(Received for publication, August 5, 1996, and in revised form, January 15, 1997)
From the Klinisches Institut für Herzinfarktforschung an der Medizinischen Universitätsklinik Heidelberg, Bergheimerstraße 58, Heidelberg 69115, Germany
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 GDPS
(guanosine 5
-O-(2-thiodiphosphate)), pertussis toxin and
COOH-terminal anti-G
i1-2 or the corresponding peptide.
Consistently, manganese could be replaced by micromolar concentrations
of GTPS (guanosine 5
-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2
generation by 20-40%. Insulin shifted the dose response curve for
GTP
S to the left (>10-fold) and increased the maximal response. In
the presence of 10 µM GTP
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-Gi and anti-insulin receptor
-subunit
(anti-IR
) affinity columns. Partially purified insulin receptor
preparations contained G
s, G
i2, and
G
(but no G
i1 or G
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 G
i, but not by G
o or G
,
indicating that insulin's signal was transduced via
G
i2. Thus, NADPH oxidase is the first example of an
effector system that is coupled to the insulin receptor via a
heterotrimeric G protein.
The insulin receptor is a heterotetrameric transmembrane protein
consisting of two - and
-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 -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, GTPS, 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 GTPS 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.
Materials
Synthetic peptides corresponding to residues 345-354 of
Gi1-2, 345-354 of G
i3, and 385-394 of
G
s and the corresponding COOH-terminal antibodies,
recombinant
-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
subunits and
i/
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-G and protein A-agarose were
from Upstate Biotechnology Inc. (Lake Placid, NY). Rabbit polyclonal
anti-G
i (I-20) was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit insulin receptor antibody (
-subunit) was from Transduction Laboratories (Lexington, KY). Antisera raised against internal decapeptide sequences of G
i1 (MIKKI),
G
i2 (IINES), or against the COOH terminus of
G
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, GTP
S, GDP
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 GhostsExperimental 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 GhostsA 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 ToxinsFor 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 GTPS (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, GTPS 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 ReceptorsThe 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 AnalysesProteins 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 Gs and different forms of
G
i, G
, or IR
, 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 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 Gi or directed against the
-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. G
or IR
and associated proteins were eluted with 0.1 M
glycin buffer, pH 2.75, containing 0.1 M NaCl.
Purified
or recombinant Gi/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 GTP
S at room
temperature for 60 min (27). GDP-ligated G
i/o was
prepared under identical conditions except that GTP
S was replaced by
500 µM GDP.
To determine the effects of activated Gi/o-subunits on
NADPH-dependent H2O2 generation,
60-70 µg of membrane protein was incubated with 0.5 µM
nucleotide-ligated G protein
-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 GTP
S or GDP,
respectively, corresponding to the concentrations of free nucleotides
carried over by the addition of G
i/o-GTP
S or
G
i/o-GDP. Incubations were terminated by washing
followed by determination of NADPH-dependent
H2O2 generation as described above.
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 GDPS. 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 GTP
S (Table I, Fig. 1).
|
Fig. 1 shows dose-response curves for GTPS in the absence and
presence of a maximal concentration of insulin (5 nM).
GTP
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 GTP
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 GTPS,
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.
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 GTPS or Mn2+ was used as a cofactor (Fig.
3).
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).
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 Gi2
by approximately one-third (Fig. 5).
Cholera toxin catalyzes the ADP-ribosylation of Gs
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 G
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).
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).
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 -subunits and
synthetic peptides corresponding to specific regions of the carboxyl
termini of G
. 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-G
s, anti-G
i1-2, or
anti-G
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 G
i1-2, regardless of whether insulin action was
assessed in the presence of 10 µM GTP
S or 3 mM Mn2+. Antibodies directed toward
G
i3 or G
s were without effect (Table II). Consistently, the COOH-terminal peptide from
G
i3, at concentrations up to 20 µM, failed
to affect the stimulatory effect of insulin on
NADPH-dependent H2O2 generation. In
contrast, the G
i2 peptide, at a concentration of 20 µM, inhibited insulin's stimulatory effect by
approximately 80% (Table III).
|
|
Collectively, the experiments using bacterial toxins as well as
antibodies against the COOH-terminal sequences of Gi 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.
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
Gi1 and G
i2, as well as COOH-terminal
anti-G
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, G
i2 was selectively enriched in partially
purified insulin receptor preparations. Only G
s,
G
i2, and G
were recovered after WGA
chromatography, whereas neither G
i1 nor
G
i3 could be detected. Essentially the same results were
obtained by immunoaffinity chromatography on Sepharose coupled to
antibodies directed against the
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).
Conversely, when human fat cell plasma membranes were solubilized and
immunoprecipitated with antibodies against a common N-terminal sequence
of Gi or anti-G
, the immunoprecipitates contained a
~95-kDa species that reacted with an antibody directed toward the
-subunit of the insulin receptor (data not shown). Accordingly, the
insulin receptor co-eluted with G
i and G
upon affinity chromatography on anti G
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 (G
i2 or G
) transduces the
signal to NADPH oxidase.
The Stimulatory Action of Insulin Is Mimicked by Activated G
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 G subunits were intact in functional terms, they were assayed for their capacity to be ADP-ribosylated by pertussis toxin in the presence of purified G
from brain. This is a sensitive assay for detection of functional Gi/Go heterotrimers (38). Controls contained 25 µM GDP or GTP
S, respectively, corresponding to the
concentrations of free nucleotides carried over by the addition of
nucleotide-ligated G protein
-subunits. In the presence of GTP
S,
nonstimulated rates of NADPH-dependent H2O2 generation were approximately 20% higher
than with GDP (Table IV). The addition of brain G
-GTP
S, but not
brain G
-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 G
i1,
G
i2, and G
i3 also stimulated
NADPH-dependent H2O2 generation.
G
i2 caused a greater activation than G
i1
or G
i3, whereas G
o was without effect. In
contrast to G
i-GDP, the addition of brain G
subunits resulted in inhibition of insulin-stimulated
H2O2 generation in the presence of 25 µM GTP
S, which causes a dissociation of heterotrimeric
G proteins and hence prevents a complexation of free
-subunits.
|
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 GDPS, 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
-subunits of different heterotrimeric G proteins and synthetic peptides corresponding to specific regions of the carboxyl termini of
G
i showed that the insulin-induced increase of
NADPH-dependent H2O2 generation was
mediated via G
i1-2 or a related protein. Finally,
manganese, which is active at millimolar concentrations, could be
replaced by micromolar concentrations of GTP
S. The synergistic activation of NADPH-dependent H2O2
generation by insulin and GTP
S was also blocked by GDP
S,
pertussis toxin, and anti-G
i1-2 or the corresponding
peptide, indicating that insulin activated the same G protein,
regardless of whether Mn2+ or GTP
S was used as a
cofactor.
Overall, it thus appeared that the involvement of Gi1-2
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.
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
2
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
-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
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 Gi
or IR
and survived wheat germ agglutinin chromatography as well as
immunoaffinity chromatography on Sepharose coupled to
anti-G
i or anti-IR
, respectively. Partially purified
insulin receptor preparations contained only G
i2 and
G
(but not G
i1 or G
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
Gi/G
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 GCurrent
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 (47). G protein-sensitive effectors are then
directly regulated by GTP-liganded
-subunits,
-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
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
-subunits derived from Gi2. However,
insulin's stimulatory effect was mimicked by activated G
subunits,
whereas
-subunits suppressed insulin-activated rates of
NADPH-dependent H2O2 generation.
G
i2 appeared to cause a greater activation than
G
i1 or G
i3, and G
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
G
subunits. Identification of unambiguous effectors for
G
i subunits has been difficult (50). The present
findings implicate NADPH oxidase as an effector system for activated
PTX-sensitive G
i proteins, fulfilling most of the
criteria utilized to establish adenylate cyclase as an effector for
G
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.
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 Gi2 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 Gi2, 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 G
i2 has suggested that
G
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 Gi2 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.
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 G-subunits and
Dr. G. Schultz (Freie Universität Berlin) for supplying purified G protein subunits.