Enhanced Tumorigenic Behavior of Glioblastoma Cells Expressing a Truncated Epidermal Growth Factor Receptor Is Mediated through the Ras-Shc-Grb2 Pathway

A mutant epidermal growth factor receptor (ΔEGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG.ΔEGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated ΔEGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTP-bound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.ΔEGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of ΔEGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins. A mutant epidermal growth factor receptor (ΔEGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG.ΔEGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated ΔEGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTP-bound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.ΔEGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of ΔEGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins. INTRODUCTIONThe EGF 1The abbreviations used are: EGFepidermal growth factorEGFRepidermal growth factor receptorBrdUrd5-bromodeoxyuridineTricineN-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycinePTBphosphotyrosine-binding domainGSTglutathione S-transferase. receptor family of tyrosine kinases has been implicated in a wide variety of human tumors, where they are typically overexpressed by gene amplification or increased transcription (1Gullick W.J. Br. Med. Bull. 1991; 47: 87-98Crossref PubMed Scopus (446) Google Scholar). In glioblastoma, deletions in the EGF receptor gene are also found at high frequency. The most common deletion involves exons 2 through 7 and occurs in about 10-20% of these tumors. The potential importance of this deleted receptor is underscored by its recent detection in tumors of the breast and lung (2Ekstrand A.J. Sugawa N. James C.D. Collins V.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4309-4313Crossref PubMed Scopus (498) Google Scholar, 3Humphrey P.A. Wong A.J. Vogelstein B. Zalutsky M.R. Fuller G.N. Archer G.E. Friedman H.S. Kwatra M.M. Bigner S.H. Bigner D.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4207-4211Crossref PubMed Scopus (328) Google Scholar, 4Garcia de Palazzo I.E. Adams G.P. Sundareshan P. Wong A.J. Testa J.R. Bigner D.D. Weiner L.M. Cancer Res. 1993; 53: 3217-3220PubMed Google Scholar, 5Wikstrand C.J. Hale L.P. Batra S.K. Hill M.L. Humphrey P.A. Kurpad S.N. McLendon R.E. Moscatello D. Pegram C.N. Reist C.J. Traweek S.T. Wong A.J. Zalutsky M.R. Bigner D.D. Cancer Res. 1995; 55: 3140-3148PubMed Google Scholar). Thus, it appears that overexpression of an EGF receptor which cannot bind its ligand is advantageous to tumor growth. We (6Nishikawa R. Ji X.D. Harmon R.C. Lazar C.S. Gill G.N. Cavenee W.K. Huang H.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7727-7731Crossref PubMed Scopus (806) Google Scholar) and others (7Ekstrand A.J. Longo N. Hamid M.L. Olson J.J. Liu L. Collins V.P. James C.D. Oncogene. 1994; 9: 2313-2320PubMed Google Scholar, 8Livneh E. Prywes R. Kashles O. Reiss N. Sasson I. Mory Y. Ullrich A. Schlessinger J. J. Biol. Chem. 1986; 261: 12490-12497Abstract Full Text PDF PubMed Google Scholar, 9Montgomery R.B. Moscatello D.K. Wong A.J. Cooper J.A. Stahl W.L J. Biol. Chem. 1995; 270: 30562-30566Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 10Batra S.K. Castelino-Prabhu S. Wikstrand C.J. Zhu X. Humphrey P.A. Friedman H.S. Bigner D.D. Cell Growth and Differ. 1995; 6: 1251-1259PubMed Google Scholar) have demonstrated that when this deletion mutant of EGF receptor (de 2-7 EGFR, here called ΔEGFR) is overexpressed in U87MG glioblastoma cells, NIH 3T3 fibroblasts, or Chinese hamster ovary cells it becomes phosphorylated on tyrosine residues, although the extent of phosphorylation is less than that observed for acutely EGF-stimulated full-length receptor. Thus, it would appear that either a small number of receptors become fully phosphorylated, or that a large number of receptors are phosphorylated, but at low stoichiometry. In any case, this constitutive phosphorylation is associated with the ability of the mutant receptor to confer sustained enhancement of the tumorigenic capacity of U87MG glioblastoma cells (6Nishikawa R. Ji X.D. Harmon R.C. Lazar C.S. Gill G.N. Cavenee W.K. Huang H.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7727-7731Crossref PubMed Scopus (806) Google Scholar).A great deal of progress has been made recently in understanding the mechanisms by which tyrosine kinase receptors signal. Following ligand binding, receptors dimerize and become phosphorylated on tyrosine residues which act as docking sites for proteins containing phosphotyrosine binding domains (11Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar). Two such domains have been identified, the SH2 (Src homology 2) domain and the PTB (phosphotyrosine-binding domain) (12Kavanaugh W.M. Turck C.W. Williams L.T. Science. 1995; 268: 1177-1179Crossref PubMed Scopus (222) Google Scholar) also termed the phosphotyrosine interaction domain (13Bork P. Margolis B. Cell. 1995; 80: 693-694Abstract Full Text PDF PubMed Scopus (171) Google Scholar). Many proteins which interact with the EGF receptor contain SH2 domains including Shc, Grb2, phospholipase C-γ, GAP, STAT 1α, STAT 3, and phophotyrosine phosphatase SH-PTP2. The Shc adapter protein also contains a PTB which appears to be responsible for its major interaction at tyrosine 1148 (14Okabayashi Y. Kido Y. Okutani T. Sugimoto Y. Sakaguchi K. Kasuga M. J Biol Chem. 1994; 269: 18674-18678Abstract Full Text PDF PubMed Google Scholar). Both Shc and Grb2 have been shown to be important for recruitment of Ras to activated receptors. Grb2 can bind directly to phosphotyrosines present in the consensus sequence YLN or indirectly through phosphorylated Shc (15Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar, 16Salcini A.E. McGlade J. Pelicci G. Nicoletti I. Pawson T. Pelicci P.G. Oncogene. 1994; 9: 2827-2836PubMed Google Scholar). One of the SH3 domains of Grb2 associates with the nucleotide exchange factor for Ras, Sos (17Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (925) Google Scholar, 18Rozakis-Adcock M. Fernley R. Wade J. Pawson T. Bowtell D. Nature. 1993; 363: 83-85Crossref PubMed Scopus (835) Google Scholar), thereby promoting increased Ras activation which initiates a cascade of activation of serine/threonine kinases and ultimately to an increase in gene transcription in the nucleus.Since Ras is a key component of mitogenic signaling pathways, we sought to determine whether it is responsible for the increased tumorigenicity of U87MG.ΔEGFR cells. Here we demonstrate by measurement of the concentration of GTP-bound Ras, that it is indeed activated in the U87MG.ΔEGFR cells relative to the parental U87MG cells. Moreover, inhibition of Ras function by microinjection of a neutralizing antibody into U87MG and U87MG.ΔEGFR cells reduces DNA synthesis to the same low background level suggesting that Ras plays a role in the increased replication of cells expressing the ΔEGFR. We also demonstrate that both Grb2 and Shc constitutively interact with the phosphorylated ΔEGF receptor expressed in U87MG.ΔEGFR cells, in a similar manner to wild-type EGF receptor, thereby providing a potential mechanism for the activation of Ras. Together these results suggest that Shc, Grb2, and Ras are involved in the enhanced mitogenesis and tumor forming potential of cells expressing the ΔEGF receptor.DISCUSSIONThe first transforming mutant EGFR to be identified was the product of the avian erythroblastosis virus v-erbB gene which encodes an EGFR with N- and C-terminal truncations as well as point mutations. Removal of the extracellular domain of EGFR has been shown to result in a weak but constitutive oncogenic activation of the receptor, while ligand binding to the full-length receptor results in a much stronger, EGF-dependent activation (28Khazaie K. Dull T.J. Graf T. Schlessinger J. Ullrich A. Beug H. Vennstrom B. EMBO J. 1988; 7: 3061-3071Crossref PubMed Scopus (78) Google Scholar). Consistent with these observations, the v-erbB, which encodes an EGF receptor displays much weaker tyrosine kinase activity than that of ligand activated wild-type receptor. A mutation involving deletion of 801 base pairs of the EGFR gene encoding a portion of the extracellular domain has been observed in human tumors of the brain, breast, and lung (2Ekstrand A.J. Sugawa N. James C.D. Collins V.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4309-4313Crossref PubMed Scopus (498) Google Scholar, 3Humphrey P.A. Wong A.J. Vogelstein B. Zalutsky M.R. Fuller G.N. Archer G.E. Friedman H.S. Kwatra M.M. Bigner S.H. Bigner D.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4207-4211Crossref PubMed Scopus (328) Google Scholar, 4Garcia de Palazzo I.E. Adams G.P. Sundareshan P. Wong A.J. Testa J.R. Bigner D.D. Weiner L.M. Cancer Res. 1993; 53: 3217-3220PubMed Google Scholar, 5Wikstrand C.J. Hale L.P. Batra S.K. Hill M.L. Humphrey P.A. Kurpad S.N. McLendon R.E. Moscatello D. Pegram C.N. Reist C.J. Traweek S.T. Wong A.J. Zalutsky M.R. Bigner D.D. Cancer Res. 1995; 55: 3140-3148PubMed Google Scholar). This mutant receptor displays low level ligand independent phosphorylation in NIH 3T3 cells, NR6, Chinese hamster ovary, or U87MG glioblastoma cells. We have used the U87MG cell line as the host for the expression of the mutant receptor since the ΔEGFR mutation spontaneously occurs in this type of cell in vivo. Expression of ΔEGFR in these cells promotes tumor formation in mouse brain (6Nishikawa R. Ji X.D. Harmon R.C. Lazar C.S. Gill G.N. Cavenee W.K. Huang H.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7727-7731Crossref PubMed Scopus (806) Google Scholar), and results in increased DNA synthesis of serum-starved cells. Here we have investigated the possible signaling pathways which may contribute to the increased tumorigenic properties of cells expressing the truncated EGFR.Recent studies have pointed to a key role for Ras in signaling pathways downstream of most receptors including tyrosine kinases, heterotrimeric G-protein-coupled receptors, and cytokine receptors (29Marshall M.S. FASEB J. 1995; 9: 1311-1318Crossref PubMed Scopus (271) Google Scholar). Moreover, it appears that the adapter proteins Shc and/or Grb2 are almost universally involved in the initial recruitment of the exchange factor for Ras to activated receptors. It was therefore the objective of this study to determine whether these key elements of the normal EGFR signaling pathway played a role in the enhanced DNA synthesis and tumorigenesis in U87MG cells overexpressing the ΔEGFR.U87MG.ΔEGFR cells were shown to possess an approximately 2-fold increase in the proportion of Ras in its active GTP-bound form relative to the parental U87MG cells. Under the starvation conditions described these cells also showed a 2-fold increase in DNA synthesis which could be abolished by the introduction of an inhibitory Ras antibody into the cells. These data illustrate that the enhanced growth of U87MG.ΔEGFR cells is dependent on Ras This is by a recent study in which expression of the ΔEGFR in NIH 3T3 cells was shown to activation of elements of the protein kinase cascade R.B. Moscatello D.K. Wong A.J. Cooper J.A. Stahl J. Biol. Chem. 1995; 270: 30562-30566Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). this activation of the Ras pathway is primarily responsible for enhanced tumorigenesis to be have also demonstrated that the truncated receptor the full-length wild-type receptor with to its interaction with Shc and Grb2, and to both in an when expressed in U87MG cells. The major sites of phosphorylation were and as has been shown for the full-length receptor. A low level of phosphorylation of a mutant receptor containing sites and was by the receptor was first with an a similar low level of phosphorylation was also on a receptor of the major phosphorylation Grb2 to containing and Shc in cells to with Tyr-1148, although a small to a mutant with with for the full-length receptor (14Okabayashi Y. Kido Y. Okutani T. Sugimoto Y. Sakaguchi K. Kasuga M. J Biol Chem. 1994; 269: 18674-18678Abstract Full Text PDF PubMed Google Scholar, T. Y. Kido Y. Sugimoto Y. Sakaguchi K. K. T. Kasuga M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, D. Schlessinger J. Cell. Biol. 1994; PubMed Google of Shc domains demonstrated that the SH2 domain of Shc can bind to of the truncated receptor, its PTB to or in is an which has been demonstrated to be the consensus for the (12Kavanaugh W.M. Turck C.W. Williams L.T. Science. 1995; 268: 1177-1179Crossref PubMed Scopus (222) Google Scholar, W.J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, P. S. Gish R. D. Shoelson S. Pawson T. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). is the which contains an binding for the and a which the domain Z. Shoelson S.E. McGlade J. P. Pawson T. M. H. Hanafusa H. T. R. D. Ratnofsky S. Cantley L.C. Cell. Biol. 1994; PubMed Scopus Google Scholar) C-terminal to the association data in cells, it would appear that the interaction site is through Tyr-1148, binding of the to could a binding to the same or receptor through its SH2 U87MG cells expressing the mutant form tumors in at a similar to the parental cell suggesting that with this site are for promoting tumor growth. M. K. H. R. H. S. and W. K. One such interaction be with the domain since the mutant is of binding to as we have demonstrated in A mutant which contains also has tumor promoting suggesting that this site is in to others to to a growth binding of Shc through both SH2 and PTB may be in to which are for DNA synthesis or which may the same In of this microinjection of the domain into NIH 3T3 cells overexpressing full-length EGFR DNA synthesis that with this domain as well as with the PTB are important for mitogenesis N. K. S. S. K. M. Oncogene. 1995; Google Scholar). a binding site for a protein the PTB and SH2 domains are A number of proteins have been shown to with Shc in to in to Grb2 I. Bowtell D. R. M.R. J. 1994; Google Scholar, McGlade J. Pelicci G. Pawson T. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, T. Downward J. Oncogene. 1994; 9: Google Scholar, L. J.E. G. Cell. Biol. 1994; PubMed Scopus Google Scholar). Shc could be involved in receptors. This may be of importance to truncated receptors than full-length receptors which dimerize in the of a ligand binding In the of the full-length EGFR it has been that of phosphorylation sites the kinase activity or mitogenic properties of the receptor E. Prywes R. Kashles O. Reiss N. Sasson I. Mory Y. Ullrich A. Schlessinger J. J. Biol. Chem. 1986; 261: 12490-12497Abstract Full Text PDF PubMed Google Scholar, A. Dull T.J. F. E. D. A. Ullrich A. Schlessinger J. EMBO J. 1988; 7: PubMed Scopus Google Scholar, A. Dull T.J. D. A. R. Ullrich A. Schlessinger J. EMBO J. 1988; 7: PubMed Scopus Google Scholar). This may be by ability of the full-length receptor to and through that mutant receptors sites were to Shc to extent is also This has also been observed for full-length EGFR and phosphorylation sites N. Schlessinger J. Margolis B. Oncogene. 1994; 9: Google Scholar, L. G. Oncogene. 1994; 9: Google Scholar, A. L. R. G. P.G. Pelicci P.G. O. Oncogene. 1995; Google Scholar, N. A. K. Y. S. S. T. Y. M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). Shc phosphorylation was and therefore for which were to bind It is possible that the large number of truncated receptors on the of the cell Shc to on the of the and these at low frequency. In where Shc was phosphorylated it also Grb2 which could presumably to Ras, the of the active was The of Grb2 associated with the receptor, to be directly to since to bind almost as much Grb2 as the but bind as Thus, it is possible that the Shc to and is which may Grb2 and may or may be of key which to be is the mutant EGF receptors are for of this portion of the extracellular One may in the that results in a increase in autophosphorylation relative to the full-length receptor, this phosphorylation at the same major the truncated receptor to form the same as the wild-type receptor. or this the of the domain such a growth is recent that a in may also be an important of the growth activity of this mutant from the wild-type receptor is by EGF binding and the is by protein kinase and In full-length receptors can with family which could a and cell Cell. 1995; 80: Full Text PDF PubMed Scopus Google Scholar). The extracellular domain may also interact with on cells such as EGF which could the of the cells K. R. E. J. Cell Biol. 1995; PubMed Scopus Google Scholar). the of the EGF binding potential from the of the truncated receptor which are of for cell INTRODUCTIONThe EGF 1The abbreviations used are: EGFepidermal growth factorEGFRepidermal growth factor receptorBrdUrd5-bromodeoxyuridineTricineN-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycinePTBphosphotyrosine-binding domainGSTglutathione S-transferase. receptor family of tyrosine kinases has been implicated in a wide variety of human tumors, where they are typically overexpressed by gene amplification or increased transcription (1Gullick W.J. Br. Med. Bull. 1991; 47: 87-98Crossref PubMed Scopus (446) Google Scholar). In glioblastoma, deletions in the EGF receptor gene are also found at high frequency. The most common deletion involves exons 2 through 7 and occurs in about 10-20% of these tumors. The potential importance of this deleted receptor is underscored by its recent detection in tumors of the breast and lung (2Ekstrand A.J. Sugawa N. James C.D. Collins V.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4309-4313Crossref PubMed Scopus (498) Google Scholar, 3Humphrey P.A. Wong A.J. Vogelstein B. Zalutsky M.R. Fuller G.N. Archer G.E. Friedman H.S. Kwatra M.M. Bigner S.H. Bigner D.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4207-4211Crossref PubMed Scopus (328) Google Scholar, 4Garcia de Palazzo I.E. Adams G.P. Sundareshan P. Wong A.J. Testa J.R. Bigner D.D. Weiner L.M. Cancer Res. 1993; 53: 3217-3220PubMed Google Scholar, 5Wikstrand C.J. Hale L.P. Batra S.K. Hill M.L. Humphrey P.A. Kurpad S.N. McLendon R.E. Moscatello D. Pegram C.N. Reist C.J. Traweek S.T. Wong A.J. Zalutsky M.R. Bigner D.D. Cancer Res. 1995; 55: 3140-3148PubMed Google Scholar). Thus, it appears that overexpression of an EGF receptor which cannot bind its ligand is advantageous to tumor growth. We (6Nishikawa R. Ji X.D. Harmon R.C. Lazar C.S. Gill G.N. Cavenee W.K. Huang H.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7727-7731Crossref PubMed Scopus (806) Google Scholar) and others (7Ekstrand A.J. Longo N. Hamid M.L. Olson J.J. Liu L. Collins V.P. James C.D. Oncogene. 1994; 9: 2313-2320PubMed Google Scholar, 8Livneh E. Prywes R. Kashles O. Reiss N. Sasson I. Mory Y. Ullrich A. Schlessinger J. J. Biol. Chem. 1986; 261: 12490-12497Abstract Full Text PDF PubMed Google Scholar, 9Montgomery R.B. Moscatello D.K. Wong A.J. Cooper J.A. Stahl W.L J. Biol. Chem. 1995; 270: 30562-30566Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 10Batra S.K. Castelino-Prabhu S. Wikstrand C.J. Zhu X. Humphrey P.A. Friedman H.S. Bigner D.D. Cell Growth and Differ. 1995; 6: 1251-1259PubMed Google Scholar) have demonstrated that when this deletion mutant of EGF receptor (de 2-7 EGFR, here called ΔEGFR) is overexpressed in U87MG glioblastoma cells, NIH 3T3 fibroblasts, or Chinese hamster ovary cells it becomes phosphorylated on tyrosine residues, although the extent of phosphorylation is less than that observed for acutely EGF-stimulated full-length receptor. Thus, it would appear that either a small number of receptors become fully phosphorylated, or that a large number of receptors are phosphorylated, but at low stoichiometry. In any case, this constitutive phosphorylation is associated with the ability of the mutant receptor to confer sustained enhancement of the tumorigenic capacity of U87MG glioblastoma cells (6Nishikawa R. Ji X.D. Harmon R.C. Lazar C.S. Gill G.N. Cavenee W.K. Huang H.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7727-7731Crossref PubMed Scopus (806) Google Scholar).A great deal of progress has been made recently in understanding the mechanisms by which tyrosine kinase receptors signal. Following ligand binding, receptors dimerize and become phosphorylated on tyrosine residues which act as docking sites for proteins containing phosphotyrosine binding domains (11Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar). Two such domains have been identified, the SH2 (Src homology 2) domain and the PTB (phosphotyrosine-binding domain) (12Kavanaugh W.M. Turck C.W. Williams L.T. Science. 1995; 268: 1177-1179Crossref PubMed Scopus (222) Google Scholar) also termed the phosphotyrosine interaction domain (13Bork P. Margolis B. Cell. 1995; 80: 693-694Abstract Full Text PDF PubMed Scopus (171) Google Scholar). Many proteins which interact with the EGF receptor contain SH2 domains including Shc, Grb2, phospholipase C-γ, GAP, STAT 1α, STAT 3, and phophotyrosine phosphatase SH-PTP2. The Shc adapter protein also contains a PTB which appears to be responsible for its major interaction at tyrosine 1148 (14Okabayashi Y. Kido Y. Okutani T. Sugimoto Y. Sakaguchi K. Kasuga M. J Biol Chem. 1994; 269: 18674-18678Abstract Full Text PDF PubMed Google Scholar). Both Shc and Grb2 have been shown to be important for recruitment of Ras to activated receptors. Grb2 can bind directly to phosphotyrosines present in the consensus sequence YLN or indirectly through phosphorylated Shc (15Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (825) Google Scholar, 16Salcini A.E. McGlade J. Pelicci G. Nicoletti I. Pawson T. Pelicci P.G. Oncogene. 1994; 9: 2827-2836PubMed Google Scholar). One of the SH3 domains of Grb2 associates with the nucleotide exchange factor for Ras, Sos (17Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (925) Google Scholar, 18Rozakis-Adcock M. Fernley R. Wade J. Pawson T. Bowtell D. Nature. 1993; 363: 83-85Crossref PubMed Scopus (835) Google Scholar), thereby promoting increased Ras activation which initiates a cascade of activation of serine/threonine kinases and ultimately to an increase in gene transcription in the nucleus.Since Ras is a key component of mitogenic signaling pathways, we sought to determine whether it is responsible for the increased tumorigenicity of U87MG.ΔEGFR cells. Here we demonstrate by measurement of the concentration of GTP-bound Ras, that it is indeed activated in the U87MG.ΔEGFR cells relative to the parental U87MG cells. Moreover, inhibition of Ras function by microinjection of a neutralizing antibody into U87MG and U87MG.ΔEGFR cells reduces DNA synthesis to the same low background level suggesting that Ras plays a role in the increased replication of cells expressing the ΔEGFR. We also demonstrate that both Grb2 and Shc constitutively interact with the phosphorylated ΔEGF receptor expressed in U87MG.ΔEGFR cells, in a similar manner to wild-type EGF receptor, thereby providing a potential mechanism for the activation of Ras. Together these results suggest that Shc, Grb2, and Ras are involved in the enhanced mitogenesis and tumor forming potential of cells expressing the ΔEGF receptor.