependent H2O2 generation were membrane-delimited and independent of the receptor kinase. The present findings confirmed that this alternative pathway of insulin receptor signaling exists and demonstrated that the transduction of insulin's signal to NADPH oxidase takes place via Galpha i2 whose participation had been obscured in former studies because NADPH oxidase was assayed in the presence of manganese, which is generally believed to be essential for a ligand-induced activation of receptor protein-tyrosine kinases in cell-free preparations. Thus, NADPH oxidase joins an expanding list of effector molecules that are influenced, possibly directly, by heterotrimeric G proteins and is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.


FOOTNOTES

*   This work was supported by a grant from the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, FRG.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1   The abbreviations used are: G protein, regulatory guanine nucleotidebinding protein; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); GDPbeta S, guanosine 5'-O-(2-thiodiphosphate); MOPS, 3-(N-morpholino)propanesulfonic acid; PTX, pertussis toxin; CTX, cholera toxin; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; MES, 4-morpholineethanesulfonic acid; WGA, wheat germ agglutinin; IRbeta , insulin receptor beta -subunit; IGF, insulin-like growth factor.
2   H. I. Krieger-Brauer and H. Kather, unpublished results.

ACKNOWLEDGEMENTS

We are indebted to Brigitte Sattel for expert technical assistance, and we thank Dr. Y. J. Ohisalo (University of Helsinki, Finland) for supplying antibodies against Galpha -subunits and Dr. G. Schultz (Freie Universität Berlin) for supplying purified G protein subunits.


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