Carboxy-terminal truncations of epidermal growth factor (EGF) receptor affect diverse EGF-induced cellular responses.

The binding of epidermal growth factor (EGF) to its receptor induces tyrosine phosphorylation of phospholipase C gamma (PLC gamma), which appears to be necessary for its activation leading to phosphatidyl inositol (PI) hydrolysis. Moreover, EGF-receptor (EGF-R) activation and autophosphorylation results in binding of PLC gamma to the tyrosine phosphorylated carboxy-terminus of the receptor. To gain further insights into the mechanisms and interactions regulating these processes, we have analyzed transfected NIH-3T3 cells expressing two EGF-R carboxy-terminal deletion mutants (CD63 and CD126) with reduced capacity to stimulate PI hydrolysis, Ca2+ rises, and DNA synthesis. In fact, the CD126 mutant lacking 126 carboxy-terminal amino acids, including four tyrosine autophosphorylation sites, was unable to stimulate PI hydrolysis or Ca2+ rise in response to EGF. Surprisingly, EGF binding to the cell lines expressing CD63 or CD126 mutants was followed by similar stimulation of tyrosine phosphorylation of PLC gamma. Our results suggest that although necessary, tyrosine phosphorylation of PLC gamma may not be sufficient for stimulation and PI hydrolysis. It is clear, however, that the carboxy-terminal region of EGF-R is involved in regulation of interactions with cellular targets and therefore plays a crucial role in postreceptor signaling pathways.     

Dissociation of cellular responses to epidermal growth factor using anti-receptor monoclonal antibodies.

Three biologically active monoclonal antibodies against the human epidermal growth factor (EGF) receptor (2E9, 2D11 and 2G5) have been used to analyse the interrelationship between various cellular responses to EGF. Antibody 2E9 (IgG1) is directed against the protein core of the receptor, close to or at the EGF binding site, while 2D11 (IgG3) and 2G5 (IgG2a) recognize blood-group A-related carbohydrate determinants of the receptor. These antibodies have EGF-like effects in that they can activate the receptor tyrosine kinase both in vitro and in vivo. Cross-linking of the receptor-bound antibodies by a second antibody mimics EGF in inducing a rapid aggregation of receptors on the cell surface. However, all three antibodies fail to mimic EGF in raising cytoplasmic pH and free Ca2+ and do not stimulate DNA synthesis in quiescent fibroblasts, even after external cross-linking of the occupied receptors. It is concluded that EGF-R tyrosine kinase activity as well as substrate specificity can be modulated by ligands other than EGF, even if they bind to sites distinct from the EGF binding domain; activation of the receptor tyrosine kinase, receptor clustering and induction of the ionic signals are causally unrelated events; and tyrosine kinase activation and receptor cross-linking are not sufficient for stimulation of DNA synthesis.     

Neuronal differentiation in response to epidermal growth factor of transfected murine P19 embryonal carcinoma cells expressing human epidermal growth factor receptors

The human epidermal growth factor receptor (hEGF-R) was introduced into murine P19 embryonal carcinoma (EC) cells, which do not express endogenous EGF-R. Undifferentiated stable P19 EC transfectants containing multiple copies of the hEGF-R complementary DNA were isolated. These cells express functional EGF-R, exhibiting characteristic biphasic EGF binding and intrinsic tyrosine protein kinase activity. Whereas normally EGF induces the expression of multiple nuclear protooncogenes, only junB expression is induced by EGF in the HER-transfected cells. This indicates that undifferentiated P19 EC cells contain at least part of a signal transduction machinery capable of coupling to the ectopically expressed hEGF-R. Interestingly, neuronal differentiation is induced in these cells in response to EGF under culture conditions resembling those during early preimplantation embryogenesis. These results indicate that neuronal differentiation of pluripotent P19 EC cells can be induced via activation of a tyrosine protein kinase signaling pathway.     

Egf binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3.

The epidermal growth factor receptor (EGF-R) and the erbB-2 proto-oncogene product protein are closely related by their structural homology and their shared enzymatic activity as autophosphorylating tyrosine kinases. We show that in mammary tumor cells (SK-BR-3) EGF causes a rapid increase in tyrosine phosphorylation of the erbB-2 protein. Phosphorylation of erbB-2 does not occur in cells lacking the EGF-R (MDA-MB-453). Phosphorylation of erbB-2 in SK-BR-3 cells is blocked if EGF is prevented from interacting with its receptor by specific monoclonal antibodies. While EGF induces the down-regulation of its receptor in SK-BR-3 cells, EGF has no effect on the stability of the erbB-2 protein. This result suggests that the erbB-2 protein is a substrate of the EGF-R and indicates the possibility of communication between these two proteins early in the signal transduction process.     

Early events in the antiproliferative action of tumor necrosis factor are similar to the early events in epidermal growth factor growth stimulation

The process of TNF-induced cytotoxicity is complex but appears to be mediated through a TNF-specific cell surface receptor. Recent evidence suggests that TNF action on tumor cells may be antagonized by epidermal growth factor (EGF) and other EGF-receptor modulatory peptides implicating a role for EGF-R in the process of TNF-induced cytotoxicity. In the present report, we investigated the biochemical actions of TNF on several biochemical events known to occur in the process of EGF signal transduction in intact cells. The actions of TNF were compared directly to those of EGF in both TNF-sensitive and -resistant tumor cell lines. In TNF-sensitive ME-180 cervical carcinoma cells, TNF (20 ng/ml) stimulated the tyrosine protein kinase activity of the EGF-receptor (EGF-R) fivefold when measured by receptor autophosphorylation in an immune complex kinase assay. TNF activation of EGF-R kinase activity in ME-180 was measurable 10 min after TNF incubation and enzymatic activity remained elevated 20 min after TNF addition. Activation of the receptor by TNF correlated with increased 32P incorporation into EGF-R protein when receptor was immunoprecipitated from 32P-equilibrated cells following a 20 min incubation with TNF. Acid hydrolysis of EGF-R protein isolated from TNF-treated ME-180 cells demonstrates an increase in the phosphotyrosine content of EGF-R when compared to receptor isolated from untreated cells. The results suggest that TNF increased EGF-R tyrosine protein kinase activity and the state of EGF-receptor tyrosine phosphorylation in a manner similar to that reported for EGF. However, TNF does not appear to be structurally related to EGF since TNF was unable to directly activate EGF-R when incubated with extensively washed immunoprecipitates of EGF-R. In TNF-resistant T24 bladder carcinoma cells, TNF failed to alter EGF-R tyrosine protein kinase activity although both EGF and phorbol ester were shown to modulate the enzymatic activity of the receptor in these cells. These results indicate that the ability of TNF to modulate EGF-R kinase in target cells may correlate with its cytotoxic actions on TNF-sensitive tumor cells. Other biochemical activities associated with the induction or regulation of cellular growth were examined in TNF- or EGF-treated tumor cells. EGF stimulated a rapid 8-16-fold increase in the expression of the proto-oncogene c-myc when analyzed by dot-blot analysis of total cellular RNA or Northern blot hybridization of polyadenylated RNA. TNF treatment failed to alter c-myc expression in ME-180 cells when analyzed by either technique.(ABSTRACT TRUNCATED AT 400 WORDS)     

A highly conserved tyrosine residue at codon 845 within the kinase domain is not required for the transforming activity of human epidermal growth factor receptor.

Epidermal growth factor receptor (EGF-R) is a widely expressed ligand-dependent tyrosine kinase. The tyrosine residue at 845 in EGF-R corresponds to Y416 of v/c-src kinase, which is highly conserved and functionally important in many tyrosine kinases. To clarify the functional role of Y845, we constructed a mutant human EGF-R in which this tyrosine was replaced with phenylalanine and transfected it to NIH3T3 cells. EGF-R F845 induced EGF-dependent cellular transformation and revealed tyrosine-autophosphorylation of a 170 kDa protein, and initiated DNA synthesis similar to the wild-type EGF-R. We conclude here that Y845 is dispensable in the above mentioned functions of EGF-R tyrosine kinase.     

Signal transduction by the epidermal growth factor receptor is attenuated by a COOH-terminal domain serine phosphorylation site.

It has been proposed that the acute desensitization of epidermal growth factor receptor (EGF-R) function can be accounted for, in part, by the effect of EGF to increase phosphorylation of the receptor at Ser1046/7 (Countaway, J.L., Nairn, A.C., and Davis, R.J. (1992) J. Biol. Chem. 267, 1129-1140). Here, we show that the mutational removal of this phosphorylation site causes an activation of EGF-R function and a potentiation of signal transduction. The mechanism of potentiation results from 1) defective down-regulation of the EGF-R when cells are incubated with high concentrations of EGF; and 2) increased EGF-stimulated tyrosine phosphorylation. The increased EGF-stimulated phosphorylation is associated with an alteration of the apparent specificity of tyrosine phosphorylation and is independent of the down-regulation defect. Together, these data strongly support the hypothesis that Ser1046/7 is a biologically significant site of regulatory phosphorylation of the EGF-R.     

Activation of phospholipase C-gamma 1 through transfected platelet-derived growth factor receptor enhances interleukin 2 production upon antigen stimulation in a T-cell line.

Phospholipase C-gamma 1 (PLC gamma 1) plays an important role in the signal transduction pathway by producing second messengers. However, the activation mechanism of PLC gamma 1 and the role of the phosphatidylinositol pathway for interleukin 2 (IL-2) production in T lymphocytes remain to be determined. To analyze the functional role of this pathway in T cells, we expressed an epidermal growth factor receptor (EGF) or platelet-derived growth factor (PDGF) receptor (EGF-R or PDGF-R), both of which are known to directly activate PLC gamma 1 in fibroblasts, into a murine T-cell hybridoma. Both receptors were expressed on the cell surface and caused tyrosine phosphorylation of multiple substrates, including the receptor itself, upon ligand binding. While EGF stimulation did not either cause phosphorylation of PLC gamma 1 or induce Ca2+ mobilization in the EGF-R transfectant in this system, PDGF treatment induced tyrosine phosphorylation of PLC gamma 1 and Ca2+ mobilization in the PDGF-R transfectant. Stimulation through PDGF-R enhanced IL-2 production upon antigen stimulation of the transfectants, although PDGF treatment alone did not induce IL-2 production. These results suggest that activation of the phosphatidylinositol pathway affects the downstream pathway to IL-2 production but is not sufficient to produce IL-2 and that cooperation with signals from tyrosine kinase cascades is required for IL-2 production.     

EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate

EGF-R [EGF (epidermal growth factor) receptor] ligands can promote or inhibit cell growth. The biological outcome of receptor activation is dictated, at least in part, by ligand-specified patterns of endocytic trafficking. EGF-R trafficking downstream of the ligands EGF and TGF-alpha (transforming growth factor-alpha) has been investigated extensively. However, less is known about EGF-R fates induced by the ligands BTC (betacellulin) and AR (amphiregulin). We undertook comparative analyses to identify ligand-specific molecular events that regulate EGF-R trafficking and degradation. EGF (17 nM) and BTC (8.5 nM) induced significant EGF-R degradation, with or without ectopic expression of the ubiquitin ligase Cbl. Human recombinant AR (17 nM) failed to affect receptor degradation in either case. Notably, levels of ligand-induced EGF-R ubiquitination did not correlate strictly with receptor degradation. Dose-response experiments revealed that AR at a saturating concentration was a partial agonist at the EGF-R, with approx. 40% efficacy (relative to EGF) at inducing receptor tyrosine phosphorylation, ubiquitination and association with Cbl. EGF-R down-regulation and degradation also were compromised upon cell stimulation with AR (136 nM). These outcomes correlated with decreased degradation of the Cbl substrate and internalization inhibitor hSprouty2. Downstream of the hSprouty2 checkpoint in AR-stimulated cells, Cbl-free EGF-R was incorporated into endosomes from which Cbl-EGF-R complexes were excluded. Our results suggest that the AR-specific EGF-R fate results from decreased hSprouty2 degradation and reduced Cbl recruitment to underphosphorylated EGF-R, two effects that impair EGF-R trafficking to lysosomes.     

EGF-induced EGF-receptor and MAP kinase phosphorylation in goat cumulus cells during in vitro maturation

EGF has been shown to influence meiotic maturation and development competence of oocyte in various mammalian species. We previously reported, in goat, that the EGF receptor (EGF-R) was present both on cumulus cells and oocytes. Here, EGF-induced signaling was investigated during the in vitro maturation process in goat cumulus-oocyte complexes (COCs). Cumulus cells and oocytes were subjected to Western immunoblotting analysis using anti-MAP kinase, anti-phosphotyrosine, anti-phospho MAP kinase, and anti-phospho EGF-R antibodies. We demonstrated that treatment with EGF during the in vitro maturation process induced rapid tyrosine phosphorylation of EGF-R in a time and concentration dependent manner in cumulus cells. A similar pattern of activation by phosphorylation was observed for MAP kinase upon EGF stimulation. AG 1478, an inhibitor of the EGF kinase, suppressed EGF-stimulated phosphorylation of EGF-R and also affected the MAP kinase activation. Treatment with the MEK inhibitor PD 98059 abolished EGF-induced MAP kinase activation. We did not observe oocyte EGF-R phosphorylation in our experiments during the in vitro maturation process. Our data indicate, in goat cumulus cells, that activation of EGF-R by EGF triggers signaling through the MAP kinase pathway during in vitro maturation. This supports the hypothesis that the major site of action for EGF, that regulates oocyte maturation, is the cumulus cell.     

Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.

Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product.     

Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized cells, all antibodies were able to activate the EGF-R tyrosine kinase, as measured by EGF-R autophosphorylation and phosphorylation of other substrates on tyrosine residues. EGF-R tyrosine kinase activation correlated strongly with the induction of EGF-R dimerization. (i) Both processes specifically occurred in a narrow antibody concentration range; (ii) both processes required the presence of detergent; and (iii) both processes depended on antibody bivalence since monovalent Fab fragments were inactive yet regained full activity after cross-linking by a second bivalent antibody. These data demonstrate that antibody bivalence is essential and sufficient for EGF-R activation and that activation occurs regardless of the EGF-R epitope recognized. Finally, EGF-R dimerization was shown not to depend on receptor autophosphorylation since it still occurred in the absence of ATP. Also, partial inhibition of the tyrosine kinase activity by the specific EGF-R tyrosine kinase inhibitor tyrphostin AG 213 did not affect formation of EGF-R dimers. Taken together these results demonstrate that induction of EGF-R dimerization is sufficient and in case of antibody action, essential, for activation of the EGF-R tyrosine kinase and thus provide strong support for an intermolecular mechanism of EGF-R tyrosine kinase activation.     

Role of EGF receptor tyrosine kinase activity in antiapoptotic effect of EGF on mouse hepatocytes

The apoptotic Fas pathway is potentially involved in the pathogenesis of liver diseases. Growth factors, such as epidermal growth factor (EGF), can protect cells against apoptosis induced by a variety of stimuli, including Fas receptor stimulation. However, the underlying mechanisms of this protection have yet to be determined. We investigated the involvement of EGF receptor (EGFR) tyrosine kinase (TK) activity in the antiapoptotic effect of EGF on primary mouse hepatocyte cultures. Cells undergoing apoptosis after treatment with anti-Fas antibody were protected by EGF treatment. This protection was significantly but partially decreased when cells were treated with two specific inhibitors of the TK activity of EGFR. Evaluation of the efficacy of these compounds indicated that they were able to abolish EGFR autophosphorylation and postreceptor events such as activation of mitogen-activated protein kinases and the phosphatidylinositol 3'-kinase pathways as well as increases in Bcl-x(L) mRNA and protein levels. This leads us to postulate that EGF exerts its antiapoptotic action partially through the TK activity of EGFR. In addition, our results suggest that Bcl-x(L) protein upregulation caused by EGF is linked to the TK activity of its receptor.     

Mechanisms of extracellularly regulated kinases 1/2 activation in adrenal glomerulosa cells by lysophosphatidic acid and epidermal growth factor

The regulation of adrenal function, including aldosterone production from adrenal glomerulosa cells, is dependent on a variety of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). In many cell types, GPCR-mediated MAPK activation is mediated through transactivation of RTKs, in particular the epidermal growth factor (EGF) receptor (EGF-R). However, the extent to which this cross-communication between GPCRs and RTKs is operative in the adrenal glomerulosa has not been defined. Bovine adrenal glomerulosa cells express receptors for lysophosphatidic acid (LPA) and EGF. In cultured bovine adrenal glomerulosa cells, LPA, which is predominantly coupled to Gi and partially to Gq/protein kinase C alpha and epsilon, caused phosphorylation of Src (at Tyr416), proline-rich tyrosine kinase (Pyk2 at Tyr402), EGF-R, protein kinase B/Akt, extracellularly regulated signal kinases 1/2, and their dependent protein, p90 ribosomal S6 kinase. Overexpression of dominant negative mutants of Ras or EGF-R, and selective inhibition of EGF-R kinase with AG1478, significantly reduced LPA-induced ERK1/2 phosphorylation. However, this was not impaired by inhibition of matrix metalloproteinase (MMP) and heparin-binding EGF. LPA-induced ERK1/2 activation occurs predominantly through EGF-R transactivation by Gi/Src and partly through activation of protein kinase C, which acts downstream of EGF-R and Ras. In contrast, LPA-induced phosphorylation of Shc and ERK1/2 in clonal hepatocytes (C9 cells) was primarily mediated through MMP-dependent transactivation of the EGF-R. These observations in adrenal glomerulosa and hepatic cells demonstrate that LPA phosphorylates ERK1/2 through EGF-R transactivation in a MMP-dependent or -independent manner in individual target cells. This reflects the ability of GPCRs expressed in cell lines and neoplastic cells to utilize distinct signaling pathways that can elicit altered responses compared with those of native tissues.     

Epidermal growth factor induces tyrosine phosphorylation of epidermal growth factor receptors not occupied with ligand in intact A431 cells.

The mechanism of epidermal growth factor receptor (EGF-R) autophosphorylation in intact A431 cells was studied. We detected epidermal growth factor (EGF) induced tyrosine phosphorylation of EGF-R not occupied with ligand. Cell monolayers were subjected to irradiation after incubation with photoreactive derivative of EGF and uncoupled EGF was extracted by acidic treatment. Subsequent immunoprecipitation with antiphosphotyrosine antibodies resulted in precipitation of both EGF-R complexes with EGF and EGF-R with unoccupied ligand-binding site. The fact of precipitation of EGF-R with unoccupied ligand-binding site in conjunction with our finding of rapid dephosphorylation of EGF-R after EGF extraction by acidic treatment, strongly supports the interpretation that cross-phosphorylation of EGF-R may take place in intact cells.     

The epidermal growth factor receptor tyrosine kinase in liver epithelial cells. The effect of ligand-dependent changes in cellular location

Epidermal growth factor (EGF) activates the intrinsic tyrosine-specific protein kinase of its receptor (EGF-R). We studied the effect of EGF-dependent EGF-R internalization on receptor autophosphorylation and on the appearance of tyrosine phosphoproteins in rat liver epithelial cells. Peak receptor autophosphorylation activity (3- to 6-fold over basal) was found in homogenates of EGF-treated cells at times when the majority of receptors (greater than 90%) had been internalized but not yet degraded (15 to 30 min). Stimulated activity persisted for at least 2 h if EGF-R degradation was blocked by methylamine or 18 degrees C incubation. Detection of stimulated autophosphorylation in homogenates of cells treated with EGF in culture required detergent in the assay. Detergent was not necessary to detect stimulated autophosphorylation when EGF was added directly to homogenates of untreated cells. Immunoblots using antibodies against phosphotyrosine (p-Tyr) demonstrated that EGF treatment of intact cells increased the p-Tyr content of at least seven proteins (EGF-R, 115, 100, 75, 66, 57, and 52 kDa) within 5 s. Incubation of intact cells with EGF at 0 degrees C to prevent endocytosis still resulted in tyrosine phosphorylation of these seven proteins. In contrast, several substrates (120, 78, and 38 kDa) showed delayed increases (45-90 s) in tyrosine phosphorylation at 37 degrees C; their phosphorylation was even slower at 18 degrees C and did not occur at 0 degrees C. In cells incubated with EGF at 18 degrees C or in the presence of methylamine, EGF-R p-Tyr in the intact cell was lost by 2 h even though receptor was not degraded and still exhibited enhanced autophosphorylation in the homogenate assay. These findings suggest that tyrosine phosphorylation in response to EGF occurs predominantly during the initial stages of endocytosis and is mediated for the most part by ligand-receptor complexes at the cell surface. A subset of phosphorylations may require intracellular movement.     

Polyoma middle T antigen or v-src desensitizes human epidermal growth factor receptor function and interference by a monensin-resistant mutation in mouse Balb/3T3 cells.

Epidermal growth factor (EGF)-induced down-regulation of its receptor is an obligatory pathway for cellular regulation of EGF-specific receptor (EGF-R) in normal and malignant cells. BNER4 cells are mouse Balb/3T3 cells transfected with the human EGF-R complementary DNA (cDNA). Polyoma middle T antigen-transfectants of BNER4, B4/MT-2, B4/MT-13, B4/MT-23, and B4/MT-24, showed diminished down-regulation of cell surface human EGF-R in response to EGF relative to the parental BNER4 cells. Also, the v-src-transfectants B4/SRC-13 and B4/SRC-24 showed much less down-regulation than BNER4 cells, whereas H-ras-transfectants of BNER4, B4/RAS-24 and B4/RAS-25, showed EGF-induced down-regulation of the cell surface EGF-R similar to that of BNER4. EGF induced DNA synthesis more than 20-fold in BNER4, but induced only about a 1.5- to 6-fold increase in the middle T antigen- and v-src-transfectants. EGF-Rs of the middle T antigen-transfectants were metabolically stable in the presence of EGF in comparison with their parental BNER4 cells. EGF-Rs of BNER4 cells degraded with half-lives of about 2 h in the presence of EGF, but those of the middle T antigen transformants were found to be highly stabilized in the presence of EGF. On the other hand, transfection with polyoma middle T antigen (MTAg) cDNA causes malignant transformation of Balb/3T3 cells, but not its monensin (an ionophoric antibiotic)-resistant mutant MO-5 cells, which have no significant EGF binding activity. Transfection of human EGF-R cDNA into MO-5 leads to the expression of high levels of human EGF-R in MNER31. Unlike the polyoma MTAg transfectants of BNER4, EGF-R in polyoma MTAg cDNA-transfectants into MNER31, M31/MT-13 and M31/MT-14, were down-regulated to levels similar to those of their parental MNER31. Exposure to EGF induced a more than 10-fold increase in DNA synthesis of quiescent BNER4, MNER31, M31/MT-13, and M31/MT-14 cells. Polyoma middle T antigen or v-src appears to modulate EGF-induced down-regulation of EGF-R, possibly through interaction of the receptor with the viral oncogenes, and this interaction may be altered in the mutant.     

A chimeric EGF-R-neu proto-oncogene allows EGF to regulate neu tyrosine kinase and cell transformation.

The neu oncogene, characterized by Weinberg and colleagues, is a transforming gene found in ethylnitrosourea-induced rat neuro/glioblastomas; its human proto-oncogene homologue has been termed erbB2 or HER2 because of its close homology with the epidermal growth factor receptor (EGF-R) gene (c-erbB1). Expression of the rat neu oncogene is sufficient for transformation of mouse NIH 3T3 fibroblasts in culture and for the development of mammary carcinomas in transgenic mice, but the neu proto-oncogene has not been associated with cell transformation. We constructed a vector for expression of a chimeric cDNA and hybrid protein consisting of the EGF-R extracellular, transmembrane and protein kinase C-substrate domains linked to the intracellular tyrosine kinase and carboxyl terminal domain of the rat neu cDNA. Upon transfection with the construct, NIH 3T3 cells gave rise to EGF-R antigen-positive cell clones with varying amounts of specific EGF binding. Immunofluorescence and immunoprecipitation using neu- and EGF-receptor specific antibodies demonstrated a correctly oriented and positioned chimeric EGF-R-neu protein of the expected apparent mol. wt on the surface of these cells. EGF or TGF alpha induced tyrosine phosphorylation of the chimeric receptor protein, stimulated DNA synthesis of EGF-R-neu expressing cells and led to a transformed cell morphology and growth in soft agar. In contrast, the neu proto-oncogene did not show kinase activity or transforming properties when expressed at similar levels in NIH 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)