Regulation of phosphorylation of the c-erbB-2/HER2 gene product by a monoclonal antibody and serum growth factor(s) in human mammary carcinoma cells.

Monoclonal antibody (MAb) 4D5 was used to analyze the phosphorylation of p185HER2, the gene product of c-erbB-2/HER2, in SK-BR-3 cells. Culture in the continuous presence of 4D5 reduced the in vivo steady-state levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4D5 may have interfered with the activation of phosphorylation of p185HER2. The observed MAb-mediated reduction of p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were grown under serum-free conditions, the steady-state levels of p185HER2 phosphorylation were reduced by 56%, and addition of 4D5 further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure to increasing concentrations of newborn calf serum in these cultures, there was a linear increase in tyrosine-specific phosphorylation of p185HER2, reaching a 5.4-fold increase with 10% newborn calf serum. Phosphorylation of p185HER2 in the presence of newborn calf serum was not attributable to stimulation of the epidermal growth factor receptor by epidermal growth factor or by transforming growth factor-alpha. Extension of these observations to two other mammary carcinoma cell lines. MDA-MB-453 and BT-474, also demonstrated a significant capacity of serum to induce p185HER2 phosphorylation. The demonstration of antibody-mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma cells may also produce and secrete a factor or factors which may activate p185HER2. Our observation that growth-inhibitory MAb 4D5 is able to reduce the phosphorylation of p185HER2 by newborn calf serum and by a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HER2 as a signal transduction pathway to regulate cell proliferation.     

Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells.

The HER2/neu gene encodes a receptor tyrosine kinase that is highly homologous to the epidermal growth factor receptor. Overexpression of the receptor in mammary and ovarian carcinoma correlates with poor patient prognosis. To determine how the overexpression of a normal receptor leads to the generation of an oncogenic signal, we compared the patterns of tyrosine phosphorylation in tumor-derived human cell lines expressing high levels of p185HER2/neu. In intact SKBR3 cells, basal phosphorylation of p185HER2/neu was not detected. However, pretreatment of cells with the tyrosine phosphatase inhibitor, sodium orthovanadate, led to the detection of phosphotyrosine on phospholipase C-gamma (PLC-gamma), GTPase-activating protein but not on the RAF-1 kinase. Strikingly, PLC-gamma was detected in a complex which contained multiple tyrosine-phosphorylated polypeptides. This complex was detected only in cytoplasmic fractions and had a distinct composition in different p185HER2/neu-overexpressing cell lines. Although GTPase-activating protein has been found previously in association with proteins of 190 and 62 kDa in fibroblasts, in SKBR3 cells it was found associated with multiple additional tyrosine-phosphorylated polypeptides. These experiments show that SKBR3 cells possess high levels of protein tyrosine phosphatase that can act upon p185HER2/neu. Moreover, they reveal, for the first time, the presence of PLC-gamma and GTPase-activating protein in cytosolic complexes containing a variety of other tyrosine-phosphorylated polypeptides. These observations suggest novel possibilities for the specific definition of receptor-generated signals in tumor cells.     

p185HER2 signal transduction in breast cancer cells.

A partially agonistic monoclonal antibody, 4D5, known to bind to the extracellular domain of p185HER2 and shown to inhibit long term growth of p185HER2-overexpressing breast cancer cells, was used to study signal transduction and phosphotyrosyl protein substrates associated with this receptor. Normal breast epithelial cells and breast carcinoma cells expressing low levels of p185HER2 were not affected by 4D5. HER2/neu-overexpressing breast cancer cells (BT-474 and SK-Br-3) exposed to 4D5 exhibited rapid phosphorylation of both p185HER2 and an associated 56-kDa phosphotyrosyl protein (ptyr56). Paralleling the 4D5- stimulated phosphorylation of p185HER2 and ptyr56 was a 5-10-fold induction of c-fos mRNA and phosphatidylinositol 4-kinase activity and a 2-fold induction of inositol 1,4,5-trisphosphate 3'-kinase activity. The increased phosphatidylinositol 4-kinase activity immunoprecipitated with p185HER2 and also co-eluted with ptyr56 from an antiphosphotyrosine immunoaffinity column. These results indicate that short term (less than 6 h) 4D5 activation of p185HER2 in overexpressing breast cancer cells produces agonistic-like signaling typical of homologous tyrosine kinase growth factor receptors such as epidermal growth factor receptor. The data also suggest that ptyr56 represents a novel phosphorylated substrate associated with 4D5-stimulated p185HER2.     

p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor.

The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.     

Role of HER2 gene overexpression in breast carcinoma

The HER2 proto-oncogene encodes a transmembrane glycoprotein of 185 kDa (p185(HER2)) with intrinsic tyrosine kinase activity. Amplification of the HER2 gene and overexpression of its product induce cell transformation. Numerous studies have demonstrated the prognostic relevance of p185(HER2), which is overexpressed in 10% to 40% of human breast tumors. Recent data suggest that p185(HER2) is a ligand orphan receptor that amplifies the signal provided by other receptors of the HER family by heterodimerizing with them. Ligand-dependent activation of HER1, HER3, and HER4 by EGF or heregulin results in heterodimerization and, thereby, HER2 activation. HER2 overexpression is associated with breast cancer patient responsiveness to doxorubicin, to cyclophosphamide, methotrexate, and fluorouracil (CMF), and to paclitaxel, whereas tamoxifen was found to be ineffective and even detrimental in patients with HER2-positive tumors. In vitro analyses have shown that the role of HER2 overexpression in determining the sensitivity of cancer cells to drugs is complex, and molecules involved in its signaling pathway are probably the actual protagonists of the sensitivity to drugs. The association of HER2 overexpression with human tumors, its extracellular accessibility, as well as its involvement in tumor aggressiveness are all factors that make this receptor an appropriate target for tumor-specific therapies. A number of approaches are being investigated as possible therapeutic strategies that target HER2: (1) growth inhibitory antibodies, which can be used alone or in combination with standard chemotherapeutics; (2) tyrosine kinase inhibitors (TKI), which have been developed in an effort to block receptor activity because phosphorylation is the key event leading to activation and initiation of the signaling pathway; and (3) active immunotherapy, because the HER2 oncoprotein is immunogenic in some breast carcinoma patients.     

Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo

Monoclonal antibodies specific for the p185HER2/neu growth factor receptor represent a significant advance in receptor-based therapy for p185HER2/neu-expressing human cancers. We have used a structure-based approach to develop a small (1.5 kDa) exocyclic anti-HER2/neu peptide mimic (AHNP) functionally similar to an anti-p185HER2/neu monoclonal antibody, 4D5 (Herceptin). The AHNP mimetic specifically binds to p185HER2/neu with high affinity (KD=300 nM). This results in inhibition of proliferation of p185HER2/neu-overexpressing tumor cells, and inhibition of colony formation in vitro and growth of p185HER2/neu-expressing tumors in athymic mice. In addition, the mimetic sensitizes the tumor cells to apoptosis when used in conjunction with ionizing radiation or chemotherapeutic agents. A comparison of the molar quantities of the Herceptin antibody and the AHNP mimetic required for inhibiting cell growth and anchorage-independent growth showed generally similar activities. The structure-based derivation of the AHNP represents a novel strategy for the design of receptor-specific tumor therapies.     

Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies

The HER2 protooncogene encodes a receptor tyrosine kinase, p185^HER2. The overexpression of p185^HER2 has been associated with a worsened prognosis in certain human cancers. In the present work we have screened a variety of different tumor cell lines for p185^HER2 expression using both enzyme-linked immunosorbent and fluorescence-activated cell sorting assays employing murine monoclonal antibodies directed against the extracellular domain of the receptor. Increased levels of p185^HER2 were found in breast (5/9), ovarian (1/6), stomach (2/3) and colorectal (5/16) carcinomas, whereas all kidney and submaxillary adenocarcinoma cell lines tested were negative. Some monoclonal antibodies directed against the extracellular domain of p185^HER2 inhibited growth in monolayer culture of breast and ovarian tumor cell lines overexpressing p185^HER2, but had no effect on the growth of colon or gastric adenocarcinomas expressing increased levels of this receptor. The most potent growth-inhibitory anti-p185^HER2 monoclonal antibody in monolayer culture, designated mumAb 4D5 (a murine IgG1 antibody), was also tested in soft-agar growth assays for activity against p185^HER2-overexpressing tumor cell lines of each type, with similar results. In order to increase the spectrum of tumor types potentially susceptible to monoclonal antibody-mediated anti-p185^HER2 therapies, to decrease potential immunogenicity issues with the use of murine monoclonal antibodies for human therapy, and to provide the potential for antibody-mediated cytotoxic activity, a mouse/human chimeric 4D5 (chmAb 4D5) and a humanized 4D5 (rhu)mAb 4D5 HER2 antibody were constructed. Both engineered antibodies, in combination with human peripheral blood mononuclear cells, elicited antibody-dependent cytotoxic responses in accordance with the level of p185^HER2 expression. Since this cytotoxic activity is independent of sensitivity to mumAb 4D5, the engineered monoclonal antibodies expand the potential target population for antibody-mediated therapy of human cancers characterized by the overexpression of p185^HER2.     

Hyaluronan promotes CD44v3-Vav2 interaction with Grb2-p185(HER2) and induces Rac1 and Ras signaling during ovarian tumor cell migration and growth

In this study we initially examined the interaction between CD44v3 (a hyaluronan (HA) receptor) and Vav2 (a guanine nucleotide exchange factor) in human ovarian tumor cells (SK-OV-3.ipl cell line). Immunological data indicate that both CD44v3 and Vav2 are expressed in SK-OV-3.ipl cells and that these two proteins are physically linked as a complex in vivo. By using recombinant fragments of Vav2 and in vitro binding assays, we have detected a specific binding interaction between the SH3-SH2-SH3 domain of Vav2 and the cytoplasmic domain of CD44. In addition, we have observed that the binding of HA to CD44v3 activates Vav2-mediated Rac1 signaling leading to ovarian tumor cell migration. Further analyses indicate that the adaptor molecule, growth factor receptor-bound protein 2 (Grb2) that is bound to p185(HER2) (an oncogene product), is also associated with the CD44v3-Vav2 complex. HA binding to SK-OV-3.ipl cells promotes recruitment of both Grb2 and p185(HER2) to the CD44v3-Vav2 complex leading to Ras activation and ovarian tumor cell growth. In order to determine the role of Grb2 in CD44v3 signaling, we have transfected SK-OV-3.ipl cells with Grb2 mutant cDNAs (e.g. Delta N-Grb2 that has a deletion in the amino-terminal SH3 domain or Delta C-Grb2 that has a deletion in the carboxyl-terminal SH3 domain). Our results clearly indicate that the SH3 domain deletion mutants of Grb2 (i.e. the Delta N-Grb2 (and to a lesser extent the Delta C-Grb2) mutant) not only block their association with p185(HER2) but also significantly impair their binding to the CD44v3-Vav2 complex and inhibit HA/CD44v3-induced ovarian tumor cell behaviors. Taken together, these findings strongly suggest that the interaction of CD44v3-Vav2 with Grb2-p185(HER2) plays an important role in the co-activation of both Rac1 and Ras signaling that is required for HA-mediated human ovarian tumor progression.     

Binding-induced activation of overexpressed p185HER2 is essential in triggering neuronal differentiation of PC12 cells

To determine whether p185^HER2 overexpression per se triggers p185^HER2 cellular signaling or whether an extracellular signal is required, we transfected PC12 cells with the human erbB-2 proto-oncogene, and established a cell line that overexpresses p185^HER2. PC12-HER2 cells, maintained in suspension culture or plated on a collagen layer, showed the same morphology and growth rate as PC12 and PC12 mock-transfected control cells. When treated with monoclonal antibody (MAb) MGr6 or other anti-p185^HER2 MAbs, PC12–HER2 cells specifically underwent neuronal differentiation comparable to that induced by nerve growth factor (NGF), and the differentiation-inducing effect of the MAb was dramatically enhanced by the addition of a second anti-mouse IgG. MAb-induced cell differentiation correlated with p185^HER2 phosphorylation, recruitment of Shc and Grb-2 transducer molecules into complexes, and MAPK phosphorylation. These data indicate the requirement for a specific binding-induced activation of the overexpressed p185^HER2 receptor in inducing PC12 cell differentiation. PC12-HER2 cells represent a suitable system for selection of p185^HER2-activating ligands (peptides, phage-displayed peptides or proteins) or specific inhibitors of its tyrosine kinase activity. J. Cell. Biochem. 67:316–326, 1997. © 1997 Wiley-Liss, Inc.     

Modulation of a Mr 175,000 c-neu receptor isoform in G8/DHFR cells by serum starvation

The neu proto-oncogene product has been found to exist in two interconvertible forms in G8/DHFR mouse fibroblasts. The 185-kilodalton form (p185) present in growing cells is replaced by a 175-kilodalton form (p175) under conditions of serum starvation. This low molecular weight form accounts almost exclusively for the phosphotyrosine content of the receptor and is associated with increased tyrosine kinase activity. Addition of serum, platelet-derived growth factor or tumor promoter induces conversion of p175 to p185 within minutes, and this increase in molecular weight is associated with phosphorylation of serine and threonine; removal of serum growth factors is followed by replacement of p185 with p175 over several hours. Unlike G8/DHFR cells, the human breast cancer cell line SK-Br-3 expresses a high molecular weight neu/HER2 receptor with unchanged phosphotyrosine content in both serum-starved and serum-stimulated cultures. These findings indicate that activation of the neu proto-oncogene product in G8/DHFR cells may be regulated in part by protein kinase C-mediated receptor transmodulation rather than by ligand availability alone.     

The presence of c-erbB-2 gene product-related protein in culture medium conditioned by breast cancer cell line SK-BR-3

The Mr 185,000 glycoprotein encoded by human c-erbB-2/neu/HER2 gene, termed c-erbB-2 gene product, shows a close structural similarity with epidermal growth factor receptor and is now regarded to be a growth factor receptor for an as yet unidentified ligand. Abundant c-erbB-2 mRNA was demonstrated by Northern blot studies in the human breast cancer cell line SK-BR-3. Cellular radiolabeling experiments followed by immunoprecipitation with three different anti-c-erbB-2 gene product antibodies, recognizing extracellular domain, kinase domain, and carboxyl-terminal portion, respectively, demonstrated the production of a large amount of c-erbB-2 gene product which had the capacity to be phosphorylated. Immunization of mice with concentrated culture medium conditioned by SK-BR-3 cells always generated antibodies against c-erbB-2 gene product, demonstrating that this culture medium contained substance(s) immunologically indistinguishable from c-erbB-2 gene product. This observation was supported by the successful development of a monoclonal antibody against c-erbB-2 gene product, GFD-OA-p185-1, by immunizing mice with this culture medium. The biochemical nature of the substance(s) present in the culture medium was further characterized. When the culture medium conditioned by [35S]cysteine-labeled SK-BR-3 cells was immunoprecipitated by three different anti-c-erbB-2 gene product antibodies, only the antibody recognizing extracellular domain precipitated the [35S]-labeled protein with a molecular weight of 110,000, namely p110. The newly developed monoclonal antibody also immunoprecipitated this protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterodimerization of c-erbB2 with different epidermal growth factor receptor mutants elicits stimulatory or inhibitory responses.

Ligand-induced dimerization of growth factor receptors is crucial for stimulation of their intrinsic protein tyrosine kinase activity promoting receptor autophosphorylation by an intermolecular mechanism. Moreover, the suppressive and negative dominant action of defective epidermal growth factor receptor (EGFR) was shown to be caused by formation of inactive heterodimers with normal EGFR leading to diminished biological signaling. In this report we explore the structural requirements and functional significance of heterodimerization between EGFR and HER2. HER2 (also called c-erbB-2 or neu) is a member of the EGFR family whose natural ligand is still unknown. We show that in response to EGF, wild type EGFR and various EGFR mutants were able to undergo heterodimerization with HER2. Addition of EGF to transfected cells co-expressing HER2 with a kinase negative point mutant of EGFR (K721A) stimulated heterodimer formation, tyrosine phosphorylation of K721A and HER2, and tyrosine phosphorylation of one of their known substrates, phospholipase C gamma. However, the binding of EGF to transfected cells co-expressing HER2 together with another EGFR mutant CD533 (a deletion mutant lacking most of the cytoplasmic domain of EGFR) caused heterodimerization and inhibition of tyrosine kinase activity. It appears therefore that EGF-induced heterodimerization of EGFR and HER2 can promote either stimulatory or inhibitory influences on kinase activity. We propose that the nature of receptor interactions on the cell surface can either activate or inhibit the initiation of growth factor-controlled cellular signaling.     

A high-throughput migration assay reveals HER2-mediated cell migration arising from increased directional persistence

Human epidermal growth factor receptor 2 (HER2) overexpression has been associated with increased invasiveness in mammalian breast cancer cell lines, but the effects of overexpression on key underlying cell migration properties such as translational speed and directional persistence are not understood. Moreover, the differential effect of HER2 activation through heterodimerization with epidermal growth factor receptor versus human epidermal growth factor receptor 3 (HER3) on cell speed and persistence has not been studied. To investigate these issues, we developed a high-throughput wound closure assay in which individual cell locomotion and wound closure kinetics were quantified in human mammary epithelial cells with varying levels of HER2 under epidermal growth factor or heregulin (a HER3 ligand) stimulation. Increasing levels of HER2 elevated wound closure with closure kinetics dependent on ligand treatment. Cell speed increased with HER2 levels under epidermal growth factor treatment, but decreased under heregulin treatment. In contrast, directional persistence increased with HER2 levels under both ligand treatments. Increasing persistence quantitatively accounted for observed elevated wound closure, as measured by the effective diffusion of the cells. Taken together, the data show that the HER2 overexpression mediates cell migration through differential control of translational speed and directional persistence dependent on epidermal growth factor receptor-HER2 versus HER2-HER3 heterodimerization. Observed consistent increases in persistence associated with HER2 overexpression indicate a prospective mechanism for invasiveness previously documented in HER2-overexpressing human breast tumors.     

All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor

Activation of the epidermal growth factor (EGF) receptor kinase leads to autophosphorylation and to the phosphorylation of various cellular substrates. The three known autophosphorylation sites of EGF receptor are located at the carboxyl-terminal tail where they probably act to compete with and thus modulate substrate phosphorylation. Mutational analysis and microsequencing techniques have been used to localize and identify new autophosphorylation site(s) of the EGF receptor. We have compared the phosphopeptide maps of human EGF receptor, and two deletion mutants lacking 63 and 126 amino acids from the carboxyl-terminal tail with the phosphopeptide maps of HER/neu and a chimeric EGF receptor containing the carboxyl-terminal tail of HER2/neu. HER2/neu is highly homologous to the EGF receptor, and it probably functions as a growth factor receptor for as yet unidentified growth factor. On the basis of this analysis, we have concluded that all autophosphorylation sites of EGF receptor and HER2/neu are located in their carboxyl-terminal tails. Utilizing the EGF receptors with carboxyl-terminal deletions, we were also able to identify tyr1086 as an additional autophosphorylation site of EGF receptor. Direct microsequencing of a phosphorylated tryptic peptide from the human EGF receptor confirmed this assignment.     

Antibody-induced activation of p185HER2 in the human lung adenocarcinoma cell line Calu-3 requires bivalency

In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185^HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by theHER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185^HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amount of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185^HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185^HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185^HER2 receptor.     

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.     

HER2 cytoplasmic domain generates normal mitogenic and transforming signals in a chimeric receptor.

We have investigated the biological function of an unidentified human growth factor, the ligand of the putative HER2 receptor, by characterizing the signalling properties of its receptor. HER2 (or c-erbB-2), the human homolog of the rat neu proto-oncogene, encodes a transmembrane glycoprotein of the tyrosine kinase family that appears to play an important role in human breast carcinoma. Since a potential ligand for HER2 has not yet been identified, it has been difficult to analyze the biochemical properties and biological function of this cell surface protein. For this reason, we replaced the HER2 extracellular domain with the closely related ligand binding domain sequences of the epidermal growth factor (EGF) receptor, and examined the ligand-induced biological signalling potential of this chimeric HER1-2 protein. This HER1-2 receptor is targetted to the cell surface of transfected NIH 3T3 cells, forms high and low affinity binding sites, and generates normal mitogenic and cell transforming signals upon interaction with EGF or TGF alpha. The constitutive activation of wild-type HER2 in transfected NIH 3T3 cells suggests the possibility that these cells synthesize the as yet unidentified HER2 ligand and activate HER2 by an autocrine mechanism.