©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Localization of the Insulin-like Growth Factor I Receptor Binding Sites for the SH2 Domain Proteins p85, Syp, and GTPase Activating Protein (*)

(Received for publication, December 6, 1994; and in revised form, May 19, 1995)

B. Lynn Seely (§) Donna R. Reichart Patricia A. Staubs Byung H. Jhun David Hsu Hiroshi Maegawa (1) Kim L. Milarski (2) Alan R. Saltiel (2) Jerrold M. Olefsky

From the (1)Department of Medicine, Division of Endocrinology and Metabolism, University of California at San Diego, La Jolla, California 92093, the Veterans Administration Medical Center, Medical Research Service, San Diego, California 92161, the Whittier Diabetes Program, La Jolla, California 92037, Shiga University of Medical Science, Shiga, 520-21, Japan, and the (2)Department of Signal Transduction, Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Potential signaling substrates for the insulin-like growth factor I (IGF-I) receptor are SH2 domain proteins including the p85 subunit of phosphatidylinositol 3-kinase, the tyrosine phosphatase Syp, GTPase activating protein (GAP), and phospholipase C- (PLC-). In this study, we demonstrate an association between the IGF-I receptor and p85, Syp, and GAP, but not with PLC- in lysates of cells overexpressing the human IGF-I receptor. We further investigated these interactions using glutathione S-transferase (GST) fusion proteins containing the amino-terminal SH2 domains of p85 or GAP, or both SH2 domains of Syp or PLC- to precipitate the IGF-I receptor from purified receptor preparations and from whole cell lysates. p85-, Syp-, and GAP-GSTs precipitated the IGF-I receptor, whereas the PLC--GST did not. Using phosphopeptides corresponding to IGF-I receptor phosphorylation sites, we determined that the p85- and Syp-GST association with the IGF-I receptor could be inhibited by a carboxyl-terminal peptide containing pY1316 and that the GAP-GST association could be inhibited by a NPXY domain peptide. The GAP-GST binding site was confirmed by showing that a mutant IGF-I receptor with a deletion of the NPXY domain including tyrosine 950 was poorly precipitated by the GAP-GST. We conclude that p85 and Syp may bind directly to the IGF-I receptor at tyrosine 1316, and that GAP may bind to the IGF-I receptor at tyrosine 950. An association between the IGF-I receptor and PLC- was not evident. p85, Syp, and GAP are potential modulators of IGF-I receptor signal transduction.


INTRODUCTION

The insulin-like growth factor I (IGF-I) (^1)receptor is a member of the large family of tyrosine kinase growth factor receptors which undergo autophosphorylation upon ligand binding(1, 2) . The activated receptor is then able to transduce this signal intracellularly by interacting with specific downstream protein substrates, ultimately resulting in hormonal effects such as cellular growth and differentiation(3, 4) . Autophosphorylation of the IGF-I receptor is followed immediately by phosphorylation of insulin receptor substrate-1 (IRS-1)(5, 6, 7, 8) .

The tyrosine phosphorylation of IRS-1 transforms this 185-kDa molecule into a ``docking'' protein, facilitating its interaction with a number of other signaling molecules containing Src homology 2 (SH2) domains(9, 10) . SH2 domains are conserved regions of 100 amino acids that promote association with phosphorylated tyrosines surrounded by specific amino acid motifs such as those found in IRS-1 and activated growth factor receptors(11, 12) . Although IRS-1 is clearly an important signaling molecule for the insulin and IGF-I receptors (5, 6, 7, 8, 13, 14) , recent evidence gained from IRS-1 knockout mice has suggested that an alternative signaling pathway(s) exists(15, 16) . We hypothesize that one such pathway may result from the direct association of potential signaling molecules with the IGF-I receptor.

Recently, Van Horn et al.(17) , demonstrated that the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) interacts directly with the insulin receptor, and we have localized this insulin receptor binding site to the YXXM motif at tyrosine 1322 (18) . Although direct association of p85 with the IGF-I receptor has not previously been demonstrated, PI 3-kinase activity increases after IGF-I stimulation. Furthermore, Yamamoto et al.(19) showed that PI 3-kinase activity associates with immobilized IGF-I receptors and can be competed away with increasing concentrations of a p85 fusion protein.

Syp (PTP1D, SHPTP2) is a protein tyrosine phosphatase that contains two SH2 domains. Syp is the homologue of corkscrew, an SH2 phosphatase in Drosophila known to enhance signal transduction of the receptor tyrosine kinase torso(20, 21, 22, 23) . Microinjection experiments indicate that Syp is involved in positive signal transduction of receptor kinases as inhibition of Syp leads to a decrease in insulin- and IGF-I-stimulated mitogenesis(24, 25) . Syp is known to bind to IRS-1(26) , but we and others have also demonstrated that a Syp-glutathione S-transferase (GST) fusion protein can associate directly with the insulin receptor at tyrosine 1322(18, 27) . It can also directly associate with the epidermal growth factor (28) and platelet-derived growth factor (29) receptors. It is not known if Syp can interact directly with the IGF-I receptor.

GTPase-activating protein (GAP) is another SH2 domain protein involved in tyrosine kinase growth factor receptor signal transduction. GAP inactivates ras by stimulating the hydrolysis of GTP-ras, thereby functioning as a negative regulator of mitogenic signal transduction(30) . GAP associates directly with the activated insulin receptor (31) at tyrosine 960(18) , but it is not known if GAP interacts with the IGF-I receptor. Phospholipase C- (PLC-) is another SH2 domain protein which is recruited directly to the activated epidermal and platelet-derived growth factor receptors and is an important mitogenic signaling molecule(32) . No previous association between PLC- and the insulin or IGF-I receptors has been reported.

In this study, we investigate the direct association of p85, Syp, GAP, and PLC- with the IGF-I receptor and map the sites of receptor interaction utilizing SH2 domain-containing fusion proteins and phosphopeptides modeled after phosphotyrosine-containing domains of the IGF-I receptor.


EXPERIMENTAL PROCEDURES

Cell Lines and Materials

Chinese hamster ovary (CHO) cells were transfected as described (33) with the expression vector, Rldn, containing the full-length human IGF-I receptor cDNA previously reported by Ullrich et al.(34) . The transfected cells were selected with 400 µg/ml G418 (Life Technologies, Inc.), and a clone expressing 1.5 10^5 receptors per cell, CHO, was used in these studies. A similarly transfected cell line overexpressing the human IGF-I receptor with a deletion of the NPXY domain (CHO^Y) was also used in some experiments(33) . Transfected CHO cells were maintained in Ham's F-12 from Life Technologies, Inc. supplemented with 10% fetal calf serum (Gemini Bioproducts, Calabasas, CA), 0.5% gentamicin (Gemini Bioproducts), 1% GlutaMAX (Life Technologies, Inc.), and 400 µg/ml G418 (Life Technologies, Inc.). Insulin, IGF-I, I-insulin, and I-IGF-I were generously provided by Eli Lilly. Electrophoresis equipment and reagents were from Bio-Rad. The antiphosphotyrosine, anti-p85, anti-Syp, and anti-PLC- antibodies were from Transduction Laboratories (Lexington, KY), the anti-IGF-I receptor antibody was from Oncogene Science (Uniondale, NY), and the anti-GAP antiserum was from UBI (Lake Placid, NY). The goat anti-mouse IgG-horseradish peroxidase antibody and the enhanced chemiluminescence detection reagents were from Amersham. Nitrocellulose was from Schleicher and Schuell. Immunoprecipitin was from Life Technologies, Inc., the Protein G- and Protein A-Sepharose were from Sigma, and the glutathione-Sepharose 4B was from Pharmacia Biotech. Phosphopeptides were from Quality Controlled Biochemicals (Hopkinton, MA) and were modeled after the amino acid sequences of specific IGF-I receptor tyrosine phosphorylation sites. These include the triple tyrosine kinase domain (pYpYpY), the NPXY domain (NPXpY and RPXpY), and the carboxyl-terminal tyrosine, pY1316. The sequences are: pYpYpY, DIpYETDpYpYRK; NPXpY, ASVNPEpYFSA; RPXpY, ASVRPEpYFSA; and pY1316, ASFDERQPpYAHMNGG. The peptide NPXpY was not used because repeat high performance liquid chromatography analysis of this peptide produced two peaks indicating conformational changes in the peptide which we believe prevented consistent phosphotyrosine binding.

GST-Fusion Protein Purification

The molecular cloning of the SH2 domain proteins used in this study is described elsewhere(35) . The p85 fusion protein used contained residues 321 to 440 from p85, including the amino-terminal SH2 domain. The Syp-GST contained residues 1 to 216 of Syp, including both SH2 domains. The GAP-GST contained amino acids 177 to 278 of GAP, including the amino-terminal SH2 domain, and the PLC--GST included residues 550 to 756 of PLC-, including the amino- and carboxyl-terminal SH2 domains. The expression and purification of fusion proteins has been described previously(18, 36) .

Receptor Association Assays

IGF-I receptors were purified from CHO cells by wheat germ agglutinin affinity chromatography, and I-IGF-I binding studies were performed as described previously(37) . Approximately 500 fmol of partially purified IGF-I receptors were incubated with 10 µl of kinase buffer (10 mM HEPES, 0.05% Triton X-100, pH 7.4 at 4 °C), 2 mM sodium orthovanadate, and 100 ng/ml IGF-I for 30 min at 4 °C. 20 µl of an activating mixture (50 µM ATP, 10 mM MnCl(2) in kinase buffer) was then added at room temperature, and the reaction was allowed to proceed for 30 min before it was terminated with 20 µl of lysis buffer (50 mM HEPES, 1% Triton X-100, 10 mM EDTA, 150 mM sodium chloride, 2 mM phenylmethylsulfonyl fluoride, 10% glycerol, 4 mM sodium vanadate, 200 mM sodium fluoride, and 20 mM sodium pyrophosphate, pH 7.4 at 4 °C). 50 µl of a 50% suspension of prewashed glutathione-Sepharose in lysis buffer plus increasing concentrations of the fusion proteins were added. Where indicated, phosphopeptides were also added at this time. The samples were rotated for 90 min at 4 °C. After spinning, the supernatants were collected, and the Sepharose was washed twice with lysis buffer. The samples were boiled in solubilizing fluid (6% SDS, 2 mM EDTA, 20% glycerol, 5% 2-mercaptoethanol, 125 mM Tris, 0.01% bromphenol blue, pH 6.8) and fractionated by gel electrophoresis. After transfer to nitrocellulose, blots were developed with a monoclonal antiphosphotyrosine antibody using the enhanced chemiluminescence system. Where indicated, whole cell lysates were used instead of purified receptors. After overnight starvation in serum-free Ham's F-12 media, whole cell monolayers were stimulated with IGF-I, 100 ng/ml, for 1 min at 37 °C. Pretreatment with phenylarsine oxide (PAO) was performed where indicated by preincubating the cells with 25 µM PAO for 5 min at 37 °C. After stimulation with IGF-I for 1 min at 37 °C, the media were removed, and 400 µl of lysis buffer was added. 100 µl of the cell lysate was then incubated with the fusion protein of interest and glutathione-Sepharose as described above.

Immunoprecipitation and Immunoblotting

The IGF-I receptor was immunoprecipitated from whole cell lysates with a monoclonal anti-IGF-I receptor antibody at a dilution of 1:100. After an overnight incubation, the antibody complex was collected with Protein G-Sepharose. IRS-1 immunoprecipitation was performed with a monoclonal antibody (38) at a dilution of 1:100 and was precipitated with prewashed Immunoprecipitin after an overnight incubation at 4 °C. The anti-GAP immunoblot was performed using an anti-GAP antiserum at a concentration of 1:1000, the anti-Syp, anti-p85, and anti-PLC- immunoblots were performed at 1:250, and antiphosphotyrosine immunoblots at 1:2000. Phosphotyrosine immunoprecipitation was performed with the antiphosphotyrosine antibody at a dilution of 1:200. Following a 4-h incubation at 4 °C, antibody complexes were collected with Protein G-Sepharose.


RESULTS

IGF-I Stimulated Tyrosine Phosphorylation

Fig.1A demonstrates the different proteins which are tyrosine-phosphorylated in CHO in response to IGF-I stimulation. Predominant phosphoprotein bands in IGF-I-stimulated cells are seen at 185 kDa, 120 kDa, and 105 kDa. Immunoprecipitation experiments identified the 185-kDa band as IRS-1 (Fig.1B) and the 105-kDa band as the IGF-I receptor (Fig.1C). Although GAP was identified in the total whole cell lysates by an anti-GAP immunoblot (Fig.1D, lane 1), GAP was not detected in immunoprecipitates using an antiphosphotyrosine antibody (lanes 2 and 3). Even after prolonged exposure, GAP was not seen in the antiphosphotyrosine antibody precipitates. GAP is, therefore, present in the whole cell lysates (lane 1), but is not the tyrosine-phosphorylated protein seen at 120 kDa (lane 5). This protein is tyrosine-phosphorylated both in the stimulated and in the unstimulated cells, and its identity is not known.


Figure 1: Protein identification. Monolayers of CHO were incubated at 37 °C for 1 min in the absence or presence of IGF-I, 100 ng/ml. A shows whole cell lysates analyzed by 7.5% SDS-PAGE, then transferred to nitrocellulose and immunoblotted with an antiphosphotyrosine antibody. Stimulated cell lysates were also immunoprecipitated with an anti-IRS-1 antibody (B) or with an anti-IGF-I receptor antibody (C) as described under ``Experimental Procedures.'' The precipitates were analyzed by 7.5% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an antiphosphotyrosine antibody. D, lanes 1-4, depict an anti-GAP immunoblot. The first lane shows the total cell lysate. The unstimulated sample in lane 2 and the IGF-I-stimulated sample in lane 3 were first precipitated with an antiphosphotyrosine antibody, then immunoblotted with an anti-GAP antiserum. Lane 4 shows the antibody alone without cell lysate immunoblotted with the anti-GAP antiserum. Lane 5 shows the cell lysate both precipitated and immunoblotted with the antiphosphotyrosine antibody.



IGF-I Receptors Associate in Whole Cell Lysates with p85, Syp, and GAP, but Not with PLC-

Fig.2shows precipitation experiments performed to demonstrate an interaction between the IGF-I receptor and various signaling molecules in whole cell lysates. In Fig.2, whole cell monolayers of CHO were stimulated with IGF-I for five minutes at 37 °C. Lysates were then precipitated overnight with an anti-IGF-I receptor antibody, and antibody complexes were collected with Protein G-Sepharose. Immunoblotting was performed with either a polyclonal anti-p85 antibody, a monoclonal anti-Syp antibody, an anti-GAP antiserum, or a monoclonal PLC- antibody. The two isoforms of p85 can be seen in lane 1, the 74-kDa Syp protein can be seen in lane 2, and GAP (lane 3) was precipitated by the IGF-I receptor antibody when the whole cell monolayers were pretreated with phenylarsine oxide. The use of this protein tyrosine phosphatase inhibitor was suggested by work on the insulin receptor previously reported by Pronk et al.(31) . Despite prolonged exposure, PLC- was not detected in the IGF-I receptor antibody precipitates (lane 4).


Figure 2: IGF-I receptor association with p85, Syp, and GAP. Whole cell lysates from monolayers of CHO were pretreated with 25 µM PAO for 5 min (lane 3 only), stimulated with IGF-I, 100 ng/ml, for 1 min at 37 °C, then precipitated with an anti-IGF-I receptor antibody. After analysis by 7.5% SDS-PAGE and transfer to nitrocellulose, the membranes were immunoblotted with an anti-p85 antibody (lane 1), an anti-Syp antibody (lane 2), an anti-GAP antiserum (lane 3), or an anti-PLC- antibody.



These data indicate that p85, Syp, and GAP, but not PLC-, are precipitated along with the IGF-I receptor. It is not clear whether these proteins are associating directly with the IGF-I receptor or indirectly with other proteins which interact with the IGF-I receptor such as IRS-1. To further explore the possibility of direct IGF-I receptor-SH2 domain protein interactions, we studied the ability of SH2 domain-GST fusion proteins to interact directly with the IGF-I receptor.

IGF-I Receptors in Whole Cell Lysates Associate with p85 and Syp, but Not with GAP and PLC- Fusion Proteins

Increasing concentrations of GST fusion proteins containing either the amino-terminal SH2 domain of p85, both SH2 domains of Syp, the amino-terminal SH2 domain of GAP, or both SH2 domains of PLC- were incubated with IGF-I-stimulated CHO whole cell lysates and glutathione-Sepharose. In each experiment, negligible amounts of receptor precipitated in the presence of the glutathione-Sepharose alone (lane 1, Fig.3, A-D). The p85-GST was able to precipitate IRS-1, p120, and the IGF-I receptor from whole cell lysates (Fig.3A). Similar results were achieved when the Syp-GST was used in place of p85 (Fig.3B). The identity of the p120 precipitated by the p85- and the Syp-GST is unknown. A p120 was also precipitated by the Syp-GST from NIH 3T3 cells transfected with and overexpressing the human insulin receptor and a dominant-negative mutant Syp rendered catalytically inactive by a cysteine to serine point mutation(25) . IRS-1 was precipitated by both the p85- and the Syp-GST at a lower concentration than was the IGF-I receptor, indicating that these fusion proteins may have a higher affinity for IRS-1 than for the IGF-I receptor.


Figure 3: SH2 domain-GST precipitation of the IGF-I receptor from whole cell lysates. Whole cell lysates from CHO stimulated with IGF-I, 100 ng/ml, for 1 min at 37 °C were incubated with increasing concentrations of p85- (A), Syp- (B), GAP- (C), or PLC-- (D) GST-fusion proteins and glutathione-Sepharose for 90 min. The precipitates were collected, analyzed by 7.5% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an antiphosphotyrosine antibody.



The IGF-I receptor was not precipitated by the GAP-GST or by the PLC--GST (Fig.3, C and D). No phosphorylated proteins were precipitated by the PLC--GST (Fig.3D), but the GAP-GST was able to precipitate a 120-kDa, an 80-kDa, and a 62-kDa phosphoprotein from whole cell lysates. A very low molecular mass protein can be seen in the last two lanes of the Syp- and the PLC--GST immunoblots. This band is the molecular mass of the added fusion proteins. It may represent either the tyrosine phosphorylation of the fusion proteins or nonspecific binding by the secondary horseradish peroxidase antibody used in the detection assay.

Partially Purified IGF-I Receptors Associate with the p85-, Syp-, and GAP-GSTs, but Not with PLC--GST

Similar experiments were performed using IGF-I-stimulated wheat germ agglutinin purified receptors from CHO. These preparations do not contain IRS-1, therefore, allowing us to test whether IRS-1 is a necessary intermediate in the association of the IGF-I receptor with the p85- and Syp-GSTs. Fig.4, A and B, clearly show that the p85- and the Syp-GSTs are both able to precipitate the IGF-I receptor in the absence of IRS-1. These data indicate that p85 and Syp can interact directly with the IGF-I receptor. In contrast to the results obtained from the cell lysate experiments, the GAP-GST precipitated the IGF-I receptor from the purified receptor preparations (Fig.4C). The PLC--GST did not precipitate any phosphoproteins (Fig.4D), although this fusion protein does precipitate activated nerve growth factor and the epidermal growth factor receptor (38) .


Figure 4: SH2 domain-GST precipitation of the IGF-I receptor from purified receptor preparations. IGF-I receptors were purified from CHO, stimulated with IGF-I, 100 ng/ml, and activated in the presence of 50 µM ATP and 10 mM MnCl(2). The samples were incubated with increasing concentrations of p85- (A), Syp- (B), GAP- (C), or PLC-- (D) GST-fusion proteins and glutathione-Sepharose for 90 min at 4 °C. The precipitates were collected, analyzed by 7.5% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an antiphosphotyrosine antibody.



IGF-I Receptors in Whole Cell Lysates Associate with the GAP-GST after Preincubation with PAO

It was of interest that the GAP-GST did not precipitate IGF-I receptors from whole cell lysates, but did precipitate purified IGF-I receptors. We preincubated cell monolayers with PAO, a phosphotyrosine phosphatase inhibitor, prior to and during IGF-I stimulation in an attempt to potentiate IGF-I receptor phosphorylation to enhance potential interactions between the GAP-GST and the IGF-I receptor in cell lysates. Under these conditions, the IGF-I receptor is precipitated by the GAP-GST. There is also a marked increase in the phosphoprotein bands visualized at 120, 80, and 62 kDa (Fig.5A, lanes 3 and 4). The identities of these proteins are not known. No phosphoproteins are precipitated by the PLC--GST despite preincubation with PAO (Fig.5A, lanes 5 and 6).


Figure 5: Precipitation of the IGF-I receptor from whole cell lysates by the GAP-GST after PAO pretreatment. A, whole cell monolayers of CHO were preincubated in the absence or presence of 25 µM PAO for 5 min at 37 °C. The cells were then stimulated in the absence or presence of IGF-I, 100 ng/ml, and warmed at 37 °C for 1 min. Cell lysates were incubated with 1.25 µM GAP- (lanes 3 and 4) or PLC--GST (lanes 5 and 6) and glutathione-Sepharose for 90 min at 4 °C. The precipitates were then analyzed by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with an antiphosphotyrosine antibody. Lanes 1 and 2 represent total cell lysates without and with IGF-I stimulation. B, CHO whole cell monolayers were stimulated with IGF-I at 37 °C for 5 (lanes 1 and 3) and 60 min (lanes 2 and 4). The samples were immunoprecipitated with an antiphosphotyrosine antibody, fractionated by 7.5% SDS-PAGE, and then immunoblotted with an anti-GAP antiserum.



To determine if some of the increase in the phosphorylated protein band at 120 kDa was from tyrosine phosphorylation of GAP, whole cell monolayers of CHO were preincubated with PAO, then immunoprecipitated with the antiphosphotyrosine antibody and immunoblotted with an anti-GAP antiserum. Fig.1D demonstrates that GAP is not tyrosine-phosphorylated in whole cell lysates. However, after PAO preincubation, tyrosine phosphorylation of GAP can be clearly detected (Fig.5B, lanes 1 and 2). In fact, after 60 min of IGF-I stimulation in the presence of PAO, >90% of the total cellular GAP protein is precipitated by the antiphosphotyrosine antibody (Fig.5B, lanes 2 and 4). GAP can, therefore, become tyrosine-phosphorylated in IGF-I-stimulated whole cells pretreated with PAO.

p85-GST Associates with the IGF-I Receptor at pY1316

The data presented above suggest a direct association between p85, Syp, and GAP fusion proteins and the IGF-I receptor. We next conducted a series of experiments to localize the specific SH2 domain binding sites on the receptor. We accomplished this by examining the ability of three tyrosine-phosphorylated peptides to compete with the IGF-I receptor for binding to each GST fusion protein. The phosphopeptides were modeled after three regions of the IGF-I receptor with important autophosphorylation sites. These include the triple tyrosine region of the kinase domain(39) , the NPXY domain(33) , and the carboxyl-terminal region including tyrosine 1316(40) . The triple tyrosine peptide is modeled after amino acids 1129 to 1138 in the IGF-I receptor tyrosine kinase domain and includes the three tyrosines important in receptor autophosphorylation. The NPXY peptide was patterned after amino acids 944 to 953 from the IGF-I receptor's transmembrane region. Because this phosphopeptide did not maintain a stable conformation, the similar RPXpY peptide was used in its place. pY1316 is derived from the carboxyl terminus of the IGF-I receptor and includes amino acids 1308 to 1322.

Fig.6A shows that p85-GST binding to the IGF-I receptor is inhibited by increasing concentrations of the pY1316. No receptor is precipitated by the p85-GST in the presence of 750 µM pY1316 (Fig.6A, lane 6). In contrast, neither the triple tyrosine peptide nor the NPXY peptide was able to inhibit the p85-GSTbulletIGF-I receptor interaction at a concentration of up to 500 µM.


Figure 6: IGF-I receptor binding to the p85-GST is inhibited by phosphopeptide pY1316. A, partially purified receptors were stimulated with IGF-I in the presence of ATP and were then incubated with a 1.0 µM concentration of the p85-GST, increasing concentrations of the phosphopeptide pY1316, and glutathione-Sepharose for 90 min at 4 °C. Precipitates were analyzed by 7.5% SDS-PAGE and immunoblotted with an antiphosphotyrosine antibody. B, this experiment was similar to that described in A, except that phosphopeptides pYpYpY (lanes 2-4) and RPXpY (lanes 5-7) were used instead of pY1316.



Syp-GST Associates with the IGF-I Receptor at pY1316

Similar experiments were performed with the Syp fusion protein. IGF-I receptor precipitation was inhibited in a dose-dependent manner by pY1316 with significant inhibition seen at a concentration of 50 µM (Fig.7A, lanes 1-3). In contrast, up to 500 µM concentrations of the triple tyrosine phosphopeptide (Fig.7B, lanes 2-4) or of the RPXpY peptide (lanes 5-7) did not inhibit IGF-I receptor precipitation by the Syp-GST. Taken together, Fig.6and Fig. 7indicate that both p85 and Syp associate with the phosphorylated IGF-I receptor at tyrosine 1316.


Figure 7: IGF-I receptor binding to the Syp-GST is inhibited by phosphopeptide pY1316. Partially purified receptors were stimulated with IGF-I in the presence of ATP and were then incubated with a 1.0 µM concentration of the Syp-GST, increasing concentrations of the phosphopeptide pY1316 (A) or pYpYpY and RPXpY (B), and glutathione-Sepharose for 90 min at 4 °C. Precipitates were analyzed by 7.5% SDS-PAGE and immunoblotted with an antiphosphotyrosine antibody.



GAP-GST Associates with the IGF-I Receptor at pY950

Fig.8A demonstrates that the GAP-GSTbulletIGF-I receptor interaction can be inhibited by increasing concentrations of the RPXpY phosphopeptide (lanes 8-10). In contrast, no inhibition was seen with the triple tyrosine or the carboxyl-terminal phosphopeptides (lanes 1-7). To confirm this finding, a cell line overexpressing mutant IGF-I receptors with deletion of the NPXY sequence motif (residues 947 to 950) was used(33) . Fig.8B (lanes 4 and 5) demonstrates that this mutant receptor is readily precipitated by the p85-GST at both 0.1 and 1.0 µM. In contrast, no mutant receptor is precipitated at a 0.1 µM concentration of the GAP-GST, and only a small amount of receptor is precipitated at 1.0 µM GAP-GST. As seen earlier in Fig.4, the p85 and GAP-GST proteins are relatively equivalent in their abilities to precipitate the wild-type IGF-I receptor.


Figure 8: IGF-I receptor binding to the GAP-GST is inhibited by phosphopeptide RPXpY, and the GAP-GST does not bind to an NPXY deletion receptor mutant. A, partially purified receptors were stimulated with IGF-I in the presence of ATP and were then incubated with a 1.0 µM concentration of the GAP-GST, increasing concentrations of the phosphopeptides pY1316 (lanes 2-4), pYpYpY (lanes 5-7), and RPXpY (lanes 8-10), and glutathione-Sepharose for 90 min at 4 °C. Precipitates were analyzed by 7.5% SDS-PAGE and immunoblotted with an antiphosphotyrosine antibody. B, partially purified receptors from CHO^Y cells were activated as described above and were incubated with increasing concentrations of the GAP-GST (lanes 1-3) or the p85-GST (lanes 4 and 5) and glutathione-Sepharose for 90 min at 4 °C. Precipitates were fractionated by 7.5% SDS-PAGE and immunoblotted with an antiphosphotyrosine antibody.




DISCUSSION

IRS-1 is known to be important in insulin and IGF-I receptor signal transduction. However, recent work in transgenic IRS-1 knockout mice has suggested the existence of IRS-1-independent, alternate signaling pathways(15, 16) . In this report, we provide evidence to suggest a direct interaction between p85, Syp, and GAP and the IGF-I receptor. We have also localized the probable sites of SH2 domain binding to the IGF-I receptor utilizing phosphopeptides derived from important autophosphorylation sites on the IGF-I receptor.

A monoclonal anti-IGF-I receptor antibody was able to precipitate p85 from IGF-I-stimulated whole cell lysates prepared from a monolayer of CHO. Although it cannot be definitively proven in these experiments, this most likely represents a direct and independent interaction between p85 and the IGF-I receptor, rather than an interaction between p85 and IRS-1 in a binary IGF-I receptorbulletIRS-1 complex. Although the nature of the IGF-I receptorbulletIRS-1 interaction is poorly defined, it is quite evanescent. IRS-1 does not co-precipitate with the IGF-I receptor when an IGF-I receptor antibody is used(41) , nor does the IGF-I receptor co-precipitate with IRS-1 when an IRS-1 antibody is used(8) . The inability to detect IGF-I receptorbulletIRS-1 complexes in this fashion makes it unlikely, but not impossible, that the presence of p85 in the anti-IGF-I receptor precipitate results from an IGF-I receptorbulletIRS-1bulletp85 interaction. We interpret these data to suggest a direct interaction between p85 and the IGF-I receptor.

Backer et al.(42) have previously shown that p85 could associate with either IRS-1 or directly with the insulin receptor, although as much as 70% of the total cellular p85 in their studies was associated with IRS-1 in insulin-stimulated cells suggesting that the p85bulletIRS-1 interaction is the primary route by which PI 3-kinase is activated upon insulin stimulation(8, 9, 41, 42, 43, 44) . However, transgenic mice with a knockout mutation of the IRS-1 gene are not diabetic despite their inability to signal through IRS-1, suggesting alternate pathways to PI 3-kinase activation including a direct receptorbulletp85 interaction(15, 16) .

The interaction of the IGF-I receptor with p85 either directly or via IRS-1 has been suggested previously. PI 3-kinase activity can associate with immobilized IGF-I receptors and can be competed away with increasing concentrations of a p85 fusion protein(19) . The activated IGF-I receptor is also known to phosphorylate recombinant IRS protein in vitro on sites identical with those phosphorylated by the insulin receptor, including those in the YMXM motif known to bind p85(8, 9, 26, 45, 46, 47) .

The preferred binding sequence for the SH2 domains of p85 is pYMXM, and the methionine in the +3 position is critical for p85 SH2 binding(26, 44) . We have previously reported that the p85-GST containing the amino-terminal SH2 domain of p85 can bind to the carboxyl terminus of the insulin receptor at the binding motif pYTHM(18) . In this report, we describe the direct binding of the p85-GST to the IGF-I receptor and localize a binding site to a similar binding motif, pYAHM, in the carboxyl terminus. We cannot exclude the possibility that IGF-I receptor phosphotyrosines other than those we studied here may participate in p85 binding. Although IRS-1 and the IGF-I receptor can both associate with p85 after IGF-I stimulation, the relative contribution of each interaction to biological signaling remains unknown.

Syp is an SH2-containing tyrosine phosphatase which binds preferentially to phosphotyrosines in a pY-hydrophobic-X-hydrophobic motif(26, 27, 48, 49, 50) . We describe here that an anti-IGF-I receptor antibody can precipitate Syp. As described above for p85, we believe this suggests a direct SypbulletIGF-I receptor interaction rather than an IGF-I receptorbulletIRS-1bulletSyp interaction because of the evanescent nature of the IGF-I receptorbulletIRS-1 association. Furthermore, we have also shown in vitro that a Syp-GST may bind directly to the IGF-I receptor at phosphotyrosine 1316 in a pYAHM binding sequence. We have not excluded the possibility that other receptor phosphotyrosyl residues not studied may be involved in Syp binding as well. Recent studies examining the crystal structures of amino-terminal SH2 domain of Syp in separate complexes with two high affinity peptides demonstrate the importance of a hydrophobic residue in the +5 position (50) . The IGF-I receptor has a hydrophobic amino acid, glycine, in the +5 position from tyrosine 1316. The biological relevance of the SypbulletIGF-I receptor versus the SypbulletIRS-1 interaction has not been defined, but microinjection studies have implicated Syp as a positive regulator of insulin- and IGF-I-induced mitogenesis(24, 25) .

GAP is shown here to associate with IGF-I receptors precipitated from whole cell lysates prepared from monolayers of CHO pretreated with phenylarsine oxide. A GAP-GST can associate with purified IGF-I receptors in vitro and with IGF-I receptors from whole cell lysates pretreated with phenylarsine oxide. Using phosphopeptides, we have determined that the SH2 domain of the GAP-GST interacts with the IGF-I receptor at tyrosine 950. The motif consists of a sequence with a hydrophobic amino acid in the +1 and +3 positions and is consistent with GAP binding sites previously described for the platelet-derived growth factor (28, 51) and the insulin receptor(18) .

It is surprising that GAP could be precipitated along with the IGF-I receptor only from cells pretreated with phenylarsine oxide. A possible explanation for this result is that the degree of phosphorylation achieved after pretreatment with this protein tyrosine phosphatase inhibitor is greater than in its absence. Insulin receptors in a cell-free system, for example, phosphorylate to a greater extent than do receptors in whole cells(52, 53, 54) . We believe we were able to increase the overall tyrosine phosphorylation of the IGF-I receptors in whole cells by pretreatment with a phosphatase inhibitor, thereby allowing detection of the IGF-I receptorbulletGAP association. Pronk et al.(31) similarly found an association between the insulin receptor and GAP in whole cell lysates only after pretreatment with PAO.

Consistent with the association of GAP and the IGF-I receptor, we have also found that IGF-I stimulation leads to tyrosine phosphorylation of GAP protein. This IGF-I-stimulated GAP tyrosine phosphorylation was only detectable in the presence of the phosphatase inhibitor. In the untreated cell, this tyrosine phosphorylation may be undetectable because of rapid dephosphorylation. These data further indicate that GAP may interact with the IGF-I receptor in vivo under certain conditions and may be a component of the IGF-I receptor signaling cascade. This interaction may be tightly regulated by PAO-sensitive phosphatases. The IGF-I receptor does not associate with PLC-, even in the presence of PAO or when the PLC--GST is used at high concentrations.

In this report, we show that the IGF-I receptor may directly associate with SH2 domain proteins including p85, Syp, and GAP. We have mapped the IGF-I receptor binding site for p85 and Syp to tyrosine 1316 in the carboxyl terminus and the binding site for GAP to tyrosine 950 in the NPXY domain. The association between these proteins and the IGF-I receptor may provide an alternate mechanism to directly activate these signaling molecules.


FOOTNOTES

*
This work was funded by National Institutes of Health Grants NIH DK 33651 (to J. M. O.) and NIH DK 02162-02 (to B. L. S.), Juvenile Diabetes Foundation Grant JDF 1921047 (to B. L. S.), and grants from the Sankyo Diabetes Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: 9500 Gilman Dr. (0673), La Jolla, CA 92093. Tel.: 619-534-8228; Fax: 619-534-7181.

^1
The abbreviations used are: IGF, insulin-like growth factor; PLC, phospholipase C; GAP, GTPase activating protein; GST, glutathione S-transferase; IRS, insulin receptor substrate; PI, phosphatidylinositol; PAGE, polyacrylamide gel electrophoresis; PAO, phenylarsine oxide.


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