Insulin and Insulin-like Growth Factor I Receptors Utilize Different G Protein Signaling Components
2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês
10.1074/jbc.m010884200
ISSN1083-351X
AutoresStéphane Dalle, William Ricketts, Takeshi Imamura, Péter Vollenweider, Jerrold M. Olefsky,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoWe 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 Gαi inhibitor (pertussis toxin) or microinjection of the Gβγ inhibitor (glutathioneS-transferase-βARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Gαi and Gβ were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Gαi increased concomitantly with a decrease in Gβ association. No association of Gαi was found with either the insulin or EGF receptor. Microinjection of anti-β-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. β-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Gαi, βγ subunits, and β-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. 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 Gαi inhibitor (pertussis toxin) or microinjection of the Gβγ inhibitor (glutathioneS-transferase-βARK) inhibited IGF-I and lysophosphatidic acid-stimulated mitogenesis but had no effect on epidermal growth factor (EGF) or insulin action. In basal state, Gαi and Gβ were associated with the IGF-I receptor (IGF-IR), and after ligand stimulation the association of IGF-IR with Gαi increased concomitantly with a decrease in Gβ association. No association of Gαi was found with either the insulin or EGF receptor. Microinjection of anti-β-arrestin-1 antibody specifically inhibited IGF-I mitogenic action but had no effect on EGF or insulin action. β-Arrestin-1 was associated with the receptors for IGF-I, insulin, and EGF in a ligand-dependent manner. We demonstrated that Gαi, βγ subunits, and β-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. insulin-like growth factor I IGF-I receptor insulin-sensitive glucose transporter mitogen-activated protein insulin receptor glutathione S-transferase lysophosphatidic acid epidermal growth factor receptor tyrosine kinase G protein-coupled receptor tetramethyl rhodamine isothiocyanate Dulbecco's modified Eagle's medium fetal calf serum bromodeoxyuridine phosphate-buffered saline polyacrylamide gel electrophoresis pertussis toxin 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 (1Cheatman B. Khan C.R. Endocr. 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Hawes B.E. Koch W.J. Touhara K. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 16495-16498Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Furthermore, recent work from our laboratory has shown that Gαq/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 Gαq/11 and that this G protein is necessary for insulin stimulation of GLUT4 translocation and glucose transport. In addition, a constitutively active form of Gαq/11 was able to mimic the effects of insulin by stimulating GLUT4 translocation and glucose transport on its own (9Imamura T. Vollenweider P. Egawa K. Clodi M. Ishibashi K. Nakashima N. Ugi S. Adams J.W. Brown J.H.B. Olefsky J.M. Mol. Cell. Biol. 1999; 19: 6765-6774Crossref PubMed Scopus (145) Google Scholar). 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. β-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, Gβ, Gαi 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. HIRcB cells, which are rat 1 fibroblasts overexpressing the human form of the IR, were maintained as previously described (26McClain D.A. Maegawa H. Lee J. Dull T.J. Ullrich A. Olefsky J.M. J. Biol. Chem. 1987; 262: 14663-14671Abstract Full Text PDF PubMed Google Scholar) 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% CO2environment. 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. 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. Cells were injected and photographed as previously described (27Haruta T.A. Morris A.J. Rose D.W. Nelson J.G. Mueckler M. Olefsky J.M. J. Biol. Chem. 1995; 270: 27991-27994Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). 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 of GLUT4 was performed as previously described (28Vollenweider P. Martin S.S. Haruta T. Morris A.J. Nelson J.G. Cormont M. Marchand-Brustel Y.L. Rose D.W. Olefsky J.M. Endocrinology. 1997; 138: 4941-4949Crossref PubMed Scopus (68) Google Scholar). 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. 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. 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 mmNa3VO4, 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). 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 mmNa2HPO4, 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 ofl-[3H]deoxyglucose as described previously (29Klip A. Li G. Logan W.J. Am. J. Physiol. 1984; 247: E291-E296PubMed Google Scholar). Nonspecific uptake was assessed by the addition of 0.1 mml-glucose containing 0.2 μCi ofl-[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. 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. Fig.1 A 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 Gαi signaling, whereas insulin- and EGF-stimulated mitogenic signaling do not. Luttrel et al. (25Luttrell L.M. Van Biesen T. Hawes B.E. Koch W.J. Touhara K. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 16495-16498Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar) have shown that Gβγ subunits are necessary for IGF-I-induced MAP kinase activation. Thus, in our conditions, we assessed the role of Gβγ in growth factor-stimulated mitogenesis. We utilized a glutathioneS-transferase (GST) fusion protein containing the C-terminal portion of the β-adrenergic receptor kinase (GST-βARK), which binds to and sequesters Gβγ subunits and behaves as a dominant negative inhibitor of Gβγ signaling (30Garcia-Higuera I. Mayor Jr., F. J. Clin. Invest. 1994; 93: 937-943Crossref PubMed Scopus (35) Google Scholar). We microinjected GST-βARK into HIRcB cells and measured insulin, IGF-I, LPA, and EGF stimulated BrdU incorporation. As shown in Fig. 1 B, microinjection of GST-βARK blocks IGF-I- and LPA-induced BrdU incorporation but not that induced by insulin or EGF. As seen in Fig.2 A, Gαiimmunoprecipitated with the IGF-I receptor in the basal state, and the amount of receptor-associated Gαi increased markedly after IGF-I stimulation. No association of Gαi with either the insulin receptor or EGF receptors was found in HIRcB cells (Fig. 2 A). Time course studies showed that the IGF-I-induced association between Gαi and IGF-IR proceeds with a maximum from 2 to 5 min (Fig. 2, B and C). Total Gαi expression in cell lysates was the same at all time points (Fig. 2, A and B). Because tyrosine phosphorylation has been shown for Gαi, we determined whether or not Gαi-tyrosine phosphorylation was enhanced by IGF-I treatment. HIRcB cells were stimulated with IGF-I for various periods, followed by phosphotyrosine antibody or Gαiantibody immunoprecipitation and Western blotting with Gαi or phosphotyrosine antibodies, respectively. IGF-I did not lead to tyrosine phosphorylation of Gαi (data not shown). To further assess Gβγ subunit involvement in the mitogenic effect of IGF-I, we investigated whether endogenous Gβ subunits interact with the IGF-I receptor. Immunoprecipitation studies show an association between the IGF-IR and Gβ, which decreased 35% in response to IGF-I (maximum decrease at 2 min) (Fig. 2, D andE). Total Gβ expression in cell lysates was the same at all time points (Fig. 2 D). The process of receptor internalization plays an essential role in receptor-mediated mitogenic signaling (31Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (822) Google Scholar,32Luttrell L.M. Ferguson S.S.G. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-660Crossref PubMed Scopus (1255) Google Scholar). Because it has been shown that β-arrestin-1 is necessary for GPCR and IGF-IR internalization (33Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (900) Google Scholar, 34Pippig S. Andexinger S. Daniel K. Puzicha M. Caron M.G. Lefkowitz R.J. Lohse M.J. J. Biol. Chem. 1993; 268: 3201-3208Abstract Full Text PDF PubMed Google Scholar, 35Ferguson S.S. Downey 3rd, W.E. Colapietro A.M. Barak L.S. Menard L Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (841) Google Scholar, 36Lin F.T. Daaka Y. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 31640-31643Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), we tested the hypothesis that this protein was involved in the mitogenic effects elicited by the receptors coupled to Gαi. We inhibited endogenous β-arrestin-1 function by single cell microinjection of anti-β-arrestin-1 antibody, followed by measurement of BrdU incorporation. As shown in Fig. 3, microinjection of the β-arrestin-1 antibody into NIH3T3 cells inhibited DNA synthesis in response to IGF-I and LPA but not to insulin or EGF. Overexpressed β-arrestin-1 can interact with IGF-I receptors in HEK 293 cells (36Lin F.T. Daaka Y. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 31640-31643Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), and, therefore, we determined whether endogenous levels of β-arrestin-1 could interact with endogenous IGF-IRs in our system. As shown in Fig.4 (A and B), in the basal state, β-arrestin-1 is associated with the IGF-IR, and this association increases >2 fold at 5 min of IGF-I stimulation. β-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, β-arrestin-1 also immunoprecipitated with the EGFR, but the time course of association was more rapid and transient (Fig. 4,E and F). Using 3T3 L1 adipocytes that express endogenous levels of IRs and IGF-I Rs, we studied the involvement of Gαi, Gβγ, or β-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 β-arrestin-1 was involved in this action of insulin using anti-β-arrestin-1 antibody microinjection followed by insulin stimulation and immunofluorescence staining for GLUT4 translocation. As shown in Fig. 5 B, inhibition of β-arrestin-1 function had no effect on insulin-stimulated GLUT4 translocation. As we previously reported (37Imamura T. Ishibashi K.I. Dalle S. Ugi S. Olefsky J.M. J. Biol. Chem. 1999; 274: 33691-33695Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), we also show in this system that GST-βARK microinjection did not inhibit insulin-induced GLUT4 translocation (Fig. 5 B). Taken together, our results show that, in 3T3L1 adipocytes, there is no involvement of a Gαi/Gβ pathway or β-arrestin-1 in insulin signaling leading to glucose uptake and GLUT4 translocation. 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 Gβγ subunits and β-arrestin-1 were involved in insulin or IGF-I signaling leading to MAP kinase phosphorylation, we microinjected GST-βARK and anti-β-arrestin-1 antibody and measured MAP kinase phosphorylation by immunofluorescence staining of cells. Microinjection of GST-βARK and anti-β-arrestin-1 antibody inhibited IGF-I-induced MAP kinase phosphorylation but not that induced by insulin (Fig. 5, Eand F). These results clearly indicate that in 3T3L1 adipocytes, IGF-I signaling leading to MAP kinase phosphorylation requires Gαi, Gβ, and β-arrestin-1, whereas insulin-induced MAP kinase signaling does not. These results also show that these reagents (PTX, GST-βARK, and anti-β-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. Because 15% of the endogenous IGF-I receptors form hybrids with the transfected human insulin receptors in HIRcB cells (38Seely B.L. Reichart D. Takata Y. Yip C. Olefsky J.M. Endocrinology. 1995; 136: 1635-1641Crossref PubMed Scopus (67) Google Scholar, 39Sasaoka T. Ishiki M. Sawa T. Ishihara H. Takata Y. Imamura T. Usui I. Olefsky J.M. Kobayashi M. Endocrinology. 1996; 137: 4427-4434Crossref PubMed Scopus (99) Google Scholar), this may have influenced our β-arrestin-1 or Gαi immunoprecipitation experim
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