Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor.

Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context. We have investigated the mechanism by which full-length hGrb10gamma inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of hGrb10gamma in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10gamma to both delay and reduce phosphorylation of Akt at Thr(308) and Ser(473) in response to insulin stimulation. Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10gamma directly associates with the IR kinase regulatory loop. However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10. In cells, hGrb10gamma inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr(972) in the juxtamembrane region and Tyr(1158/1162/1163) in the regulatory domain. We conclude that binding of hGrb10gamma to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.

The insulin receptor (IR) 1 transmits signals through the actions of its intrinsic receptor tyrosine kinase. Ligand binding results in the autophosphorylation of the IR on multiple tyrosine residues (1,2). Once phosphorylated, these residues serve as docking sites for specific intracellular effectors central to the transmission of the insulin signal. The insulin receptor substrate (IRS) family (IRS-1 through IRS-4) is one such group of proteins that recognizes the phosphorylated IR via their phosphotyrosine binding domains (PTB) (3,4). The IRS-PTB domain associates with the NPXY motif surrounding tyrosine 972 in the juxtamembrane region of the IR, which promotes the IR-IRS interaction (5). Once in contact with the receptor, IRS is phosphorylated on multiple tyrosine residues by the IR tyrosine kinase. Numerous adaptor proteins and enzymes then associate with tyrosine-phosphorylated IRS via their Src homology 2 (SH2) domains and convey the insulin signal downstream (6,7).
The insulin receptor-binding protein Grb10 is of considerable interest due to its potential to positively or negatively affect receptor-tyrosine kinase signaling. Grb10 is a member of a superfamily of adaptor proteins, which includes Grb7 and Grb14. This family of proteins shares several structural features, including a SH2 and a Pleckstrin homology (PH) domain (8). The C-terminal SH2 domain has been shown to associate with the IR (9,10), and mutation of a critical arginine residue in this region of full-length Grb10 disrupts IR-Grb10 association in cells (11). A second domain termed the BPS (for between the Pleckstrin and SH2) has also been suggested as a second independent IR-interacting domain (9). Grb10 interacts with the regulatory kinase loop of the IR (10,12,13), although studies have also reported a Grb10-interacting region in the IR C terminus (14,15). Several human Grb10 isoforms exist and most likely arise from alternative splicing. Two isoforms of hGrb10 differ in the PH domain. hGrb10␥ encodes for the full-length protein, while Grb10␣ (previously named Grb-IR) lacks an intact PH domain as it contains a 46-amino acid deletion in this region (16,17). A third isoform, hGrb10␤ is identical to hGrb10␥ with the exception of 58 amino acids at the extreme N terminus of the ␥ isoform (12,17).
The functional role for Grb10 in insulin signaling remains controversial. Grb10 has been shown to positively stimulate insulin-induced mitogenesis (18). Microinjection of a peptide fragment including the BPS and SH2 domain of Grb10 inhibited insulin-stimulated DNA synthesis (13,18), which, if functioning as a dominant negative, is consistent with Grb10 as a positive regulator for cell growth. Several studies have indicated an inhibitory role for Grb10 as well. Overexpression of hGrb10␣ negatively regulates insulin receptor-mediated tyrosine phosphorylation of GTPase-activated protein (GAP)-associated protein p60 and IRS-1 (16). Furthermore, binding of Grb10 via its BPS/SH2 domains has been shown to inhibit IR catalytic activity in vitro (19 -21).
Stable expression of hGrb10␣ in CHO/IR reduces insulin-dependent phosphorylation of IRS-1 (16), although a direct effect of full-length Grb10 on IRS-1/2 and downstream effectors of the insulin signal, such as Akt, has not been reported. However, several studies indicate that Grb10 may regulate downstream events in various signaling pathways. Overexpression of hGrb10␥ (Grb10) in rat hepatocytes inhibits insulin-stimulated glycogen synthase activity, through a proposed novel pathway outside of the classical PI 3-kinase to Akt/glycogen synthase kinase-3 signaling (21). Grb10 has also been found to associate with tyrosine-phosphorylated c-kit receptor, and synergistically promote Akt activation. This study indicated Grb10 forms a constitutive complex with Akt and proposed that Grb10 positively influenced Akt activity by promoting its translocation to the cell membrane leading to the phosphorylation and activation of this kinase (22).
Insulin-stimulated tyrosine phosphorylation of IRS proteins induces the association of the p85 regulatory subunit of PI 3-kinase and subsequently results in an increase in PI 3-kinase activity (23). As an IR-interacting protein, Grb10 has the potential to influence signaling through this IRS/PI 3-kinase pathway and moderate the actions of downstream effectors such as Akt. This study investigates the molecular mechanisms linked to the effects of Grb10 on insulin signaling. We find that hGrb10␥ both delays and decreases Akt phosphorylation. This inhibition stems from a rapid and sustained inhibition of insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Our studies suggest that the mechanism for Grb10-mediated inhibition of insulin signaling results from a physical disruption of IRS association with phosphorylated residues of the IR kinase domain and/or the NPXY motif (Tyr 972 ). The SH2 domain of Grb10 is essential for IR/IRS disruption. In addition, this region is required to facilitate Grb10 association with the IR and inhibition of IRS tyrosine phosphorylation in cells. This physical disruption of IR/IRS interaction by Grb10 functions to slow signaling relayed through tyrosine-phosphorylated IRS proteins.

MATERIALS AND METHODS
Cell Lines and Reagents-Chinese hamster ovary (CHO) cells expressing both the human insulin receptor and hGrb10␥ have been previously described (16,17). 3T3-L1 cells were from ATCC. Anti-HA antibody was obtained from BABCO. Monoclonal anti-Myc antibody was from Santa Cruz Biotechnology, Inc. Antibodies recognizing IRS-1 and IRS-2 were from Upstate Biotechnology. Akt and phospho-Akt (Thr 308 and Ser 473 ) antibodies were from New England Biolabs. The anti-phosphotyrosine (RC-20) antibody was from Transduction Laboratories. Phosphospecific antibodies to Tyr 972 , Tyr 1158 , and Tyr 1162/1163 of the insulin receptor were obtained from BIOSOURCE International. Secondary antibodies conjugated to alkaline phosphatase and horseradish peroxidase were from Promega.
Plasmids-cDNAs encoding HA-tagged Grb10␥ and Myc-tagged Akt have been previously described (17,24). HA-tagged Grb10␥ R520G was generated by single-stranded site-directed mutagenesis according to the protocol described by Kunkel et al. (25). The forward PCR primer 5Ј-GCTCTAGATGGCCTTGCTGTCCCCGTTC-3Ј and the reverse PCR primer 5Ј-CGGAATTCCACTCGGATGCAGTGGTG-3Ј were used to amplify the BPS/SH2 fragment using pBex/Grb10␥ or pBex/Grb10␥ R520G as template. The reverse PCR primer 5Ј-GCGAATTCGTGCTGTGTC-CTGTGAATC-3Ј was used to amplify the ⌬SH2 fragment. The PCR products were inserted in-frame with the HA tag using the XbaI and EcoRI restriction sites of the mammalian expression vector pBex (16). The forward PCR primer 5Ј-TGCTCTAGACAGTGCTACAGAGC-CAAC-3Ј and reverse PCR primer 5Ј-GATCGGTCGACGTGCTGTGTC-CTGTG-3Ј were used to amplify a 1.5-kb fragment from hGrb10␥ (region encoding for amino acids 1-491). The forward PCR primer 5Ј-GATCGGTCGACCCCCCAAATCCTTCTCTT-3Ј and the reverse primer 5Ј-CGGAATTCCACTTTCCGATCCACGGG-3Ј were used to amplify a 0.4-kb (SH2 domain) fragment from pRK/Shc-myc (plasmid was a generous gift from Dr. Ben Margolis). The individual PCR products were separately subcloned into an intermediate vector using the XbaI/SalI and SalI/EcoRI restriction sites. The resulting chimeric fragment was inserted in-frame with the HA tag using the XbaI/EcoRI restriction sites of pBex. A plasmid encoding Myc-tagged IRS-1 was a generous gift from Dr. Richard Roth (26). A plasmid encoding full-length human IRS-2 was a generous gift from Dr. Klein-Hitpass (27).
Adenoviruses-Adenoviruses encoding green fluorescence protein (GFP, as an adenovirus control) and hGrb10␥ were produced by using the AdEasy system (The AdEasy system is a generous gift of Drs. Tong-Chuan He and Bert Vogelstein, Ref. 28). The cDNA encoding HA-tagged Grb10␥ was cloned into the pAdTrack-CMV transfer vector using EcoRV and XbaI restriction sites. The hGrb10␥-HA encoding sequence was then transferred into the pAdEasy viral DNA plasmid by homologous recombination in the BJ5183 Escherichia coli strain. The recombinant adenoviral construct was transfected into HEK293 cells to produce viral particles. Adenoviruses were purified by CsCl gradient centrifugation. The infection efficiency was estimated for GFP expression using an Olympus IX70 immunofluorescence microscope.
Cell Culture, Immunoprecipitation, and Western Blot-CHO/IR cells were maintained in Ham's F12 media supplemented with 10% newborn calf serum and 1% penicillin/streptomycin. Cells were grown to 60 -80% confluency 24 h prior to transfection using LipofectAMINE reagent according to the manufacturer's protocol (Invitrogen). 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were grown to confluency and then differentiated with the addition of 500 M isobutylmethylxanthine, 25 M dexamethasone, and 4 g/ml insulin for 3 days, followed by 3 days in supplemental media with 4 g/ml insulin added. Cells were cultured an additional 3-4 days in supplemental media to complete differentiation. For adenovirus experiments, cells were transduced with GFP (control) or Grb10␥ viral stocks at a multiplicity of infection of 20. Twenty-four hours post-transfection/posttransduction cells were serum-starved for 4 -18 h, treated with 10 nM (CHO/IR) or 100 nM (adipocytes) insulin, and lysed in ice-cold Buffer A (50 mM HEPES, pH 7.6, 150 mM NaCl, 1% Triton-X, 10 mM NaF, 20 mM sodium pyrophosphate, 20 mM ␤-glycerophosphate, 1 mM sodium orthovanadate, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 M microcystin-LR, and 1 mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged (10,000 ϫ g, 4°C, 10 min), and the supernatants used for immunoprecipitation or Western blot experiments. For immunoprecipitation, cell lysates were incubated with specific antibodies bound to either protein A or protein G-Sepharose beads (Amersham Biosciences) for 6 -18 h at 4°C with gentle rotation. Immunoprecipitates were washed extensively with ice-cold Buffer B (50 mM HEPES, pH 7.6, 150 mM NaCl, 0.1% Triton X). Proteins bound to beads were eluted by heating at 95°C for 4 min in SDS sample loading buffer. Eluted proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected with specific antibodies. Quantitation of the relative increase in Akt phosphorylation was performed by analyzing Western blots using the Scion Image program and normalized to the level of Akt expression in each experiment.
Yeast Transformation and Interaction Assay-The yeast MATCH-MAKER LexA two-hybrid system reagents were purchased from Clontech. The yeast Saccharyomyces cerevisiae strain GUSEGY48 (Mat␣, trp1, his3, urs3, 6LexAop-LEU2, LYS2) and the pLexA-IR, and various pB42AD Grb10 constructs were generous gifts from Dr. Thomas A. Gustafson. GUSEGY48 was sequentially transformed with plasmid constructs by the polyethylene glycol/lithium acetate method according to the Clontech protocol. Transformants were grown on SD-HisTrp agar plates for 3 days at 30°C. Four independent colonies were streaked onto SD-HisTrp agar plates, grown overnight, replica-plated onto SD galactose/raffinose -HisLeuTrp agar plates and incubated for 5 days at 30°C to induce expression of B42 fusion proteins and to determine interacting partners.

Grb10␥ Inhibits Akt Phosphorylation in Response to Insulin
Stimulation-In the insulin signaling cascade, elevated phospholipid levels result from PI 3-kinase activity and provide for the activation of downstream mediators, such as Akt (29,30). To test the effects of hGrb10␥ on Akt activation, Myc-tagged Akt was expressed in CHO/IR cells together with HA-tagged hGrb10␥. Insulin-stimulated Akt phosphorylation was detected using phosphospecific antibodies to Akt T308 and Akt S473 . Significant phosphorylation on both Akt T308 and Akt S473 was seen with a 5-min insulin stimulation (Fig. 1A, lanes 1 and 2, first and third panel), which is consistent with published results (31). The presence of hGrb10␥ greatly reduced phosphorylation on Akt T308 (Fig. 1A, lanes 3 and 4, top panel). Phosphorylation at Akt S473 was also decreased with hGrb10␥ overexpression (Fig. 1A, lanes 3 and 4, third panel). Quantitative analyses indicated that hGrb10␥ reduced Akt S473 phospho-rylation by ϳ50 -60% and virtually abolished Akt T308 phosphorylation (Fig. 1B).
Previous reports indicated that insulin rapidly induces phosphorylation of Akt T308 and that this is stable during extended stimulation (31,32). Furthermore, limited Akt S473 phosphorylation in the basal state rapidly increases following growth factor stimulation (33). Since hGrb10␥ significantly inhibits Akt T308 and partially inhibits insulin-stimulated Akt S473 phosphorylation, we hypothesized that hGrb10␥ might have a temporal effect on Thr 308 and Ser 473 phosphorylation. Akt was overexpressed in CHO/IR cells in the absence or presence of hGrb10␥. Cells were treated with 10 nM insulin for increasing times, and phosphorylation was detected in whole cell lysates using phosphospecific antibodies. hGrb10␥ delayed Akt phosphorylation at both sites, while concurrently decreasing the overall extent of phosphorylation for up to 1 h of insulin treatment (Fig. 1C).
Full-length Grb10 Inhibits Insulin-stimulated Tyrosine Phosphorylation of IRS-1 and IRS-2-The IRS proteins are rapidly phosphorylated in response to insulin stimulation. Since tyrosine-phosphorylated IRS-1/2 increases PI 3-kinase activity in the cell by binding the regulatory subunit of PI 3-kinase (7), we considered whether Grb10-mediated inhibition of Akt phosphorylation might arise from decreased signaling through IRS-1/2. A partial inhibition of insulin-stimulated tyrosine phosphorylation of IRS has been observed with hGrb10␣ (the isoform that lacks a PH-domain) (16), although the effects of full-length hGrb10␥ have not been investigated. hGrb10␥ and IRS-1 were co-expressed in CHO/IR cells and insulinstimulated IRS-1 tyrosine phosphorylation was measured. In the absence of Grb10, IRS-1 was tyrosine-phosphorylated after 5 min of insulin stimulation ( Fig. 2A, lanes 1 and 2). Insulinstimulated tyrosine phosphorylation of IRS-1 was substantially inhibited when hGrb10␥ was co-expressed ( Fig. 2A, lanes 3 and  4). IRS-1 tyrosine phosphorylation was inhibited in a dose-dependent manner (Fig. 2B, lane 2 versus lanes 3-6). We also examined the effects of hGrb10␥ on IRS-2 phosphorylation. Since little endogenous IRS-2 was detected in CHO/IR cells (data not shown), we co-expressed IRS-2 and hGrb10␥ in CHO/IR cells. We found that treatment of cells with insulin led to a significant increase in IRS-2 tyrosine phosphorylation (Fig.  2C, lane 2 versus lane 1). The insulin-stimulated IRS-2 tyrosine phosphorylation was greatly inhibited by overexpressing hGrb10␥ (Fig. 2C, lane 4 versus lane 2). These findings demonstrate that the Grb10-mediated inhibition is not specific for IRS-1.
hGrb10␥ Inhibits Endogenous IRS-1 and Akt Phosphorylation in Differentiated Adipocytes-To determine if hGrb10␥ inhibits phosphorylation of endogenous IRS and Akt in physiologically relevant cells, we used an adenovirus system to express hGrb10␥ in differentiated 3T3-L1 adipocytes. Infection efficiency was 85-90% as assessed by visualization of green fluorescence emitted from co-expressed GFP (data not shown). Insulin-stimulated phosphorylation of endogenous IRS-1 was inhibited (Fig. 3A), and Akt Thr 308 and Ser 473 phosphorylation significantly reduced in adipocytes expressing hGrb10␥ (Fig. 3B).
Grb10 Binding to IR Correlates with Inhibited IRS-1 Phosphorylation-Because hGrb10␥ delayed and sustained reduced levels of insulin-induced Akt phosphorylation (Fig. 1C), we investigated the temporal effects of hGrb10␥ on IRS-1 tyrosine phosphorylation. Time course studies indicated that hGrb10␥ inhibited IRS-1 tyrosine phosphorylation as early as 30 s after insulin stimulation, and that hGrb10␥-mediated inhibition continued through an hour of insulin stimulation (Fig. 4A, top  panel). Since hGrb10␥ inhibits insulin-stimulated IRS-1 tyro-FIG. 1. Effects of Grb10␥ on insulin-stimulated phosphorylation of Akt. A, full-length Grb10 decreases insulin-stimulated phosphorylation of Akt. CHO/IR cells were transiently transfected with Myc-tagged Akt and either the empty vector or an expression construct encoding full-length Grb10. Cells were serum-starved and stimulated with 10 nM insulin for 5 min. Cell lysates were run on a 10% SDS-PAGE gel and insulin-stimulated phosphorylation of Akt at Thr 308 and Ser 473 was detected using phosphospecific antibodies. The expression level of Akt was detected by antibody to the Myc tag and Grb10␥ expression detected by antibody recognizing the HA tag. Experiments were performed at least three times with similar results. B, quantitative analysis of Akt phosphorylation. Akt phosphorylation was quantified using Scion Image and normalized to Akt expression levels. The mean fold stimulation expressed as a percentage of the maximum Ϯ S.E. from three independent experiments is shown. C, insulin-stimulated phosphorylation of Akt is delayed by the presence of Grb10␥. CHO/IR cells were transfected and blotted as described in A, except cells were stimulated with 10 nM insulin for the indicated time periods. sine phosphorylation over an extended period, we studied the kinetics of hGrb10␥ binding to the IR. CHO/IR cells transiently expressing HA-tagged hGrb10␥ were stimulated with insulin for varying times, and hGrb10␥ was immunoprecipitated from the cell lysates with antibody to the HA tag. Bound IR was detected using an antibody specific to the ␤-subunit of IR. hGrb10␥ rapidly associated with the IR within 30 s of insulin stimulation, and the association persisted through 60 min of hormonal treatment (Fig. 4B), which mirrors the speed and duration of hGrb10␥ inhibition of insulin stimulated IRS-1 tyrosine phosphorylation (Fig. 4A). We observed a decrease in Grb10␥ association following a 10-min insulin stimulation, which may result from subsequent dephosphorylation of the IR by activated tyrosine phosphatases.
Autophosphorylation of IR Is Not Affected by Grb10 Expression-IRS interacts directly with the tyrosine-phosphorylated NPEY 972 (34) motif in the IR juxtamembrane (4). Studies have indicated that autophosphorylation of the IR initiates at tyrosines 1158, 1162, and 1163 in the core catalytic domain (35), and phosphorylation in the C-terminal and juxtamembrane domains rapidly follows. Since Grb10 associates with the regulatory kinase domain of the IR (10, 12, 13), we considered that the effects of Grb10 on IRS tyrosine phosphorylation might arise from a decrease or loss in receptor autophosphorylation. The insulin receptor was immunoprecipitated from cell lysates of either CHO/IR or CHO/IR stably expressing hGrb10␥ and the extent of insulin-stimulated phosphorylation at Tyr 972 , FIG. 2. Full-length Grb10 inhibits insulin-stimulated tyrosine phosphorylation of IRS. A, Grb10␥ inhibits insulin-stimulated tyrosine phosphorylation of overexpressed IRS-1 in CHO/IR cells. CHO/IR cells were transiently transfected with mammalian expression constructs encoding Myc-tagged IRS-1 and HA-tagged Grb10␥. Cells were serumstarved, stimulated with 10 nM insulin for 10 min, lysed, and IRS-1 immunoprecipitated with antibody to the Myc tag. Insulin-stimulated tyrosine phosphorylation of IRS-1 was detected by Western blot with a phosphotyrosine-specific antibody. Membranes were then stripped and reblotted for IRS-1 using an antibody to the Myc tag. Grb10␥ expression was detected in cell lysates using an antibody to the HA tag. B, Grb10 inhibits insulin-stimulated IRS tyrosine phosphorylation in cells in a dose-dependent manner. Myc-tagged IRS-1 was co-transfected with increasing amounts (1, 2, 4, or 8 g) of HA-tagged Grb10␥ into CHO/IR cells. Cells were serum-starved, stimulated for 5 min with 10 nM insulin and lysed. Tyrosine phosphorylation of IRS-1 in cell lysates was determined as in A. C, full-length Grb10 inhibits IR-mediated tyrosine phosphorylation of IRS-2 overexpressed in CHO/IR cells. CHO/IR cells were transiently transfected with an expression construct encoding IRS-2, and either cotransfected with empty vector or an expression construct for Grb10␥. Twenty-four hours post-transfection cells were serum-starved and stimulated with 10 nM insulin for 10 min. Cell lysates were run on a 7.5% SDS-PAGE gel and tyrosine phosphorylation of overexpressed IRS proteins detected with a phosphotyrosine-specific antibody. Membranes were stripped and IRS-2 was detected with an antibody specific to the protein.
The presence of Grb10 in the lysate was determined by probing with an antibody to the HA tag.

FIG. 3. Grb10␥ inhibits endogenous IRS-1 and Akt phosphorylation in differentiated adipocytes.
A, Grb10␥ inhibits insulin-stimulated tyrosine phosphorylation of endogenous IRS-1. 3T3-L1 adipocytes were infected with either GFP or Grb10 adenovirus. Postinfection, cells were serum-starved and stimulated with 100 nM insulin for 5 min. Insulin stimulated tyrosine phosphorylation of endogenous IRS-1 was detected in the lysates with a phosphotyrosine-specific antibody. Membranes were stripped and endogenous IRS-1 detected using an antibody specific to the protein. The amount of adenovirally expressed HA-tagged Grb10␥ was detected in the lysates using an antibody to the tag. B, Grb10␥ inhibits phosphorylation of endogenous Akt. 3T3-L1 adipocytes were treated and lysates prepared as in A. Insulinstimulated phosphorylation of Akt at Thr 308 and Ser 473 was detected using phosphospecific antibodies. Endogenous Akt was detected using an antibody specific to the protein. Data is representative of three independent experiments.
Tyr 1158 , and Tyr 1162/1163 was determined using phosphospecific antibodies. Phosphorylation at Tyr 972 was not significantly reduced in cells that stably expressed hGrb10␥ (Fig. 5, lanes 2  versus 4, first panel), suggesting that the reduction in IRS tyrosine phosphorylation is not a direct consequence of an impaired IRS docking site on the IR. Phosphorylation of the IR on the three critical tyrosine residues in the regulatory loop were similarly unaffected by expression of hGrb10␥ (Fig. 5, lanes 2 versus 4, second and third panels). While the presence of Grb10 has been shown to inhibit tyrosine phosphorylation of IR substrates in vitro (19 -21), it is unclear whether this is directly related to impaired catalytic activity. We considered that if Grb10␥ does not significantly impair IR catalytic activity in cells, then an alternative mechanism for Grb10-mediated inhibition of substrate phosphorylation may exist. The results in Fig. 5 illustrate that the catalytic activity of IR, at least toward itself, is not significantly hindered by Grb10␥ overexpression in cells.
Grb10 Disrupts the Association of IRS-1/2 with the Insulin Receptor-To determine the mechanism behind Grb10-mediated inhibition of IRS-1/2 tyrosine phosphorylation, a yeast tri-hybrid disruption assay was performed using IR as bait, IRS-1 or IRS-2 as prey and various Grb10 constructs (Fig. 6A) as disrupters. The interaction between IR and IRS-1 (36) and IRS-2 (37,38) in yeast, as determined by growth on selective media, has been well characterized. Co-expression of fulllength hGrb10␤ in the yeast tri-hybrid system prevented growth on selective media, indicating that IR/IRS-1/2 interactions were inhibited (Fig. 6B). Since interactions between Grb10 and IR are mediated by two separate "domains," the BPS and SH2 (9), we set out to determine which is responsible for the disruption of IR/IRS-1/2 interactions. A truncated construct containing the BPS and SH2 domains (342-536) blocked growth on selective media (Fig. 6B), indicating that the N terminus and PH domain are not required for inhibition of FIG. 4. Grb10 binding to IR correlates with inhibited IRS-1 phosphorylation. A, IRS-1 tyrosine phosphorylation is rapidly and stably inhibited by Grb10␥. CHO/IR cells were co-transfected with Myc-tagged IRS-1 and empty vector or HA-tagged Grb10␥. Cells were serum-starved and stimulated with 10 nM insulin for the indicated times prior to cell lysis. Lysates were separated by SDS-PAGE and insulin-stimulated tyrosine phosphorylation detected using a phosphotyrosine-specific antibody. Blots were stripped and IRS-1 expression determined by blotting with ␣-Myc antibody. Grb10␥ expression was detected by Western blot with antibody to the HA tag. B, Grb10␥ rapidly associates with IR. CHO/IR cells were transiently transfected with an expression construct for Grb10␥. Cells were serum-starved and stimulated with 10 nM insulin for the indicated times. Grb10 was immunoprecipitated from the cell lysates using an antibody to the HA tag and samples separated by SDS-PAGE. Co-immunoprecipitated IR was detected by Western blot using an antibody specific to the IR␤ subunit. hGrb10␥ expression was detected by Western blot with an antibody to the HA tag. Data are representative of three independent experiments.

FIG. 5. Insulin receptor autophosphorylation is unaffected by
Grb10␥. CHO/IR cells alone or stably expressing Grb10␥ (HA-tagged) were serum-starved and stimulated with 10 nM insulin for 5 min. The IR was immunoprecipitated from the cell lysates, and samples were separated by SDS-PAGE. IR tyrosine phosphorylation was detected using phosphospecific antibodies to Tyr 972 , Tyr 1158 , and Tyr 1162/1163 . Grb10␥ and IR expression levels were detected in whole cell lysates using antibodies specific for the HA tag and the IR␤ subunit, respectively.

FIG. 6. Grb10 disrupts the interaction between IRS proteins and the insulin receptor.
A, yeast two-hybrid: interaction between the insulin receptor (bait) and various Grb10 constructs (prey) was determined by the ability to induce growth on selective media (ϩ). Grb10 constructs that do not interact with the insulin receptor do not induce growth on selective media (Ϫ). B, Yeast tri-hybrid: disruption of the insulin receptor (bait) and IRS-1 or IRS-2 (prey) interaction by various Grb10 constructs (disruptants) was determined by the inhibition of yeast growth on selective media. IR/IRS-1/2 binding. To block SH2 domain function, the critical arginine residue at 462 was replaced with alanine (R462A) in the full-length and truncated (BPS/SH2) hGrb10␤ proteins. Both constructs interacted with IR ( Fig. 6A and Ref. 9) and both also disrupted the IR/IRS-1/2 interactions similar to wildtype Grb10 (Fig. 6B). These data suggest that the BPS domain may be responsible for inhibiting IR/IRS-1/2 binding. However, the BPS (358 -412) or SH2 (413-536) domains alone, which interact with IR in yeast two-hybrid experiments (Fig. 6A and Ref. 9), do not prevent yeast growth on selective media, indicating that the domains by themselves are unable to disrupt IR/IRS-1/2 interactions (Fig. 6B). Deleting the SH2 domain (Grb10␤ 1-412 aa) or replacing it with the SH2 domain of Shc(p52) (Grb10␥ 1-491aa :ShcSH2) does not inhibit association with the IR in yeast two hybrid experiments (Fig. 6A); however, removing or swapping the SH2 domain of Grb10 blocks the ability of Grb10 to inhibit the binding of IR/IRS-1/2 (Fig. 6B). Furthermore, an inactive hGrb10␤ SH2 domain (413-536, R462A), which cannot associate with the IR in yeast two hybrid experiments (Fig. 6A and Ref. 9), can support interaction and disruption when combined with the BPS domain (Fig. 6, A and  B). Therefore, BPS directed disruption of IR/IRS-1/IRS-2 requires the structural presence of an active or inactive (R462A) Grb10 SH2 domain, which may physically block binding of IRS-1/2 to the NPXY 972 motif.
Complete Inhibition of IRS Phosphorylation Requires an Intact Grb10 SH2 Domain-Results of the yeast tri-hybrid system indicated that disruption of IR/IRS interactions required both the BPS and SH2 domains. Furthermore, the Grb10 SH2 domain appeared to be essential and disruption occurred regardless of whether the SH2 domain was binding-competent. To determine if the BPS/SH2 region was necessary for inhibition of insulin-stimulated IRS phosphorylation in mammalian cells, we co-expressed full-length or BPS/SH2 fragments of wild-type or mutated forms of hGrb10␥ along with IRS-1 in CHO/IR cells. The hGrb10␤ (yeast tri-hybrid studies) and hGrb10␥ (mammalian cell studies) isoforms are identical outside of a 58-amino acid extension at the protein's N terminus (12,17). hGrb10␤ R462 corresponds to hGrb10␥ R520 (Fig. 7A). As previously observed, insulin-stimulated IRS-1 phosphorylation was inhibited in the presence of full-length wild-type hGrb10␥ (Fig. 7B). Consistent with our yeast tri-hybrid findings, coexpression of the BPS/SH2 fragment in cells similarly decreased insulin-stimulated IRS-1 phosphorylation (Fig. 7B,  first panel, lane 4). Inhibition of IRS-1 tyrosine phosphorylation correlated with Grb10 association with the IR, and the BPS/SH2 fragment was sufficient to maintain this interaction (Fig. 7B, third panel, lanes 3 and 4). Interestingly, IRS-1 tyrosine phosphorylation was partially restored with either the BPS/SH2 R520G fragment or full-length Grb10␥ R520G (Fig. 7B,  first panel, lanes 5 and 8). These findings suggest that an active SH2 domain is necessary for complete inhibition of IRS tyrosine phosphorylation in cells. Studies have indicated that the BPS and SH2 domains may act cooperatively to facilitate the interaction of Grb10 with the IR (9). Full-length Grb10␥ with an inactive SH2 domain is unable to co-immunoprecipitate detectable levels of the IR (Fig. 7B, lane 8, third panel and Ref. 11). Similarly, the BPS/SH2 R520G fragment was unable to coimmunoprecipitate detectable amounts of the IR (Fig. 7B, lane  5, third panel). Our results suggest that the BPS domain is necessary, but not sufficient to mediate a tight association with the IR in cells. In the absence of an intact SH2 domain, the interaction between Grb10 and the IR may be transient and therefore undetectable by co-immunoprecipitation (Fig. 7B,  third panel, lanes 5 and 8 versus 3 and 4). However, this transient association is sufficient to affect IR/IRS-1 interac-tions as indicated by the partial inhibition of IRS-1 tyrosine phosphorylation (Fig. 7B). To confirm the necessity of the SH2 domain for Grb10-IR association and its effect on the inhibition of insulin-stimulated IRS tyrosine, we tested two additional Grb10␥ constructs. By either replacing the Grb10 SH2 domain with the SH2 domain of Shc(p52) or by deleting this domain altogether, IRS-1 tyrosine phosphorylation was restored, 41 and 30%, respectively (Fig. 7B, first panel, lanes 6 and 7). Immunoprecipitation with either the Grb10␥ 1-491 :ShcSH2 chimera or Grb10␥⌬SH2 revealed that the BPS domain alone can not maintain a sufficient interaction to co-immunoprecipitate the IR (Fig. 7B, lanes 6 and 7, third panel). Taken together with the yeast tri-hybrid results, the SH2 domain makes dual contributions to Grb10-mediated inhibition of IRS phosphorylation: in cells, the SH2 domain stabilizes the interaction of Grb10 with the IR, and with a stable Grb10/IR interaction, the SH2 domain sterically hinders IRS access to its binding motif on the IR.

DISCUSSION
Grb10 has been shown to play both a positive and negative role in insulin signaling, and these differences appear to depend both on cell type and on the Grb10 splice variant being FIG. 7. Complete inhibition of IRS-1 tyrosine phosphorylation requires an intact SH2 domain. A, schematic diagram of Grb10 constructs used in B. Full-length Grb10␤ is shown for a comparison. B, Myc-tagged IRS-1 was co-expressed in CHO/IR cells with the indicated Grb10␥ construct. Twenty-four hours post-transfection cells were serum-starved and stimulated with 10 nM insulin for 5 min. Cells were lysed and the lysates split for IRS-1 immunoprecipitation using an antibody to the Myc tag and IR co-immunoprecipitation with Grb10 using an antibody to the HA tag. Insulin-stimulated tyrosine phosphorylation of IRS-1 was detected with a phosphotyrosine-specific antibody, and the level of IRS-1 determined by reblotting the stripped membrane with Myc antibody. IR was detected with antibody to the ␤-subunit. Grb10 expression was detected in cell lysates using an antibody to the HA tag. Data are representative of three independent experiments. studied. To understand these functional differences, we investigated the molecular mechanism of Grb10 action at the insulin receptor. Here, we show that full-length human Grb10 has an inhibitory effect on insulin-stimulated signaling through the IRS/PI 3-kinase pathway to Akt. Using a yeast tri-hybrid system, we found Grb10 can disrupt the association of IRS proteins with IR. The Grb10 SH2 domain is essential for this disruption and for complete inhibition of IRS tyrosine phosphorylation in cells. Our findings support a mechanism for Grb10mediated inhibition of IRS/PI 3-kinase/Akt signaling, in which association of IRS proteins with the IR kinase domain and/or the juxtamembrane NPXY motif (Tyr 972 ) is sterically hindered by Grb10.
Previous studies have concluded that blocking IR catalytic activity by either full-length Grb10 or domain fragments results in decreased substrate phosphorylation (19,20). In agreement with these studies, we have observed a Grb10-mediated decrease in phosphorylation of IR substrates, including IRS-1/2 (this study) (16), p62 dok (16,39), and Shc (data not shown). However, Mournier et al. (21) reported that overexpression of hGrb10 did not affect IRS tyrosine phosphorylation, PI 3-kinase activity or Akt activity in rat hepatocytes, although IR kinase activity and IR autophosphorylation were reportedly reduced. Our results in CHO/IR cells show that the catalytic activity of the RTK toward itself is not significantly impaired by Grb10. To a degree this is intuitive since tyrosine phosphorylation of the IR is necessary for Grb10 association. The rapid association of Grb10 with the IR could suggest that if Grb10 blocked the enzymatic activity of the RTK, autophosphorylation of the IR would be reduced in CHO/IR/hGrb10␥ stable cells. However, we found that the overall tyrosine phosphorylation of the IR was also unaffected by the presence of hGrb10␥ (data not shown). It is possible that the effects of Grb10 on catalytic activity arise from conformational changes that sterically hinder access of the kinase with its substrates. While we can not exclude inhibition of enzymatic activity of the insulin receptor for IRS-1/2 as a cause for decreased substrate phosphorylation, the observation that hGrb10 physically disrupts the interaction between IR and IRS proteins using the yeast tri-hybrid system supports structural interference as a mechanism for Grb10-mediated inhibition. The interaction of IRS proteins with the IR is transient in nature, and several negative feedback mechanisms exist in cells to prompt rapid dissociation of tyrosine-phosphorylated IRS proteins with the receptor (40 -42). As a result, we were unable to detect an interaction between IR and IRS proteins by co-immunoprecipitation (data not shown), and cannot completely assess physical disruption as an alternative mechanism for Grb10-mediated inhibition in mammalian cells.
Although both the BPS and SH2 domains can independently interact with the IR, the two regions together are necessary for disruption of interactions between IRS-1/2 and IR. Our results with the yeast tri-hybrid system indicate that the BPS domain with either an active or inactive SH2 domain is sufficient for disruption. This would suggest that the BPS domain mediates the interaction with the IR and the SH2 domain facilitates a physical disruption of IR/IRS interaction. When the Grb10 SH2 domain is deleted or replaced with the Shc SH2 domain, the IR/IRS interaction is not disrupted although both constructs interact with the IR in yeast two-hybrid assay. These findings support a structural role for the Grb10 SH2 domain in hindering IR/IRS interactions. However, Grb10 molecules lacking an intact SH2 domain cannot support complete inhibition of IRS tyrosine phosphorylation in mammalian cells (Fig. 7B). Interestingly, while the BPS domain with either an active or inactive SH2 domain disrupted IR/IRS interactions in the yeast tri-hybrid studies, only Grb10 (full-length and BPS/SH2 fragment) with an active SH2 domain inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in mammalian cells. Complete inhibition appeared to correlate with the ability of Grb10 to co-immunoprecipitate the IR. In contrast with results using yeast two-hybrid, in which the BPS domain is sufficient for interaction with the IR, regardless of an active SH2 domain (Ref. 9 and Fig. 6), only Grb10 with an active SH2 domain could co-immunoprecipitate the IR in cells. The high sensitivity of the yeast system may allow for the detection of the BPSmediated IR interaction and may override the need for an active SH2 domain. In mammalian cells, the SH2 domain may be needed to facilitate or strengthen BPS-mediated association with the IR. For this reason, we could not fully assess the structural requirement of the SH2 domain in physically blocking IRS-1/2 access to the IR in cells. In addition, we were unable to express fragments encoding only the BPS or SH2 domains, most likely due to their instability in mammalian cells. While we were unable to detect Grb10 association with the IR when the SH2 was inactivated or deleted, partial inhibition of IRS tyrosine phosphorylation with overexpression of these mutants suggests that some interaction via the BPS domain is likely.
Full-length hGrb10␥ significantly reduced phosphorylation of Akt on both Thr 308 and Ser 473 in CHO/IR cells. A similar inhibitory effect on phosphorylation of endogenous Akt was seen with adenoviral expression of hGrb10␥ in differentiated adipocytes. Our findings differ from a recent study reporting mGrb10 functioned as a positive regulator of Akt in the c-kit signaling pathway by promoting relocalization to the membrane (22). We do not find hGrb10 to co-immunoprecipitate with Akt in CHO/IR cells (data not shown). Protein interactions with Grb10 may be species-specific. Receptor-associated variations in recruited signaling pathways may also account for the observed differences in Grb10 function. IRS signaling is directly impaired by Grb10 interactions with IR and inhibition of Akt activity by hGrb10␥ may stem from a specific requirement for IRS proteins and their supporting role in the activation of PI 3-kinase in response to insulin stimulation.
In this study, the mechanism for Grb10-mediated inhibition of insulin signaling was investigated. We found that full-length hGrb10 could significantly inhibit insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2 and subsequently delay signaling to the downstream effector, Akt. Autophosphorylation of the IR is not impaired by the binding of Grb10 to the receptor, which indicates functional activity of the RTK in cells. hGrb10␥ inhibits insulin-stimulated tyrosine phosphorylation of IRS in a dose-dependent manner, and the interaction between IRS and the IR is disrupted when full-length Grb10 is used in the yeast tri-hybrid system. These studies describe a new molecular mechanism for Grb10-mediated inhibition. By structurally hindering access to the IR kinase domain and/or the NPXY motif (Tyr 972 ), Grb10 inhibits tyrosine phosphorylation of IRS-1 and IRS-2 and functions to decrease intracellular insulin signaling through the IRS/PI 3-kinase pathway.