The Role of the Tyrosine Kinase Domain of the Insulin-like Growth Factor-I Receptor in Intracellular Signaling, Cellular Proliferation, and Tumorigenesis*

Insulin and insulin-like growth factor (IGF-I) recep- tors are heterotetrameric proteins consisting of two (cid:97) and two (cid:98) -subunits and members of the transmembrane tyrosine kinase receptors. Specific ligand binding to the receptor triggers a cascade of intracellular events, which begins with autophosphorylation of several tyro- sine residues of the (cid:98) -subunit of the receptor. The triple cluster in the tyrosine kinase domain of the (cid:98) -subunit is the earliest and major autophosphorylation site. Previ- ous studies have shown that substitutions of these three tyrosines by phenylalanines of both insulin and IGF-I receptors practically abolish any activation of cellular signaling pathways. We have studied the effect of double tyrosine mutations on IGF-I-induced receptor autophosphorylation, activation of Shc and IRS-1 pathways, and cell proliferation and tumorigenicity. Substitution of tyrosines 1131/1135 blocks any detectable autophospho- rylation, whereas substitution of tyrosines 1131/1136 or 1135/1136 only reduces autophosphorylation levels in some clones by (cid:59) 50%. Nevertheless, all the cells expressing IGF-I receptors with double tyrosine substitutions demonstrated markedly reduced signaling through Shc and IRS-1 pathways. In addition, they were unable to respond to IGF-I-stimulated cell growth in culture, and tumor formation in nude mice was abrogated. These data suggest that the presence of tyrosine 1131

Insulin and insulin-like growth factor (IGF-I) receptors are heterotetrameric proteins consisting of two ␣and two ␤-subunits and members of the transmembrane tyrosine kinase receptors. Specific ligand binding to the receptor triggers a cascade of intracellular events, which begins with autophosphorylation of several tyrosine residues of the ␤-subunit of the receptor. The triple cluster in the tyrosine kinase domain of the ␤-subunit is the earliest and major autophosphorylation site. Previous studies have shown that substitutions of these three tyrosines by phenylalanines of both insulin and IGF-I receptors practically abolish any activation of cellular signaling pathways. We have studied the effect of double tyrosine mutations on IGF-I-induced receptor autophosphorylation, activation of Shc and IRS-1 pathways, and cell proliferation and tumorigenicity. Substitution of tyrosines 1131/1135 blocks any detectable autophosphorylation, whereas substitution of tyrosines 1131/1136 or 1135/1136 only reduces autophosphorylation levels in some clones by ϳ50%. Nevertheless, all the cells expressing IGF-I receptors with double tyrosine substitutions demonstrated markedly reduced signaling through Shc and IRS-1 pathways. In addition, they were unable to respond to IGF-I-stimulated cell growth in culture, and tumor formation in nude mice was abrogated. These data suggest that the presence of tyrosine 1131 or 1135 essential for receptor autophosphorylation, whereas the presence of each of these tyrosines is necessary for a fully functional receptor.
The multiple physiological actions, including cell growth and differentiation of the insulin-like growth factors (IGFs) 1 are mediated by the IGF-I receptor. While the IGF-I receptor and the structurally related insulin receptor are members of the type II receptor tyrosine kinase family, their in vivo biological functions are quite separate. Both the IGF-I and insulin receptors are heterotetrameric proteins composed of two extracellular ␣-subunits and two membrane-spanning ␤-subunits linked by disulfide bonds (1)(2)(3). Sequences found in the ␣-subunits of each receptor are important for determining ligand specificity. The amino-terminal and carboxyl-terminal portions of the ␣ subunit of the insulin receptor are critical for high affinity insulin binding, while the cysteine-rich domain of the IGF-I receptor determines high affinity IGF-I binding (4 -6). Likewise, the ␤-subunits contain a number of structurally distinct domains including the extracellular, transmembrane, juxtamembrane, tyrosine kinase, and carboxyl-terminal regions. Binding of ligand to the ␣-subunit activates the tyrosine kinase activity of the ␤-subunit resulting in autophosphorylation on distinct tyrosine residues. The triple tyrosine cluster within the kinase domain (1131, 1135, and 1136 tyrosines in the IGF-I receptor and the equivalent residues in the insulin receptor; numbering system of Ullrich et al. (2)) is the earliest and major site of autophosphorylation. Phosphorylation of these three tyrosine residues is necessary for activation of the kinase toward other substrates (7,8). When the triple tyrosine cluster is substituted by phenylalanine residues, the receptor loses all ligand-induced biological actions (9,10).
Whereas the function of the triple tyrosine cluster in the tyrosine kinase domain of the insulin receptor has been well characterized (11)(12)(13), much less is known about the corresponding tyrosines of the IGF-I receptor. Single substitutions of tyrosine 1131 or 1135 have relatively small effects on receptor and endogenous substrate phosphorylation or on cell proliferation (14,15). In contrast, substitution of tyrosine 1136 apparently has an inhibitory effect on those functions (14). In addition, it has been demonstrated, at least in the case of the insulin receptor, that in intact cells, bis phosphorylation of the kinase domain at Tyr-1158 and either Tyr-1162 or Tyr-1163 comprises 80% of phosphorylated receptors (7, 16 -18). Thus, a study involving the substitution of combinations of double tyrosines may be more instructive. To further characterize the role of these tyrosines in IGF-I receptor function, we have performed substitutions of combinations of two tyrosines in the kinase domain of the IGF-I receptor. We transfected NIH-3T3 cells to study receptor autophosphorylation and post-receptor signaling pathways as well as biological functions of the receptor including cell growth and tumorigenicity.

EXPERIMENTAL PROCEDURES
Materials-Restriction endonucleases were purchased from New England Biolabs, Boehringer Mannheim, and Life Technologies, Inc. Cell culture media and reagents were purchased from Biofluids, Inc. (Rockville, MD) and Advanced Biotechnologies (Columbia, MD). Insulin-free bovine serum albumin (fraction V) was obtained from Armour (Kankakee, IL). Monoclonal antiphosphotyrosine antibody (clone 4G10) was purchased from Upstate Biotechnology, Inc. Recombinant antiphosphotyrosine RC20H horseradish peroxidase-conjugated, polyclonal anti-Shc, monoclonal anti-Grb2, and monoclonal anti-PTP1D antibodies were purchased from Transduction Laboratories (Lexington, KY). Recombinant human IGF-I, fetal bovine serum (FBS), monoiodinated 125 I-IGF-I, horseradish peroxidase-conjugated anti-mouse immunoglobulin, and the ECL detection kit were purchased from Amersham Corp. Prestained high molecular weight protein standards and 3-[4,5-* 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.
Construction of the Mutant IGF-I Receptor DNA-The wild-type human IGF-I receptor expression vector has been previously described (19). Mutation of the human IGF-I receptor cDNA at amino acid residues 1131, 1135, and 1136 (numbering system is that of Ullrich et al. (2)) was performed by in vitro site-directed mutagenesis using the Double Take Mutagenesis Kit (Stratagene) and a pBluescript II plasmid containing an EcoRI-BamHI fragment of the human IGF-I receptor (previously described by Kato et al. (19)). The tyrosines at positions 1131, 1135, and 1136 were designated as being the first (1), second (2), or third (3) in the triple tyrosine cluster. The following double tyrosine mutations were generated: DYF12 (tyrosines 1131 and 1135 mutated to phenylalanines 1131 and 1135), DYF13 (tyrosines 1131 and 1136 mutated to phenylalanines) and DYF23 (tyrosines 1135 and 1136 mutated to phenylalanines). The sequence of the bridging primer was 5Ј-GC-CGCCACCGCGGTGGAGCTCCAATTCGCC-3Ј (the SacI site used for mutagenesis is underlined), and the sequence of the extension primer was 5Ј-AGCTCCACCGCGGTGGCGGCCGCT-3Ј. The sequence of the mutagenic primer to generate DYF12 was 5Ј-CTTTCCGGTA-AAAGTCTGTTTCGAAGATATCTCGCGT-3Ј. The sequence of the mutagenic primer to generate DYF13 was 5Ј-CTCCTTTCCGGAAAT-AGTCTGTCTCAAAGATATCTC-3Ј. The sequence of the mutagenic primer to generate DYF23 was 5Ј-TGCCTCCTTTCCGGAAAAAGTCT-GTCTCATA-3Ј. In the mutagenic primers, codons mutated from tyrosine to phenylalanine are underlined. Nucleotide sequences mutated to generate a restriction enzyme site are presented in bold type. A new SfuI site was introduced in the cDNA for DYF12. A new AccIII site was introduced in the cDNA for both DYF13 and DYF23. The cDNA sequences of all the mutations were confirmed by dideoxy sequencing. The mutated cDNAs in pBluescript II were excised with SalI and NotI and cloned into a bovine papilloma virus-derived mammalian expression vector (pBPV; Pharmacia Biotech Inc.) that had been linearized with XhoI and NotI.
Cell Culture and Transfection-All NIH-3T3 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM glutamine in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. NIH-3T3 cells were cotransfected with 20 g of wild-type or mutant expression vector or insert-less pBPV plus 1 g of pMCINeo (Clontech) in Lipofectamine reagent (Life Technologies, Inc.). Selection was carried out as described previously (19). Clones overexpressing IGF-I receptors were selected based on results of IGF-I binding assays as described previously (19). Stably transfected cells were maintained in the media described above supplemented with 500 g/ml G418 (Geneticin, Life Technologies, Inc.). Serum-free medium containing 0.1% bovine serum albumin, 20 mM Hepes, pH 7.5, and antibiotics was used in phosphorylation assays of the receptor and cellular substrates.
Receptor Autophosphorylation-Confluent cells in 100-mm plates were serum-starved overnight and then incubated either with or without IGF-I (100 ng/ml) for 1 min at 37°C. The cells were washed rapidly with ice-cold phosphate-buffered saline and lysed in the presence of 50 nM Hepes, pH 7.9, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, 4 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 10 mM sodium fluoride. Cell lysates were cleared by centrifugation. Protein content was determined by the method of Bradford using a protein assay kit (Bio-Rad). Equal amounts of protein (up to 40 g) were reduced by ␤-mercaptoethanol and fractionated by 7.5% SDS-PAGE. Resolved proteins were electrophoretically transferred to nitrocellulose membrane (Hybond, ECL, Amersham). The amount of IGF-I receptor present was determined by immunoblotting with monoclonal anti-IGF-I receptor ␤-subunit antibody (Siddle 1-2) at a 1:500 dilution at 4°C overnight; this antibody was detected with horseradish peroxidase-conjugated anti-mouse immunoglobulin (1:5000 dilution). The blots were then developed with the ECL system. Based on these immunoblots, equal amounts of receptors were fractionated by SDS-PAGE as described above. Blots for tyrosine phosphorylation were immunoblotted with monoclonal antiphosphotyrosine antibody (clone 4G10) (1:1000 dilution) and detected with horseradish peroxidase-conjugated anti-mouse immunoglobulin (1:5000 dilution) using the ECL system. The level of phosphorylation of the ␤-subunits was quantitated by digitalizing the signal from the x-ray film and analyzing the signal using NIH image version 1.55 software and calculated according to receptor density assessed using the Siddle 1-2 antibody (15).
MTT Cellular Proliferation Assay-3 ϫ 10 3 cells were plated in each well of 96 multi-well plates and allowed to recover overnight in DMEM plus 10% FBS. Thereafter, cell growth was continued in either 1% FBS or 1% FBS supplemented with 100 ng/ml IGF-I for 7 days. Media were replenished at 72 h of growth. The cells were processed at varying time points as described previously (20), and the cellular number was calculated daily.
Tumorigenicity Assay-The ability of various cell lines to form tumors was determined by injecting 1 ϫ 10 7 cells subcutaneously in the lower dorsal region of nude mice. Mice were housed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. After 4 weeks, mice were examined weekly to monitor tumor formation, and measurements were taken by using a caliber. Tumor volume was calculated by the following formula: length ϫ width 2 /2 (V ϭ a ϫ b 2 /2).
Statistics-Statistical significance between groups was tested using the Student's t test.

Expression of Wild-type and Mutant
Receptors-Two separate clones overexpressing each mutant or wild-type IGF-I receptor were used in this study. DYF12 designates substitution of the tyrosine residues with phenylalanine at positions 1131 and 1135. Substitution of tyrosine residues with phenylalanine at positions 1131 and 1136 is designated as DYF13, and substitution of tyrosine residues at positions 1135 and 1136 is designated as DYF23. WT43 and WT50 are clones overexpressing wild-type human IGF-I receptor (15). pNeo is a clone cotransfected with a neomycin-resistant plasmid and the pBPV vector (15). All clones overexpressing IGF-I receptor had similar numbers of cell surface IGF-I receptors as determined by Scatchard analysis (Table I) (21).
Receptor Autophosphorylation-One of the first events after IGF-I binding to the IGF-I receptor is the autophosphorylation of tyrosine residues of the ␤-subunit of the receptor. The triple tyrosine cluster in the tyrosine kinase domain is considered to be the major autophosphorylation site of the IGF-I receptor (10). To study the autophosphorylation capability of the mutant Phosphorylation of Endogenous Substrates-The two major signal transduction pathways following IGF-I receptor stimulation characterized thus far are those mediated by IRS-1 and Shc. We therefore studied the activation of IRS-1 and Shc pathways by IGF-I in wild-type and double tyrosine mutants. To evaluate IRS-1 pathway activation, cells were stimulated with 100 ng/ml IGF-I for 1 min at 37°C, and IRS-1 immunoprecipitates were assayed for tyrosine phosphorylation of IRS-1 and Grb2 and PTP1D association. Stripping and reblotting the membranes with a polyclonal anti-IRS-1 antibody confirmed that similar amounts of IRS-1 protein were immunoprecipitated in all samples (data not shown). A typical result of these experiments is presented in Fig. 2. Panel A shows IRS-1 phosphorylation immunoblotted with a phosphotyrosine antibody RC20H. The ability of IGF-I to stimulate IRS-1 tyrosine phosphorylation was reduced in cells expressing the double mutant receptors as compared to wild-type clones. In addition, the IGF-I-stimulated association of PTP1D and Grb2 with IRS-1 occurred to a much lower extent in double tyrosine mutants when compared with stimulated levels in wild-type clones (panels B and C, respectively). This effect was most dramatic in studies of IRS-1/PTP1D association.
To evaluate Shc pathway activation, cells were stimulated with 100 ng/ml IGF-I for 5 min at 37°C as described above, and Shc immunoprecipitates were assayed for Shc phosphorylation and Grb2 association. Similar amounts of Shc proteins were detected when the membranes were reblotted with a polyclonal anti-Shc antibody (data not shown). A typical experiment is shown in Fig. 3, A and B, respectively. IGF-I stimulation of tyrosine phosphorylation of the 52-and 46-kDa isoforms of Shc protein (Fig. 3A) was observed in wild-type clones, whereas cells overexpressing the double tyrosine mutant receptors pre-sented very low levels of Shc phosphorylation. Shc-Grb2 association following IGF-I stimulation was similarly diminished in mutant clones with respect to the wild-type clones (Fig. 3B). These results suggest that activation of both the IRS-1 and Shc pathways by IGF-I are at least partially blocked in cells overexpressing double tyrosine mutant receptors as compared with cells overexpressing a similar number of wild-type receptors.
In Vitro Cell Growth-To determine whether the differential receptor autophosphorylation levels in IGF-I receptor double tyrosine mutants resulted in alterations in IGF-I-induced biological effects, we studied the growth rate of both wild-type and mutant cell lines in culture using the MTT assay. Cells were cultured either in DMEM with 1% FBS or DMEM plus 1% FBS supplemented with 100 ng/ml IGF-I. The results of a typical assay are shown in Fig. 4A. The mean and standard deviations of three separate experiments are plotted for the 120-h time point in Fig. 4B. Both wild-type and mutant clones failed to grow in DMEM with 1% FBS (data not shown). In contrast, wild-type clones grew well when stimulated with 100 ng/ml IGF-I. All mutant clones demonstrated a flat growth response to IGF-I and were not different than the pNeo clone (p Ͼ 0.05), whereas their growth response was significantly less than the wild-type clones (p Ͻ 0.01).
Tumor Formation-Overexpression of wild-type IGF-I receptors in NIH-3T3 cells confers tumorigenicity (22). We therefore tested the double tyrosine mutants for tumor formation in nude mice. 1 ϫ 10 7 cells of each clone were injected per mouse, and tumor measurements were performed as described under "Experimental Procedures." Cells overexpressing wild-type receptors typically started to form measurable tumors five and a half weeks after injection (Fig. 5). The size of these tumors increased progressively until the animals died or had to be sacrificed. In contrast, none of the cells expressing the double tyrosine mutants formed large tumors. Only the DYF23 (no. 1) clone gave rise to some tumors that were very small in comparison to the wild-type clones.

DISCUSSION
The earliest post-binding event following the interaction of insulin and IGF-I with their specific receptors is the autophosphorylation of the triple tyrosine cluster within the ␤-subunit tyrosine kinase domain (7,8). Mutational analysis of the insulin receptor tyrosine kinase domain has provided interesting, and at the same time, controversial information about the regulatory role of these three tyrosines in receptor kinase activity. The most dramatic effect has been shown with insulin receptors where these three tyrosine residues (tyrosines 1158, 1162, and 1163) have been mutated to phenylalanine. Insulinstimulated autophosphorylation and cell signaling by these mutant receptors are impaired (13,23). Single substitutions of any one of these tyrosine residues with phenylalanine (i.e. Y1158F, Y1162F, or Y1163F) may reduce in vitro autophosphorylation of the receptor ␤-subunit on the remaining tyrosines and reduce tyrosine kinase activity, although, in contrast, Zhang and Roth (6) reported that the Y1158F mutation had no effect on autophosphorylation or on insulin-stimulated exogenous tyrosine kinase activity. Data on thymidine incorporation is also controversial. In general, however, substitutions of any single tyrosine have only slight effects on insulin-stimulated thymidine incorporation (13,23). A more substantial reduction in insulin-induced tyrosine kinase activity was observed when double tyrosine substitutions were studied (23). Generally, the triple tyrosine substitution results in even more reduction in thymidine incorporation, although HTC cells expressing the triple tyrosine mutant receptor exhibit normal thymidine incorporation (24). Despite these discrepancies, the data overall are consistent with the idea that autophosphorylation of ty- Although IGF-I and insulin receptors are structurally similar, their in vivo biological actions are different. At which level this divergence of function occurs is not yet defined. Thus, analyses of the structural and functional aspects of the IGF-I receptor is of considerable scientific interest. Studies using mutational analyses of the IGF-I receptor are considerably less complete than of the insulin receptor. We and others have reported previously that substitution of the triple tyrosine cluster with phenylalanine has similar effects as seen with the insulin receptor, i.e. essentially all of the functions of the IGF-I receptor were abrogated (9,10). A single substitution of tyrosine residue 1131 reduces autophosphorylation and receptor internalization, whereas IRS-1 phosphorylation, thymidine incorporation, and cell proliferation were unaffected (15). Li et al. (14) showed that single substitutions of tyrosines 1131 or 1135 did not affect mitogenicity and only slightly reduced autophosphorylation. In contrast, they reported that substitution of tyrosine 1136 abrogated autophosphorylation and cell growth.
In the present study, we show that substitutions of tyrosines 1131 and 1135 reduced ␤-subunit autophosphorylation to levels below the level of detectability by our antibody, suggesting that the presence of either tyrosine 1131 or 1135 is required for full ␤-subunit autophosphorylation. Substitutions of 1131/1136 and 1135/1136 affect autophosphorylation to a lesser degree, suggesting that tyrosine 1136 is not crucial for IGF-I-stimulated autophosphorylation. The crystal structure of the tyrosine kinase domain of the human insulin receptor has recently been characterized (25). Apparently, the phosphorylation of tyrosine 1162 (the second of the three tyrosines) is a key step in the receptor kinase activation. The apo structure shows that the hydroxyl group of tyrosine 1162 is bound in the active site autoinhibiting the tyrosine kinase of the receptor. This finding suggests that phosphorylation of this tyrosine would disengage it from the active site. Our results, in the present study, suggest an important role not only for tyrosine 1135 (equivalent to tyrosine 1162 in insulin receptor) but also for tyrosine 1131 in autophosphorylation of the IGF-I receptor.
Of particular interest in our study is the finding that all the cells expressing double tyrosine substitutions failed to respond mitogenically to IGF-I stimulation, and tumor formation was reduced compared to cells overexpressing wild-type IGF-I receptors, despite the fact that substitution of tyrosines 1131/ 1136 or 1135/1136 only slightly reduced receptor autophosphorylation. These findings suggest that perhaps a relatively high threshold of autophosphorylation is required to fully activate the signaling pathways. The inability of all double mutant receptors to mediate biological activities could be explained by the absence of IGF-I-induced tyrosine phosphorylation of the two major IGF-I signaling pathways, IRS-1 and Shc. IRS-1 is considered an adapter protein between the insulin and the IGF-I receptors and the network of their signaling pathways (26 -29). IRS-1 is phosphorylated on multiple tyrosine residues upon receptor stimulation (30). This provides multiple sites of interaction for proteins with SH2 (src-homology 2) domains (31). Several SH2-containing proteins have been shown to associate with IRS-1: PI3-kinase (32)(33)(34), Nck (35), Grb2 (36 -41), and PTP1D (30,39). PTP1D is a tyrosine phosphatase that binds to IRS-1 at tyrosine 1172; this binding provides a potential mechanism for its activation (40). Evidence suggests that PTP1D is involved in stimulation of mitogenesis, and regulation of Ras and mitogen-activated protein kinase activation (41,42). Grb2 is thought to stimulate p21 ras activity through a monovalent interaction with p21 ras GDP-GTP exchange factor, mSOS, resulting in stimulation of a serine/threonine phosphorylation cascade leading to activation of the mitogen-activated protein kinase pathway implicated in cell growth and metabolism. Thus, the binding of the Grb2-mSOS complex to IRS-1 after insulin and IGF-I stimulation links insulin and IGF-I receptor tyrosine kinases and p21 ras signaling pathways. An alternative and possible redundant pathway that links insulin and IGF-I signaling with p21 ras activation is through Shc (43)(44)(45)(46)(47). Shc is tyrosine phosphorylated upon insulin and IGF-I stimulation (44 -46). The phosphorylation of Shc provides a binding site for Grb2, resulting in the formation of the Shc-Grb2-mSOS complex and probably leading to activation of the mitogen-activated protein kinase pathway. Therefore, diminished stimulation of the IRS-1/PTP1D and IRS-1/Grb2 as well as Shc-Grb2 pathways by IGF-I in our cells expressing receptors with double tyrosine substitutions is associated with decreased IGF-I-stimulated cell growth response of cells expressing these receptors. We cannot exclude the possible involvement of other signaling pathways, such as Crk, that are also involved in IGF-I-induced signaling (48).
In summary, we have demonstrated that the presence of both tyrosines 1131 and 1135 of the IGF-I receptor are necessary for full IGF-I-stimulated autophosphorylation of the ␤-subunit. However, the absence of any of the tyrosine residues of the triple tyrosine cluster significantly reduced signal trans- duction through IRS-1 and Shc activation, thus abrogating IGF-I-stimulated cell growth and IGF-I receptor-mediated tumor formation of transfected fibroblasts.