Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells

The epidermal growth factor receptor (EGFR) gene is frequently amplified and/or overexpressed in human malignancies. To investigate the biological effects of its overexpression, we constructed a eukaryotic vector containing human EGFR cDNA. Introduction of this construct led to reconstitution of functional EGF receptors in NR6 mutant cells, which are normally devoid of this receptor. Transfection of NIH 3T3 resulted in no significant alterations in growth properties. However, EGF addition led to the formation of densely growing transformed foci in liquid culture and colonies in semisolid medium. NIH 3T3-EGFR clonal lines, which expressed the EGF at 500- to 1000-fold levels over control NIH 3T3 cells, demonstrated a marked increase in DNA synthesis in response to EGF. Thus EGF receptor overexpression appears to amplify normal EGF signal transduction. Finally, high levels of EGFR expression, which conferred a transformed phenotype to NIH 3T3 cells in the presence of ligand, were demonstrated in representative human tumor cell lines that contained amplified copies of the EGFR gene.     

Transmembrane TGF-alpha precursors activate EGF/TGF-alpha receptors

TGF-alpha and EGF are structurally related factors that bind to and induce tyrosine autophosphorylation of a common receptor. Proteolytic cleavage of the transmembrane TGF-alpha precursor's external domain releases several TGF-alpha species. However, membrane-bound TGF-alpha forms remain on the surface of TGF-alpha-expressing cell lines. To evaluate the biological activity of these forms, we modified two cleavage sites in the TGF-alpha precursor coding sequence, making processing into the 50 amino acid TGF-alpha impossible. Overexpression of this cDNA in a receptor-negative cell line, partial purification, and N-terminal sequence analysis indicate the existence of two transmembrane TGF-alpha forms. These solubilized precursors induce tyrosine autophosphorylation of the EGF/TGF-alpha receptor in intact receptor-overexpressing cells, and anchorage-independent growth of NRK fibroblasts. Cell-cell contact between TGF-alpha precursor-overexpressing cells and cells expressing high numbers of receptors also resulted in receptor activation. These findings suggest a role for transmembrane TGF-alpha forms in intercellular interactions in proliferating tissues.     

Cytotoxic activities of a fusion protein comprised of TGF alpha and Pseudomonas exotoxin

A cDNA encoding transforming growth factor type alpha (TGF alpha) was fused to the 5' end of a gene encoding a modified form of Pseudomonas exotoxin A (PE), which is devoid of the cell recognition domain (domain Ia). The chimeric molecule, termed TGF alpha-PE40, was expressed in Escherichia coli and isolated from the periplasm or inclusion bodies depending on the construction expressed. TGF alpha-PE40 was found to be extremely cytotoxic to cells displaying epidermal growth factor (EGF) receptors. Comparison with a similar molecule in which TGF alpha was placed at the carboxyl end of PE40 demonstrated the importance of the position of the cell recognition element; TGF alpha-PE40 was found to be about 30-fold more cytotoxic to cells bearing EGF receptors than PE40-TGF alpha. In addition, TGF alpha-PE40 was shown to be extremely cytotoxic to a variety of cancer cell lines including liver, ovarian, and colon cancer cell lines, indicating high levels of EGF receptor expression in these cells.     

Abelson transformed fibroblasts lacking the EGF receptor are not tumourigenic in nude mice.

Cells transformed by the v-abl-oncogene produce large amounts of the tumour growth factor alpha TGF. alpha TGF is homologous to the epidermal growth factor (EGF) and stimulates cell growth via the EGF receptor pathway. To separate metabolic events in the v-abl-transformed cells mediated by alpha TGF as opposed to the v-abl-encoded protein-tyrosine kinase, we have employed the Swiss 3T3 variant cell line NR6 which lacks a functional EGF receptor. v-abl was found to transform efficiently NR6 cells in vitro. These transformed NR6 cells displayed a variety of in vitro properties which were indistinguishable from transformed wild-type fibroblast lines. However, in contrast to the wild-type lines, v-abl-transformed NR6 cells failed to form tumours when injected into athymic nude mice. These results imply an important function for alpha TGF and the EGF receptor in the establishment of the v-abl-induced fibrosarcomas.     

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.     

Human esophageal carcinoma cells have fewer, but higher affinity epidermal growth factor receptors

Squamous cell carcinomas have recently been shown to contain increased numbers of epidermal growth factor (EGF) receptors. Since EGF has an important role in epithelial growth and differentiation, it is possible that modulation of its receptor may have an important role in neoplasia. In an attempt to further explore the relationship of EGF receptor expression to malignant transformation, we examined 14 squamous cell carcinoma cell lines of the esophagus for the number and affinity of EGF receptors. Seven cell lines were newly isolated by this laboratory and recently characterized. The seven additional cell lines were obtained from Japan (4 cell lines) and South Africa (3 cell lines). Surprisingly, we found that esophageal carcinomas contained lowered quantities of surface EGF receptors (2- to 100-fold) and that the affinity of the EGF receptor was increased (6- to 100-fold) when compared to normal esophageal epithelial cells. Moreover, the biologic response of esophageal carcinoma cells to EGF differed markedly from that of other squamous cell tumor cells exhibiting elevated numbers of receptors, such as A431 and SCC-15. Human esophageal carcinoma cells were maximally stimulated by the addition of 5 ng/ml of EGF, similar to normal esophageal keratinocytes, but in contrast to normal cells were not inhibited by the higher concentrations tested (up to 40 ng/ml). On the other hand, addition of any EGF to the medium (beyond that normally present in serum) was found to dramatically inhibit the growth of A431 and SCC-15 cells. Our findings indicate that squamous cell neoplasia is not dependent upon increased numbers of cell surface EGF receptors, that EGF receptor number may have a determinant role in EGF cell toxicity, and that the stimulatory response of cells to EGF may reflect a complex function of EGF receptor number, affinity, and occupancy.     

Effects of epidermal growth factor on transferrin receptor phosphorylation and surface expression in malignant epithelial cells

The transferrin (Tf) receptor is a major transmembrane protein which provides iron for normal and malignant cell growth. Epidermal growth factor (EGF) has been reported to rapidly and transiently alter the number of surface Tf receptors in normal and transformed epithelial cells. To investigate mechanisms of EGF-induced changes in surface Tf display, EGF effects on surface Tf receptors were compared in two cell lines which differ in their number of EGF receptors and growth responses to EGF. In cloned A431 cells with high receptor numbers which are growth-inhibited by EGF, EGF caused a 50% decrease in Tf receptor expression after 30 min. In contrast, EGF induced a rapid, transitory increase (within 5 min) in the number of surface Tf receptors on KB carcinoma cells which returned to basal levels by 15 min. The observed changes in Tf receptor display were due to altered receptor distribution and not changes in ligand affinity or total cellular transferrin receptor pools. Anti-EGF receptor monoclonal antibody blocked effects of EGF on transferrin receptor expression. Since the antibody is internalized and causes EGF receptor down-regulation, effects on transferrin receptor expression were independent of these events. EGF-induced alterations in Tf receptor display occurred even when cells were pretreated with colchicine, suggesting that changes in surface Tf binding were not mediated by cytoskeletal components. Na orthovanadate, which mimics some early cellular effects of EGF, duplicated EGF's effects on A431 Tf receptors, but had no effect on KB cells, suggesting these responses occur by differing mechanisms. To determine whether EGF caused changes in Tf receptor phosphorylation, 32P-labelled Tf receptors were immunoprecipitated after EGF treatment. After exposure to EGF, A431 cells showed no change in Tf phosphorylation, but KB cells showed a transient, 6-fold increase in transferrin receptor phosphorylation on serine residues. In both A431 and KB cells, phorbol ester (PMA) also increased phosphorylation on transferrin receptors, but had little effect on surface Tf receptor expression. In malignant cell lines, EGE induces rapid, variable changes in transferrin receptor expression and phosphorylation which differ from the effects of PMA. These early responses to EGF appear to differ with the cell type and correlate poorly with alterations in Tf receptor phosphorylation. These results suggest Tf receptor phosphorylation does not regulate Tf receptor display in all cells.     

ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes

The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these growth factor receptors.     

EGF receptor activity and mitogenic response of Balb/3T3 cells expressing Ras and Myc oncogenes. EGF receptor activity in oncogene transformed cells.

EGF receptors are found on the surface of most cells, usually with high and low binding affinities. To investigate functional relationships between EGF (EGF-like growth factors) and oncogenes we have characterized the expression of the epidermal growth factor receptor (EGFr) in H-Ras, v-Myc, and H-Ras-v-Myc transformed Balb/3T3 cells. H-Ras cells show a marked decrease in the number of EGFr molecules per cell compared to parental cells. v-Myc and H-Ras-v-Myc transformed cells express an intermediate level of receptors. The majority of the EGF receptors on the parental and oncogene transformed cells bind EGF with low affinity and this low affinity receptor is down-regulated by oncogene transformation. v-Myc expression, in the H-Ras-v-Myc transformed cells, abrogates the receptor down-regulation seen with H-Ras transformation. The mechanism of abrogation is not a result of a change in the p21-Ras concentration in the H-Ras-v-Myc transformed cells. In addition, the mitogenic response to EGF was examined. H-Ras and H-Ras-v-Myc transformed cells do not respond to EGF mitogenically. In contrast, EGF stimulates DNA synthesis in parental cells and v-Myc transfected cells; this result suggests that growth promoting signals from the EGF receptor may not be required in H-Ras transformed cells.     

A mutant epidermal growth factor receptor with defective protein tyrosine kinase is unable to stimulate proto-oncogene expression and DNA synthesis.

Cultured NIH-3T3 cells devoid of endogenous epidermal growth factor (EGF) receptors were transfected with cDNA expression constructs encoding either normal human EGF receptor or a receptor mutated in vitro at Lys-721, a residue that is thought to function as part of the ATP-binding site of the kinase domain. Unlike the wild-type EGF-receptor expressed in these cells, which exhibited EGF-dependent protein tyrosine kinase activity, the mutant receptor lacked protein tyrosine kinase activity and was unable to undergo autophosphorylation and to phosphorylate exogenous substrates. Despite this deficiency, the mutant receptor was normally expressed on the cell surface, and it exhibited both high- and low-affinity binding sites. The addition of EGF to cells expressing wild-type receptors caused the stimulation of various responses, including enhanced expression of proto-oncogenes c-fos and c-myc, morphological changes, and stimulation of DNA synthesis. However, in cells expressing mutant receptors, EGF was unable to stimulate these responses, suggesting that the tyrosine kinase activity is essential for EGF receptor signal transduction.     

Kinetics of tyrosine phosphorylation and internalization of human EGF receptors overexpressed in NIH 3T3 fibroblasts

Binding of epidermal growth factor (EGF) to cells rapidly induces tyrosine phosphorylation of its receptor which is followed by its internalization and dephosphorylation. The kinetics of these processes differs widely in time from minutes to hours according to cell types. In this paper we analyzed EGF receptor phosphorylation and down-regulation in NIH 3T3 cells transfected with the recombinant hEGF-R cDNA which express 4 X 10(5) receptors/cell. In the presence of EGF receptor phosphorylation reached a maximum after 1 min and was then maintained for about 1 h, while during this time the number of EGF-binding sites was reduced to 40% of the initial number. Detailed analysis of the fate of a population of receptors previously activated and autophosphorylated at 4 degrees C, after warming to 37 degrees C in the absence of the ligand, showed that internalization of the cell surface-associated EGF and dephosphorylation of the receptor were rapid (t1/2 15 min) and followed a similar kinetics. Our data indicate that at any given time only a fraction of the total cell surface receptors is phosphorylated on tyrosine and that dephosphorylation occurs at the cell surface or very rapidly after internalization. In addition the data also suggest that a certain recycling of previously internalized receptors may occur in these cells during EGF treatment.     

Reduction of epidermal growth factor receptor affinity by heterologous ligands: evidence for a mechanism involving the breakdown of phosphoinositides and the activation of protein kinase C

The tetradecapeptide bombesin converts epidermal growth factor (EGF) receptors on Swiss 3T3 cells from a high affinity state (KD = 9.8 X 10(-11)M) to a lower affinity state (KD = 1.8 X 10(-9)M). This conversion occurs when the cells are incubated with bombesin at 37 degrees C but not when incubated at 4 degrees C. Previously, a number of other (chemically unrelated) cell growth-promoting peptides and polypeptides have been shown to induce a similar indirect, temperature-dependent reduction of EGF receptor affinity. We have now demonstrated that hormones and growth factors which cross-regulate EGF receptor affinity in Swiss 3T3 cells have a common ability to stimulate the breakdown of phosphoinositides in these cells. We propose that the reduction of EGF receptor affinity is a consequence of the activation of protein kinase C by the diacylglycerol generated by this breakdown. In support of this proposal we have found that exogenously added diacylglycerol reduces the affinity of the Swiss 3T3 cell EGF receptor.     

Amino acid determinants of alpha 7 nicotinic acetylcholine receptor surface expression

Transient transfection has not been a successful method to express the alpha7 nicotinic acetylcholine receptor such that these receptors are detected on the cell surface. This is not the case for all ligand-gated ion channels. Transient transfection with the 5-hydroxytryptamine type 3 subunit cDNA results in detectable surface receptor expression. Cell lines stably expressing the alpha7 nicotinic acetylcholine receptor produce detectable, albeit variable, levels of surface receptor expression. alpha7 nicotinic acetylcholine receptor surface expression is dependent, at least in part, on cell-specific factors. In addition to factors provided by the cells used for receptor expression, we hypothesize that the surface expression level in transfected cells is an intrinsic property of the receptor protein under study. Employing a set of alpha7-5-hydroxytryptamine type 3 chimeric receptor subunit cDNAs, we expressed these constructs in a transient transfection system and quantified surface receptor expression. We have identified amino acids that control receptor distribution between surface and intracellular pools; surface receptor expression can be manipulated without affecting the total number of receptors. These determinants function independently of the cell line used for expression and the transfection method employed. How these surface expression determinants in the alpha7 nicotinic acetylcholine receptor might influence synaptic efficacy is discussed.     

Modulation of the epidermal growth factor receptor by brain-derived growth factor in Swiss mouse 3T3 cells

Incubation of Swiss mouse 3T3 cells at 37 degrees C with bovine brain-derived growth factor (BDGF) decrease the cell surface 125I-EGF binding activity of these cells by 70-80%. This down-modulation of the EGF receptor by BDGF was time, temperature, and dose dependent. Scatchard plot analysis indicated that BDGF binding led to a selective decrease in the number of high-affinity EGF receptors. The BDGF-induced down-modulation of the EGF receptor was completely blocked by protamine, a potent inhibitor of receptor binding and mitogenic activities of BDGF. BDGF down-modulated the EGF receptor in phorbol myristic acetate (PMA)-pretreated cells, as well as in control cells. Furthermore, PMA-pretreated cells responded mitogenically to BDGF, whereas PMA itself failed to stimulate the mitogenic response of PMA-pretreated cells. This BDGF-induced down-modulation of the EGF receptor in PMA-desensitized cells suggests that BDGF down-regulates the EGF receptor by a mechanism distinct from that of PMA. Incubation of cells with compounds which are known to inhibit pinocytosis blocked the down-modulation induced either by BDGF or by platelet-derived growth factor (PDGF) but had no effect on the PMA-induced down-modulation. Incubation of cells with inhibitors of receptor recycling enhanced the BDGF-induced down-modulation of the EGF receptor. These results suggest that BDGF and PDGF induce down-modulation of the EGF receptor by increasing the internalization of cell surface high-affinity receptors and that the internalization process may not be required for down-modulation induced by PMA.     

Mitogenic response to epidermal growth factor: Relationship to number,affinity, and down-regulation of EGF receptors in three murine embyro cell lines

Swiss 3T3 and C3H-M2 cells have a greater mitogenic response to epidermal growth factor (EGF) than do C3H-10T1/2 cells. The latter cell line, however, has a number of EGF receptors per cell intermediate between the two cell lines that have a more vigorous response to EGF. Scatchard analysis of binding data indicate that all three cell lines have one class of EGF receptor, with indistinguishable affinity for the ligand. When exposed to 10-nM EGF all three cell lines “down-regulate” their EGF receptors with the same time course, and to the same precentage of initial receptors.     

Tumorigenic transformation of NIH 3T3 cells by the autocrine synthesis of transforming growth factor alpha

A variety of cancer cells overexpress transforming growth factor alpha (TGF alpha), a mitogenic peptide. A cDNA sequence coding for the full-length human TGF alpha precursor protein was subcloned into a retroviral expression vector and introduced into clone 7 NIH 3T3 cells, which have low numbers of endogenous epidermal growth factor receptors (EGFRs). The autocrine synthesis of TGF alpha by these cells resulted in their focal transformation. In contrast, control NIH 3T3 cells treated in a paracrine manner with exogenous, saturating concentrations of the mature form of TGF alpha, though stimulated to divide, remained morphologically untransformed. The addition of saturating quantities of soluble, mature TGF alpha to NIH 3T3 cells expressing the transferred TGF alpha gene actually suppressed their growth and focal transformation. The transformation induced by the TGF alpha gene remained an EGFR-dependent process, since the degree of transformation was correlated with EGFR expression in NIH 3T3 cells and since NR6 cells, which are Swiss 3T3 cells devoid of endogenous EGFRs, were transformed by the TGF alpha vector only when exogenous EGFR genes were also introduced. When inoculated into nude mice, the TGF alpha-expressing cells rapidly gave rise to tumors that grew progressively, whereas control cells did not form tumors. We conclude that in certain circumstances autocrine TGF alpha can be more oncogenic than paracrine and that paracrine TGF alpha can suppress this effect.     

Epidermal growth factor induced membrane changes in 3T3 cells.

Epidermal growth factor (EGF) is a mitogen for Swiss 3T3 cells. Short incubation periods with physiological concentrations of EGF induced increased binding of Swiss 3T3 cells to Con A-coated nylon fibers. This effect was not induced in an EGF non-responsive 33 variant, in the transformed murine XC cells or in Swiss SV3T3 cells. The increase in Con A fiber-binding seems to be specific for EGF, since it was not observed in response to insulin, prostaglandin F2alpha or a higher serum concentration, which also initiate cell devision of confluent quiescent 3T3 cells. EGF also reduced Con A-mediated hemadsorption to 3T3, but had no effect on hemadsorption by the EFG non-responsive 3T3 variant. There was no change in the number of Con A-receptors on 3T3 cells after EGF treatment. Binding to WGA-coated fibers and WGA-mediated hemadsorption were not effected by preincubation with EGF.     

Growth regulation of ovarian cancer cells by epidermal growth factor and transforming growth factors alpha and beta 1.

Regulation of ovarian cancer growth is poorly understood. In this study, the effects of EGF, TGF alpha and TGF beta 1 on two ovarian cancer cell lines (OVCAR-3 and CAOV-3) were investigated. The results showed that EGF/TGF alpha stimulated cell growth and DNA synthesis in OVCAR-3 cells, but inhibited cell proliferation and DNA synthesis in CAOV-3 cells. TGF beta 1 invariably inhibited cell proliferation and DNA synthesis in both cell lines. These effects on growth factors are dose dependent. The interaction of TGF beta 1 and EGF/TGF alpha was antagonistic in OVCAR-3 cells. In contrast, EGF/TGF alpha and TGF beta 1 had an additive inhibitory effect on CAOV-3 cells. Our results demonstrated that mature and functional EGF receptors are present in both cell lines and that they are capable of ligand binding, internalization, processing and ligand-enhanced autophosphorylation. Both high- and low-affinity binding are present in these cell lines, with CAOV-3 cells having about 2-3-fold higher total receptors than OVCAR-3 cells. These results together with those from our previous studies show that these cells express TGF alpha, TGF beta 1 and EGF receptors and that cell growth may be modulated by these growth factors in an autocrine and paracrine manner. This report presents evidence supporting the important roles of growth factors in ovarian cancer growth and provides a foundation for further study into the mechanism of growth regulation by growth factors in these cell lines.     

Interactions between growth factor receptors and corresponding monoclonal antibodies in human tumors

Monoclonal antibodies (MAbs) to the human epidermal growth factor (EGF) receptor, the type I insulin-like growth factor (IGF) receptor, and the nerve growth factor (NGF) receptor were used to study the growth regulation of malignant cells. Anti-EGF receptor MAb 425 inhibited the growth of A 431 squamous carcinoma cells which express high numbers of EGF receptors on their surfaces. Growth inhibition induced by MAb 425 was accompanied by alterations of the cell-cycle distribution of these cells, indicating the ability of a monoclonal antibody to act as a biologically active ligand. Growth stimulation of melanoma cells by EGF was unrelated to EGF receptor expression on the cell surface. Insulin- and IGF-I-induced growth stimulation of melanoma cells was inhibited by MAb alpha IR-3 which reacts with the type I IGF receptor. This result indicates that the type I IGF receptor mediated growth stimulation not only by IGF-I but also by insulin. Normal melanocytes and cells of all stages of tumor progression expressed in tissue culture the receptor for NGF, but no effect on the growth of these cells has been observed.     

Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.