Insulin and Insulin-like Growth Factor I Receptors
Utilize Different G Protein Signaling Components*
Stephane
Dalle,
William
Ricketts,
Takeshi
Imamura,
Peter
Vollenweider, and
Jerrold M.
Olefsky
From the Department of Medicine, Division of Endocrinology and
Metabolism, University of California, San Diego, La Jolla,
California 92093-0673, the Whittier Institute for Diabetes, La Jolla,
California 92037 and the San Diego Veterans Administration Medical
Center, San Diego, California 92161
Received for publication, December 1, 2000, and in revised form, January 30, 2001
 |
ABSTRACT |
We examined the role of heterotrimeric G protein
signaling components in insulin and insulin-like growth factor I
(IGF-I) action. In HIRcB cells and in 3T3L1 adipocytes, treatment with the G
i inhibitor (pertussis toxin) or
microinjection of the G
inhibitor (glutathione
S-transferase-
ARK) inhibited IGF-I and lysophosphatidic
acid-stimulated mitogenesis but had no effect on epidermal growth
factor (EGF) or insulin action. In basal state, G
i and
G
were associated with the IGF-I receptor (IGF-IR), and after ligand
stimulation the association of IGF-IR with G
i increased concomitantly with a decrease in G
association. No association of
G
i was found with either the insulin or EGF receptor.
Microinjection of anti-
-arrestin-1 antibody specifically inhibited
IGF-I mitogenic action but had no effect on EGF or insulin action.
-Arrestin-1 was associated with the receptors for IGF-I, insulin,
and EGF in a ligand-dependent manner. We demonstrated that
G
i, 
subunits, and
-arrestin-1 all play a
critical role in IGF-I mitogenic signaling. In contrast, neither
metabolic, such as GLUT4 translocation, nor mitogenic signaling by
insulin is dependent on these protein components. These results suggest
that insulin receptors and IGF-IRs can function as G protein-coupled
receptors and engage different G protein partners for downstream signaling.
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INTRODUCTION |
Although the insulin-like growth factor I receptor
(IGF-IR)1 and the insulin
receptor (IR) are structurally and functionally related
heterotetrameric proteins and share many of the same signaling molecules, they modulate different responses within the cell. IGF-I has
been implicated mostly in mitogenic functions and insulin in metabolic
actions (1, 2). The insulin and IGF-I receptors consist of two
extracellular
-subunits and two transmembrane
-subunits and are
members of the receptor tyrosine kinase (RTK) class of membrane
localized receptors (2-4). Ligand binding activates the tyrosine
kinase activity of the
subunits (2-4) leading to
autophosphorylation, as well as tyrosine phosphorylation of a variety
of endogenous substrates, such as the IRS proteins (5), Shc (6, 7),
Gab1 (8), and G
q/11 (9). It is thought that these
endogenous substrates then go on to mediate the biologic effects of
these two hormones through a variety of mechanisms (5-9). Heptahelical
receptors are another broad class of membrane receptors, and this
receptor class is often referred to as G protein-coupled receptors
(GPCRs), because they exert their biologic effects by interacting with
a family of heterotrimeric G protein signaling molecules (10-12).
Although it is often thought that the RTKs and heptahelical/GPCRs
represent structurally and functionally different classes of receptor
types, recent evidence indicates that this may not be strictly the case
(13-16). For example, it has now been established that specific
heptahelical receptor biologic responses, such as activation of MAP
kinase (16-19), can be mediated by tyrosine kinase events initiated
through activation of Src kinase (20-24). Further, several reports
have appeared that show a strict dependence of insulin receptor and
IGF-I signaling on heterotrimeric G proteins (9, 25). Evidence exists
to indicate that the IGF-I receptor requires a heterotrimeric G protein
containing G
i for some of its biologic effects. Thus,
treatment with pertussis toxin (an inhibitor of
G
i-mediated signaling) blocks IGF-I-induced activation of MAP kinase, and inhibition of G
function also impairs MAP kinase signaling from the IGF-I receptor (25). Furthermore, recent work
from our laboratory has shown that G
q/11 plays a key
role in insulin-induced GLUT4 translocation and stimulation of glucose
transport in 3T3-L1 adipocytes. In these studies, we showed that the IR
physically associates with and phosphorylates G
q/11 and
that this G protein is necessary for insulin stimulation of GLUT4
translocation and glucose transport. In addition, a constitutively active form of G
q/11 was able to mimic the effects of
insulin by stimulating GLUT4 translocation and glucose transport on its own (9). Based on these studies, one can suggest that the term GPCR is
a broader functional definition, rather than a structural one referring
to heptahelical receptors specifically. Because of the extensive
structural homology between the IR and IGF-IR, we have now directly
studied the involvement of different G protein signaling components in
these RTK action pathways.
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EXPERIMENTAL PROCEDURES |
Materials--
-Arrestin-1 and horseradish peroxidase-
conjugated phosphotyrosine (RC-20) antibodies were from Transduction
Laboratories (Lexington, KY). Horseradish peroxidase-linked
anti-rabbit, anti-mouse antibodies, protein A/G-plus agarose, IR,
IGF-IR, G
, G
i antibodies were purchased from Santa
Cruz Laboratories (Santa Cruz, CA). Rabbit polyclonal anti-GLUT4
antibody (F349) was kindly provided by Dr. Michael Mueckler (Washington
University, St. Louis, MO). Mouse monoclonal phospho-p44/42 MAP kinase
antibody was purchased from New England Biolabs (Beverly, MA), and
rabbit monoclonal phospho-p44/42 MAP kinase antibody was from Promega
(Madison, MA). Sheep IgG, tetramethyl rhodamine isothocyanate (TRITC)-
and fluorescein isothiocyanate-conjugated anti- rabbit, -mouse, -goat, and -sheep IgG antibodies were from Jackson Immunoresearch Laboratories Inc. (West Grove, PA). Dulbecco's modified Eagle's medium (DMEM) and
fetal calf serum (FCS) were purchased from Life Technologies, Inc.
Polyvinylidene difluoride membranes (Immobilon-P) were from Millipore
(Bedford, MA). All other reagents were purchased from Sigma.
Cell Culture--
HIRcB cells, which are rat 1 fibroblasts
overexpressing the human form of the IR, were maintained as previously
described (26) in DMEM/Ham's F-12 medium with 50 units/ml penicillin, 50 µg/ml streptomycin, 10% FCS, 0.5% glutamax, and 0.5%
methothrexate in a 5% CO2 environment. NIH/3T3 cells,
established from NIH Swiss mouse embryo cultures, were grown in DMEM
with 4.5 g/l glucose, 50 units/ml penicillin, 50 µg/ml streptomycin,
and 10% calf serum (Colorado Serum, Co.), in a 10% CO2
environment. Cultures were never allowed to become completely
confluent. 3T3-L1 adipocytes were maintained in DMEM with 4.5 g/liter
glucose, 50 units/ml penicillin, 50 µg/ml streptomycin, and 10% FCS,
in a 10% CO2 environment. 3T3-L1 were differenciated 2 days post-confluency by the addition of the same media containing 500 µM isobutylxanthine, 25 µM dexamethasone, and 4 µg/ml insulin. After 3 days, the medium was removed and replaced with DMEM containing 10% FCS, 5 mM glucose,
glutamax, and 1% penicillin-streptomycin. Seven days after the
addition of the differentiation medium, the cells were plated in 6-well dishes at a density of 8 × 105 cells/dish. The medium
was changed every second day until use, 10-12 days
post-differentiation. Approximately 90% of the cells exhibited large
lipid droplets indicative of adipocytes. 24 h prior to the start
of experiments, cells were given fresh DMEM containing 10% FCS, 5 mM glucose without antibiotics.
Bromodeoxyuridine (BrdU) Incorporation--
HIRcB cells were
grown on 6 well plates to 50-70% confluency and then starved in
serum-free DMEM/Ham's F-12 medium for 24 h. Cells were stimulated
with either 100 ng/ml insulin, 100 ng/ml IGF-1, 10 ng/ml epidermal
growth factor (EGF), or 10 µM lysophosphatidic acid
(LPA). 12 h later, BrdU was added, and cells were incubated for an
additional 4 h. Cells were then fixed for 20 min in 3.7% formaldehyde and incubated with rat anti-BrdU antibody followed by
incubation with fluorescein isothiocyanate- labeled donkey anti-rabbit
IgG and rhodamine-labeled donkey anti-rat IgG. Results were analyzed on
an Axiphot fluorescence microscope (Carl Zeiss, Inc.). Positive cells
in Fig. 1 represent the ratio of a total population of cells in a given
area (nucleus stained cells using blue Dye) compared with cells which
incorporate BrdU corresponding to a new DNA synthesis.
Microinjection--
Cells were injected and photographed as
previously described (27). Briefly, HIRcB cells were grown on glass
coverslips to 50% confluency. Cells were starved in serum-free
DMEM/Ham's F-12 medium for 24 h. 1 h after the
microinjection, cells were stimulated with either 100 ng/ml insulin,
100 ng/ml IGF-I, 10 ng/ml EGF, or 10 µM LPA. 12 h
later, BrdU incorporation was performed as already described. For GLUT4
translocation experiments, 3T3L1 adipocytes were trypsinized on day 7 post-differentiation and reseeded on acid-washed coverslips in
preparation for microinjection on days 10-12. Microinjection of the
various reagents was carried out using a semiautomatic Eppendorf
microinjection system. All reagents for microinjection were dissolved
in microinjection buffer containing 5 mM sodium phosphate,
pH 7.2, 100 mM KCl. Antibodies were coinjected into the
cytoplasm of the cell with 5 mg/ml sheep IgG to allow identification of
the injected cells.
Immunostaining for GLUT4--
Immunostaining of GLUT4 was
performed as previously described (28). 3T3L1 adipocytes were fixed in
3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min at
room temperature. After washing, the cells were permeabilized and
blocked with 0.1% Triton X-100 and 2% FCS in PBS for 10 min. The
cells were then incubated with anti- GLUT4 antibody in PBS with 2% FCS
overnight at 4 °C. After washing, GLUT4 and injected IgG were
detected by incubation with TRITC-conjugated donkey anti-rabbit IgG
antibody and fluorescein isothiocyanate-conjugated donkey anti-sheep
antibody, respectively. Immunofluorescence observed by microscope was
used to evaluate the results. Injected cells were scored as positive for GLUT4 translocation if they were observed to have a ring of fluorescence at the cell periphery. In all counting experiments, the
observer was blinded to the experimental condition of each coverslip.
Immunostaining for Phospho-p44/42 MAP Kinase in 3T3L1
Adipocytes--
Cells were fixed in 3.7% formaldehyde in PBS for 10 min, washed, and permeabilized with 0.1 M Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100 (TBST buffer). After
incubation in blocking buffer (5% normal horse serum in TBST buffer)
for 60 min at room temperature, cells were exposed to the
anti-phospho-p44/42 MAP kinase primary antibody in 5% bovine serum
albumine TBST buffer overnight at 4 °C (1:400 dilution). After
washing, phospho-p44/42 MAP kinase was detected by incubation with
TRITC-conjugated donkey anti-mouse or rabbit IgG secondary antibody in
1% bovine serum albumine TBST buffer (1:250 dilution). Mouse
monoclonal and rabbit polyclonal phospho-p44/42 MAP kinase antibodies
react specifically with phosphorylated MAP kinase and do not
cross-react with nonphosphorylated MAP kinase by Western blotting.
Western Blotting and Immunoprecipitation--
HIRcB cells or
3T3L1 adipocytes plated in 6-well dishes were treated as described in
figure legends. After stimulation, cells were lysed for 30 min at
4 °C in a solubilizing buffer containing 50 mM HEPES, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 30 mM PyroPO4, 10 mM NaF, and 1 mg/ml bacitracin. Cells lysates were centrifuged at 14,000 rpm
for 30 min to remove insoluble materials. For Western blot analysis,
the supernatants (25-50 µg of protein/lane) were denatured by
boiling for 3 min in Laemmli's sample buffer in reduced or nonreduced
conditions (with or without 100 mM dithiothreitol and 2-mercaptoethanol) and resolved by SDS-polyacrylamide gel
electrophoresis (PAGE). For immunoprecipitation, the supernatants
(200-350 µg of total protein) were incubated with primary antibody
as indicated in each experiments for 4 h at 4 °C.
Immunocomplexes were precipitated from the supernatant with protein
A/G-plus agarose, washed three times with ice-cold cell lysis buffer
and boiled for 3 min in Laemmli's sample buffer containing (or not)
100 mM dithiothreitol and 2-mercaptoethanol and resolved by
SDS-PAGE gel electrophoresis. Gels were transferred to polyvinylidene
difluoride membrane (Immobilon-P; Millipore, Bedford, MA) by using
Transblot apparatus (Bio-Rad). For immunoblotting, membranes were
blocked and probed with specified antibodies according to the
manufacturer's instructions. Following incubation with horseradish
peroxidase-linked second antibody, proteins were visualized by ECL
detection (Pierce).
2-Deoxyglucose Uptake in 3T3L1 Adipocytes--
Before the
uptake, 12 days post-differenciation 3T3L1 adipocytes were placed in
DMEM containing 5 mM glucose for 1 h at 37 °C.
Cells were then washed with KRPH buffer (5 mM
Na2HPO4, 20 mM HEPES, pH 7.4, 1 mM MgSO4, 1 mM CaCl2,
136 mM NaCl, 4.7 mM KCl, and 0.1%
bovine serum albumine) and either untreated or stimulated as described
in figure legends. Glucose transport was determined by the addition of
0.1 mM 2-deoxyglucose containing 0.2 µCi of L-[3H]deoxyglucose as described previously
(29). Nonspecific uptake was assessed by the addition of 0.1 mM L-glucose containing 0.2 µCi of
L-[3H]glucose. The reaction was stopped after
10 min by aspiration, and extraneous glucose was removed by four washes
with ice-cold PBS. Cells were lysed in 1 N NaOH and uptake
was assessed by scintillation counting. Samples were normalized for
protein content by Bradford protein assay.
Statistical Analysis--
Values are expressed as the means ± S.E. Results were analyzed by using Student's t test. A
value of p < 0.05 was considered significant.
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RESULTS |
Effect of G
i and G
Subunits on Insulin-,
IGF-I-, and EGF-induced Mitogenesis in HIRcB Cells--
Fig.
1A depicts the effects of PTX
treatment of HIRcB cells on ligand-stimulated BrdU incorporation
mediated by insulin, IGF-I, EGF, or LPA. IGF-I- and LPA-induced BrdU
incorporation was blocked by PTX treatment, whereas insulin or EGF
stimulated BrdU incorporation was not affected. These results indicate
that the pathways for IGF-I- and LPA-stimulated DNA synthesis require
G
i signaling, whereas insulin- and EGF-stimulated
mitogenic signaling do not. Luttrel et al. (25) have shown
that G
subunits are necessary for IGF-I-induced MAP kinase
activation. Thus, in our conditions, we assessed the role of G
in
growth factor-stimulated mitogenesis. We utilized a glutathione
S-transferase (GST) fusion protein containing the C-terminal
portion of the
-adrenergic receptor kinase (GST-
ARK), which binds
to and sequesters G
subunits and behaves as a dominant negative
inhibitor of G
signaling (30). We microinjected GST-
ARK into
HIRcB cells and measured insulin, IGF-I, LPA, and EGF stimulated BrdU
incorporation. As shown in Fig. 1B, microinjection of
GST-
ARK blocks IGF-I- and LPA-induced BrdU incorporation but not
that induced by insulin or EGF.

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Fig. 1.
Effect of PTX treatment and microinjection of
GST- ARK on ligand-induced BrdU incorporation
in HIRcB cells. A, HIRcB cells (50-70% confluent)
were incubated overnight in serum-free medium in the presence or
absence of PTX (100 ng/ml) prior to determination of agonist-induced
BrdU incorporation. BrdU incorporation in control (solid
bars) and PTX treated cells (open bars) were determined
following stimulation with 100 ng/ml insulin, 100 ng/ml IGF-I, 10 ng/ml
EGF, or 10 µM LPA as described under "Experimental
Procedures." B, to test the effect of microinjected
GST- ARK on ligand-induced BrdU incorporation, serum-starved HIRcB
cells were incubated with insulin, IGF, EGF, or LPA, after 1 h of
sheep IgG microinjection used as control, or GST- ARK microinjection.
After 12 h, BrdU incorporation was performed and cells were fixed.
Data are presented as the means ± S.E. for four separate
experiments. *, p < 0.05; **, p < 0.01, versus ligand-stimulated control cells or
versus ligand-stimulated IgG microinjected control
cells.
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Selective G
i Interaction with Receptor Tyrosine
Kinases--
As seen in Fig.
2A, G
i
immunoprecipitated with the IGF-I receptor in the basal state, and the
amount of receptor-associated G
i increased markedly
after IGF-I stimulation. No association of G
i with
either the insulin receptor or EGF receptors was found in HIRcB cells
(Fig. 2A). Time course studies showed that the IGF-I-induced
association between G
i and IGF-IR proceeds with a
maximum from 2 to 5 min (Fig. 2, B and C). Total
G
i expression in cell lysates was the same at all time
points (Fig. 2, A and B). Because tyrosine
phosphorylation has been shown for G
i, we determined
whether or not G
i-tyrosine phosphorylation was enhanced by IGF-I treatment. HIRcB cells were stimulated with IGF-I for various
periods, followed by phosphotyrosine antibody or G
i
antibody immunoprecipitation and Western blotting with
G
i or phosphotyrosine antibodies, respectively. IGF-I
did not lead to tyrosine phosphorylation of G
i (data not
shown). To further assess G
subunit involvement in the mitogenic
effect of IGF-I, we investigated whether endogenous G
subunits
interact with the IGF-I receptor. Immunoprecipitation studies show an
association between the IGF-IR and G
, which decreased 35% in
response to IGF-I (maximum decrease at 2 min) (Fig. 2, D and
E). Total G
expression in cell lysates was the same at all time points (Fig. 2D).

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Fig. 2.
Selective association of
G i with endogenous tyrosine-kinase
receptors in HIRcB cells. A, HIRcB cells were starved
for 24 h in serum-free DMEM/Ham's F-12 medium with 0.1% bovine
serum albumine. Cells were then stimulated with IGF-I (100 ng/ml),
insulin (10 ng/ml), and EGF (10 ng/ml) at 37 °C for 5 min, washed
with ice-cold PBS, lysed, and subjected to immunoprecipitation
(IP) in reduced conditions with either IGF-I or insulin
receptor subunit antibodies and with an antibody specific to the
the C terminus of the EGF receptor. Immunoprecipitated proteins were
resolved by 10% SDS-PAGE and blotted with anti-G i
antibody that recognizes rat G i-1, G i-2,
or G i-3 (A and B). Relative
quantities of the IGF-I, insulin, and EGF receptors determined by
reblots in these immunoprecipitations experiments are also shown.
B, starved HIRcB cells were stimulated with IGF-I (100 ng/ml) at 37 °C for various time as indicated. Lysates were
immunoprecipitates with IGF-IR subunit antibody. Representative
exposures are shown. G i content within total cell
lysates is also shown (A and B). C- Temporal
pattern of IGF-IR and G i association deduced from
quantitative results of five representative IGF-IR immunoprecipitation
experiments obtained using NIH Image program. D, to
determine the temporal pattern of G subunit-IGF-IR association,
serum-starved HIRcB cells were stimulated with 100 ng/ml IGF-I at
37 °C for various time as indicated. Cell lysates were
immunoprecipitated with anti-IGF-IR antibody, resolved by 10%
SDS-PAGE, and blotted with anti-G antibody. A typical exposure
representative of three experiments is shown. G content within total
cell lysates is also shown. E, temporal pattern of IGF-IR
and G association deduced from quantitative results of three
representative IGF-IR immunoprecipitation experiments obtained using
NIH Image program.
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Effects of
-Arrestin-1 on Mitogenic Signaling by Insulin, IGF-I,
and EGF in NIH 3T3 Cells--
The process of receptor internalization
plays an essential role in receptor-mediated mitogenic signaling (31,
32). Because it has been shown that
-arrestin-1 is necessary for
GPCR and IGF-IR internalization (33-36), we tested the
hypothesis that this protein was involved in the mitogenic effects
elicited by the receptors coupled to G
i. We inhibited
endogenous
-arrestin-1 function by single cell microinjection of
anti-
-arrestin-1 antibody, followed by measurement of BrdU
incorporation. As shown in Fig. 3,
microinjection of the
-arrestin-1 antibody into NIH3T3 cells inhibited DNA synthesis in response to IGF-I and LPA but not to insulin
or EGF.

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Fig. 3.
Effect of microinjection of
-arrestin-1 antibody on agonist-induced BrdU
incorporation from NIH3T3 cells Serum-starved NIH3T3 cells were
incubated in the presence of 100 ng/ml insulin, 100 ng/ml IGF-I, 10 ng/ml EGF, or 10 µM LPA for 20 min, after 1 h of
sheep IgG, used as control, or -arrestin-1 antibody cytoplasm
microinjection. BrdU incorporation was performed as described under
"Experimental Procedures." Data are presented as the means ± S.E. for four separate experiments. *, p < 0.05; **,
p < 0.01, versus ligand-stimulated IgG
microinjected control cells.
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Interaction between Insulin, IGF-I, EGF Receptors, and
-Arrestin-1 in HIRcB Cells--
Overexpressed
-arrestin-1 can
interact with IGF-I receptors in HEK 293 cells (36), and, therefore, we
determined whether endogenous levels of
-arrestin-1 could interact
with endogenous IGF-IRs in our system. As shown in Fig.
4 (A and B), in the
basal state,
-arrestin-1 is associated with the IGF-IR, and this
association increases >2 fold at 5 min of IGF-I stimulation.
-Arrestin-1 was also observed in anti- IR immunoprecipitates, and
the time course of this association was similar to that observed for
the IGF-IR (Fig. 4, C and D). Interestingly,
-arrestin-1 also immunoprecipitated with the EGFR, but the time
course of association was more rapid and transient (Fig. 4,
E and F).

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Fig. 4.
Interaction between endogenous receptor
and -arrestin-1 in HIRcB cells. Starved
HIRcB cells were stimulated with 100 ng/ml IGF-I (A), 10 ng/ml insulin (C), or 10 ng/ml EGF (E) at
37 °C for various times as indicated, lysed for 30 min at 4 °C in
the solubilizing buffer and subjected to immunoprecipitation (200-350
µg of total protein) in nonreduced conditions for 4 h at 4 °C
with IGF-I, with insulin receptor -subunit antibodies, or with the
anti-EGF receptor antibody. Immunoprecipitated proteins were resolved
by 10% SDS-PAGE and blotted with anti- -arrestin-1 antibody.
Representative exposures are shown. Identical amounts of proteins were
analyzed as described under "Experimental Procedures." Temporal
pattern of receptors and -arrestin-1 association deduced from
quantitative results of five representative IGF-IR or three IR and EGFR
immunoprecipitation experiments obtained using NIH Image program are
also shown (B, D, and F).
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Effect of PTX, GST-
ARK, and
-Arrestin-1 on the Signaling
Pathways Elicited by Insulin and IGF-I in 3T3L1 Adipocytes--
Using
3T3 L1 adipocytes that express endogenous levels of IRs and IGF-I Rs,
we studied the involvement of G
i, G
, or
-arrestin-1 in the metabolic and mitogenic actions of insulin and
IGF-I. To accomplish this, we measured insulin-stimulated glucose
uptake and GLUT4 translocation. As shown in Fig.
5 (A and B), PTX
pretreatment did not inhibit the effects of insulin on glucose uptake
or GLUT4 translocation. We also determined whether
-arrestin-1 was
involved in this action of insulin using anti-
-arrestin-1 antibody
microinjection followed by insulin stimulation and immunofluorescence
staining for GLUT4 translocation. As shown in Fig. 5B,
inhibition of
-arrestin-1 function had no effect on
insulin-stimulated GLUT4 translocation. As we previously reported (37),
we also show in this system that GST-
ARK microinjection did not
inhibit insulin-induced GLUT4 translocation (Fig. 5B). Taken
together, our results show that, in 3T3L1 adipocytes, there is no
involvement of a G
i/G
pathway or
-arrestin-1 in
insulin signaling leading to glucose uptake and GLUT4
translocation.

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Fig. 5.
Effects of PTX treatment,
GST- ARK and
-arrestin-1 antibody microinjection on insulin and
IGF-I MAP kinase activation and on insulin-stimulated GLUT4
translocation from 3T3-L1 adipocytes. A, for glucose uptake,
differentiated 3T3L1 adipocytes were pretreated overnight with
(open bars) or without (solid bars) 100 ng/ml of
PTX. Cells were then stimulated with 100 ng/ml of insulin for 20 min.
The initial rate of 2-[3H]deoxyglucose was determined as
described under "Experimental Procedures." B, for GLUT4
translocation experiments, serum-starved 3T3L1 adipocytes on coverslips
were stimulated with insulin (100 ng/ml) for 5 min after pretreatment
with PTX or microinjection of sheep IgG, GST- ARK protein, or
-arrestin-1 antibody, mixed with sheep IgG, as described under
"Experimental Procedures." Fixed cells were stained for GLUT-4 with
rabbit anti-GLUT4 antibody followed by incubation with TRITC-conjugated
anti-rabbit IgG antibody. The percentage of cells positive for GLUT4
translocation was calculated by counting at least 100-160 cells at
each point. C, serum-starved (24 h) 3T3L1 adipocytes were
incubated overnight with (open bars) or without (solid
bars) 100 ng/ml of PTX. Cells were stimulated with insulin or
IGF-I (100 ng/ml) at 37 °C for 5 min, washed with ice-cold PBS, and
lysed for 30 min at 4 °C in the solubilizing buffer. Lysates
(200-350 µg of total protein) were resolved by 10% SDS-PAGE in
reduced conditions and blotted with mouse phospho-p44/42 MAP kinase
monoclonal antibody. Above the graph, a typical exposure representative
of three experiments is shown. D-F, serum-starved (24 h)
3T3L1 adipocytes on coverslips were incubated overnight with or without
100 ng/ml of PTX as indicated or subjected to microinjection of sheep
IgG (Control), GST- ARK protein, or -arrestin-1
antibody, mixed with sheep IgG as marker, as described under
"Experimental Procedures," and then stimulated with insulin or
IGF-I (100 ng/ml) for 5 min. After fixation, cells were stained for
phospho-p44/42 MAP kinase as described under "Experimental
Procedures." The percentage of cells positive for phospho-p44/42 MAP
kinase staining was calculated by counting at least 100 cells at each
point. Errors bars represent the means ± S.E. for
three independent experiments. *, p < 0.05; **,
p < 0.01.
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We also measured insulin- and IGF-I-stimulated MAP kinase activity in
these same cells, and these experiments were performed in two ways.
First, we assess phospho-MAP kinase immunoblots of cell lysates.
Secondly, we developed a single cell assay for microinjected cells by
immunostaining of coverslips with the same antibody to determine the
percentage of cells positive for phospho MAP kinase. As shown in Fig. 5
(C and D), MAP kinase phosphorylation induced by
IGF-I was blocked by PTX pretreatment, whereas insulin stimulated MAP
kinase phosphorylation was unaffected. To determine whether G
subunits and
-arrestin-1 were involved in insulin or IGF-I signaling
leading to MAP kinase phosphorylation, we microinjected GST-
ARK and
anti-
-arrestin-1 antibody and measured MAP kinase phosphorylation by
immunofluorescence staining of cells. Microinjection of GST-
ARK and
anti-
-arrestin-1 antibody inhibited IGF-I-induced MAP kinase
phosphorylation but not that induced by insulin (Fig. 5, E
and F). These results clearly indicate that in 3T3L1
adipocytes, IGF-I signaling leading to MAP kinase phosphorylation
requires G
i, G
, and
-arrestin-1, whereas
insulin-induced MAP kinase signaling does not. These results also show
that these reagents (PTX, GST-
ARK, and anti-
-arrestin-1 antibody)
were effective at inhibiting IGF-I induced MAP kinase activation in
these cells, indicating that the lack of effect on insulin metabolic
actions was specific.
Selective Interaction between Insulin and IGF-I Receptors and
G
i, G
, and
-Arrestin-1 in 3T3 L1
Adipocytes--
Because 15% of the endogenous IGF-I receptors form
hybrids with the transfected human insulin receptors in HIRcB cells
(38, 39), this may have influenced our
-arrestin-1 or
G
i immunoprecipitation experiments. To rule out this
possibility, we conducted similar experiments in 3T3 L1 adipocytes that
express endogenous levels of all these proteins. Consistent with the
HIRcB cell results, G
i and G
immunoprecipitated with
the IGF-I receptor in the basal state; the amount of
receptor-associated G
i increased markedly after 5 min of
IGF-I stimulation, whereas the amount of receptor-associated G
decreased significantly. No interaction of IR with G
i
was found.
-Arrestin-1 associated with both receptors, and this was increased 2-3-fold following 5 min of ligand stimulation (Fig. 6). In previous studies, we have shown
that the IR can physically associate with G
q/11 protein
and couples into this G protein to stimulate glucose transport (9). In
the current studies, we find no association of the IGF-IR with
G
q/11 (data not shown), indicating that IRs and IGF-IRs
utilize specific G protein partners to mediate their biologic
effects.

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|
Fig. 6.
Selective interaction between endogenous
receptor and G i,
G q-11, G and
-arrestin-1 in 3T3-L1 adipocytes.
Differentiated 3T3L1 adipocytes were stimulated with 100 ng/ml IGF-I or
10 ng/ml insulin for 5 min at 37 °C. Lysates were immunoprecipitated
(IP) with receptors antibodies as already described,
separated by 10% SDS-PAGE, and blotted with anti-G i,
G , or anti- -arrestin-1 antibodies. Typical autoradiograph
representative of two experiments are shown.
|
|
 |
DISCUSSION |
The insulin and IGF-I receptors are very similar heterotetrameric
RTKs whose signaling cascades share many of the same molecular components, at least qualitatively. Both receptors can stimulate mitogenesis and metabolic events, although insulin is far more potent
at activating metabolic pathways and IGF-I is a more potent mitogen (1,
2). However, the molecular explanation for these relative differences
in mitogenic versus metabolic potencies remains poorly
understood. Recently, evidence has accrued suggesting that some RTKs
may also couple into heterotrimeric G proteins, similar to the
signaling events elicited by classical heptahelical GPCRs (9, 25). In
the current studies, we find that insulin and IGF-I receptors differ
strikingly in their ability to engage specific heterotrimeric G
proteins, and we propose that this explains, at least in part, the
relative potency differences between these two receptors with respect
to metabolic versus mitogenic signaling.
Our studies show that the IGF-I receptor displays the full range of
properties attributed to classical heptahelical GPCRs. Thus, the
mitogenic signaling effects of IGF-I are inhibitable by treatment of
cells with pertussis toxin and are also blocked by microinjection of
the G
subunit sequestrant GST-
ARK. In addition, inhibition of
-arrestin-1 also blocks IGF-I receptor signaling. Finally, we find
that the IGF-I receptor physically associates with G
i
and releases G
subunits in a ligand-dependent manner.
In contrast, no role for G
i in insulin signaling was detected. Our results with the IGF-I receptor are fully consistent with
the work of Luttrell et al. (25) and Lin et al.
(36). Thus, these workers have shown that IGF-I-stimulated MAP kinase activation is PTX- and GST-
ARK-sensitive and that IGF-I receptors associate with
-arrestin-1. Taken together, it is clear that the
IGF-I receptor can couple into G
i and that this coupling property is essential for mitogenic signaling. Thus, from a functional point of view, the IGF-I receptor behaves as a GPCR. Heptahelical receptors serve as the classical GPCRs, and, in the past, these two
terms have been used interchangeably to refer to this category of
receptors. Because the IGF-I receptor is G protein-coupled, it would
appear that receptors from at least two structural classes (heptahelical receptors and RTKs) can be classified as GPCRs.
For classical GPCRs, the role of
-arrestin-1 in signaling is fairly
well understood. After G protein-coupled receptor kinase phosphorylation of the GPCR,
-arrestin-1 binds to the receptor. The
receptor bound
-arrestin-1 recruits a Src family member tyrosine kinase to the receptor complex and this leads to activation of Src with
downstream phosphorylation of Shc and MAP kinase activation (12, 14,
32, 40). In addition to mediating Src kinase signaling,
-arrestin-1
facilitates heptahelical GPCR endocytosis (33-35, 41), and Daaka
et al. (42) and Pierce et al. (43) have provided
evidence that endocytosis of these receptors is required to fully
activate the Ras/MAP kinase signaling pathway downstream of Ras. Thus,
-arrestin-1 facilitates mitogenic signaling in at least two ways: 1)
the initial tyrosine kinase mediated signaling events that lead to
p21ras activation and 2) endocytosis mediated signaling events that
propagate activation steps downstream of Ras. Because the IGF-I
receptor is itself a tyrosine kinase, the role of
-arrestin-1 in
IGF-I signaling is unclear. Our results showing coprecipitation of
G
i with
-arrestin-1 raise the possibility that
-arrestin-1 may function as a scaffolding protein bringing G
i to the IGF-I receptor complex. It is possible that
-arrestin-1 may provide a scaffold to dock other molecules to the
IGF-I receptor complex, which may be involved in signaling functions.
Alternatively, it has been shown that
-arrestin-1 mediates
endocytosis of GPCRs, and recent data indicate that
-arrestin-1
facilitates IGF-I receptor internalization (36) and that
internalization of the receptor is necessary for MAP kinase activation
and mitogenic signaling (44). In this event, the functional inhibition
of
-arrestin-1 induced by microinjection of anti-
-arrestin-1
antibodies could block mitogenic signaling by inhibiting receptor
endocytosis. Our results also show that sequestration of G
subunits (GST-
ARK microinjection) inhibits IGF-I signaling. For GPCR
systems, G
subunits serve to dock G protein-coupled receptor
kinases in the region of the receptor, which initiates the process that
ultimately leads to recruitment of Src and tyrosine kinase activation
(12, 45). For IGF-I receptor signaling, however, the role of G
subunits is less clear. It has been reported that G
subunits can
bind to PH domains of certain signaling molecules (46, 47). Thus, one
possibility is that in IGF-I signaling the G
subunits associate
with PH domain containing signaling molecules that convey signals from
the IGF-I receptor to p21ras and subsequently to MAP kinase.
We have also found evidence that the IR can behave as a GPCR. Thus, the
IR is associated with
-arrestin-1 in the basal state and, after
insulin stimulation, a 2-3-fold increase in
-arrestin-1-IR association occurs. In contrast to the IGF-I receptor, however, we did
not find any evidence for G
i involvement in IR
signaling. Thus, the actions of insulin were unaffected by PTX
treatment or microinjection of GST-
ARK or anti-
-arrestin-1
antibody. Furthermore, no G
i was observed in IR
immunoprecipitates. Earlier studies indicated that there might be some
G
i dependence of IR signaling because Luttrell et
al. (25) showed partial PTX sensitivity of insulin-stimulated MAP
kinase activity in HIRc cells. However, this difference in results is
more apparent than real, because these workers used high concentrations
of insulin, which can cross over into the IGF-I receptor. Thus, it is
likely that the partial PTX sensitivity was due to inhibition of
insulin activating MAP kinase through the IGF-I receptor. Although the
IR does not appear to be coupled to G
i in our systems,
we have reported previously that insulin-stimulated GLUT4 translocation
is dependent on G
q/11. Thus, we found that
G
q/11 associates with the IR and undergoes tyrosine
phosphorylation after insulin stimulation. Inhibition of
G
q/11 function blocks insulin stimulated glucose
transport, and constitutively active G
q/11 stimulates
glucose transport and GLUT4 translocation in the absence of insulin
(9). Taken together with the data in the current paper, we would
conclude that the IR can behave as a GPCR, but unlike the IGF-I
receptor, the relevant G protein is G
q/11. In contrast,
the IGF-I receptor does not associate with G
q/11 or
utilize this G protein in its signaling pathways.
In summary, our results suggest that both the IGF-IR and IR can couple
to G proteins to mediate biologic signals. The IGF-IR utilizes
G
I, whereas the IR does not. In contrast, the IR signals through G
q/11, whereas the IGF-IR does not.
G
i mediates mitogenic signaling, whereas
G
q/11 mediates metabolic actions. This provides a
potential explanation for the fact that insulin is a more potent metabolic hormone than IGF-I, whereas IGF-I is a more potent mitogen. The insulin and IGF-I receptors are heterotetrameric RTKs that share a
great deal of structural similarity. Each of these receptors can couple
into specific G proteins and associate with
-arrestin-1. Thus, like
the general class of heptahelical receptors, these two RTKs can behave
as GPCRs. This raises the possibility that a broader number of RTKs may
be G protein-coupled and that this is a more general paradigm, as it is
for the heptahelical receptors. In this event, these two structural
classes of surface receptors would engage a number of common downstream
signaling components. We suggest that this will be the case and that
analogous to the heptahelical receptors, the G proteins used will be
receptor specific and possibly cell context specific.
 |
ACKNOWLEDGEMENTS |
We thank Dr. R. J. Lefkowitz for helpful
discussion and for the gift of
-arrestin-1 antibody. We also thank
Elizabeth Hansen for editorial assistance.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant DK 33651 and by the Whittier Institute for Diabetes.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.
To whom correspondence should be addressed: Dept. of Medicine
(0673), University of California, San Diego, 9500 Gilman Dr., La Jolla,
CA 92093-0673. Tel.: 858-534-6651; Fax: 858-534-6653.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M010884200
 |
ABBREVIATIONS |
The abbreviations used are:
IGF-I, insulin-like
growth factor I;
IGF-IR, IGF-I receptor;
GLUT4, insulin-sensitive
glucose transporter;
MAP, mitogen-activated protein;
IR, insulin
receptor;
GST, glutathione S-transferase;
LPA, lysophosphatidic acid;
EGF, epidermal growth factor;
RTK, receptor
tyrosine kinase;
GPCR, G protein-coupled receptor;
TRITC, tetramethyl
rhodamine isothiocyanate;
DMEM, Dulbecco's modified Eagle's medium;
FCS, fetal calf serum;
BrdU, bromodeoxyuridine;
PBS, phosphate-buffered
saline;
PAGE, polyacrylamide gel electrophoresis;
PTX, pertussis
toxin.
 |
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