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

Open AccessPublished:March 20, 1998DOI:https://doi.org/10.1074/jbc.273.12.6885
      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 β (PDGFβ) receptor (PDGFβR), 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 PDGFβR and results in the recruitment of phosphatidylinositol 3-kinase to the PDGFβR. In R1hER cells, which overexpress the EGFR, we find ligand-independent tyrosine phosphorylation of the PDGFβR 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/PDGFβR association is ligand-independent in all cell lines tested. We also demonstrate that the fraction of PDGFβR 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.
      Receptor tyrosine kinases have been divided into subfamilies based on common structural features (
      • Ullrich A.
      • Schlessinger J.
      ). The epidermal growth factor (EGF)
      The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PDGFβR, PDGFβ receptor; PI, phosphatidylinositol; SH2, Src homology 2; IP, immunoprecipitation.
      1The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PDGFβR, PDGFβ receptor; PI, phosphatidylinositol; SH2, Src homology 2; IP, immunoprecipitation.
      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 (
      • van der Geer P.
      • Hunter T.
      ). The PDGF receptor subfamily includes the PDGFαR, PDGFβR, 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 (
      • Heldin C-H.
      ,
      • Weiss F.U.
      • Daub H.
      • Ullrich A.
      ). For example, heterodimerization has been well described between members of the EGFR subfamily (
      • Earp H.S.
      • Dawson T.L.
      • Li X.
      • Yu H.
      ). The addition of EGF to a number of cell lines results in EGFR-dependent tyrosine phosphorylation of ErbB2 (
      • Akiyama T.
      • Saito T.
      • Ogawara H.
      • Toyoshima K.
      • Yamamoto T.
      ,
      • King C.R.
      • Borrello I.
      • Bellot F.
      • Comoglio P.
      • Schlessinger J.
      ,
      • Kokai Y.
      • Dobashi K.
      • Weiner D.B.
      • Myers J.N.
      • Nowell P.C.
      • Greene M.I.
      ,
      • Stern D.F.
      • Kamps M.P.
      ). EGF can induce dimerization of EGF receptors and ErbB2 in transfected NR6 and NIH3T3 cells (
      • Qian X.C.
      • LeVea M.
      • Freeman J.K.
      • Dougall W.C.
      • Greene M.I.
      ,
      • Wada T.
      • Qian X.
      • Greene M.I.
      ) and also in SKBR-3 cells, which overexpress the ErbB2 receptor (
      • Goldman R.B.
      • Levy N.
      • Peles E.
      • Yarden Y.
      ). Similarly, heregulin may induce heterodimeric complexes between ErbB2 and ErbB3 or ErbB4 (
      • Plowman G.D.
      • Green J.M.
      • Culouscou J.M.
      • Carlton G.W.
      • Rothwell V.M.
      • Buckley S.
      ,
      • 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 III, K.L.
      ). A number of heterodimeric combinations of these receptors have been described, and some combinations are favored over others (
      • Tzahar E.
      • Waterman H.
      • Chen X.
      • Levkovitz G.
      • Karunagaran D.
      • Lavi S.
      • Ratzkin B.J.
      • Yarden Y.
      ). It is important to note that heterodimerization between the EGFR and ErbB2 proteins can be detected even in the absence of ligand (
      • Qian X.C.
      • LeVea M.
      • Freeman J.K.
      • Dougall W.C.
      • Greene M.I.
      ,
      • Qian X.
      • Dougall W.C.
      • Hellman M.E.
      • Greene M.I.
      ).
      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 (
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ). 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 (
      • Fedi P.
      • Pierce J.H.
      • Di Fiore P.P.
      • Kraus M.H.
      ,
      • Prigent S.A.
      • Gullick W.G.
      ,
      • Soltoff S.P.
      • Carraway III, K.L.
      • Prigent S.A.
      • Gullick W.G.
      • Cantley L.C.
      ). 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 (
      • Soltoff S.P.
      • Carraway III, K.L.
      • Prigent S.A.
      • Gullick W.G.
      • Cantley L.C.
      ,
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Lammers R.
      • Ullrich A.
      • Schlessinger J.
      ). 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 (
      • Soltoff S.P.
      • Carraway III, K.L.
      • Prigent S.A.
      • Gullick W.G.
      • Cantley L.C.
      ,
      • Carraway K.C.
      • Cantley L.C.
      ), or by using chimeric receptors in fibroblasts (
      • Fedi P.
      • Pierce J.H.
      • Di Fiore P.P.
      • Kraus M.H.
      ). 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 (
      • Karunagaran D.
      • Tzahar E.
      • Beerli R.B.
      • Chen X.
      • Graus-Porta D.
      • Ratzkin B.J.
      • Seger R.
      • Hynes N.E.
      • Yarden Y.
      ). Recently, transactivation of the EGFR has also been described in response to stimulation of G-protein-coupled receptors (
      • Daub H.
      • Weiss F.U.
      • Wallasch C.
      • Ullrich A.
      ). In the PDGFR subfamily, the different isoforms of PDGF induce different dimeric forms of the receptors; e.g. PDGF-AA induces αα homodimers only while PDGF-BB induces all three combinations of receptors (i.e. αα, ββ, and αβ (
      • Claesson-Welsh L.
      )). 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 (
      • Schlessinger J.
      • Ullrich A.
      ). 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 (
      • Pawson T.
      ). 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-γ1 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 (
      • Kapeller R.
      • Cantley L.C.
      ). 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 (
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • del Rosario M.
      • McCormick F.
      • Williams L.T.
      ,
      • Kazlauskas A.
      • Kashishian A.
      • Cooper J.A.
      • Valius M.
      ). 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 (
      • Valius M.
      • Kazlauskas A.
      ). Other functions of PI 3-kinase (reviewed in Ref.
      • Carpenter C.L.
      • Cantley L.C.
      ) 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 (
      • Wrann M.
      • Fox C.F.
      ). 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 (
      • Bowen-Pope D.F.
      • Dicorleto P.E.
      • Ross R.
      ,
      • Friedman B.A.
      • Frackelton R.A.
      • Ross H.R.
      • Connors J.M.
      • Fujiki H.
      • Sugimura T.
      • Rosner M.R.
      ,
      • Hunter T.
      • Ling N.
      • Cooper J.A.
      ). 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 (
      • Countaway J.L.
      • Girones N.
      • Davis R.J.
      ,
      • Davis R.J.
      • Czech M.P.
      ). 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 PDGFβ receptor.

      DISCUSSION

      In this study, we describe interactions between the EGF and the PDGFβ 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 PDGFβ 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 PDGFβR 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 PDGFβR. 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 PDGFβR following EGF stimulation. This suggests the following order of events. Activation of the EGFR leads to tyrosine phosphorylation and activation of the PDGFβR. This leads to the association of PI 3-kinase with the PDGFβR. Although the EGF-induced increase in PI 3-kinase activity associated with the PDGFβR 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 (
      • Ridley A.J.
      • Paterson H.F.
      • Johnston C.L.
      • Diekmann D.
      • Hall A.
      ). 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 PDGFβR. We looked for a similar association between the PDGFβR and other SH2 domain-containing proteins such as phospholipase C-γ1, 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 PDGFβR, mimicking the effect of EGF stimulation in untransfected cells. In addition, there is a substantial constitutive association of the PDGFβR 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 PDGFβR 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 PDGFβR 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 PDGFβR 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 PDGFβR may serve a similar function. Secondly, overexpression of the EGFR in R1hER cells mimics the effect of EGF stimulation on the PDGFβR 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 PDGFβR.
      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 PDGFβR 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 PDGFβR 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 (
      • Qian X.C.
      • LeVea M.
      • Freeman J.K.
      • Dougall W.C.
      • Greene M.I.
      ,
      • Qian X.
      • Dougall W.C.
      • Hellman M.E.
      • Greene M.I.
      ). Secondly, in R1hER cells the fraction of PDGFβ 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.

      Acknowledgments

      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.

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