The Epidermal Growth Factor Receptor Associates with and Recruits Phosphatidylinositol 3-Kinase to the Platelet-derived Growth Factor beta  Receptor*

Amyn A. HabibDagger , Thorbergur Högnason, Jane Ren, Kári Stefánsson§, and Rajiv R. Ratan

From the Department of Neurology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115 and § DeCode Genetics, Lynghals 1, Reykjavik, Iceland

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Receptor tyrosine kinases are classified into subfamilies, which are believed to function independently, with heterodimerization occurring only within the same subfamily. In this study, we present evidence suggesting a direct interaction between the epidermal growth factor (EGF) receptor (EGFR) and the platelet-derived growth factor beta  (PDGFbeta ) receptor (PDGFbeta R), members of different receptor tyrosine kinase subfamilies. We find that the addition of EGF to COS-7 cells and to human foreskin Hs27 fibroblasts results in a rapid tyrosine phosphorylation of the PDGFbeta R and results in the recruitment of phosphatidylinositol 3-kinase to the PDGFbeta R. In R1hER cells, which overexpress the EGFR, we find ligand-independent tyrosine phosphorylation of the PDGFbeta R and the constitutive binding of a substantial amount of PI-3 kinase activity to it, mimicking the effect of ligand in untransfected cells. In support of the possibility that this may be a direct interaction, we show that the two receptors can be coimmunoprecipitated from untransfected Hs27 fibroblasts and from COS-7 cells. This association can be reconstituted by introducing the two receptors into 293 EBNA cells. The EGFR/PDGFbeta R association is ligand-independent in all cell lines tested. We also demonstrate that the fraction of PDGFbeta R bound to the EGFR in R1hER cells undergoes an EGF-induced mobility shift on Western blots indicative of phosphorylation. Our findings indicate that direct interactions between receptor tyrosine kinases classified under different subfamilies may be more widespread than previously believed.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Receptor tyrosine kinases have been divided into subfamilies based on common structural features (1). The epidermal growth factor (EGF)1 subfamily includes the ErbB1/EGFR, ErbB2/HER2/Neu, ErbB3/HER3, and ErbB4/HER4 receptors and is characterized by the presence of two extracellular cysteine-rich domains and an uninterrupted kinase domain (2). The PDGF receptor subfamily includes the PDGFalpha R, PDGFbeta R, stem cell factor receptor (Kit), colony-stimulating factor receptor (Fms), and Flk2 receptors and is characterized by the presence of five extracellular immunoglobulin-like domains. Members of this subfamily contain a kinase insert in which a regulatory region has been inserted into the conserved kinase domain.

It is established that growth factors bind to specific receptors. Binding of the growth factor to its receptor may result in homodimerization or heterodimerization between members of a receptor subfamily (3, 4). For example, heterodimerization has been well described between members of the EGFR subfamily (5). The addition of EGF to a number of cell lines results in EGFR-dependent tyrosine phosphorylation of ErbB2 (6-9). EGF can induce dimerization of EGF receptors and ErbB2 in transfected NR6 and NIH3T3 cells (10, 11) and also in SKBR-3 cells, which overexpress the ErbB2 receptor (12). Similarly, heregulin may induce heterodimeric complexes between ErbB2 and ErbB3 or ErbB4 (13, 14). A number of heterodimeric combinations of these receptors have been described, and some combinations are favored over others (15). It is important to note that heterodimerization between the EGFR and ErbB2 proteins can be detected even in the absence of ligand (10, 16).

Heterodimerization may have important functional consequences. Phosphatidylinositol (PI) 3-kinase has been shown to bind directly to activated receptors at domains that are autophosphorylated on tyrosine and contain a Tyr-X-X-Met motif (17). The EGFR lacks the binding motifs for the SH2 domains of the PI 3'-kinase, while the ErbB3 receptor may have little or no kinase activity but has multiple copies of the binding motif for PI 3-kinase (18-20). Stimulation of cells with EGF is known to increase the activity of PI 3-kinase. Although some studies have shown an increase in EGFR-associated PI 3-kinase activity upon ligand stimulation, this is considerably less than the increase associated with other activated receptors (20, 21). This suggested that the mechanism of this increase may be a recruitment by the activated EGFR of other members of the EGFR subfamily, such as ErbB3. This has been demonstrated in A431 cells, where stimulation of cells with EGF increases ErbB3-associated PI 3-kinase activity (20, 22), or by using chimeric receptors in fibroblasts (18). It should be noted that EGF does not bind to ErbB3. In addition, different receptor heterodimers may alter ligand binding kinetics, with resultant variations in signal characteristics (23). Recently, transactivation of the EGFR has also been described in response to stimulation of G-protein-coupled receptors (24). In the PDGFR subfamily, the different isoforms of PDGF induce different dimeric forms of the receptors; e.g. PDGF-AA induces alpha alpha homodimers only while PDGF-BB induces all three combinations of receptors (i.e. alpha alpha , beta beta , and alpha beta (25)). However, for polypeptide growth factors, heterodimerization is generally believed to be limited to members of the same subfamily of receptor tyrosine kinases. Thus, although receptors from different subfamilies may use common substrates, they are believed to function independently, without directly influencing each other and directly interacting only with members of the same subfamily.

The engagement of a receptor tyrosine kinase by its ligand results in dimerization, activation of the kinase activity of the receptor, and autophosphorylation (26). Autophosphorylation of an activated receptor results in the creation of docking sites for SH2 and phosphotyrosine-binding domain-containing proteins that associate with the receptor (27). These include proteins believed to have adaptor functions, such as Shc and Grb2. Another class of SH2 domain-containing proteins that bind to autophosphorylated receptor tyrosine kinases have intrinsic catalytic activity. These include proteins such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase, which associate with the activated receptor. PI 3-kinase is composed of two subunits, an 85-kDa regulatory or adaptor subunit and a 110-kDa catalytic subunit (28). The p85 subunit has two SH2 domains in its carboxyl-terminal half. The enzyme is capable of phosphorylating the D-3 position on phosphatidylinositol, phosphatidylinositol 4-phosphate, or phosphatidylinositol 4,5-biphosphate. PI 3-kinase may be an important mediator of mitogenic signaling in certain cell types. Studies with mutant PDGF receptors lacking association sites for PI 3-kinase show a decrease in DNA synthesis upon PDGF stimulation (29, 30). Restoring the PI 3-kinase binding site to a mutant PDGFR deficient in mitogenic signaling restores the ability of the receptor to initiate DNA synthesis (31). Other functions of PI 3-kinase (reviewed in Ref. 32) include cellular trafficking and cytoskeletal alterations induced by growth factor stimulation and a role in cell survival.

PDGF induces a heterologous down-regulation of EGF receptors (33). Stimulation by PDGF or phorbol esters results in a decrease in the affinity of the EGF receptor for its ligand without influencing the number of receptors and results in a decrease in the kinase activity of the EGF receptor (34-36). Stimulation of fibroblasts with PDGF or with phorbol esters leads to phosphorylation of the EGF receptor at threonine 654, which is a site of protein kinase C phosphorylation. This led to the suggestion that protein kinase C activation mediated by the PDGFR leads to phosphorylation of the EGFR at threonine 654, and this phosphorylation is responsible for transmodulation. However, subsequent studies have suggested that neither activation of protein kinase C nor phosphorylation at threonine 654 are required for the transmodulation induced by PDGF (37, 38). The mechanism of PDGFR-mediated transmodulation remains unknown. An influence of the EGFR on PDGFR receptor signaling, to our knowledge, has not previously been described. In this study, we present evidence suggesting that the EGFR associates with and directly influences signaling through the PDGFbeta receptor.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Lines, Growth Factors, and Transfection-- Hs27, COS-7, Rat-1 R1hER, B82L, and 293 EBNA cells (Invitrogen) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. R1hER cells are Rat-1 fibroblasts transfected with a human EGFR construct. Human recombinant PDGF-BB and human recombinant EGF were used at a concentration of 50 and 100 ng/ml, respectively, for 5 min unless specified otherwise. A human PDGFbeta receptor construct was cloned into pcDNA 3.1 (+) vector using standard molecular cloning techniques. A human EGFR construct was cloned into pcDNA 3.1 (-) vector. The empty vectors were purchased from Invitrogen. Transient transfections were performed using the calcium phosphate method (39). Expression of transfected genes was confirmed by Western blotting. For transient transfection experiments, cells were harvested 24 h after transfection.

Western Blotting, Immunoprecipitation, and Antibodies-- Standard protocols were used for immunoprecipitation and Western blotting in different experiments (39). Quantitation of proteins was performed by using a Bio-Rad detergent-compatible protein assay kit and confirmed by Coomassie staining of aliquots subjected to SDS-polyacrylamide gel electrophoresis. For immunoprecipitation, cells were serum-starved and exposed to growth factor for the times indicated. Cells were subsequently lysed in a modified radioimmune precipitation buffer (150 mM NaCl, 1% Nonidet P-40, 0.25% deoxycholate, 1 mM EGTA, 1 mM NaF, 50 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate), and equal amounts of protein were incubated with the primary antibody for 90 min. Protein A-Sepharose or Protein G-agarose beads were subsequently added to the lysates and incubated overnight at 4 °C. The beads were subsequently washed and solubilized in SDS sample buffer and then boiled and analyzed by SDS-polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose. Western blots were developed with ECL reagents (Amersham Corp.). In experiments where cell lysates were examined directly, cells were lysed in SDS sample buffer, boiled, and subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting. The phosphotyrosine antibody used in all of the experiments described in this study was PY-20, obtained from Transduction Laboratories (Lexington, KY). Other antibodies used for the different experiments were as follows.

For detecting tyrosine phosphorylation of the PDGFbeta R in response to EGF in Hs27 and COS-7 cells, rabbit polyclonal PDGFbeta R antibodies raised against the carboxyl tail of the PDGFbeta R (Pharmingen, San Diego, CA, catalog number 15746E; Ref. 31) or rabbit polyclonal antibodies raised against a synthetic peptide corresponding to amino acids 1013-1025 of the human PDGFbeta R (Upstate Biotechnology, Inc., Lake Placid, NY, catalog number 06-498; Ref. 40) were used for immunoprecipitation. A peptide control for this antibody was also used (Upstate Biotechnology, 12-162). The same antibodies were used to demonstrate tyrosine phosphorylation of the PDGFbeta R in R1hER cells and for immunoprecipitating the PDGFbeta R in other experiments. These two antibodies were also used for Western blotting of the PDGFbeta R. The EGFR was immunoprecipitated using either a mouse monoclonal antibody against the human EGFR, which does not recognize ErbB2 (Pharmingen, 14891A; Ref. 41), or sheep polyclonal EGFR antibodies (Upstate Biotechnology, 06-128). For Western blotting of the EGFR sheep polyclonal antibodies (Upstate Biotechnology; 06-128) or rabbit polyclonal antibodies raised to residues 1005-1016 of the human EGFR (sc-03; Santa Cruz Biotechnology, Santa Cruz, CA) were used. For studies of coimmunoprecipitation, PDGFbeta R immunoprecipitates were run on a 6% polyacrylamide gel and immunoblotted with rabbit polyclonal EGFR antibodies (sc-03; Santa Cruz Biotechnology) or sheep polyclonal EGFR antibodies (UBI, 06-128), with identical results. The reverse experiment was done by immunoprecipitating the EGFR and immunoblotting with either the tail or the UBI PDGFbeta R antibodies. p85 antibodies directed against the N-SH2 domain (Upstate Biotechnology, 06-496) were used for both immunoprecipitation and immunoblotting. For experiments examining the tyrosine phosphorylation of phospholipase C-gamma 1, p120 GAP, and SHP-2, cell lysates were immunoprecipitated with the respective antibodies and immunoblotted with phosphotyrosine. Antibodies to these three proteins were obtained from Santa Cruz Biotechnology (sc-426, sc-425, and sc-280).

PI 3-Kinase Assays-- PI 3-kinase assays were done as follows (42). Cells were serum-starved for 48 h prior to treatment with PDGF-BB or EGF as indicated. The cells were incubated with growth factors at 37 °C for 5 min and then washed with ice-cold buffer A (137 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM CaCl2, 1 mM MgCl2) and 100 µM NaVO4 and lysed with in 1 ml of buffer A containing 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 100 µM NaVO4. 2 µl of anti-PDGF-R kinase insert antibody (Pharmingen, 15756E; Ref. 43) was added and incubated for 90 min on ice, after which protein A-Sepharose beads were added. Following a 1-h incubation at 4 °C, the beads were washed three times in buffer A containing 1% Nonidet P-40; twice in 100 mM Tris-HCl, pH 7.4, 500 mM LiCl; and twice in TNE (100 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA), all containing 100 µM NaVO4. The immunoprecipitates were resuspended in 50 µl of TNE, 10 µl of 100 mM MgCl2, and 10 µl of sonicated 1 mg/ml PI (Avanti Polar Lipids), dried under Argon, and resuspended in 10 mM HEPES, 1 mM EGTA (pH 7.0). The reaction volume was incubated with a 10-µl volume of ATP (10 µCi of gamma -32P, 100 mM MgCl2, 5 mM HEPES, and 0.25 µM unlabeled ATP) at 37 °C for 10 min and was stopped by adding 20 µl of 6 N HCl and 160 µl of chloroform/methanol (1:1, v/v). The aqueous and lipophilic phases were separated by centrifugation for 10 min at 14,000 rpm, and 40 µl of the lower phase was spotted onto a silica gel 60 plate (Merck), previously immersed in 1% potassium oxalate and heat-activated. After developing the plate in chloroform/methanol/H2O/NH4OH (60:47:11:1.8, v/v), the radioactive phosphate spots were detected by autoradiography, identified by comparison with phospholipid standards, and quantitated by liquid scintillation counting.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The EGF Receptor Induces Tyrosine Phosphorylation of the PDGF Receptor-- The Hs27 fibroblast cell line and COS-7 cells express receptors for both PDGF and EGF. In this study, we have examined signaling through the PDGFbeta R. Stimulation of quiescent cells with PDGF results in a rapid tyrosine phosphorylation of the PDGF receptor (Fig. 2B). We find that stimulation of cells with EGF also results in an increase in the phosphotyrosine content of the PDGFR (Figs. 1 and 2). This increase can be seen within 5 min in COS-7 cells and can be detected by immunoprecipitating cell lysates with phosphotyrosine antibodies followed by immunoblotting with a PDGFbeta R antibody (Pharmingen, 15746E), as shown in Fig. 1B. Also, if PDGFbeta R antibodies are used for immunoprecipitation of denatured cell lysates and this is followed by immunoblotting with phosphotyrosine antibodies, an EGF-induced increase in tyrosine phosphorylation of the PDGFR can be detected (Fig. 1A). Note that this antibody fails to immunoprecipitate the denatured EGFR from EGF-stimulated A431 cells (Fig. 1A). Also, stripping this blot and reprobing it with EGFR antibodies showed no staining (not shown). EGF-induced tyrosine phosphorylation of the PDGFbeta R is also noted when samples are not denatured prior to immunoprecipitation. The same results were obtained with Hs27 cells (Fig. 2, A and B). We estimate that about 5% of the PDGFbeta receptors undergo tyrosine phosphorylation in these cells in response to EGF (Fig. 2B). We are unable to detect any increase in tyrosine phosphorylation of the PDGFR in response to EGF in B82L cells, which lack the EGFR. We do not detect tyrosine phosphorylation of the EGFR in response to PDGF (not shown).


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Fig. 1.   A, tyrosine phosphorylation of the PDGFbeta R in response to EGF stimulation in COS-7 cells. Cells were serum-starved for 24 h and exposed to EGF (100 ng/ml) for 5 min. Lysates were denatured by boiling in 1% SDS for 5 min followed by immunoprecipitation with a PDGFbeta R tail antibody (Pharmingen, 15746E) and immunoblotting with a phosphotyrosine antibody (PY 20). Lanes 1 and 3 show unstimulated cells, while lanes 2 and 4 show cells stimulated with EGF. In lane 5, EGF-stimulated cells were denatured and immunoprecipitated with an isotype-matched control antibody. Lane 6 shows an EGF-stimulated denatured A431 cell lysate immunoprecipitated with the same PDGFbeta R antibodies. B, tyrosine phosphorylation of the PDGFbeta receptor in COS-7 cells. Cells were serum-starved and exposed to EGF (+) and denatured as in A, followed by immunoprecipitation with phosphotyrosine antibodies and Western blotting with the PDGFbeta R tail antibody described in the legend to panel A. Lanes 1 and 3 show unstimulated cells, while lanes 2 and 4 show EGF-stimulated cells. In lane 5, cells were stimulated with EGF and immunoprecipitated with an isotype-matched control antibody.


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Fig. 2.   A, tyrosine phosphorylation of the PDGFbeta R in response to EGF in Hs27 cells. Cell lysates were denatured as in Fig. 1A, followed by immunoprecipitation with phosphotyrosine antibodies and immunoblotting with PDGFbeta R tail antibodies. Lanes 1 and 3 show unstimulated cells, while lanes 2 and 4 show cells stimulated with EGF. B, tyrosine phosphorylation of the PDGFbeta R in response to EGF in Hs27 cells. Cell lysates were denatured as in Fig. 1A, followed by immunoprecipitation with phosphotyrosine antibodies and blotting with PDGFbeta R tail antibodies. Lane 1 shows unstimulated cells. In lane 2, cells were stimulated with PDGF, and 5% of the immunoprecipitate was run on the gel. In lane 3, cells were stimulated with EGF. In lane 4, cells were stimulated with EGF and immunoprecipitated with an isotype-matched control antibody. C, constitutive tyrosine phosphorylation of the PDGFbeta R in R1hER cells. Cells were serum-starved for 24 h, followed by denaturation and immunoprecipitation with PDGFbeta R tail antibodies and immunoblotting with phosphotyrosine antibodies. Lane 1, untransfected Rat-1 fibroblasts; lane 2, R1hER fibroblasts; lane 3, R1hER cells were immunoprecipitated with an isotype-matched control antibody.

We next studied the effect of EGFR overexpression on tyrosine phosphorylation of the PDGFbeta R. Rat-1 fibroblasts transfected with the human EGFR (R1hER) were used. These cells were obtained from Dr. Michael Weber (University of Virginia, Charlottesville, VA) and have been described elsewhere (44). These cells express about 7.5 × 105 EGF receptors/cell, which is about a 7-fold increase over the parental Rat-1 fibroblast cell line (11). In R1hER cells, tyrosine phosphorylation of the PDGFbeta R in response to PDGF is intact, suggesting that binding of the ligand to its receptor and homodimerization are unimpaired. In these cells, there is a constitutive tyrosine phosphorylation of the EGFR. Significantly, we also detect constitutive tyrosine phosphorylation of the PDGFbeta receptor (Fig. 2C), mimicking the effect of EGF stimulation in untransfected cells. Since EGF does not bind to the PDGFR, and since the ligand-dependent tyrosine phosphorylation of the PDGF receptor seen in untransfected cells is seen constitutively in cells overexpressing the EGFR, we conclude that EGF-induced tyrosine phosphorylation of the PDGFbeta R is mediated by the activated EGFR. This conclusion is supported by the observation that this EGF effect requires the presence of the EGFR. Since a physical association can be demonstrated between the two receptors (see below), we suggest that the PDGFbeta R may be a substrate of the EGFR and/or that these effects may result from dimerization or oligomerization between these two receptors.

The EGFR Recruits PI 3-Kinase to the PDGFR-- Activation of a number of receptor tyrosine kinases results in the association of PI 3-kinase with the receptor and an increase in receptor-associated PI 3-kinase activity. Although stimulation of cells with EGF leads to an increase in PI 3-kinase activity in phosphotyrosine immunoprecipitates, there is little increase in EGFR-associated activity. The mechanism of this increase may be a recruitment by the activated EGFR of other members of the EGFR subfamily, such as ErbB3. To study whether the PDGFbeta R could play a similar role to ErbB3, we examined whether exposure of cells to EGF would lead to an increase in the PI 3-kinase activity associated with the PDGFbeta R. We find that in Hs27 cells EGF induces a 2-fold increase in the PI 3-kinase activity associated with the PDGFbeta R, which is similar to the increase seen in phosphotyrosine immunoprecipitates (Fig. 3A). Also, when cells are preincubated with a low concentration of PDGF (5 ng/ml) for 30 min and this is followed by stimulation with EGF, we can detect a significant further increase in the PI 3-kinase activity associated with the PDGFbeta R (Fig. 3A), over PDGF alone. Treatment with 5 ng/ml PDGF alone for 30 min leads to about a 20-fold increase, while further stimulation with EGF increases the PI 3-kinase activity to about 30-fold compared with unstimulated cells. Similar results were seen with COS-7 cells (Fig. 3B).


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Fig. 3.   A, PI 3-kinase activity in Hs27 cells. A PDGFbeta R kinase insert antibody (Pharmingen, 15756E) was used for immunoprecipitation (lanes 1-5). Lane 1 shows unstimulated cells. Lanes 2 and 3 show cells stimulated with PDGF-BB (50 ng/ml) and EGF (100 ng/ml), respectively, for 5 min. Lane 4 shows cells stimulated with PDGF-BB (5 ng/ml) for 30 min, while lane 5 shows cells treated with EGF for 5 min after a 30-min incubation with PDGF-BB (5 ng/ml). Lanes 6 and 7 show PI 3-kinase activity associated with phosphotyrosine immunoprecipitates, with or without EGF stimulation. The magnitude of EGF-induced increase in phosphotyrosine immunoprecipitates is similar to that seen in PDGFbeta R immunoprecipitates from cells stimulated with EGF. The experiment shown is representative of three independent experiments. B, PI 3-kinase activity associated with the PDGFbeta R in COS-7 cells exposed to EGF for 5 min. The experiment shown is representative of three independent experiments.

In unstimulated R1hER cells, there is about an 8-fold increase in the PI 3-kinase activity in phosphotyrosine immunoprecipitates compared with parental Rat-1 cells. As can be seen in Fig. 4, the PI 3-kinase activity associated with the PDGFbeta R is also increased in unstimulated R1hER cells, and the magnitude of this increase is almost equal to that detected in phosphotyrosine immunoprecipitates. It is difficult to detect any PI 3-kinase activity associated with EGFR in either Rat-1 or R1hER cells. Adding EGF to R1hER cells results in a further increase in PDGFR-associated PI 3-kinase activity (not shown).


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Fig. 4.   PI 3-kinase activity in Rat-1 and R1hER cells. Lanes 1 and 2 show PI 3-kinase activity associated with phosphotyrosine immunoprecipitates in unstimulated cells. An increase in the PI 3-kinase activity is seen in R1hER cells. Lanes 3 and 4 show PI 3-kinase activity associated with the PDGFbeta R in unstimulated cells. There is an increase in PI 3-kinase activity in R1hER cells, which is similar to the increase seen in phosphotyrosine immunoprecipitates. No PI 3-kinase activity is detected in association with the EGFR in either R1 (lane 5) or R1hER cells (lane 6). The experiment shown is representative of three independent experiments.

We can also detect tyrosine-phosphorylated PDGFbeta R in p85 immunoprecipitates from Hs27 or COS-7 cells stimulated with EGF. This was observed by immunoprecipitating p85 from cells stimulated with EGF followed by immunoblotting with a PDGFbeta R antibody. A band was seen at 180 kDa, which comigrated with a more intense band seen in cells stimulated with PDGF (not shown). Stripping the blot and reprobing with phosphotyrosine antibodies confirmed that the PDGFbeta R was tyrosine-phosphorylated. A substantial amount of PDGFbeta R coimmunoprecipitates with p85 in R1hER cells in the absence of ligand (not shown). We next investigated if a similar constitutive recruitment to the PDGFR could be detected for other SH2 domain-containing proteins such as p120GAP, phospholipase C-gamma 1 or SHP-2. No such association was detected for these three proteins in R1hER cells even upon stimulation with EGF.

The EGFR Coimmunoprecipitates with the PDGFR-- Although the EGFR has been shown to heterodimerize with other members of the EGFR subfamily, heterodimerization with the PDGFR has not been described. To determine whether the tyrosine phosphorylation of the PDGFbeta R in response to EGF was mediated directly by the EGFR, we looked for a physical association between the two receptors. Surprisingly, we found that the two receptors can be coimmunoprecipitated from Hs27 and COS-7 cells in the absence of ligand. This association was detected by immunoprecipitating the EGFR and staining Western blots with the PDGFbeta R (Fig. 5A). To confirm this association immunoprecipitation was then performed with PDGFbeta R antibodies followed by blotting with EGFR antibodies with the same result (Fig. 5B). About 5% of the PDGF receptors exist in a complex with the EGFR in these cells (Fig. 5C). These results were consistently obtained using a number of antibodies recognizing different epitopes on the receptors (described under "Experimental Procedures"), isotype-matched negative controls, and peptide inhibition of immunoprecipitation.


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Fig. 5.   A, association of the PDGFbeta R with the EGFR in COS-7 cells. Cells were serum-starved for 24 h, following which cells were stimulated with PDGF-BB (lane 2) or EGF (lanes 3 and 4) followed by immunoprecipitation with an EGFR antibody (Pharmingen, 14891A) in lanes 1-3. An isotype-matched control antibody was used for immunoprecipitation in lane 4. The Western blot was probed with PDGFbeta R tail antibodies. The two bands represent the mature processed form of the PDGFbeta R and the precursor form. Identical results were obtained in Hs27 cells. No increase in the association is seen with growth factor addition. B, same as A except that PDGFbeta R antibodies (Upstate Biotechnology, 06-498), for which a peptide is available, were used for immunoprecipitation in lanes 1-3. In lane 4, PDGFbeta R antibodies were preincubated with specific peptide (Upstate Biotechnology) prior to immunoprecipitation. In lane 5, isotype-matched control antibodies were used for immunoprecipitation. The Western blot was probed with EGFR antibodies (Santa Cruz Biotechnology, sc-03). The same result was obtained when the PDGFbeta R tail antibodies were used for immunoprecipitation and in Hs27 cells. C, quantitation of the association between the two receptors in COS-7 cells. Cells lysates were denatured as described previously. In lane 1, cell lysates were immunoprecipitated with EGFR antibodies described in A, and 10% of the immunoprecipitate was run on the gel. In lane 2, lysates were immunoprecipitated with the PDGFR tail antibody, while in lane 3 an isotype-matched control antibody was used. The Western blot was probed with EGFR antibodies as in B. D, coimmunoprecipitation of the two receptors in R1hER cells. Cells were serum-starved for 24 h followed by immunoprecipitation using EGFR antibodies as in Fig. 5A, followed by immunoblotting with PDGFbeta R tail antibodies. Upon the addition of EGF, the coimmunoprecipitating PDGFR undergoes a mobility shift seen in lane 3. In lane 4, immunoprecipitation was performed using an isotype-matched control antibody.

To rule out cross-reactivity between the antibodies, the following experiments were undertaken. The antibodies used to immunoprecipitate the EGFR (Pharmingen, 14891A; Upstate Biotechnology, 06-128) were tested for their ability to immunoprecipitate the PDGFbeta R in B82L cells (which lack the EGFR but express PDGFR) in PDGFbeta R blots. No staining was observed. Next, antibodies used to immunoprecipitate the PDGFbeta R (Upstate Biotechnology, 06-498; Pharmingen, 15746E) were tested for their ability to immunoprecipitate the EGFR from A431 cells (which do not express the PDGFbeta R) in EGFR blots with negative results. The antibodies used for immunoprecipitation were also used for Western blotting of the PDGFR and were tested for their ability to recognize the EGFR immunoprecipitated from A431 cells in Western blots with negative results. The antibodies used for Western blotting of the EGFR (Pharmingen, 14891A; Santa Cruz Biotechnology, sc-03) were tested for their ability to recognize the PDGFR immunoprecipitated from B82L cells in Western blots, also with negative results. There is no increase in the amount of coimmunoprecipitating receptor with the addition of either PDGF or EGF.

In R1hER cells, we again detect the ligand-independent coimmunoprecipitation described above. In addition, upon the addition of EGF, we can detect a mobility shift of the fraction of the PDGFR that is bound to the EGFR (Fig. 5D). This suggests that the fraction of PDGFbeta R that is bound to the EGFR in these cells undergoes tyrosine phosphorylation in response to EGF. We do not detect EGF-induced increases in tyrosine phosphorylation of the total complement of PDGFbeta receptors in R1hER cells, although as noted earlier there is a constitutive tyrosine phosphorylation of these receptors in R1hER cells.

The PDGFbeta R and EGFR Associate in Transiently Transfected 293 Cells-- We further explored the association between the two receptors in 293EBNA cells, which normally express little or no endogenous EGFR (45). In addition, we were unable to detect the PDGFbeta R in these cells by immunoblotting. Both receptors were introduced into these cells by calcium phosphate transfection. A human PDGFbeta R cDNA construct was cloned into the PCDNA 3.1 vector. Expression in 293 cells was confirmed by immunoblotting with PDGFbeta R antibodies. A human EGFR construct was also cloned into a PCDNA 3.1 vector (Invitrogen). Expression in 293 cells was confirmed by immunoblotting with EGFR antibodies. We coexpressed the receptors using the empty vector as a control, and 24 h after transfection we immunoprecipitated cell lysates with PDGFbeta R antibodies, followed by Western blotting and staining with EGFR antibodies. The EGFR can be detected in cells where both receptors were introduced but not in cells where the the PDGFbeta R was transfected with a control vector (Fig. 6A). The reverse experiment was done by immunoprecipitating the EGFR and staining Western blots with PDGFbeta R antibodies. This again confirmed the association between the two receptors (Fig. 6B). As is the case in untransfected cells, the addition of either PDGF or EGF did not result in any increase in the amount of coimmunoprecipitating receptor.


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Fig. 6.   A, association of the EGFR with the PDGFbeta R in 293 EBNA cells. Cells were transiently transfected with a human PDGFbeta R construct (lanes 1-5). A human EGFR cDNA construct was cotransfected into cells in lanes 1, 2, 3, and 5. An empty vector was cotransfected into cells shown in lane 4. Transfections were done with calcium phosphate, and 10 µg of DNA was used for each construct in 100-mm dishes. Lanes 1-4 were immunoprecipitated using PDGFbeta R antibodies described in Fig. 5. Lane 5 was immunoprecipitated using an isotype-matched control antibody. Immunoblotting was done with an EGFR antibody (sc-03). B, 293 EBNA cells were transiently transfected with a human EGFR construct (lanes 1-3). A PDGFbeta R construct was cotransfected in lanes 1 and 3. In lane 2, an empty vector was used. Lanes 1 and 2 were immunoprecipitated using an EGFR antibody, while lane 3 was immunoprecipitated with an isotype-matched control antibody. Immunoblotting was done with PDGFbeta R tail antibodies.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In this study, we describe interactions between the EGF and the PDGFbeta receptors, which are members of different receptor tyrosine kinase subfamilies. The interactions observed between the two receptors include a physical association between the two receptors and tyrosine phosphorylation of the PDGF receptor by the activated EGFR and EGF-induced recruitment of PI 3-kinase to the PDGFR. These studies were done in Hs27 human foreskin fibroblasts and in R1hER fibroblasts cells, which overexpress the human EGFR. These effects are also seen in the COS-7 and in 293 cells, demonstrating that these interactions extend to other cell types.

Stimulation of Hs27 and COS-7 cells with EGF results in tyrosine phosphorylation of PDGFbeta receptors. EGF has previously been demonstrated to induce tyrosine phosphorylation of other members of the EGFR subfamily. As discussed earlier, the addition of EGF to certain cell lines results in tyrosine phosphorylation of the ErbB2 and ErbB3 receptors, although EGF does not bind to ErbB2 or ErbB3. Similarly, EGF does not bind to the PDGFR, and the tyrosine phosphorylation of the PDGFbeta R in response to EGF seen in Hs27 and COS-7 cells is likely to result from activation of the EGFR; also, these effects are not seen in B82L cells, where the EGFR is not expressed. Furthermore, overexpression of the EGFR in Rat-1 fibroblasts causes a substantial ligand-independent tyrosine phosphorylation of the PDGFbeta R. Tyrosine phosphorylation of the EGFR in response to PDGF, however, was not observed in any of the cell lines we tested.

The EGFR-mediated tyrosine phosphorylation of the PDGF receptor could have a number of functional consequences. It could influence the kinase activity of the PDGF receptor, leading to tyrosine phosphorylation of downstream substrates and/or result in recruitment of SH2 domain-containing proteins to the receptor. At least one of these outcomes is seen in untransfected cells, namely the association of PI 3-kinase with the PDGFbeta R following EGF stimulation. This suggests the following order of events. Activation of the EGFR leads to tyrosine phosphorylation and activation of the PDGFbeta R. This leads to the association of PI 3-kinase with the PDGFbeta R. Although the EGF-induced increase in PI 3-kinase activity associated with the PDGFbeta R is small (a 2-fold increase), even small increases in PI 3-kinase activity may be functionally significant. For example, PDGF-induced cytoskeletal changes such as membrane ruffling, which is dependent on PI 3-kinase activity, have been observed with concentrations of PDGF as low as 3 ng/ml (46). It should be noted that the activation of PI 3-kinase by PDGF is dose-dependent over a certain range. If cells are preincubated with low doses of PDGF, EGF induces a substantial further increase in the PI 3-kinase activity associated with the PDGFbeta R. We looked for a similar association between the PDGFbeta R and other SH2 domain-containing proteins such as phospholipase C-gamma 1, p120GAP, and SHP-2 in cells exposed to EGF. No association was found.

Overexpression of the EGFR in Rat-1 cells results in a constitutive tyrosine phosphorylation of the PDGFbeta R, mimicking the effect of EGF stimulation in untransfected cells. In addition, there is a substantial constitutive association of the PDGFbeta R with PI 3-kinase in R1hER cells, again consistent with results seen in untransfected cells upon the addition of EGF. The PI 3-kinase activity in phosphotyrosine immunoprecipitates is significantly increased in R1hER cells, in the absence of ligand. Almost all of this activity may be associated with the PDGFbeta R in these cells. It should also be noted that PI 3-kinase is the only SH2 domain-containing protein we can detect that binds constitutively to the PDGFbeta R in R1hER cells. These observations lead us to infer the following. First, that EGF-mediated increases in PI 3-kinase activity may involve the recruitment of the PDGFbeta R in certain cells. As noted before, the addition of EGF to A431 cells leads to an increase in PI 3-kinase activity associated with the ErbB3 receptor, another member of the EGFR subfamily. The PDGFbeta R may serve a similar function. Secondly, overexpression of the EGFR in R1hER cells mimics the effect of EGF stimulation on the PDGFbeta R in untransfected cells in a ligand-independent fashion. This suggests that although R1hER cells express high levels of the EGFR, observations made in these cells may provide clues to the interactions between the two receptors under physiologic conditions. R1hER cells may also serve as a model for interactions between the receptors in tumors that overexpress the EGFR and also express the PDGFbeta R.

What is the mechanistic basis for this influence of the EGFR on PDGFR signaling? We have shown that ligand-dependent activation of the EGFR results in tyrosine phosphorylation of the PDGFbeta R in untransfected cells, while overexpressing the EGFR leads to such an effect in the absence of ligand. This effect could be mediated directly by the EGFR or by intermediate kinases. Two observations suggest that this may be a direct interaction. First, the EGFR binds to the PDGFbeta R as detected by coimmunoprecipitation experiments. Although this association is ligand-independent, there is precedent for this. It has previously been shown that heterodimerization may occur between EGFR and ErbB2 proteins even in the absence of ligand (10, 16). Secondly, in R1hER cells the fraction of PDGFbeta receptors associated with the EGFR undergoes an EGF-induced mobility shift suggestive of phosphorylation. This again supports a direct interaction between the two receptors.

From the studies presented here, we conclude that direct interactions between receptor tyrosine kinases classified under different subfamilies may be more widespread than previously believed. This may include heterodimerization or oligomerization and/or transphosphorylation with resultant recruitment of SH2 domain-containing proteins to the activated receptor. Such a scheme would alter the signaling repertoire of the receptor depending on other receptors expressed in the same cell and has obvious implications for specificity in cellular signaling. The ability of different receptor tyrosine kinases to directly influence each other is also relevant to a better understanding of coordination of signals generated by multiple cytokines acting on the same cell.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Michael Weber and Gordon Gill for generous gifts of R1hER and B82L cells. We thank Dr. Axel Ullrich for an EGFR cDNA construct. We thank Dr. Stephen Soltoff for critically reading this manuscript. We thank Drs. Stephen Soltoff, Alex Toker, Geraint Thomas, and Anthony Couvillon for help with PI 3-kinase assays.

    FOOTNOTES

* This work was supported in part with National Institutes of Health Grant ROINS32977.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: The Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Room 807, Boston, MA 02115. Tel.: 617-667-0837; Fax: 617-667-0811; E-mail: ahabib{at}bidmc.harvard.edu.

1 The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PDGFbeta R, PDGFbeta receptor; PI, phosphatidylinositol; SH2, Src homology 2; IP, immunoprecipitation.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Ullrich, A., and Schlessinger, J. (1990) Cell 61, 203-212[Medline] [Order article via Infotrieve]
  2. van der Geer, P., and Hunter, T. (1994) Annu. Rev. Cell Biol. 10, 251-337[CrossRef]
  3. Heldin, C-H. (1995) Cell 80, 213-223[Medline] [Order article via Infotrieve]
  4. Weiss, F. U., Daub, H., and Ullrich, A. (1997) Curr. Opin. Genet. Dev. 7, 80-86[CrossRef][Medline] [Order article via Infotrieve]
  5. Earp, H. S., Dawson, T. L., Li, X., and Yu, H. (1995) Breast Cancer Res. Treat. 35, 115-132[Medline] [Order article via Infotrieve]
  6. Akiyama, T., Saito, T., Ogawara, H., Toyoshima, K., and Yamamoto, T. (1988) Mol. Cell. Biol. 8, 1019-1026[Medline] [Order article via Infotrieve]
  7. King, C. R., Borrello, I., Bellot, F., Comoglio, P., and Schlessinger, J. (1988) EMBO J. 7, 1647-1651[Abstract]
  8. Kokai, Y., Dobashi, K., Weiner, D. B., Myers, J. N., Nowell, P. C., Greene, M. I. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5389-5393[Abstract]
  9. Stern, D. F., and Kamps, M. P. (1988) EMBO J. 7, 995-1001[Abstract]
  10. Qian, X. C., LeVea, M., Freeman, J. K., Dougall, W. C., Greene, M. I. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1500-1504[Abstract]
  11. Wada, T., Qian, X., and Greene, M. I. (1990) Cell 61, 1339-1347[Medline] [Order article via Infotrieve]
  12. Goldman, R. B., Levy, N., Peles, E., and Yarden, Y. (1990) Biochemistry 29, 11024-11028[Medline] [Order article via Infotrieve]
  13. Plowman, G. D., Green, J. M., Culouscou, J. M., Carlton, G. W., Rothwell, V. M., Buckley, S. (1993) Nature 366, 473-475[CrossRef][Medline] [Order article via Infotrieve]
  14. Sliwkowski, M. X., Schaefer, G., Akita, R. W., Lofgren, J. A., Fitzpatrick, V. D., Nuijens, A., Fendly, B. M., Cerione, R. A., Vandlen, R. L., Carraway, K. L., III (1994) J. Biol. Chem. 269, 14661-14665[Abstract/Free Full Text]
  15. Tzahar, E., Waterman, H., Chen, X., Levkovitz, G., Karunagaran, D., Lavi, S., Ratzkin, B. J., Yarden, Y. (1996) Mol. Cell. Biol. 16, 5276-5287[Abstract]
  16. Qian, X., Dougall, W. C., Hellman, M. E., Greene, M. I. (1994) Oncogene 9, 1507-1514[Medline] [Order article via Infotrieve]
  17. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302[Medline] [Order article via Infotrieve]
  18. Fedi, P., Pierce, J. H., Di Fiore, P. P., Kraus, M. H. (1994) Mol. Cell. Biol. 14, 492-500[Abstract]
  19. Prigent, S. A., and Gullick, W. G. (1994) EMBO J. 13, 2831-2841[Abstract]
  20. Soltoff, S. P., Carraway, K. L., III, Prigent, S. A., Gullick, W. G., Cantley, L. C. (1994) Mol. Cell. Biol. 14, 3550-3558[Abstract]
  21. Hu, P., Margolis, B., Skolnik, E. Y., Lammers, R., Ullrich, A., Schlessinger, J. (1992) Mol. Cell. Biol. 12, 981-990[Abstract]
  22. Carraway, K. C., and Cantley, L. C. (1994) Cell 78, 5-8[Medline] [Order article via Infotrieve]
  23. Karunagaran, D., Tzahar, E., Beerli, R. B., Chen, X., Graus-Porta, D., Ratzkin, B. J., Seger, R., Hynes, N. E., Yarden, Y. (1996) EMBO J. 15, 254-264[Abstract]
  24. Daub, H., Weiss, F. U., Wallasch, C., and Ullrich, A. (1996) Nature 379, 557-560[CrossRef][Medline] [Order article via Infotrieve]
  25. Claesson-Welsh, L. (1994) J. Biol. Chem. 269, 32023-32026[Free Full Text]
  26. Schlessinger, J., and Ullrich, A. (1992) Neuron 9, 383-391[Medline] [Order article via Infotrieve]
  27. Pawson, T. (1995) Nature 373, 573-579[CrossRef][Medline] [Order article via Infotrieve]
  28. Kapeller, R., and Cantley, L. C. (1994) BioEssays 16, 565-576[Medline] [Order article via Infotrieve]
  29. Fantl, W. J., Escobedo, J. A., Martin, G. A., Turck, C. W., del Rosario, M., McCormick, F., Williams, L. T. (1992) Cell 69, 413-423[Medline] [Order article via Infotrieve]
  30. Kazlauskas, A., Kashishian, A., Cooper, J. A., Valius, M. (1992) Mol. Cell. Biol. 12, 2534-2544[Abstract]
  31. Valius, M., and Kazlauskas, A. (1993) Cell 73, 321-334[Medline] [Order article via Infotrieve]
  32. Carpenter, C. L., and Cantley, L. C. (1996) Curr. Opin. Cell. Biol. 8, 153-158[CrossRef][Medline] [Order article via Infotrieve]
  33. Wrann, M., and Fox, C. F. (1980) Science 210, 1363-1365[Medline] [Order article via Infotrieve]
  34. Bowen-Pope, D. F., Dicorleto, P. E., Ross, R. (1983) J. Cell Biol. 96, 679-683[Abstract]
  35. Friedman, B. A., Frackelton, R. A., Ross, H. R., Connors, J. M., Fujiki, H., Sugimura, T., Rosner, M. R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3034-3038[Abstract]
  36. Hunter, T., Ling, N., and Cooper, J. A. (1984) Nature 311, 480-483[Medline] [Order article via Infotrieve]
  37. Countaway, J. L., Girones, N., and Davis, R. J. (1989) J. Biol. Chem. 264, 13642-13647[Abstract/Free Full Text]
  38. Davis, R. J., and Czech, M. P. (1987) J. Biol. Chem. 262, 6832-6841[Abstract/Free Full Text]
  39. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Struhl, K. (1991) Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York
  40. Claesson-Welsh, L., Eriksson, A., Moren, A., Severinsson, L., Ek, B., Ostman, A., Betsholtz, C., and Heldin, C. H. (1988) Mol. Cell. Biol. 8, 3476-3486[Medline] [Order article via Infotrieve]
  41. Waterfield, M. D., Mayes, E. L., Stroobant, P., Bennet, P. L., Young, S., Goodfellow, P. N., Banting, G. S., Ozanne, B. (1982) J. Cell. Biochem. 20, 149-161[Medline] [Order article via Infotrieve]
  42. Whitman, M., Kaplan, D. R., Schaffhausen, B., Cantley, L., and Roberts, T. M. (1985) Nature 315, 239-242[Medline] [Order article via Infotrieve]
  43. Kazlauskas, A., Durden, D. L., and Cooper, J. A. (1991) Cell Regul. 2, 413-425[Medline] [Order article via Infotrieve]
  44. Wasilenko, W. J., Payne, D. M., Fitzgerald, D. L., Weber, M. J. (1991) Mol. Cell. Biol. 11, 309-321[Medline] [Order article via Infotrieve]
  45. Chan, C., and Gill, G. N. (1996) J. Biol. Chem. 271, 22619-22623[Abstract/Free Full Text]
  46. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., Hall, A. (1992) Cell 70, 401-410[Medline] [Order article via Infotrieve]


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