Binding of epidermal growth factor from man, rat and mouse to the human epidermal growth factor receptor

Human, rat and mouse epidermal growth factors (EGF) bind to the same receptor on human placenta, but the binding characteristics differ. The apparent affinity constant (KA) for human EGF is higher (15 X 10(9) l/mol) than KA for rat EGF (10 X 10(9) l/mol). Mouse EGF binds with the lowest KA (5 X 10(9) l/mol). The pH optimum differs so that human and rat EGF bind with a pH optimum of 8.0, whereas mouse EGF binds with an optimum of pH 7.4. Half maximal dissociation is 130, 50 and 25 min for human, rat and mouse EGF, respectively. The structures of human, rat and mouse EGF differ somewhat. At least 11 of the first 24 residues differ. The N-terminal sequence of rat EGF is: Ala/Ser-Gly-X-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-X-Lys-Asp-Gly-Gly-Val-X-Met-Ty r-Val -Glu.     

Roles of epidermal growth factor (EGF) in the growth and differentiation of human endometrium

Endometrial tissues undergo drastic changes during menstrual cycle. After menstruation, they proliferate and differentiate into cells with secretory activity in the preparation for egg implantation. Although sex steroids play an important role in the development of endometrial tissues, sequential events occurring in the endometrium can not be fully explained by the direct actions of sex steroids. In this study, we offer evidences that EGF is released from endometrial cells and they possess the receptor for EGF. These findings prompted us to explore the biological roles of EGF in endometrial tissues. Here we clearly demonstrate that EGF is involved in the proliferation of endometrial cells. Moreover, EGF is found to enhance both glycogenesis and glycogenolysis, thus increasing the supply of glucose for blastocysts. We further set forth that EGF augments the capacity of progestin receptor and release of prostaglandins in endometrial cells. In summary, this study emphasizes that EGF may participate in the development of human endometrial tissues in concert with sex steroids, thus contributing to the acquisition of receptivity of eggs in the endometrium.     

Peptide mimics of epidermal growth factor (EGF) with antagonistic activity

Epidermal growth factor is a potent growth-promoting factor for a variety of tissue cells in vivo and in vitro. Epidermal growth factor binds, phosphorylates, and activates epidermal growth factor receptors on the cell surface. In this study, we attempted to design functional peptide mimics by panning a phage display library on the anti-epidermal growth factor monoclonal antibody. By using anti-epidermal growth factor monoclonal antibody as a mold of the structure of the binding site of epidermal growth factor, high-efficiency probing was expected. From a random peptide phage display library, phage clones that bind to the anti-epidermal growth factor monoclonal antibody were isolated. One of the phage clones also exhibited binding activity to the epidermal growth factor receptor. The amino acid sequence of this phage clone showed slight similarity to the primary sequence of epidermal growth factor. We synthesized this motif to a 9-amino-acid intramolecularly disulfide-linked peptide. This synthetic peptide inhibited mitogenesis as well as epidermal growth factor receptor tyrosine phosphorylation, which is induced by epidermal growth factor. The present results suggest that the peptide synthesized in this study may mimic the epidermal growth factor receptor-binding region in epidermal growth factor.     

Sarcoma growth factor (SGF): specific binding to epidermal growth factor (EGF) membrane receptors

Cells transformed by murine sarcoma viruses (MSV) produce and release into their tissue culture media several polypeptide growth stimulating factors. One of these has been partially purified using Bio-Gel P-60 column chromatography followed by DEAE-cellulose chromatography. This growth factor was assigned the name sarcoma growth factor (SGF), and is here shown to require the epidermal growth factor (EGF) receptor in order to function as a growth factor. DEAE-cellulose chromatography yielded a product that was several-fold purer than the material present in the Bio-Gel P-60 column pool II. The biologically active material from the DEAE-cellulose column, when labeled with 125I, showed specific binding to EGF membrane receptors. The specific binding could be prevented with the addition of either unlabeled EGF or SGF. Both radiolabeled SGF and EGF will bind to live or fixed cells. We were able to bind 125I-SGF as well as 125I-EGF to fixed cells and elute the bound material from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed cells. A 3T3 clone lacking EGF receptors was unable to respond to either EGF or SGF, whereas it responded well to serum and several other purified growth factors. The SGF isolated using DEAE-cellulose chromatography was unable to compete in a radioimmune assay using 125I-EGF and antibody to purified mouse submaxillary gland EGF; it also was not precipitated by anti-EGF antibody. From these studies it appears that the SGF produced and released by these MSV-transformed cells combines with and requires the EGF receptor in order to exert its biological effects. The peptide, however, is antigenically distinct from mouse submaxillary gland EGF.     

Cytotoxic properties of DAB486EGF and DAB389EGF, epidermal growth factor (EGF) receptor-targeted fusion toxins

Elevated expression of the receptor for epidermal growth factor (EGF) is a characteristic of several malignancies including those of the breast, bladder, prostate, lung, and neuroglia. To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor-expressing tumor cells, we have constructed a hybrid gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF. The resulting fusion toxins, DAB486EGF and DAB389EGF, bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM. Comparisons of DAB486EGF and DAB389EGF showed that DAB389EGF was consistently 10- to 100-fold more cytotoxic than DAB486EGF. Like diphtheria toxin, the cytotoxic action of DAB389EGF results from ADP-ribosylation of elongation factor-2 and is sensitive to the action of chloroquine. Studies of the kinetics of cellular intoxication showed that a 15-min exposure of EGF receptor-expressing A431 cells to DAB389EGF results in complete protein synthesis inhibition within 4 h. Furthermore, inhibition of protein synthesis results in elimination of human tumor cell colonies. These findings show that DAB389EGF is a potential therapeutic agent for a wide variety of EGF receptor-expressing solid tumors.     

Growth modulation by epidermal growth factor (EGF) in human colonic carcinoma cells: constitutive expression of the human EGF gene

The functional role of epidermal growth factor (EGF) in epithelium-derived human colonic carcinoma cells was investigated by transfection with plasmid pUCDS3, which contained synthetic human EGF encoding sequences, into two human colonic carcinoma cell types with dissimilar phenotypic properties: the moderately differentiated and growth factor-responsive Moser and the highly metastatic KM12SM cells. The Moser cells exhibited a proliferative response to treatment with exogenous EGF, while the KM12SM cells did not. The constitutive expression of the human EGF gene in these colonic carcinoma cell types resulted in elevated expression of EGF mRNA, with concurrent production and secretion of a large amount of EGF, and downmodulation of transforming growth factor-alpha (TGF-alpha) secretion. Growth stimulation and down-modulation of both high and low affinity EGF receptors were observed in the EGF-transfected Moser clones. Results of experiments using anti-EGF and anti-EGF-receptor antibody to block the proliferation of EGF-transfected Moser clones suggested that autocrine stimulatory mechanisms involving both EGF and TGF-alpha were operative in these cells. By comparison, a growth-inhibitory effect, with no apparent EGF receptor modulation, was observed in the EGF-transfected KM12SM clones. Both the parental and EGF-transfected KM12SM clones possessed fewer EGF receptors than the Moser cells, and anti-EGF or anti-EGF-receptor antibody did not affect the cells' growth properties. These results suggested that the mechanisms of growth inhibition in the EGF-transfected KM12SM clones were non-autocrine or intracellular in nature. Thus, constitutive expression of the human EGF gene in two phenotypically different, epithelium-derived human colonic carcinoma cells resulted in divergent altered growth characteristics.     

Immunoreactivities for epidermal growth factor (EGF) and for EGF receptors in rats with gastric ulcers

Summary The present study was aimed at assessing whether epidermal growth factor (EGF) and its receptors are present in the gastric mucosa during the healing of gastric ulcers. Immunohistochemical, immunochemical and functional studies were performed in rats after induction of ulcers in the oxyntic mucosa. Controls, which included non-operated and sham-operated animals, displayed only rare cells in the bottom of the oxyntic glands showing EGF-like immunoreactivity. Within one day after ulcer induction, a markedly increased number of chief cells in undamaged mucosa showed intense staining. Concomitantly, there was an increased immunoreactivity for EGF receptors in the mucous neck cells. Maximal immunostaining for both compounds was observed at 3 days after ulcer induction; augmented staining was still demonstrable after 3 weeks. RIA revealed significantly increased EGF concentration in the oxyntic mucosa three days after ulcer induction, and at this stage stimulated gastric acid secretion, measured in a parallel group of chronic fistula rats, indicated significant inhibition. The transient increases in EGF-like and EGF receptor immunoreactivities may stimulate gland cell proliferation. The local release of EGF-like substances may also serve to reduce gastric acidity and thereby promote ulcer healing.     

Epithelioid and fibroblastic rat kidney cell clones: epidermal growth factor (EGF) receptors and the effect of mouse sarcoma virus transformation.

Fibroblastic and epithelioid clones have been isolated from the normal rat kidney line, NRK. These clones were studied for their ability to bind epidermal growth factor (EGF), susceptibility to transformation by mouse sarcoma virus (MSV), and alteration in EGF binding upon sarcoma virus transformation. The epithelioid clones bound much more EGF than the fibroblastic clones; Scatchard plots on two of these clones, one epithelioid and one fibroblastic, showed that the higher EGF binding (1.3 x 10(5) molecules per cell for the epithelioid clone and 1.3 x 10(4) molecules per cell for the fibroblastic clone) was due to a greater number or receptors on the epithelioid cells rather than to a difference in the apparent affinity constant. When the clones were transformed by Moloney murine sarcoma virus the EGF binding decreased, the effect being greater with the fibroblastic clones. In 20 out of 20 independently isolated sarcoma virus transformed fibroblastic clones, the level of EGF binding was either greatly reduced or completely eliminated. In contrast to EGF, another growth factor, multiplication stimulating activity (MSA), bound to a greater extent to the fibroblastic clones than the epithelioid clones, and its binding was not decreased by sarcoma virus transformation. The results show that loss of EGF binding ability correlates with expression of the murine sarcoma virus transformation.     

Binding of epidermal growth factor (EGF) and insulin to human liver microsomes and Golgi fractions

Microsomes and Golgi fractions were isolated from 13 human liver samples without local malignancy. Binding of insulin to microsomes (per cent per 0.5 mg protein) was 14.4 +/- 7.9% with two classes of receptors: K1 = 1.4 nM, R1 = 0.28 pmol/mg; K2 = 8.1 nM, R2 = 0.62 pmol/mg. The binding was insignificantly lower than in rats. Binding of EGF was only 3.4 +/- 1.7% with two classes of receptors: K1 = 1.4 nM, R1 = 0.06 pmol/mg; K2 = 10.8 nM, R2 = 0.22 pmol/mg; the binding was much lower than in rats (26.3 +/- 5.8%). Binding of insulin to Golgi fraction (per cent per 0.1 mg protein) was 5.5 +/- 0.4% with straight line Scatchard plot; Kd = 5.6 nM, Ro = 3.06 pmol/mg; it was only half of that found in rats. In one case of hepatoma, the binding of insulin to microsomes was normal but that of EGF very low.     

Characteristics of specific binding of epidermal growth factor (EGF) on human tumor cell lines

Using seventeen human tumor cell lines derived from a variety of tissues, specific binding sites for epidermal growth factor (EGF), a mouse submandibular gland-derived growth factor, has been characterized. A significant amount of membrane-bound EGF receptors, although considerably varied, was demonstrated in all the tumor cell lines studied. Epidermoid carcinoma appeared to have more EGF receptors than adenocarcinoma. One small cell carcinoma of the lung, one choriocarcinoma of the stomach and three bone tumors also possessed EGF receptors comparable to those of epidermoid carcinoma, while one adenoacanthoma of the stomach had less EGF receptors comparable to adenocarcinoma. Among a variety of phorbol esters tested, tetradecanoyl phorbol acetate, a potent tumor promotor, was shown to be the most effective compound in inhibiting 125I-labeled EGF binding to its receptors. Our results indicate that human tumor cells contain varying amounts of membrane-bound receptors for EGF and that phorbol esters interact with these EGF receptor sites. However, the relationship between EGF receptor sites on tumor cells and cellular proliferation and/or differentiation awaits further study.     

In vivo evaluation of epidermal growth factor (EGF) receptor density on human tumor xenografts using radiolabeled EGF and anti-(EGF receptor) mAb 425

 In order to study the potential of non-invasive scintigraphic evaluation of the epidermal growth factor (EGF) receptor status in vivo, the biokinetics and tumor binding of ¹²⁵I-EGF and anti-(EGF receptor) mAb 425 were investigated in nude mice bearing human tumor xenografts with different EGF-receptor densities as determined by a radioreceptor assay. The results demonstrated a tumor uptake for both substances depending on the receptor level. The EGF receptor status, however, was reflected slightly better by the binding of EGF to tumor tissue compared to the mAb. The rapid blood clearance of EGF with a plasma half-life of less than 1 min led to a tumor-to-blood ratio of approximately 3 within 6 h after injection in tumors with a high receptor expression. A similar ratio for the mAb was not obtained before day 6 after injection. The absolute concentration of EGF, however, was low compared to the mAb. Therefore, it can be concluded that the EGF receptor status as a target for (radio)immunotherapy can be evaluated in vivo with EGF labeled with a short-life positron-emitting radionuclide or with monoclonal antibodies to the EGF receptor or their fragments. Received: 14 September 1995 / Accepted: 6 December 1995     

Synthesis and secretion of an epidermal growth factor (EGF) by human fibroblast cells in culture

Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.     

Transient epidermal growth factor (EGF)-dependent suppression of EGF receptor autophosphorylation during internalization.

To study the activity of the epidermal growth factor (EGF) receptor during EGF-directed internalization, liver epithelial cells were exposed to EGF at 37 degrees C for various periods of time, washed, and homogenized at 0 degrees C. EGF receptor autophosphorylation was assessed in homogenates using [gamma-32P]ATP. Autophosphorylation was stimulated 3- to 6-fold in homogenates of cells incubated with EGF (100 ng/ml) for 15 min but was at or below basal levels in homogenates of cells treated with EGF for 2.5-5 min. This was surprising because immunoblotting revealed that EGF receptor phosphotyrosine (P-Tyr) content in intact cells was near maximal from 30 s to 5 min after EGF treatment. Excess EGF (1 microgram/ml), added after homogenization but prior to the assay, increased autophosphorylation in homogenates of cells that had not been treated with EGF, but failed to increase activity in homogenates of cells treated with EGF in culture for 2.5-5 min. Suppression of tyrosine phosphorylation of an exogenous kinase substrate was also observed at times paralleling the suppression of EGF receptor autophosphorylation. The transient suppression of receptor autophosphorylation in the cell-free assay was not explained by persistent occupation of autophosphorylation sites by phosphate added in the intact cells. The sites were greater than 80% dephosphorylated during the homogenization. Additionally phosphatase inhibition that prevented the normal loss of EGF receptor P-Tyr in intact cells at 15 min did not affect the pattern of early (2.5-5 min) suppression and later (15 min) stimulation of autophosphorylation measured in the cell-free assay. The suppression was not explained by activation of protein kinase C in that depletion of greater than 95% of cellular protein kinase C activity by an 18-h incubation of cells with 10 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the early suppression of autophosphorylation in EGF-treated cells. Moreover, under the conditions tested, activation of protein kinase C by short-term treatment (0.5-10 min) with TPA or angiotensin II did not appreciably alter subsequent autophosphorylation in the cell-free assay. In contrast, a 30 degrees C preincubation of homogenates from cells with suppressed EGF receptor autophosphorylation led to the recovery of the ability of EGF to stimulate EGF receptor autophosphorylation. These results suggest that a rapid reversible protein kinase C-independent process prevents detection of EGF receptor kinase activity during an early phase of EGF-dependent receptor internalization.     

Central role of epidermal growth factor (EGF) receptor density in anchorage-independent growth of normal rat kidney cells

The gamma-carboxyglutamic acid (Gla) content of several variants of human prothrombin has been measured by using capillary electrophoresis and laser-induced fluorescence (CE-LIF). Both plasma-derived prothrombin and recombinant prothrombin contain ten residues of Gla per molecule of protein. In contrast, a variant of human prothrombin (containing the second kringle domain of bovine prothrombin) was separated into two populations that differed in their Gla content. Direct measurement of the Gla content showed an association with the presence or absence of the calcium-dependent conformational change that is required for prothombinase function. Thus, the CE-LIF assay is useful in determining the carboxylation status of recombinant proteins.     

Comparison of specific binding of human epidermal growth factor (EGF) to sinusoidal and bile canalicular membranes isolated from rat liver

The binding characteristics of human epidermal growth factor (EGF) were compared between highly purified canalicular (CMV) and sinusoidal (basolateral) rat liver plasma membrane (SMV) preparations. The dissociation constants (2-3 nM) for these membranes were comparable, while the binding capacity for CMV was approximately half that for SMV. The binding capacity for CMV was too high to be accounted for only by the contamination with sinusoidal membranes, since the measurements of specific activities of various enzymes (Na+,K+-ATPase, alkaline phosphatase, and leucine aminopeptidase) indicated that the extents of the cross contamination with other membrane fractions were at most 10%. Although the physiological function of specific binding of EGF to bile canalicular membrane domain remains to be determined, it may have a role in biliary excretion of EGF. The specific binding of EGF to bile canalicular membranes from rat liver was identified for the first time.     

A trophic effect of epidermal growth factor (EGF) on rat colonic mucosa in organ culture

The development of an organ-culture system for rat colonic mucosa has enabled a direct assessment of the effect of epidermal growth factor (EGF) on cell division. An augmented mitotic index (AIm) has been employed to identify changes in cell proliferation. Explants of colonic mucosa from four animals were maintained in a medium containing serum for five days. On the fifth day of culture, half of the explants received fresh medium containing EGF (40 ng/ml) and the remainder (controls) fresh medium only. At 6, 12, 24 and 48 hr thereafter groups of both experimental and control explants received the metaphase-arresting drug vincristine (4 micrograms/ml) for 3 hr prior to fixation. The proportions of vincristine-arrested metaphases within the explants were determined. Analysis of the data indicates that when serum is present exogenous EGF exerts a trophic effect which increases with time (P less than 0.001). In a second experiment colonic explants from four animals were maintained for five days in a serum-free medium and were then divided into groups, each of which received one of a range of concentrations of EGF. The AIm was determined for each group after 36 hr. It was found that increasing concentrations of EGF produce a small but significant increase in cell proliferation (P less than 0.01). This effect, however, was less pronounced than that seen when serum was present. These results suggest that EGF has a trophic action on the colon and interacts with additional factors found in serum.     

Metabolic effects induced by epidermal growth factor (EGF) in cells expressing EGF receptor mutants

The epidermal growth factor (EGF) receptor tyrosine kinase activity is required for both the earliest EGF-stimulated post-binding events (enhancement of inositol phosphate formation and Ca2+ influx, activation of Na+/H+ exchange), and the ultimate EGF-induced mitogenic response. To assess the role of EGF receptor kinase in EGF-induced metabolic effects (2-deoxyglucose and 2-aminoisobutyric acid uptake), we used NIH3T3 cells (clone 2.2), which do not possess endogenous EGF receptors and which were transfected with cDNA constructs encoding either wild type or kinase-deficient human EGF receptor (HER). In addition, we tested the importance of three HER autophosphorylation sites (Tyr-1068, Tyr-1148, and Tyr-1173) in transduction of EGF-stimulated 2-deoxyglucose uptake. Taking our data together, we conclude the following: (i) HER tyrosine kinase activity is required to elicit EGF stimulation of both 2-deoxyglucose and 2-aminoisobutyric acid uptake; (ii) mutations on individual HER autophosphorylation sites, Tyr-1068, Tyr-1148, and Tyr-1173 do not impair EGF-stimulated 2-deoxyglucose uptake.     

A trophic effect of epidermal growth factor (EGF) on rat colonic mucosa in organ culture

Abstract. The development of an organ-culture system for rat colonic mucosa has enabled a direct assessment of the effect of epidermal growth factor (EGF) on cell division. An augmented mitotic index (AI_m) has been employed to identify changes in cell proliferation.
Explants of colonic mucosa from four animals were maintained in a medium containing serum for five days. On the fifth day of culture, half of the explants received fresh medium containing EGF (40 ng/ml) and the remainder (controls) fresh medium only. At 6,12,24 and 48 hr thereafter groups of both experimental and control explants received the metaphase-arresting drug vincristine (4 μ g/ml) for 3 hr prior to fixation. The proportions of vincristine-arrested metaphases within the explants were determined. Analysis of the data indicates that when serum is present exogenous EGF exerts a trophic effect which increases with time ( P < 0.001).
In a second experiment colonic explants from four animals were maintained for five days in a serum-free medium and were then divided into groups, each of which received one of a range of concentrations of EGF. The AI_m was determined for each group after 36 hr. It was found that increasing concentrations of EGF produce a small but significant increase in cell proliferation ( P < 0.01). This effect, however, was less pronounced than that seen when serum was present.
These results suggest that EGF has a trophic action on the colon and interacts with additional factors found in serum.     

Influence of epidermal growth factor (EGF) on ruffling activity, pinocytosis and proliferation of cultivated human glia cells

Normal human glia cells in culture were studied with respect to ruffling activity, macro-pinocytosis and cell proliferation under standard culture conditions with 10% serum in the medium, in serum-free medium and after addition of epidermal growth factor (EGF) or serum to previously serum-free medium. Pinocytotic uptake of droplets of medium occurred only in relation to well developed ruffling membranes. Omitting the serum from the medium led to a drastic reduction in thymidine incorporation. The cells became slender under these conditions, and soon after the change of medium their ruffling activity and pinocytosis were almost completely abolished. Following the change to a medium containing 2 ng EGF/ml a rapid reappearance of ruffling and pinocytosis was observed. DNA synthesis, however, was not demonstrated until after 20 h, showing that ruffling and pinocytosis occurred before DNA synthesis had started. Thus EGF may initially induce conformational changes of the plasma membrane, resulting in its internalization due to formation of endocytotic vacuoles. The observed relationship between occurrence of well developed ruffling membranes, macro-pinocytosis and cell multiplication indicates that one of the functions of growth-promoting factors may be stimulation of plasma membrane turnover.     

Effects of epidermal growth factor (EGF) on adult mouse small intestine in vivo and in organ culture

1. Exogenous administration of epidermal growth factor (EGF) has not modified the protein and DNA content, nor several brush border enzymes activities of duodenum, jejunum and ileum of intact and fasted adult mice. 2. Exogenous administration of EGF has not stimulated the DNA synthesis in the three regions of the small intestine of intact adult mice. 3. EGF has a stimulatory effect on DNA synthesis of fasted mice intestine 12 hr after injection. 4. In organ culture, EGF has not altered at any concentration (10, 50, 100, 200, 800 ng/ml), the same parameters in duodenal and jejunal explants taken from animals fasted 24 hr before being killed. 5. These last results suggest that the increase of DNA synthesis observed in vivo was not a direct effect of EGF administration. 6. Finally, the EGF content of serum af adult male mice was measured in fed and fasted mice and in the organ culture media.