Synergistic interaction of p185c-neu and the EGF receptor leads to transformation of rodent fibroblasts

The protein product of the rodent neu oncogene, p185neu, is a tyrosine kinase with structural similarity to the epidermal growth factor receptor (EGFR). Transfection and subsequent overexpression of the human p185c-erbB-2 protein transforms NIH 3T3 cells in vitro. However, NIH 3T3 cells are not transformed by overexpressed rodent p185c-neu. NIH 3T3 transfectants overexpressing EGF receptors are not transformed unless incompletely transformed. Several groups have recently demonstrated EGF-induced, EGFR-mediated phosphorylation of p185c-neu. During efforts to characterize the interaction of p185c-neu with EGFR further, we created cell lines that simultaneously overexpress both p185c-neu and EGFR and observed that these cells become transformed. These observations demonstrate that two distinct, overexpressed tyrosine kinases can act synergistically to transform NIH 3T3 cells, thus identifying a novel mechanism that can lead to transformation.     

GM3 content modulates the EGF-activated p185c-neu levels, but not those of the constitutively activated oncoprotein p185neu

The functional relationship between ganglioside GM(3) and two tyrosine-kinase receptors, the normal protein p185(c-neu) and the mutant oncogenic protein p185(neu), was examined in HC11 cells and in MG1361 cells, respectively. In the former, p185(c-neu) expression and activation are controlled by EGF addition to the culture medium and by epidermal growth factor receptor (EGFR) activity, whereas the latter express unchangingly high levels of constitutively activated p185(neu). Studies were carried out using (+/-)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ganglioside biosynthesis resulting in ganglioside depletion, and addition of exogenous GM(3) to the culture medium. In HC11 cells treated with only [D]-PDMP, p185(c-neu) levels remain similar to control cells, whereas levels of tyrosine-phosphorylated p185(c-neu) increase after treatment with [D]-PDMP in combination with EGF. When exogenous GM(3) is added in combination with [D]-PDMP and EGF, the enhanced phosphorylated-p185(c-neu) returns to control levels. Interestingly, EGFR levels also vary and, analogously to phosphorylated-p185(c-neu), the increase of EGFR content consequent to the [D]-PDMP and EGF addition is reversed by exogenous GM(3). In contrast, the addition of neither [D]-PDMP nor exogenous GM(3) modifies expression and tyrosine-phosphorylation levels of p185(neu) in MG1361 cells. These findings indicate that changes in GM(3) content modulate the tyrosine-phosphorylated p185(c-neu) levels in a reversible manner, but this is not specific for p185(c-neu) because EGFR levels are also modified. Furthermore, these data suggest that GM(3) may play a functional role by affecting the internalisation pathway of p185(c-neu)/EGFR heterodimers, but not of p185(neu) homodimers.     

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.     

Anomalous binding of epidermal growth factor to A431 cells is due to the effect of high receptor densities and a saturable endocytic system

This study was conducted to determine how extraordinarily high numbers of epidermal growth factor receptors (EGF-R) affected the binding and internalization of EGF in the transformed cell line A431. I found that at low EGF concentrations, the kinetics of binding behaved as a nonsaturable, first-order process showing no evidence of multiple-affinity classes of receptors. However, EGF dissociation rates were strongly dependent on the degree of receptor occupancy in both intact cells and isolated membranes. This occupancy-dependent dissociation appears to be due to diffusion-limited binding. EGF-induced receptor internalization was rapid and first order when the absolute number of occupied receptors was below 4 x 10(3) min-1. However, at higher occupancies the specific internalization rate progressively declined to a final limiting value of 20% normal. The saturation of EGF-R endocytosis was specific since internalization of transferrin receptors was not affected by high concentrations of either transferrin or EGF. Saturation of EGF-R endocytosis probably involves a specific component of the endocytic pathway since fluid phase endocytosis increased coordinately with EGF-R occupancy. I conclude that there are several aspects of EGF-R dynamics on A431 cells are neither similar to the behavior of EGF-R in other cell types nor similar to the reported behavior of other hormone receptors. Although A431 cells have an extraordinary number of EGF-R, they do not seem to have corresponding levels of at least two other crucial cell surface components: one that mediates EGF-induced rapid receptor internalization and one that attenuates EGF-induced membrane responses. These factors, in addition to the presence of diffusion-limited binding at low EGF concentrations, are probably responsible for the appearance of multiple-affinity classes of receptors in this cell type.     

EGF receptor regulation in normal mouse mammary gland.

Estrogen (E), progesterone (P), and epidermal growth factor (EGF) are known to regulate growth and development of the normal mammary gland, and it is possible that EGF may interact with E and/or P. Estrogen (ER), progesterone (PR), and EGF receptors (EGF-R) have been detected in both mammary epithelial and stromal cells, and the relative roles of the various cells types in hormone-dependent growth regulation are not known. The present studies were undertaken to determine if E and/or P influence EGF action by exerting a regulatory effect on EGF-R levels and which cell types are affected. The comparative effects of ovariectomy and hormone treatments on EGF-R levels were examined in immature, pubertal 5-week-old and sexually mature 10-week-old female mice. EGF-R were characterized as a single class of high affinity sites and EGF-R concentration was 2-fold higher in glands of 5-week-old mice. Ovariectomy had no significant effect on EGF-R concentration in either age group, and treatment with E and/or P had no effect on EGF-R levels in either epithelial or stromal cells in 5-week-old mice. In contrast, E+P treatment caused a 2-fold increase in receptor concentration in 10-week-old mice in the mammary epithelium. Thus it appears that the developmental state of the gland may determine the nature and extent of the interaction of of EGF, E, and P.     

Internalization and down-regulation of the human epidermal growth factor receptor are regulated by the carboxyl-terminal tyrosines

The C terminus of the epidermal growth factor receptor (EGF-R) contains three tyrosines (Y1068, Y1148, and Y1173) which correspond to the major autophosphorylation sites. To investigate the role of the tyrosines in internalization and down-regulation of the EGF-R, mutational analysis was performed with receptors in which 1, 2, or all 3 tyrosines were changed to phenylalanines. The triple point mutant EGF-R, expressed in NIH-3T3, exhibited low autophosphorylation in vivo, low biological and reduced kinase activities. Single and double point mutants were down-regulated, as well as wild type EGF-R in response to EGF showing a half-life of about 1 h. Degradation of the triple point mutant, however, was impaired and resulted in a half-life of 4 h in the presence of EGF. EGF-dependent down-regulation of surface receptors was decreased in the triple point mutant EGF-R as was internalization and degradation of EGF. The specific rate of internalization of the triple point mutant was reduced. By contrast, intracellular processing of ligand previously internalized at 20 degrees C was similar between wild type and mutant receptors. Taken together the data indicate that the delay in degradation observed in cells expressing the triple point mutant EGF-R can be attributed mainly to a slower removal from the cell surface. Our results show that in the full-length EGF-R all three C-terminal tyrosines are necessary for rapid internalization, suggesting that autophosphorylation is required for efficient EGF-dependent receptor endocytosis.     

Regulation of the mannose 6-phosphate/IGF II receptor expression at the cell surface by mannose 6-phosphate, insulin like growth factors and epidermal growth factor.

Mannose 6-phosphate, insulin like growth factors I and II (IGF I, IGF II), insulin and epidermal growth factor (EGF) induce a 1.5- to 2-fold increase of mannose 6-phosphate binding sites at the cell surface of human skin fibroblasts. The increase is completed within 10-15 min, is dose and temperature dependent, reversible and transient even in the presence of the effectors. It is due to a redistribution of mannose 6-phosphate/IGF II receptors from internal membranes to the cell surface, while the affinity of the receptors is not affected. Combinations of mannose 6-phosphate with IGF I, IGF II or EGF stimulate the redistribution of the receptor to the cell surface in an additive manner, while combinations of the growth factors result in a non-additive stimulation of redistribution. The redistribution is not dependent on extracellular calcium and appears also to be independent of changes of free intracellular calcium. Pre-treatment of fibroblasts with cholera toxin or pertussis toxin increases the number of cell surface receptors 2- and 1.5-fold, respectively. Neither of the toxins affects the redistribution of mannose 6-phosphate/IGF II receptors induced by the growth factors, while both toxins abolish the receptor redistribution induced by mannose 6-phosphate. These results suggest a multiple regulation of the cell surface expression of mannose 6-phosphate/IGF II receptors by Gs- and Gi-like proteins sensitive to cholera toxin and pertussis toxin and by stimulation of mannose 6-phosphate/IGF II, IGF I and EGF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Akt by EGF-R inhibitors,a possible mechanism of EGF-R inhibitor-enhanced TRAIL-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane cytokine and a potent inducer of apoptosis. Epidermal growth factor (EGF) signaling is well known to involve in tumor survival and overexpression of EGF receptor (EGF-R) attributes to decreased responsiveness to many available therapies in cancer treatment. We investigated whether EGF-R inhibitors enhance TRAIL-induced apoptosis. We exposed A549 cells to Genistein, PD153035, and PD158780 for 12h and then treated with recombinant TRAIL protein. TRAIL alone induced 25% cell death after a 3-h treatment, but in cells pretreated with EGF-R inhibitors, TRAIL induced cell death to more than 70% after 3h treatment. Genistein enhanced TRAIL-induced apoptosis in a time- and dose-dependent manner. Western blot analyses showed that pretreatment with Genistein down-regulated the protein levels of total Akt and phosphorylated active Akt. Genistein also decreased the protein level of Bcl-XL that is regulated by Akt. These molecules are well characterized to act against induction of apoptotic cell death. Therefore, our data suggest that EGF-R inhibitor may sensitize A549 cells to TRAIL-induced apoptosis by regulating expression of these proteins. EGF-R inhibitors may play an important role in the anti-cancer activity of TRAIL protein, especially in TRAIL-resistant tumors that arise by expressing constitutively active Akt.     

IL-1 and TNF transmodulate epidermal growth factor receptors by a protein kinase C-independent mechanism

The effect of the human rIL-1 alpha and rTNF-alpha on the binding of 125I-labeled epidermal growth factor ([125I]EGF) to its receptor (EGF-R) has been studied in human gingival fibroblasts (HuGi). Incubation of these cells with recombinant cytokines at 37 degrees C caused a rapid, dose-dependent decrease in their ability to subsequently bind subsaturating levels of [125I]EGF at 4 degrees C. Inhibition was evident at 5 min after addition of cytokines, reached a maximal level (60-70% reduction) after 15 to 30 min, and declined thereafter. Normal EGF binding was attained by 2 h. Half-maximal inhibition of EGF binding occurred at 10 pM IL-1 and 50 pM TNF. The two cytokines were not additive in their effect. Competition experiments at 4 degrees C showed that the cytokines did not interact directly with EGF-R; Scatchard analysis of binding of [125I]EGF to HuGi after treatment with IL-1 and TNF revealed an increase in EGF-R Kd from 0.75 nM to 2.9 nM with no change in receptor number. The effect of IL-1 and TNF on EGF-R was compared with that of the tumor-promotor PMA which is known to "transmodulate" EGF-R affinity by activating protein kinase C which then phosphorylates EGF-R. PMA caused a greater inhibition of EGF binding to HuGi (80 to 85% inhibition; ED50 = 500 pM), and recovery of binding was much slower. Importantly, in HuGi made deficient in protein kinase C by prolonged incubation with PMA, addition of fresh PMA no longer affected EGF binding, while the response to IL-1 and TNF was intact. Cytokine- but not PMA-mediated EGF-R transmodulation was partially reversed by treatment of the cells with millimolar concentrations of the kinase inhibitor amiloride. HuGi were incubated with H3 32PO4, stimulated with PMA or cytokines, and EGF-R were immunoprecipitated; IL-1 and TNF, like PMA, caused a 2- to 5-fold increase in receptor phosphorylation. We conclude that occupation of IL-1 and TNF-R activates a protein kinase, distinct from kinase C, for which EGF-R is a substrate.     

Recruitment of epidermal growth factor receptors into coated pits requires their activated tyrosine kinase

EGF-receptor (EGF-R) tyrosine kinase is required for the down- regulation of activated EGF-R. However, controversy exists as to whether ligand-induced activation of the EGF-R tyrosine kinase is required for internalization or for lysosomal targeting. We have addressed this issue using a cell-free assay that selectively measures the recruitment of EGF-R into coated pits. Here we show that EGF bound to wild-type receptors is efficiently sequestered in coated pits. In contrast, sequestration of kinase-deficient receptors occurs inefficiently and at the same basal rate of endocytosis of unoccupied receptors or receptors lacking any cytoplasmic domain. Sequestration of deletion mutants of the EGF-R that lack autophosphorylation sites also requires an active tyrosine kinase. This suggests that a tyrosine kinase substrate(s) other than the EGF-R itself, is required for its efficient ligand-induced recruitment into coated pits. Addition of a soluble EGF-R tyrosine kinase fully and specifically restores the recruitment of kinase-deficient EGF-R into coated pits providing a powerful functional assay for identification of these substrate(s).     

Immunohistochemical localization of epidermal growth factor receptor (EGF-R) in normal and diseased newborn lung tissues

The distribution of EGF receptors (EGF-R) was examined in normal, hyaline membrane diseased and pneumonic newborn lung tissues by immunohistochemical methods under the light microscope. The PAP technique with polyclonal antibodies was performed to demonstrate the EGF receptor localisation in these tissues. Strong EGF-R reactivity was observed on bronchiolar epithelium and type I and type II alveolar cells in normal newborn lung tissues; whereas, poor reactivity was observed in alveolar macrophages. On the other hand, strong immunoreactivity was detected in type I alveolar cells and alveolar macrophages in hyaline membrane disease, but no reactivity was present in type II alveolar cells. The strongest immunoreactivity was observed in alveolar macrophages of newborn pneumonic lung tissues. In conclusion, the most meaningful form of reactivity was observed in normal newborn lung tissues of airway track and respiration area. This result is related with the maturation of the lungs after birth.     

Heregulin-beta is especially potent in activating phosphatidylinositol 3-kinase in nontransformed human mammary epithelial cells

The neu differentiation factors/heregulins (HRGs) comprise a family of polypeptide growth factors that activate p185(erbB-2) through direct binding to either erbB-3 or erbB-4 receptor tyrosine kinases. We have previously shown that HRG-beta is mitogenic for various human mammary epithelial cell lines that coexpress c-erbB-2 and c-erbB-3. Phosphatidylinositol 3-kinase (PI3K) is activated by p185(erbB-2) /erbB-3 heterodimers in cells stimulated by HRG, and PI3K is constitutively activated by p185(erbB-2) /erbB-3 in breast carcinoma cells that overexpress c-erbB-2. To better understand the relative abilities of HRGs, epidermal growth factor (EGF), or insulin to activate PI3K under normal physiological conditions, we compared the levels of recruitment of the 85-kDa regulatory subunit of PI3K when activated by the type I (erbB) or type II [insulin-like growth factor (IGF)] receptor tyrosine kinases in two different nontransformed human mammary epithelial cell lines. The nontransformed H16N-2 cells isolated from normal tissue express EGFR, p185(erbB-2), and erbB-3, and are highly responsive to the mitogenic effects of HRG-beta as well as to the combination of EGF and insulin in serum-free culture. We measured the stoichiometry of p85 recruited by tyrosine-phosphorylated proteins induced in H16N-2 cells by either the alpha or the beta isoform of HRG. HRG-beta was greater than 10-fold more potent in inducing p85 recruitment than was the less biologically active HRG-alpha isoform. HRG-beta was also a more potent inducer of p85 recruited by tyrosine-phosphorylated proteins than was either EGF, insulin, or EGF and insulin combined. Furthermore, erbB-3 principally mediated the direct recruitment of p85 in cells stimulated by HRG or EGF, indicating that, in addition to the high-level activation of PI3K by p185(erbB-2) / erbB-3, EGFR/erbB-3 heterodimer interaction is essential for the weak but significant level of PI3K activated by EGF in cells that express normal EGFR levels. Studies using the PI3K inhibitor wortmannin also indicated that PI3K activation was required for the proliferation of H16N-2 cells induced by either HRG-beta or EGF and insulin in serum-free culture. Finally, HRG-beta was also an especially potent inducer of PI3K in the nontransformed MCF-10A cells, which were derived spontaneously from normal reduction mammoplasty tissue. These data show, for the first time, a side-by-side quantitative comparison of the relative degree of PI3K activated by different growth factors in nontransformed growth factor-dependent cells under precisely defined conditions in culture.     

Cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin II in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin II signaling

Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMCs). Cholesterol and sphingomyelin-enriched lipid rafts are plasma membrane microdomains that concentrate various signaling molecules. Caveolae are specialized lipid rafts that are organized by the cholesterol-binding protein, caveolin, and have been shown to be associated with EGF-Rs. Angiotensin II stimulation promotes a rapid movement of AT(1) receptors to caveolae; however, their functional role in angiotensin II signaling has not been elucidated. Here we show that cholesterol depletion by beta-cyclodextrin disrupts caveolae structure and concomitantly inhibits tyrosine phosphorylation of the EGF-R and subsequent activation of protein kinase B (PKB)/Akt induced by angiotensin II. Similar inhibitory effects were obtained with other cholesterol-binding agents, filipin and nystatin. In contrast, EGF-R autophosphorylation and activation of Akt/PKB in response to EGF are not affected by cholesterol depletion. The early Ang II-induced upstream signaling events responsible for transactivation of the EGF-R, such as the intracellular Ca(2+) increase and c-Src activation, also remain intact. The EGF-R initially binds caveolin, but these two proteins rapidly dissociate following angiotensin II stimulation during the time when EGF-R transactivation is observed. The activated EGF-R is localized in focal adhesions together with tyrosine-phosphorylated caveolin. These findings suggest that 1) a scaffolding role of caveolin is essential for EGF-R transactivation by angiotensin II and 2) cholesterol-rich microdomains as well as focal adhesions are important signal-organizing compartments required for the spatial and temporal organization of angiotensin II signaling in VSMCs.     

Nonfunctional epidermal growth factor receptor in cells transformed by Kirsten sarcoma virus

The cell membrane receptor for epidermal growth factor (EGF) appears to be a glycoprotein of Mr 170,000 and mediates the mitogenic and metabolic responses of cells with EGF receptors (EGF-R). Normal rat kidney (NRK) have about 3 X 10(5) EGF-R per cell. Upon transformation of NRK cells by Kirsten sarcoma virus, the transformed derivative (KNRK) loses the ability to bind 125I-EGF. Membranes from NRK and KNRK cells were included in EGF-dependent phosphorylation reactions to search for evidence of the EGF-R. A phosphorylated protein of Mr 170,000 was detected in both NRK and KNRK membranes. The Mr 170,000 protein was identified to be EGF-R by immunoprecipitation with monoclonal antibody to the receptor. Furthermore, two-dimensional peptide mapping using trypsin and chymotrypsin digestions of the iodinated receptors from both NRK and KNRK cells showed essentially identical patterns. These data indicate that the EGF-R is present in KNRK cells with apparently the same protein structure as the NRK counterpart.     

A novel receptor-targeted gene delivery system for cancer gene therapy

Some growth factor receptors, such as insulin like growth factor I and II receptor (IGF I R, IGF II R) and epidermal growth factor receptor (EGF R), have been proved to be over-expressed in a variety of human cancers derived from different tissue origins. Based on this molecular alteration, a polypeptide conjugate gene delivery system was designed and synthesized. It contains three essential moieties: a ligand oligopeptide (LOP) for receptor recognition, a polycationic polypeptide (PCP) such as protamine (PA) or poly-L-lysine (PL) as a backbone for DNA binding and an endosome-releasing oligopeptide (EROP) such as influenza haemagglutinin oligopeptide (HA20) for endosomolysis. These components are covalently conjugated as LOP-PCP-HA20 or in the form of a mixture of LOP-PCP and HA20-PCP. A 14 amino acid E5 was designed and synthesized as LOP for IGF I R and IGF II R, and a 16 amino acid GE7 as LOP for EGF R. Both E5 and GE7 systems could form stable complex with the plasmid DNA as E5-PCP/ DNA/PCP-HA20 and GE7-PCP/DNA/PCP-HA20. Using bacterial β-galactosidase gene (pSVβ-gal) as a reporter, the present system is able to efficiently target exogenous gene to human cancer cells of different tissue types with high efficiency bothin vitro and in implanted tumors in nude mice. It was also demonstrated that the transduced genes were highly expressed in cancer cells bothin vitro andin vivo. The present system will provide a novel effective vehicle to target therapeutic genes into cancer cells in gene therapy.     

Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body

We compared the internalization and intracellular sorting of epidermal growth factor receptor (EGF-R) and point mutant kinase-negative EGF-R separately expressed in NIH 3T3 cells lacking endogenous receptor. Both EGF-Rs internalized rapidly, but kinase-negative receptor was surface down-regulated only with monensin or at 20 degrees C. Furthermore, EGF internalized by mutant receptor alone was, in significant proportion, returned to the cell surface undegraded. Hence unlike wild-type receptor, kinase-negative EGF-R recycles. By electron microscopy the early pathways of endocytosis for the two receptors were identical; however, after 10-20 min the pathways diverged at the multivesicular body (MVB). Wild-type EGF-R, destined for degradation, localized to internal vesicles, while kinase-negative EGF-R, destined for recycling, localized to surface membranes of the MVBs and moved to small tubulovesicles. We conclude that sorting of internalized receptor for degradation or recycling can occur through spatial segregation within the MVB, and sorting of EGF-R is controlled by tyrosine kinase activity.     

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.