©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Insulin-like Growth Factor I Receptor Activated by a Transmembrane Mutation (*)

(Received for publication, January 30, 1995; and in revised form, May 24, 1995)

Katsutoshi Takahashi (§) Kazuyoshi Yonezawa (¶) Ikuo Nishimoto (**)

From theCardiovascular Research Center, Massachusetts General Hospital-East and the Department of Medicine, Harvard Medical School, Charlestown, Massachusetts 02129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We constructed mutant receptors by mutating transmembrane Val of the human insulin-like growth factor I receptor (IGF-IR). Assays of receptor kinase and autophosphorylation revealed constitutively augmented tyrosine kinase activity of V922E IGF-IR in both transient and stable expression. The constitutively active tyrosine kinase of this mutant was verified by promoted tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) in the absence of IGF-I. In CHO cells stably expressing V922E IGF-IR, both IRS-1 phosphorylation and the IRS-1-associated phosphoinositide 3-kinase activity were stimulated in the absence of IGF-I to the level attained by 1 nM IGF-I stimulation of wild-type IGF-IR, whereas the Ras-mitogen-activated protein kinase pathway was not activated under the same condition. In these CHO cells, V922E IGF-IR significantly stimulated glucose uptake but did not promote mitogenesis in the absence of IGF-I. We thus conclude that the V922E mutation of IGF-IR switches on the intrinsic tyrosine kinase and differentially activates the downstream pathways. This mutant is extremely useful in clarifying the turning-on mechanism of IGF-IR as well as the differential roles of individual downstream pathways of receptor tyrosine kinases.


INTRODUCTION

Insulin-like growth factor I (IGF-I) (^1)is a regulator of cell growth and metabolism(1) . IGF-IR, a receptor tyrosine kinase belonging to the IR superfamily, mediates most actions of IGF-I(2, 3, 4) . It consists of a 135-kDa alpha subunit and a 95-kDa beta subunit and forms an alpha(2)beta(2) heterodimer on the cell surface(5) . The binding of IGF-I to the alpha subunit turns on the tyrosine kinase in the beta subunit(6) . IRS-1 is the major protein that undergoes tyrosine phosphorylation in response to IGF-I(7, 8) . Phosphorylated IRS-1 then switches on the downstream pathways by recruiting PI 3-kinase(8, 9) , tyrosine-specific protein phosphatase Syp(10) , and probably others. As ligand-activated IGF-IR also autophosphorylates its beta subunit, phosphorylation of both the beta subunit and IRS-1 thus provides the best index of the activation of IGF-IR.

A basic question is what physiological roles IGF-IR plays in living cells. IGF-I produces a variety of cellular responses, both mitogenic and metabolic. Which action of IGF-I is mediated by IGF-IR is, however, less defined. This is mainly because IGF-I also acts on IR, which mediates similar cellular responses(11) . Another question is how the differences in the cellular outputs between IGF-IR and IR are created in vivo. IGF-I and insulin preferentially activate mitogenesis and metabolism via IGF-IR and IR, respectively(1) . Nevertheless, these two receptors are highly homologous structurally and biochemically(1) . The downstream pathways of IGF-IR so far clarified are all shared by IR(11) . Thus, it remains unknown if each receptor has its unique signaling pathway or if such differences result from the different activation profiles of the same downstream pathways.

To address these questions, it is necessary to turn on IGF-IR selectively. One of the best ways is to construct and use a constitutively active mutant of IGF-IR. The expression of such an autoactive mutant allows the precise analysis of the downstream pathways and functional roles of IGF-IR. There are mutants of growth factor receptors whose tyrosine kinase activities are constitutively turned on in the absence of their ligands(12, 13, 14, 15) . In the present study, based on analogy with mutation-dependent activation of these receptors, we have constructed mutant IGF-IRs by substituting Glu or Ile for the transmembrane Val residue and have found that the former mutant retains increased tyrosine kinase activity and constitutively and differentially activates the downstream cascade of IGF-IR. Differential roles of IGF-IR signaling pathways in biological outputs are also discussed by comparing the metabolic and mitogenic effects of this activated mutant to its intracellular signals.


EXPERIMENTAL PROCEDURES

An EcoRI fragment of human IGF-IR cDNA containing a complete open reading frame was subcloned into pUC19. The mutagenized cDNAs were generated by the unique site elimination method (Pharmacia Biotech) using a selection primer in which an original SspI site was replaced with an EcoRV site (5`-CTTCCTTTTTCGATATCATT GAAGCATTT-3`: an EcoRV site is underlined). The sequences of mutagenic target primers were 5`-GTACAGCATAATGATCAACCCTCC-3` for V922I IGF-IR and 5`-GTACAGCATAATCTCCAACCCTCC-3` for V922E IGF-IR (the codon for Val is underlined). These mutations were confirmed by dideoxynucleotide sequencing using Sequenase (U. S. Biochemical Corp.), and IGF-IR cDNAs were cloned into pcDNA1. COS-7 and SV40-infected BALB c/3T3 cells (SV40-BALB cells, ATCC) were grown in Dulbecco's modified Eagle's medium plus 10% calf serum. CHO cells were grown in Ham F-12 plus 10% FCS. The antibodies used were: alphaIR3, monoclonal antibody against human IGF-IR (Oncogene Science); 1D6, monoclonal antibody against rat IRS-1(16) ; RC20H, anti-Tyr(P) monoclonal antibody conjugated with horseradish peroxidase (Transduction Laboratories); polyclonal antibody for the C terminus of rat IRS-1 (UBI); and rabbit antisera against C terminus (residues 350-367) of rat ERK1.

Transient transfection was performed by the lipofection method (10 µg of either WT, V922E, or V922I IGF-IR cDNA and 20 µl of LipofectAMINE (Life Technologies, Inc.)) as described(17) . After transfection, cells were treated with or without IGF-I for 5 min at 37 °C. For stable expression of IGF-IRs, CHO cells were transfected by the lipofection method using 1 µg of pSV2neo and 10 µg of WT or mutant IGF-IR cDNA. Cells were split the next day at 1:10, plated in Ham F-12 plus 10% FCS, and, 2 days later, were selected with 450 µg/ml G418. After 2-3 weeks, colonies were picked and tested for I-IGF-I binding as described(11) . The colonies with high binding levels were further subcloned by limiting dilution. Cells overexpressing WT, V922E, or V922I IGF-IR were designated as CHO-IGF-IR, CHO-V922E, or CHO-V922I, respectively.

For immunoprecipitation, cells were starved in Ham F-12 for 24 h at 37 °C and treated with IGF-I at 37 °C. Cells were lysed and precipitated with mouse IgG (Cappel), alphaIR3, or 1D6, as described (18) . The samples were electrophoresed on SDS-polyacrylamide gels, transferred to a polyvinylidene difluoride sheet, probed with RC20H or anti-IRS-1 antibody/horseradish peroxidase-conjugated anti-rabbit IgG, and detected by ECL. For labeling studies, 45 h post-transfection cells were incubated with Met- and Cys-free Dulbecco's modified Eagle's medium containing [S]Met and [S]Cys (0.1 mCi/ml) with 10% dialyzed FCS for 3 h at 37 °C. Cells were then incubated with IGF-I for 5 min and analyzed as described(19) .

The IGF-IR kinase activity was measured using Poly(Glu, Tyr) 4:1 (Sigma) as described(18) . The bands of poly(Glu, Tyr) and IGF-IR beta subunit were excised and quantitated by Cerenkov counting. The PI 3-kinase activity was measured directly in 1D6 precipitates in a reaction mixture as described(18) . IGF-I-stimulated 2-deoxy-D-[1,2-^3H]glucose uptake (20) and [^3H]methylthymidine incorporation (21) were measured as described. The cell proliferation assay using XTT (Boehringer Mannheim) was performed according to the manufacturer's instructions. Analysis of Ras-bound guanine nucleotides was performed as described(16) . The MAP kinase activity was measured by immune-complex kinase assay in anti-ERK1 immunoprecipitates and in-gel kinase assay using cell lysates. ERK1 was precipitated with anti-ERK1 rabbit sera (1:25 dilution), and its activity was measured using myelin basic protein (MBP) as described (22) . In-gel kinase assay was performed using MBP as described(23) . All radioisotopes were purchased from DuPont NEN. All experiments were reproduced at least three times (only in immune-complex MAP kinase assay, experiments were independently repeated twice).


RESULTS

We first examined in vitro kinase activity of Val mutants using an exogenous substrate. IGF-IRs transiently expressed in COS-7 cells treated with or without 100 nM IGF-I were precipitated with alphaIR3. Tyrosine kinase activity was assessed by measuring incorporation of radioactivity into poly(Glu, Tyr) 4:1. As shown in Fig.1A, poly(Glu, Tyr) phosphorylation markedly increased in the IGF-I-untreated precipitate of V922E IGF-IR but not in that of WT or V922I IGF-IR. Notably, the same figure shows that autophosphorylation of the 95-kDa beta subunit of IGF-IR (as well as the 200-kDa IGF-IR precursor) also increased in V922E IGF-IR but not in WT or V922I IGF-IR, verifying the augmented in vitro kinase activity of V922E IGF-IR. Expression levels of IGF-IRs were assumed to be similar in these experiments, as the levels of tyrosine kinase activities maximally stimulated by IGF-I were similar (Fig.1A). This was confirmed by parallel experiments, in which COS-7 cells transfected with mutant IGF-IR cDNAs were metabolically labeled. Proteins of 200, 135, and 95 kDa, corresponding to the precursor, alpha, and beta subunits of IGF-IR, respectively, were precipitated by alphaIR3 from these transfected COS-7 cells in similar amounts (Fig.1B).


Figure 1: Transient expression of Val IGF-IR mutants. A, semi-in vivo kinase activity of IGF-IRs. WT, V922E, or V922I IGF-IR transiently expressed in COS-7 cells were immunoprecipitated with alphaIR3 from lysates of cells that had been treated with or without 100 nM IGF-I for 5 min at 37 °C. Receptor tyrosine kinase activity was measured as incorporation of radioactivity into either the exogenous substrate poly(Glu, Tyr) 4:1 or the beta subunit of IGF-IR. Shown is an autoradiograph of SDS-polyacrylamide gel electrophoresis. pre, IGF-IR precursor; poly(E,Y), poly(Glu, Tyr) 4:1. B, metabolic S labeling of COS-7 cells. COS-7 cells transfected with pcDNA1 (mock), WT, V922E, or V922I IGF-IR cDNA were metabolically labeled, then treated with or without 100 nM IGF-I for 5 min at 37 °C, and their lysates were precipitated with alphaIR3 and analyzed by SDS-polyacrylamide gel electrophoresis. Shown is an autoradiograph. C, in vivo autophosphorylation of IGF-IR beta subunit in COS-7 cells. COS-7 cells transfected with WT, V922E, or V922I IGF-IR cDNA were lysed, then precipitated with alphaIR3. Precipitates were probed with anti-Tyr(P) antibody RC20H. D, in vivo tyrosine phosphorylation of IRS-1 in SV40-BALB cells. SV40-BALB cells transfected with WT, V922E, or V922I IGF-IR cDNA were lysed, then precipitated with 1D6 (anti-rat IRS-1 antibody). Precipitates were probed with RC20H.



We also examined autophosphorylation of the beta subunit of V922E IGF-IR using a different method, which represents in vivo tyrosine kinase activity of this mutant. IGF-IRs transiently expressed in COS-7 cells were precipitated by alphaIR3 and immunoblotted with anti-Tyr(P) antibody RC20H. Constitutively augmented beta-subunit autophosphorylation of V922E IGF-IR, but not WT or V922I was observed (Fig.1C).

We next examined IRS-1 phosphorylation by the V922E mutant in intact cells. As 1D6 recognizes rodent IRS-1 but not monkey IRS-1, we used SV40-BALB cells instead of COS-7 for this experiment with transient transfection. Without IGF-I treatment, 1D6 precipitated IRS-1 with enhanced tyrosine phosphorylation of IRS-1 from SV40-BALB cells transfected with V922E IGF-IR cDNA (Fig.1D). This was not observed in the parallel transfection with WT or V922I cDNA.

In an effort to confirm these results obtained from transient expression, we established CHO cell lines overexpressing IGF-IR mutants. CHO-IGF-IR, CHO-V922E (Clones 1 and 2), or CHO-V922I cells specifically bound 3.7 to 4.1 times more I-IGF-I as compared to the parental CHO cells. The expressed WT and mutant IGF-IRs had apparent K values of 1.3 to 2.7 10M for IGF-I (Fig.2A). The K value of native IGF-IR in parental CHO cells was 1.0 10M. Therefore, the stable CHO cell lines did overexpress IGF-IRs with comparable affinities for IGF-I. We also assessed the amount of IRS-1 in the stable CHO cell lines, as it has been reported that a CHO cell line overexpressing a mutant IR retains reduced amounts of IRS-1(24) . The lysates of CHO-IGF-IR, CHO-V922E (Clones 1 and 2), or CHO-V922I were precipitated with 1D6 and blotted with anti-IRS-1 antibody. The results clearly indicated that the amounts of IRS-1 were similar among these cell lines (Fig.2B).


Figure 2: Stable expression of V922 IGF-IR mutants in CHO cells. A, radioactive IGF-I binding to CHO cell lines. The binding of I-IGF-I to parent CHO (), CHO-IGF-IR (bullet), CHO-V922E (Clones 1 () and 2 (black square)), or CHO-V922I (Delta) cells was measured in the presence of the indicated concentrations of IGF-I (I-labeled plus unlabeled). Results are means ± S.D. of three independent experiments. B, amount of IRS-1 in the stable CHO cell lines. Lysates of CHO-IGF-IR, CHO-V922E (Clones 1 and 2 (C1 and C2)), or CHO-V922I were precipitated with 1D6 and blotted with a polyclonal anti-IRS-1 antibody. C and D, in vivo tyrosine kinase activities of Val mutants in CHO cell lines. CHO cell lines were treated with or without 10 nM IGF-I for 5 min at 37 °C. WT or mutant IGF-IRs and IRS-1 were immunoprecipitated with alphaIR3 (C) and 1D6 (D), respectively, from lysates of cells and then immunoblotted with anti-Tyr(P) antibody.



From stable CHO cell lines, we immunoprecipitated both IGF-IRs and IRS-1 and detected them with anti-Tyr(P) antibody immunoblotting. In the absence of IGF-I, augmented autophosphorylation of the IGF-IR beta subunit was observed in CHO-V922E (Clones 1 and 2) but not in CHO-IGF-IR or CHO-V922I (Fig.2C). Besides, tyrosine phosphorylation of IRS-1 was stimulated in the absence of IGF-I in CHO-V922E (Clones 1 and 2) but not in CHO-IGF-IR or CHO-V922I (Fig.2D). The levels of tyrosine phosphorylation of both IGF-IR beta subunit and IRS-1 maximally activated by 10 nM IGF-I were similar among these cell lines.

As the established target of IRS-1 activated by IGF-IR is PI 3-kinase, we measured IRS-1-associated PI 3-kinase activity in CHO-V922E cells. By precipitating IRS-1 from lysates of CHO-V922E cells, the PI 3-kinase activity was found to be significantly elevated in these cells (Fig.3A). We quantitated the PI 3-kinase-stimulating effect of V922E IGF-IR by comparing it with those effects of various concentrations of IGF-I in CHO-IGF-IR. The effect of V922E IGF-IR on PI 3-kinase was virtually equivalent to that of 1 nM IGF-I in CHO-IGF-IR (Fig.3A). This was also the case when the effect was assessed by tyrosine phosphorylation of IRS-1 (Fig.3B). Thus, V922E mutation turns on the IGF-IR signal as strongly as does 1 nM IGF-I stimulation of WT IGF-IR.


Figure 3: IRS-1-associated PI 3-kinase activity in CHO cell lines. CHO cell lines were treated without (lanes 1, 5, and 6) or with 0.1 nM (lane 2), 1 nM (lane 3), and 10 nM (lanes 4 and 7) IGF-I for 5 min at 37 °C. IRS-1 was immunoprecipitated with 1D6 from cell lysates. The precipitates were measured with the associated PI 3-kinase activity (A) and immunoblotted with anti-Tyr(P) antibody (B). PIP marker indicates the position of migration of PI 4-phosphate standard on TLC.



Thus, it is shown that V922E IGF-IR has autoactive intrinsic tyrosine kinase, and this causes constitutive activation of the IRS-1/PI 3-kinase cascade. Next, we investigated biological outputs of this mutant by examining metabolic and mitogenic stimulation. In CHO-IGF-IR cells, exposure to IGF-I (20 min) resulted in a dose-dependent enhancement of the uptake of radiolabeled 2-deoxyglucose (Fig.4A). CHO-V922E cells (Clone 1) exhibited significantly elevated basal uptake of glucose, as strongly as did 0.1 nM IGF-I stimulation in CHO-IGF-IR cells. As for mitogenic stimulation, thymidine uptake assay and cell proliferation assay with XTT were performed. Very interestingly, in clear contrast to glucose uptake, CHO-V922E cells (Clone 1) showed no basal elevation in thymidine uptake and XTT values and exhibited IGF-I-dependent stimulation similar to that observed in CHO-IGF-IR with both assays (Fig.4, B and C). Similarly, Clone 2 of CHO-V922E cells also showed basal elevation of glucose uptake but not of cell proliferation assays (data not shown).


Figure 4: Biological outputs of V922E IGF-IR. A, 2-deoxyglucose uptake in CHO cell lines. CHO-IGF-IR () and CHO-V922E (bullet) were stimulated with the increasing concentrations of IGF-I for 20 min at 37 °C and incubated for another 20 min in the presence of 2-deoxy-D-[1,2-^3H]glucose. The incorporated radiolabeled glucose was determined. Incorporated radiolabeled glucose in the presence of 100 nM wortmannin (CHO-IGF-IR, 5868 ± 107 cpm; CHO-V922E, 3843 ± 39 cpm) was subtracted from each value, and results were expressed as percentage of the uptake at 10M IGF-I (unsubtracted values are: CHO-IGF-IR, 11,536 ± 327; CHO-V922E, 9,507 ± 27 cpm) in each CHO cell line. Results are means ± S.E. of three independent experiments. The panel shows a representative result from three separate experiments. In all experiments presented in this figure, Clone 1 was used as CHO-V922E. *, p < 0.01, unpaired t-test; asterisks indicate significant differences of the incorporated radiolabeled glucose between CHO-V922E and CHO-IGF-IR. B, thymidine uptake in CHO cell lines. CHO-IGF-IR (WT, ) and CHO-V922E (V922E, bullet) were stimulated with IGF-I or 10% FCS for 16 h at 37 °C and incubated for another 2 h in the presence of [^3H]thymidine. Then the incorporated radiolabeled thymidine was determined. Results are means ± S.E. of three independent experiments, expressed as percentage of 10% FCS stimulation (CHO-IGF-IR, 39,121 ± 1,010 cpm; CHO-V922E, 24,072 ± 534 cpm) of each cell line. The panel shows a representative result from seven separate experiments. C, XTT assay of proliferation in CHO cell lines. CHO-IGF-IR (WT) and CHO-V922E (V922E) were cultured with IGF-I for 36 h at 37 °C and incubated for another 30 min in the presence of XTT labeling mixture. The spectrophotometrical absorbance (A nm/A nm) was measured using a microtiter plate reader. Results are means ± S.E. of three independent experiments, expressed as percentage of 10% FCS stimulation (S indicates 10% FCS stimulation). The panel shows a representative result from four separate experiments.



Finally, we made an effort to address the question of why this autoactive IGF-IR mutant fails to stimulate cellular mitogenesis. Since the Ras-MAP kinase pathway has been implicated in tyrosine kinase-induced mitogenesis(22, 25, 26, 27, 28) , we focused on this pathway and examined both Ras activation and MAP kinase activation in CHO-V922E cells. The former assay clearly showed that no basal elevation was observed in the Ras-GTP amount, while IGF-I treatment stimulated the Ras activity in CHO-V922E cells to an extent similar to that observed in CHO-IGF-IR (Fig.5A). Activities of MAP kinases were measured by immune-complex kinase assay of ERK1 (Fig.5B) and in-gel kinase assay of ERKs 1 and 2 (Fig.5C). Again, CHO-V922E cells showed no basal elevation in the MAP kinase activities, and IGF-I-dependent stimulation of MAP kinases was similar between CHO-V922E and CHO-IGF-IR cells. These data clearly indicate that the V922E mutant fails to activate the Ras-MAP kinase cascade in CHO cells in the absence of IGF-I. The activation failure in this pathway provides a reasonable explanation for the failure of constitutive mitogenic stimulation by the V922E mutant.


Figure 5: Failure of activation of the Ras-MAP kinase cascade by V922E IGF-IR in the absence of IGF-I. A, Ras activation in CHO cell lines. CHO-IGF-IR (WT) and CHO-V922E (V922E) were P-labeled for 6 h at 37 °C and stimulated with 100 nM IGF-I for 3 min at 37 °C. Ras protein was recovered by immunoprecipitation with anti-Ras monoclonal antibody Y13-259, and bound nucleotides were separated by TLC. Shown is a representative result from three independent experiments. Left, an autoradiogram of the TLC plate. The positions of GDP and GTP are indicated. Right, the right panel shows the quantification of the data in the left panel. The relative molar ratio of GTP and GDP was corrected for the number of phosphates/mol of guanosine, and the percentage of the Ras-GTP complex relative to the total amount of Ras was calculated. B, MAP kinase activation in CHO cell lines with immune-complex kinase assay. C, MAP kinase activation in CHO cell lines with in-gel kinase assay. CHO-IGF-IR (WT) and CHO-V922E (V922E) were stimulated with IGF-I for 10 min at 37 °C. MAP kinase activity was measured by immune-complex kinase assay on anti-ERK1 immunoprecipitates (B) and in-gel kinase assay on cell lysates (C). The data in B and C are from independent experiments. B, an autoradiogram of MBP phosphorylated by immunoprecipitated ERK1 is shown. The incorporated P values (cpm) into MBP were (from left to right): CHO-IGF-IR, 271, 292, 644, 795; CHO-V922E, 269, 470, 515, 1053. The panel shows a representative result from two separate experiments. C, in-gel kinase assay was subjected to quantitative analysis using a phosphoimage analyzer (Fuji BAS2000), and the radioactivity of ERK1 (44 kDa) and ERK2 (42 kDa) bands was determined. The panel shows a representative result from three separate experiments.




DISCUSSION

We have herein shown that the transmembrane V922E mutation turns on the tyrosine kinase activity of IGF-IR. As the substitution of the same Val to Ile does not turn on the kinase activity, V922E should specifically act on the receptor conformation and switch on the intrinsic tyrosine kinase. The results also show that the V922E mutant causes phosphorylation of IRS-1 and stimulation of the IRS-1-associated PI 3-kinase activity. Thus, V922E IGF-IR can turn on the consecutive downstream pathway of IGF-IR in intact cells. Mediation by IR of the action of V922E IGF-IR is less likely, because BALB/c 3T3 cells, in which this mutant can transmit the signal to IRS-1, lack detectable amounts of IR(29) . Four examples of autoactive receptor tyrosine kinases by point mutations have so far been reported: oncogenic Neu with V664E(12) , IR with V938D(14) , c-Kit with V560G or D814V(15) , and CSF-1 receptor/c-Fms with L301S or Y969F(13) . Although these mutants were shown to carry augmented tyrosine kinase activity, it has been less defined whether their downstream pathways are biochemically activated in intact cells. For the first time, this study thus shows that mutationally activated receptor tyrosine kinase turns on the downstream consecutive signaling cascade, like ligand-dependent activation.

V922E submaximally activated IGF-IR as compared to the maximal stimulation by IGF-I. The effects of V922E IGF-IR on IRS-1 and PI 3-kinase were equivalent to those of 1 nM IGF-I. As 1 nM is a concentration at which IGF-I can exert its physiological functions, this mutant should generate physiologically significant outputs of cells. Submaximal activation by V922E IGF-IR may reflect that a partial population of this mutant is activated, with each mutant being completely turned on. Alternatively, each molecule of V922E mutant may be in partially activated conformation, with all the population of this mutant being in such a half-activated state.

IGF-IR has pleiotropic outputs of cells, both mitogenic and metabolic. Recent studies prove that this receptor serves a wider range of biological roles than previously thought(30, 31, 32, 33, 34) . Also, IGF-IR has multiple sets of different downstream signaling pathways. However, little has been known about the role that each pathway plays for the cellular outputs of IGF-IR. This study clearly showed that V922E IGF-IR stimulated glucose uptake in the absence of IGF-I in CHO cells. It was also shown that V922E IGF-IR constitutively activated the IRS-1/PI 3-kinase pathway in the same cells. These data strongly suggest a close relationship between the PI 3-kinase stimulation and cellular response of glucose uptake. Accumulated evidence (35, 36, 37) has so far indicated that PI 3-kinase mediates glucose uptake stimulated by insulin. Our results are thus consistent with this notion. Further analysis of the dose-response relationship revealed that glucose uptake stimulation by V922E IGF-IR was 0.1 nM IGF-I-equivalent in expressing CHO cells. Given that PI 3-kinase activation by this mutant was 1 nM IGF-I-equivalent in the same cells, this suggests that other IGF-I-dependent machinery may potentiate PI 3-kinase-mediated glucose uptake and allow its optimal activation by IGF-I stimulation.

This study also indicated that V922E IGF-IR failed to promote mitogenesis in the absence of IGF-I. Does this signify that mitogenesis is not a physiological output of IGF-IR? We believe that mitogenesis is certainly the native output of this receptor and assumed that the V922E mutant could not turn on the native IGF-IR-responsive set of intracellular signals necessary for cell growth in the absence of IGF-I. This idea was verified by the results obtained from multiple independent assays all showing that V922E mutant could not activate the Ras-MAP kinase pathway, which is thought to be required for mitogenesis by receptor tyrosine kinases(22, 25, 26, 27, 28) . These data thus demonstrate that a transmembrane point mutation differentially turns on the downstream signaling pathways of IGF-IR. The mechanism for such differential activation of signals seems to be related to the partial tyrosine phosphorylation of IRS-1 by V922E mutant (see above). A simple interpretation of these data is that V922E-induced conformational change of IGF-IR allows its intrinsic tyrosine kinase to phosphorylate the PI 3-kinase-binding tyrosine on IRS-1 but not some of other sites on IRS-1. Alternatively, it might be related to differential phosphorylation of other IGF-IR kinase substrates such as Shc(38) . We must thus await precise analysis of V922E-dependent tyrosine phosphorylation sites of IRS-1 and comprehensive investigation of all possible IGF-IR downstream pathways turned on by this mutation.

The differential activation of the two downstream pathways by V922E IGF-IR also suggests that the physiological level of activation of PI 3-kinase does not lead to the activation of the Ras-MAP kinase cascade, signifying that PI 3-kinase may not be the native upstream machinery of the Ras system. This is consistent with multiple reports using the same CHO cells showing that a dominant negative mutant of p85, the adaptor subunit of PI 3-kinase, inhibits insulin-induced PI 3-kinase activation but not Ras activation (36) or a dominant negative mutant of tyrosine-specific phosphatase Syp and that of Ras guanylnucleotide exchange factor SOS both inhibit insulin-induced Ras activation but not PI 3-kinase activation(39, 40) . In further support of this notion, 50 nM wortmannin completely inhibits insulin stimulation of PI 3-kinase in CHO cells, whereas the same concentration of wortmannin has no effect on insulin-induced Ras activation. (^2)On the other hand, Hu et al.(41) have recently shown that expression of the constitutively active mutant of p110, the catalytic subunit of PI 3-kinase, activates Ras-MAP kinase cascade in Xenopus oocytes. The discrepancy between their and our results may be attributed to the difference of cell systems studied. Alternatively, the V922E mutant activated endogenous PI 3-kinase submaximally in our experiments, whereas constitutively active PI 3-kinase has been exogenously overexpressed in their experiments. Both stimulation intensity and spatial localization of PI 3-kinases might be potentially different between these studies. It is therefore quite interesting to compare precisely the action of V922E IGF-IR with that of the activated p110 mutant using the same experimental systems.


FOOTNOTES

*
This work was supported by Bristol-Myers Squibb. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Japan Research Foundation for Clinical Pharmacology, the Nakatomi Foundation, and the Japan Heart Foundation.

To whom correspondence may be addressed: Cardiovascular Research Center, Massachusetts General Hospital-East, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-4350; Fax: 617-724-9622; yonezawa{at}cvrc.mgh.harvard.edu.

**
To whom correspondence may be addressed: Cardiovascular Research Center, Massachusetts General Hospital-East, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-3902; Fax: 617-724-9622; nishimoto{at}helix.mgh.harvard.edu.

^1
The abbreviations used are: IGF-I, insulin-like growth factor I; IGF-IR, the receptor for IGF-I; IR, insulin receptor; IRS-1, insulin receptor substrate-1; PI 3-kinase, phosphoinositide-3-OH kinase; WT, wild type; V922X IGF-IR, IGF-IR whose Val is mutated to X; FCS, fetal calf serum; MBP, myelin basic protein; Tyr(P), phosphotyrosine; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase.

^2
K. Takahashi, K. Yonezawa, and I. Nishimoto, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. Takashi Okamoto for his indispensable technical assistance to the construction of the IGF-IR mutants and critical reading of this manuscript and Dr. Kenta Hara for his technical assistance to the MAP kinase assays and critical reading. We also indebted to Dr. Richard A. Roth for his gift of human IGF-IR cDNA and technical advice, Drs. Hisashi Umemori and Tadashi Yamamoto for their assistance to the experiments preliminary to those presented here, Drs. Yoshinori Okabayashi and Masato Kasuga for their gift of anti-ERK1 rabbit sera, and Ugo Giambarella and Dr. Dovie Wylie for expert technical assistance.


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