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. OlefskyDagger

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Galpha i inhibitor (pertussis toxin) or microinjection of the Gbeta gamma inhibitor (glutathione S-transferase-beta ARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Galpha i and Gbeta were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Galpha i increased concomitantly with a decrease in Gbeta association. No association of Galpha i was found with either the insulin or EGF receptor. Microinjection of anti-beta -arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. beta -Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Galpha i, beta gamma subunits, and beta -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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -subunits and two transmembrane beta -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 beta  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 Galpha 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 Galpha i for some of its biologic effects. Thus, treatment with pertussis toxin (an inhibitor of Galpha i-mediated signaling) blocks IGF-I-induced activation of MAP kinase, and inhibition of Gbeta gamma function also impairs MAP kinase signaling from the IGF-I receptor (25). Furthermore, recent work from our laboratory has shown that Galpha 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 Galpha 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 Galpha 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- beta -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, Gbeta , Galpha 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of Galpha i and Gbeta gamma 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 Galpha i signaling, whereas insulin- and EGF-stimulated mitogenic signaling do not. Luttrel et al. (25) have shown that Gbeta gamma subunits are necessary for IGF-I-induced MAP kinase activation. Thus, in our conditions, we assessed the role of Gbeta gamma in growth factor-stimulated mitogenesis. We utilized a glutathione S-transferase (GST) fusion protein containing the C-terminal portion of the beta -adrenergic receptor kinase (GST-beta ARK), which binds to and sequesters Gbeta gamma subunits and behaves as a dominant negative inhibitor of Gbeta gamma signaling (30). We microinjected GST-beta ARK into HIRcB cells and measured insulin, IGF-I, LPA, and EGF stimulated BrdU incorporation. As shown in Fig. 1B, microinjection of GST-beta 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-beta 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-beta 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-beta 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.

Selective Galpha i Interaction with Receptor Tyrosine Kinases-- As seen in Fig. 2A, Galpha i immunoprecipitated with the IGF-I receptor in the basal state, and the amount of receptor-associated Galpha i increased markedly after IGF-I stimulation. No association of Galpha 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 Galpha i and IGF-IR proceeds with a maximum from 2 to 5 min (Fig. 2, B and C). Total Galpha i expression in cell lysates was the same at all time points (Fig. 2, A and B). Because tyrosine phosphorylation has been shown for Galpha i, we determined whether or not Galpha i-tyrosine phosphorylation was enhanced by IGF-I treatment. HIRcB cells were stimulated with IGF-I for various periods, followed by phosphotyrosine antibody or Galpha i antibody immunoprecipitation and Western blotting with Galpha i or phosphotyrosine antibodies, respectively. IGF-I did not lead to tyrosine phosphorylation of Galpha i (data not shown). To further assess Gbeta gamma subunit involvement in the mitogenic effect of IGF-I, we investigated whether endogenous Gbeta subunits interact with the IGF-I receptor. Immunoprecipitation studies show an association between the IGF-IR and Gbeta , which decreased 35% in response to IGF-I (maximum decrease at 2 min) (Fig. 2, D and E). Total Gbeta expression in cell lysates was the same at all time points (Fig. 2D).


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Fig. 2.   Selective association of Galpha 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 beta  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-Galpha i antibody that recognizes rat Galpha i-1, Galpha i-2, or Galpha 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 beta  subunit antibody. Representative exposures are shown. Galpha i content within total cell lysates is also shown (A and B). C- Temporal pattern of IGF-IR and Galpha 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 Gbeta 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-Gbeta antibody. A typical exposure representative of three experiments is shown. Gbeta content within total cell lysates is also shown. E, temporal pattern of IGF-IR and Gbeta association deduced from quantitative results of three representative IGF-IR immunoprecipitation experiments obtained using NIH Image program.

Effects of beta -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 beta -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 Galpha i. We inhibited endogenous beta -arrestin-1 function by single cell microinjection of anti-beta -arrestin-1 antibody, followed by measurement of BrdU incorporation. As shown in Fig. 3, microinjection of the beta -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 beta -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 beta -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.

Interaction between Insulin, IGF-I, EGF Receptors, and beta -Arrestin-1 in HIRcB Cells-- Overexpressed beta -arrestin-1 can interact with IGF-I receptors in HEK 293 cells (36), and, therefore, we determined whether endogenous levels of beta -arrestin-1 could interact with endogenous IGF-IRs in our system. As shown in Fig. 4 (A and B), in the basal state, beta -arrestin-1 is associated with the IGF-IR, and this association increases >2 fold at 5 min of IGF-I stimulation. beta -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, beta -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 beta -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 beta -subunit antibodies, or with the anti-EGF receptor antibody. Immunoprecipitated proteins were resolved by 10% SDS-PAGE and blotted with anti-beta -arrestin-1 antibody. Representative exposures are shown. Identical amounts of proteins were analyzed as described under "Experimental Procedures." Temporal pattern of receptors and beta -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).

Effect of PTX, GST-beta ARK, and beta -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 Galpha i, Gbeta gamma , or beta -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 beta -arrestin-1 was involved in this action of insulin using anti-beta -arrestin-1 antibody microinjection followed by insulin stimulation and immunofluorescence staining for GLUT4 translocation. As shown in Fig. 5B, inhibition of beta -arrestin-1 function had no effect on insulin-stimulated GLUT4 translocation. As we previously reported (37), we also show in this system that GST-beta 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 Galpha i/Gbeta pathway or beta -arrestin-1 in insulin signaling leading to glucose uptake and GLUT4 translocation.


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Fig. 5.   Effects of PTX treatment, GST-beta ARK and beta -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-beta ARK protein, or beta -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-beta ARK protein, or beta -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.

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 Gbeta gamma subunits and beta -arrestin-1 were involved in insulin or IGF-I signaling leading to MAP kinase phosphorylation, we microinjected GST-beta ARK and anti-beta -arrestin-1 antibody and measured MAP kinase phosphorylation by immunofluorescence staining of cells. Microinjection of GST-beta ARK and anti-beta -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 Galpha i, Gbeta , and beta -arrestin-1, whereas insulin-induced MAP kinase signaling does not. These results also show that these reagents (PTX, GST-beta ARK, and anti-beta -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 Galpha i, Gbeta , and beta -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 beta -arrestin-1 or Galpha 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, Galpha i and Gbeta immunoprecipitated with the IGF-I receptor in the basal state; the amount of receptor-associated Galpha i increased markedly after 5 min of IGF-I stimulation, whereas the amount of receptor-associated Gbeta decreased significantly. No interaction of IR with Galpha i was found. beta -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 Galpha 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 Galpha 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 Galpha i, Galpha q-11, Gbeta and beta -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-Galpha i, Gbeta , or anti- beta -arrestin-1 antibodies. Typical autoradiograph representative of two experiments are shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Gbeta gamma subunit sequestrant GST-beta ARK. In addition, inhibition of beta -arrestin-1 also blocks IGF-I receptor signaling. Finally, we find that the IGF-I receptor physically associates with Galpha i and releases Gbeta gamma subunits in a ligand-dependent manner. In contrast, no role for Galpha 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-beta ARK-sensitive and that IGF-I receptors associate with beta -arrestin-1. Taken together, it is clear that the IGF-I receptor can couple into Galpha 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 beta -arrestin-1 in signaling is fairly well understood. After G protein-coupled receptor kinase phosphorylation of the GPCR, beta -arrestin-1 binds to the receptor. The receptor bound beta -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, beta -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, beta -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 beta -arrestin-1 in IGF-I signaling is unclear. Our results showing coprecipitation of Galpha i with beta -arrestin-1 raise the possibility that beta -arrestin-1 may function as a scaffolding protein bringing Galpha i to the IGF-I receptor complex. It is possible that beta -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 beta -arrestin-1 mediates endocytosis of GPCRs, and recent data indicate that beta -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 beta -arrestin-1 induced by microinjection of anti-beta -arrestin-1 antibodies could block mitogenic signaling by inhibiting receptor endocytosis. Our results also show that sequestration of Gbeta gamma subunits (GST-beta ARK microinjection) inhibits IGF-I signaling. For GPCR systems, Gbeta gamma 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 Gbeta gamma subunits is less clear. It has been reported that Gbeta gamma subunits can bind to PH domains of certain signaling molecules (46, 47). Thus, one possibility is that in IGF-I signaling the Gbeta gamma 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 beta -arrestin-1 in the basal state and, after insulin stimulation, a 2-3-fold increase in beta -arrestin-1-IR association occurs. In contrast to the IGF-I receptor, however, we did not find any evidence for Galpha i involvement in IR signaling. Thus, the actions of insulin were unaffected by PTX treatment or microinjection of GST-beta ARK or anti- beta -arrestin-1 antibody. Furthermore, no Galpha i was observed in IR immunoprecipitates. Earlier studies indicated that there might be some Galpha 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 Galpha i in our systems, we have reported previously that insulin-stimulated GLUT4 translocation is dependent on Galpha q/11. Thus, we found that Galpha q/11 associates with the IR and undergoes tyrosine phosphorylation after insulin stimulation. Inhibition of Galpha q/11 function blocks insulin stimulated glucose transport, and constitutively active Galpha 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 Galpha q/11. In contrast, the IGF-I receptor does not associate with Galpha 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 Galpha I, whereas the IR does not. In contrast, the IR signals through Galpha q/11, whereas the IGF-IR does not. Galpha i mediates mitogenic signaling, whereas Galpha 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 beta -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 beta -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.

Dagger 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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