Association of Heterotrimeric Gi with the Insulin-like Growth Factor-I Receptor

The insulin-like growth factor-I receptor (IGF-IR) is a key regulator of cell proliferation and survival. Activation of the IGF-IR induces tyrosine autophosphorylation and the binding of a series of adaptor molecules, thereby leading to the activation of MAPK. It has been demonstrated that pertussis toxin, which inactivates the Gi class of GTP-binding proteins, inhibits IGF-I-mediated activation of MAPK, and a specific role for Gβγ subunits in IGF-I signaling was shown. In the present study, we have investigated the role of heterotrimeric Gi in IGF-IR signaling in neuronal cells. Pertussis toxin inhibited IGF-I-induced activation of MAPK in rat cerebellar granule neurons and NG-108 neuronal cells. Gαi and Gβ subunits were associated with IGF-IR immunoprecipitates. Similarly, in IGF-IR-null mouse embryo fibroblasts transfected with the human IGF-IR, Gi was complexed with the IGF-IR. Gαs was not associated with the IGF-IR in any cell type. IGF-I induced the release of the Gβ subunits from the IGF-IR but had no effect on the association of Gαi. These results demonstrate an association of heterotrimeric Gi with the IGF-IR and identify a discrete pool of Gβγ subunits available for downstream signaling following stimulation with IGF-I.

In addition to their role in fully differentiated cells, GPCRs have been linked to mitogenesis and development (5)(6)(7)(8). A specific role for G i in the induction of mitogenesis has been highlighted by the use of pertussis toxin, which inactivates G i by ADP-ribosylation of the G ␣ subunit. However, G ␣ subunits from several classes of G-proteins are not strongly mitogenic. Rather G ␤␥ heterodimer subunits activate a series of nonreceptor tyrosine kinases, which in turn activates p21 ras and extracellular signal-regulated kinases (or MAPK). Thus, G ␤␥ subunits serve to bridge intracellular signaling of classical GPCRs and mitogenic tyrosine kinase receptors (RTKs).
G i also appears to be involved in the mitogenic actions of RTKs. Pertussis toxin variably inhibits the metabolic actions of insulin, both in vitro and in vivo (9 -16), and the insulin receptor may associate with G i (17)(18)(19). Importantly, mice with targeted knockout of G i have defects in insulin signaling (20). EGF-dependent signaling is also impaired by pertussis toxin in rat hepatocytes (21)(22)(23) and other cells (24 -27).
The insulin-like growth factor-I receptor (IGF-IR), which has strong homology to the insulin receptor, exists as an ␣ 2 -␤ 2heterodimer and contains a cytoplasmic tyrosine kinase domain (28,29). Upon activation by its ligands IGF-I and IGF-II, the IGF-IR undergoes tyrosine autophosphorylation, after which it phosphorylates key signaling molecules and leads to the sequential activation of ras, raf, and MAPK. The role of G i in signaling by the IGF-IR is controversial. In fibroblasts, pertussis toxin inhibits the IGF-I-induced opening of a calciumpermeable cation channel (30) and the activation of MAPK (31). In the latter study, Luttrell et al. (31) demonstrated that MAPK activation by IGF-I was also inhibited by G ␤␥ subunit binding proteins. Inhibitory effects of pertussis toxin have been observed for other IGF-I-dependent cellular effects (32-35) although not in all cases (36 -39).
Because IGF-I exerts profound proliferative and survival effects on many cell types, including neuronal cells, we have investigated the role of G i in IGF-I signaling in neuronal cells. We report the specific association of G i with the IGF-IR and inhibition of MAPK activation by pertussis toxin. Importantly, IGF-I stimulation induces the release of G ␤␥ subunits from the IGF-IR. The findings offer a model wherein G i heterotrimers are constitutively associated with the IGF-IR and identify a discrete pool of G ␤␥ subunits available for IGF-IR signaling.

EXPERIMENTAL PROCEDURES
Materials-Antibodies against G ␣i , G ␣s, G ␤, IGF-IR ␤ domain, and anti-phosphotyrosine (py99) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-MAPK and anti-phospho-MAPK (E10) antibodies, horseradish peroxidase-conjugated goat anti-rabbit IgG, and horse anti-mouse IgG antibodies and signal-enhanced chemiluminescence reagents were obtained from New England Biolabs (Beverly, MA). Pertussis toxin was purchased from Calbiochem (La Jolla, CA). Human recombinant IGF-I was obtained from Bachem Bioscience, Inc., (King of Prussia, PA). All other chemical and biochemicals were of the highest purity commercially available.
Cell Lines and Culture Conditions-Balb/c3T3 cells were obtained from American Type Culture Collection (Manassas, VA). Mouse embryo fibroblasts with targeted knockout of the IGF-IR (R Ϫ cells) and R Ϫ cells that express human IGF-IR (R ϩ ) cells were a gift from Dr. R. Baserga (Kimmel Cancer Center, Thomas Jefferson University). The generation and characterization of R Ϫ and R ϩ cells have been described elsewhere (40,41). Fibroblasts were passaged in Dulbecco's modified Eagle's medium supplemented with 10% normal calf serum and 2 mM glutamine. NG-108 neuroblastoma cells, a gift from Dr. I. Diamond, University of California, were cultured in Dulbecco's modified Eagle's medium containing 6% (v/v/) fetal calf serum and supplemented with 1 M aminopterin, 100 M hypoxanthine, and 16 M thymidine. Fibroblasts and NG-108 cells were cultured in serum-free medium for 18 h prior to addition of IGF-I. Where indicated, cells were treated with pertussis toxin for 4 h prior to the addition of IGF-I.
Rat cerebellar granule neurons were prepared from 7-day-old rat pups as reported previously (42,43). Briefly, cerebella were obtained from postnatal day 7 Harlan Sprague-Dawley rat pups, cross-chopped (400 ϫ 400 M), and then treated with 0.025% trypsin-EDTA (including 0.01% DNase I) for 15 min, 37°C. Cells were triturated and plated at a density of 1 ϫ 10 6 /cm 2 on poly-D-lysine-coated plates in Basal Medium Eagle (BME) containing 10% fetal bovine serum, 25 mM KCl, 100 units/ml penicillin, 100 g/ml streptomycin, and 0.25 g/ml amphotericin B. 10 M cytosine arabinoside was added after 24 h to inhibit nonneuronal replication. Cerebellar neurons were used after 7 days in vitro. For treatment with IGF-I, cells were washed twice and cultured for 2 h in BME with 5 mM KCl.
Immunoprecipitations and Western Blot Analysis-Cells were lysed in ice-cold Triton lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.5 mM EDTA, 10% glycerol, 1% Triton X-100, 100 mM NaF, 2 mM Na 3 VO 4 , 10 mM sodium PP I , 10 mM phenylmethylsulfonyl fluoride, 500 M AEBSF (Sigma), 150 nM aprotinin, 1 M E-64 (Sigma), and 1 M leupeptin). The lysates were centrifuged at 14,000 ϫ g for 10 min at 4°C, and the supernatants were collected. Protein concentrations were determined by a colorimetric assay (44). Cell lysates containing 30 g of protein were electrophoresed in 11% denaturing polyacrylamide gels. For immunoprecipitations, supernatants were diluted with 1 volume of HNTG (20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 10 mM NaF and 2 mM Na 3 VO 4 ). Equal amounts of protein (250 g) from each sample were immunoprecipitated using 0.4 g/ml of antibody to either IGF-IR or G-protein subunits and were captured using protein A-agarose beads. Immunoprecipitates were subjected to SDS-PAGE, transferred to Immobilon-P membranes and visualized by Western blotting. For Western blot analysis, the proteins were transferred to Immobilon-P membranes, blocked with 3% bovine serum albumin and probed with antibodies (1:2000 for all antibodies except 1:1000 for anti-MAPK antibodies), followed by secondary horseradish peroxidase-conjugated goat anti-rabbit IgG or horse anti-mouse IgG antibodies. Membranes were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). The figures are scanned images and are representative of 3-5 experiments.

RESULTS
The role of G i in the activation of p42/p44 MAPK by IGF-I was investigated in Balb/c 3T3 cells, rat cerebellar granule neurons, and NG-108 neuronal cells (Fig. 1). Basal MAPK phosphorylation was reduced by pertussis toxin in Balb/c 3T3 and NG-108 cells but not in cerebellar granule neurons. In agreement with a previous report (31), pertussis toxin markedly inhibited IGF-I-induced MAPK phosphorylation in all cell types. Total MAPK content was unchanged under all conditions. Pertussis treatment had no effect on cell viability. Pertussis toxin inactivates both G i and G o , but 3T3 cells lack G o . The results indicate that G i is at least partially required for MAPK activation by IGF-I.
Having established a requirement for G i in IGF-I-induced neuronal MAPK activation, we next examined the relationship between G ␣i and the IGF-IR. IGF-IR was immunoprecipitated under nondenaturing conditions and probed for G ␣i content by Western blot analysis with anti-G ␣i antibody. Fig. 2 illustrates that G ␣i is associated with the IGFR-I in both cerebellar granule cells and NG-108 cells. The IGF-IR and G ␣i were similarly complexed in Balb/c 3T3 cells (data not shown). G ␣s subunits were not detected in IGF-IR immunoprecipitates from any cell type despite copious expression in total cell lysates (not shown). The IGF-IR/G ␣i interaction was completely disrupted when the IGF-IR was immunoprecipitated under denaturing conditions (not shown). IGF-I had no effect on the association of G ␣i with the IGF-IR. The IGF-IR was active under these experimental conditions, as manifest by IGF-I-induced tyrosine autophosphorylation of the ␤ domain of the IGF-IR in both cell types (Fig. 2, bottom panel).
The anti-IGF-IR antibody did not cross-react with G ␣i . Fig. 3 demonstrates that G ␣i subunits were not identified using anti-IGF-IR antibodies in mouse embryo fibroblasts with targeted knockout of the IGF-IR (R Ϫ cells). Both proteins were immunoprecipitated by anti-IGF-IR antibody upon restoration of IGF-IR expression (R ϩ cells) A requirement for G ␤␥ subunit release in IGF-IR-induced MAPK activation was suggested by the studies of Luttrell et al. (31). In conjunction with the observation that G ␣i does not dissociate from the IGF-IR in response to IGF-I, we conjectured that IGF-I induces G ␤␥ subunit release from the IGF-IR. Fig. 4 demonstrates that G ␤ subunit coimmunoprecipitates with the IGF-IR in both cerebellar granule neurons and NG-108 cells. The amount of G ␤ subunit associated with the IGF-IR decreased dramatically in response to IGF-I. G ␤ subunit expression within total cell lysates was identical under all conditions. Fig. 4 also demonstrates that pertussis toxin prevented the release of G ␤ subunit from the IGF-IR but had no effect on the expression of IGF-IR or G ␤ subunit. We conclude that heterotrimeric G i is constitutively associated with IGF-IR and responds to IGF-I by release of free G ␤␥ subunits. DISCUSSION The use of pertussis toxin and ␤␥ subunit binding proteins has provided indirect evidence for an additional role of G i in the activation of MAPK by IGF-I (31). In the current study, we extended these findings to neuronal cell types and now provide direct evidence for the constitutive association of heterotrimeric G i with the IGF-IR complex. Both G ␣i and G ␤ subunits co-immunoprecipitate with IGF-IR in cerebellar neurons and NG-108 cells. Importantly, IGF-I induces the release of G ␤ subunits from the IGF-IR complex, whereas the association of G ␣i with the IGF-IR is unaffected. The current data do not allow us to determine whether G ␣i binds directly to the IGF-IR or to another protein in the IGF-IR complex. G ␤␥ subunits exhibit a pleckstrin homology domain which mediates binding to several molecules that are part of the IGF-IR signaling complex including IRS-1 and PI 3-kinase (3). However, these interactions are unlikely to play a role in binding of G ␤␥ subunits to the IGF-IR because IRS-1 and PI 3-kinase are not bound to the IGF-IR in its unactivated state.
RTKs for insulin, IGF-I and EGF, among others, contain SH2 binding domains which anchor adaptor proteins such as IRS-1 and Shc (28,29). Phosphorylation of these intermediaries leads to the activation of p21 ras , MAPK, and transcriptional activation. Both IRS-1 and Shc can independently lead to MAPK activation, although studies in some cell types have suggested that Shc may ultimately be more important for mitogenesis (45,46). The IGF-IR also activates PI 3-kinase, which has diverse functions, including an anti-apoptotic role mediated by its activation of Akt (47)(48)(49). However, the relative contribution of these diverse signaling pathways to functional effects must be studied individually. There is also ample experimental evidence to support a role for the IGF-IR in the maintenance of the transformed phenotype. Notably, R Ϫ cells are resistant to transformation in response to several oncogenic influences (50). Transformation is restored upon expression of the IGF-IR. In contrast, Leroith and co-workers have recently demonstrated that a constitutively active mutant of G ␣13 can transform R Ϫ cells (51).
The role of G i in IGF-I signaling likely varies among different cell types. Pertussis toxin variably inhibits biological actions of IGF-I. In 3T3 cells (30) and chondrocytes (32), pertussis toxin inhibits IGF-I-induced mobilization of intracellular calcium. Anti-G ␣i antibodies inhibit the opening of a calcium-permeable cation channel in response to IGF-I (30). Pertussis toxin also inhibits the activation of human neutrophil phagocytosis (35) and blocks IGF-I induced proliferation of myoblasts (33). By contrast, pertussis toxin failed to inhibit several IGF-I-dependent phenomena including GTP[S] binding to rat kidney epithelial cell membranes (36), DNA synthesis in MG-63 osteosarcoma cells (38), and IGF-I-induced human melanoma cell motility (39). We have also noted that pertussis toxin has no effect on the ability of IGF-I to protect cerebellar granule cells from apoptosis induced by removal of growth factors and low extracellular potassium (43,52). 2 Thus, as is the case for other signaling mediators of the IGF-IR, the specific functional role of G ␤␥ subunits in IGF-I signaling is likely dependent upon cell context. G i may also associate with the insulin receptor. G ␣i2 and G ␤ (but not G ␣i1 or G ␣i3 ) were identified in purified insulin receptor preparations (14). In a recent study, Sanchez et al. (19) reported that activation of the insulin receptor recruits G ␣i to this receptor, in a manner similar to classical GPCRs. However, this differs from our finding that G ␣i constitutively associates with the IGF-IR. Pertussis toxin inhibits metabolic and mitogenic actions of insulin in a variety of cell culture models (9 -16), and insulin enhances GTP binding to membranes (16,18). In a recent study of rat hepatoma cell membranes (19), antibodies targeted against G ␣i but not G ␣o , prevented insulin-2 H. Hallak, and R. Rubin, unpublished data. IGF-IR immunoprecipitates were obtained from cerebellar granule neurons and NG-108 cells either before or after a 10-min treatment with IGF-I (25 ng/ml), in the presence or absence of pertussis toxin. G ␤ subunit content within IGF-IR immunoprecipitates or total lysates was determined by Western blotting. G ␤ content within total lysates is also shown. stimulated GTP binding. Similarly, anti-G ␣i antibodies blocks insulin-induced NADPH-dependent generation of hydrogen peroxide in human adipocyte membranes (14).
The mechanism by which the IGF-IR induces G ␤␥ subunit release is unclear. Direct tyrosine phosphorylation of G ␣i by the insulin receptor was suggested in studies of phospholipid vesicles co-inserted with G ␣i (17). However, other data are inconsistent with the notion that G␣i is phosphorylated by the insulin receptor (53)(54)(55)(56). Thus, a pathway other than tyrosine kinase activation may account for G ␤␥ release, perhaps a configurational change induced by IGF-I. Our preliminary studies are in agreement with this view. Although we can detect tyrosine phosphate residues within G ␣i by Western blotting, IGF-I has no effect on the tyrosine phosphorylation state of the G ␣i subunit protein.
In conclusion, this study demonstrates the constitutive association of G i with the IGF-IR, and provides a mechanism for release of an IGF-I-sensitive pool of G ␤␥ subunits. In cerebellar granule cells and NG-108 cells specifically, G ␤␥ subunit release is required for MAPK activation. The findings highlight the potential role of G ␤␥ subunits in the regulation of neuronal MAPK activity, which is a key element in neuronal development and regeneration.