Number Of Epidermal Growth Factor Binding Sites Research Articles

A STOPPED-FLOW FLUORESCENCE ANISOTROPY METHOD FOR MEASURING HORMONE BINDING AND DISSOCIATION KINETICS WITH CELL-SURFACE RECEPTORS IN LIVING CELLS

ABSTRACTWe have developed a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent, stopped-flow mixing, we determined that 32D cells, murine hematopoietic precursor cells, can survive rapid mixing, even at the high flow rates necessary to achieve dwell times as short as 10 msec. In addition, 32D cells do not express any member of the ErbB family of receptors, providing a null background for studying this receptor family. We have established a series of stable, monoclonal 32D-derived cell lines that express the epidermal growth factor (EGF) receptor, ErbB2, or a combination of both at different ratios. Using these cell lines and a homogeneous fluorescent derivative of H22Y-mEGF modified with fluorescein at the amino terminus (F-EGF), we have measured association and dissociation of F-EGF with its receptor. Association was measured by following the time-dependent changes in fluorescence anisotropy after rapidly mixing cells at various cell densities with F-EGF at 1–15 nM. Dissociation was measured both by chase experiments in which unlabeled EGF was mixed with cells pre-equilibrated with F-EGF or by dilution of cells equilibrated with F-EGF. Comparison of these dissociation experiments demonstrated that little or no ligand-induced dissociation occurs in the chase dissociation experiments. For each cell line, data from a series of association experiments and dilution dissociation experiments were subjected to global analysis using a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations, even in cells bearing only the EGF receptor. Increasing the relative expression of ErbB2 leads to an increase in the fraction of high affinity class receptors observed, without altering the total number of EGF binding sites.

Effect of ErbB2 coexpression on the kinetic interactions of epidermal growth factor with its receptor in intact cells.

We have extended the use of stopped-flow mixing and fluorescence anisotropy detection to investigate in real-time the effects of ErbB2 coexpression on the kinetic interactions of epidermal growth factor (EGF) with the EGF receptor. Using stable 32D-derived cell lines expressing both the EGF receptor and ErbB2, and fluorescein-labeled H22Y murine EGF (F-EGF), a series of association and dissociation experiments were performed in which the kinetic interaction of F-EGF with cells was monitored by observing time-dependent changes in fluorescence anisotropy following rapid mixing. Data were collected at various concentrations of F-EGF and multiple cell densities, using cells that express similar levels of the EGF receptor but different levels of ErbB2, and then analyzed by fitting to a two independent receptor-class model using global analysis techniques. The recovered kinetic parameters indicated that the coexpression of ErbB2 had relatively modest effects on recovered rate constants and calculated K(d) values, but a significant effect on the fraction of receptors associated with the high-affinity receptor class. This effect on the fraction of high-affinity receptors depended on the relative expression of ErbB2, as higher ErbB2 expression levels correlated with a larger fraction of high-affinity receptors. Further, the increase in the fraction of high-affinity receptors due to the presence of ErbB2 occurred without any change in the total number of EGF binding sites per cell. Thus, we have identified modulation of the relative populations of high- and low-affinity classes of EGF receptors as a consequence of coexpression of ErbB2 with the EGF receptor.

Activation of Epidermal Growth Factor Receptor Gene Transcription by Phorbol 12-Myristate 13-Acetate Is Mediated by Activator Protein 2

The response of the epidermal growth factor (EGF) receptor gene to phorbol 12-myristate 13-acetate (PMA) was analyzed using nuclei and nuclear extracts prepared from PMA-treated KB cells. Transient transfection assays and nuclear run-off experiments showed that PMA increased EGF receptor gene transcription. Cell-free transcription with promoter mutants revealed that the region of the promoter containing nucleotides -150 to -16 was sufficient for PMA inducibility. A promoter fragment containing nucleotides -167 to -105 showed increased binding of a factor present in extracts prepared from PMA-treated cells. When this factor was partially purified by column chromatography, it showed specific PMA-dependent binding to an EGF receptor promoter fragment. This binding was competed by an SV40 fragment containing binding sites for Sp1, AP1, and AP2. Purified AP2 was used in DNase I footprinting experiments to show that this factor can bind to the EGF receptor promoter. Oligonucleotides corresponding to the AP2 binding sites found in the EGF receptor promoter showed the ability to bind AP2 and compete for the binding of a factor induced by PMA treatment. The addition of AP2 to nuclear extract resulted in increased transcription from the EGF receptor promoter. These results demonstrate that AP2 can activate EGF receptor gene expression and may mediate the PMA response of this gene.

TREATMENT WITH LUTEINIZING-HORMONE-RELEASING HORMONE ANTAGONIST SB-75 DECREASES LEVELS OF EPIDERMAL GROWTH-FACTOR RECEPTOR AND ITS MESSENGER-RNA IN OV-1063 HUMAN EPITHELIAL OVARIAN-CANCER XENOGRAFTS IN NUDE-MICE

The aim of this study was to investigate the effect of administration of LH-RH antagonist SB-75 and agonist [D-Trp(6)]LH-RH on receptors for epidermal growth factor (EGF) in OV-1063 human epithelial ovarian cancer. Female athymic nude mice bearing xenografts of OV-1063 human epithelial ovarian cancer were treated for 3 weeks with the modern LH-releasing hormone (LH-RH) antagonist [Ac-DNal(2)(1), D-Phe(4Cl)(2), D-Pal(3)(3), D-Cit(6), D-Ala(10)] LH-RH (SB-75, Cetrorelix), the agonist [D-Trp(6)]LH-RH, or bombesin/gastrin-releasing peptide antagonist RC-3095. SB-75 and [D-Trp(6)] LH-RH were injected s.c. at doses of 100 mu g/day, and RC-3095 was injected at a dose of 40 mu g/day. Tumor growth, as measured by percentage change in tumor volume, was significantly inhibited by the treatment with SB-75, but not by [D-Trp(6)] LH-RH or RC-3095. Treatment with SB-75 greatly decreased the levels of mRNA for EGF receptor and reduced the number of EGF binding sites on tumor membranes. Effects of SB-75 on EGF receptors might be related to inhibition of tumor growth. Our findings support the view that LH-RH antagonists such as SB-75 could be considered for possible hormonal therapy of epithelial ovarian cancer.

Human growth hormone augmentation of epidermal growth factor binding sites on rat granulosa cells

The effect of human GH (hGH) on the regulation of epidermal growth factor (EGF) receptor was investigated during differentiation of FSH-treated rat granulosa cells, which has been reported to be mediated by a cAMP-dependent mechanism. By measuring the binding of [125I]iodo-EGF to the intact cells, FSH was shown to cause increases in the number of EGF binding sites after culture for 72 h. When granulosa cells were cultured with hGH, the number of FSH-induced EGF binding sites was augmented, with a half-maximal effect at about 10 micrograms hGH/l and a maximal stimulatory concentration of 100 micrograms/l. The stimulatory effect of hGH was absolutely dependent on insulin which by itself showed stimulatory effects on EGF binding sites. Scatchard analysis of EGF binding sites indicated that treatment with hGH increased the number of EGF binding sites (17,200 sites/cell after treatment with FSH; 31,700 sites/cell after FSH plus hGH), but did not alter the binding affinity. The augmentation was observed after culturing for 48 h and increased progressively with time, reaching 280% of the level after FSH treatment by 120 h. Although progesterone synthesis was increased by hGH, the markers of cell differentiation such as cAMP synthesis and LH binding sites were suppressed, indicating hGH inhibition of the cAMP-mediated signal. The action of hGH on the EGF binding sites was not accompanied by cell proliferation. These findings indicate that hGH has a novel action on the regulation of rat granulosa cell EGF binding sites and that the granulosa cell may possess both cAMP-dependent and -independent mechanisms for expression of EGF binding sites.

Epidermal growth factor reduces HER-2 protein level in human ovarian carcinoma cells.

Over-expression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian, endometrial, and mammary carcinoma is an indicator of poor prognosis. Interactions between the epidermal growth factor (EGF) receptor and the HER-2 protein have been described. The aim of this study was to elucidate the effects of EGF on HER-2 expression. In the human ovarian carcinoma cell lines HTB-77, OVCAR-3, 2780, SKOV-6, SKOV-8 and 2774, and the human mammary tumor cell line SKBR-3, total cellular p185HER-2 was determined by an ELISA, whereas the surface p185HER-2 was measured with a living-cell RIA. Stimulation of these cell lines with either EGF (0.1-30 nM) or TGF-alpha (0.1-30 nM) led to a significant reduction in p185HER-2 expression. The effect was more pronounced in cells with normal HER-2 expression. A reduction of mRNA levels for p185HER-2 by EGF was observed in OVCAR-3 cells but not in the over-expressing lines HTB-77 and SKBR-3. Interestingly, the EGF-induced effect was not always associated with growth stimulation and was not correlated with the number of EGF binding sites detected by a radioligand assay. Our data indicate that EGF treatment results in a down-regulation of p185HER-2.

Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor

The epidermal growth factor (EGF) receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization.

Regulation of rat hepatocyte epidermal growth factor receptor by the liver tumor promoter ethinyl estradiol

We have previously demonstrated that the liver tumor promoter ethinyl estradiol (EE) greatly enhanced the DNA synthetic response of rat hepatocytes in primary culture to epidermal growth factor (EGF). This effect was associated with a 2-fold increase in surface EGF receptor number. In this report, we demonstrate that the increase in cell surface [125I]EGF binding caused by EE is time dependent, beginning at 8 h and reaching a plateau at 18 h. This increased EGF binding was accompanied by a comparable increase in the amount of total EGF receptor protein. In vivo, EE treatment also increased the number of EGF binding sites. EE treatment did not increase the rate of [35S]methionine incorporation into immunoprecipitated EGF receptor protein, nor did it appear to affect the steady-state levels of EGF receptor mRNA compared to that found in controls. However, EE treatment did cause an increase in the half-life of EGF receptor protein from 4.5 +/- 0.5 h in control hepatocytes to 10.4 +/- 1.8 h. Taken together, these results indicate that the EE-induced 2-fold increase in EGF receptor levels, which is associated with the potentiation of responsiveness to EGF, was brought about by stabilization of receptor protein.

Characterization of insulin-like growth factor I and epidermal growth factor receptors in meningioma

Receptors for insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) were localized and characterized in eight samples of human meningioma (four fibrous, two meningothelial, and two angioblastic types), using quantitative autoradiographic techniques. Effects of both growth factors on deoxyribonucleic acid (DNA) synthesis in the cultured meningioma cells were examined. High numbers of specific binding sites for both IGF-I and EGF were homogeneously present in tissue sections derived from fibrous and meningothelial types of meningiomas, whereas binding sites for these growth factors were not detectable in adjacent leptomeninges. While relatively large numbers of IGF-I binding sites were located in the wall of the intratumoral vasculature, the number of binding sites in the stromal component was lower in angioblastic-type meningiomas, including a low number of EGF binding sites detected only in the stromal portion. Scatchard analysis revealed the presence of a single class of high-affinity binding sites for both IGF-I and EGF in the meningiomas examined (dissociation constant (Kd) = 0.6 to 2.9 nM, and the maximum number of binding sites (Bmax) = 16 to 80 fmol/mg for IGF-I; and Kd = 0.6 to 4.0 nM, Bmax = 3 to 39 fmol/mg for EGF). Both growth factors increased the synthesis of DNA, in a dose-dependent manner, as measured by 3H-thymidine incorporation. The combination of IGF-I and EGF synergistically stimulated the synthesis of DNA, and the effects seen with 10% fetal bovine serum could be reproduced at a concentration of 10(-10) M. These observations can be interpreted to mean that both IGF-I and EGF may be involved in the growth modulation of meningiomas, possibly through paracrine or autocrine mechanisms.

Epidermal growth factor (EGF)-like transforming growth factor (TGF) activity and EGF receptors in ovine fetal tissues: possible role for TGF in ovine fetal development.

To determine whether epidermal growth factor (EGF) or EGF-like transforming growth factors (TGFs) are present in ovine fetal tissues, we have tested acid-ethanol extracts of ovine fetal kidney for the ability to induce anchorage-independent growth of normal rat kidney fibroblasts in soft agar and to compete with 125I-mouse EGF for binding to receptors in sheep liver. The fetal kidney extract (20-1000 micrograms protein/ml) stimulated a dose-dependent increase in the number of soft agar colonies of normal rat kidney cells. Approximately 70% of these colonies measured greater than or equal to 3100 microM2. The fetal kidney extract was dissolved in 1 M acetic acid and chromatographed on Bio-Gel P 10. Two peaks of TGF-like activity, with approximate MW 14.5K (peak 1) and 9K (peak 2), eluted from the column. Half-maximal effects of pooled peaks 1 and 2 on colony formation were achieved using 100 and 20 micrograms protein/ml, respectively. Peaks 1 and 2 also competed with 125I-mouse EGF for binding to EGF receptors in ovine fetal liver but had no activity in a homologous mouse EGF radioimmunoassay sensitive to 15 pg mouse EGF. Neither TGF activity nor EGF receptor binding activity was detected in Bio-Gel fractions co-eluting with 125I-mouse EGF. Specific 125I-EGF binding sites in fetal liver were detected as early as midgestation, and the number of EGF binding sites increased markedly in late gestation, exceeding the number of EGF binding sites in the livers of pregnant ewes. These findings demonstrate the presence of TGF-like activity in ovine fetal kidney and high affinity EGF receptors in ovine fetal liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP.

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.

Altered [125I] Epidermal Growth Factor Binding and Receptor Distribution in Psoriasis

Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [125I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.

Epidermal Growth Factor Receptors in Different Skin Tumors

Specific binding of 125I-labeled epidermal growth factor (EGF) was measured in 62 skin tumors of different severity. Within a group of 28 benign tumors, 11 of 15 condylomata acuminata were receptor positive, whereas the investigated mesenchymal tumors and normal skin as a control were receptor negative. 6 of 18 basal cell epitheliomas bound EGF specifically. In the group of precancerous and malignant skin tumors, 7 of 8 squamous cell carcinomas had the highest number of EGF binding sites and a high affinity state, whereas 5 malignant melanomas were receptor negative. The clinical relevance of these findings is not yet clear due to the short follow-up of the patients.