Enzyme-linked immunosorbent assay for the epidermal growth factor receptor

An enzyme-linked immunosorbent assay (ELISA) for the epidermal growth factor (EGF) receptor was developed using three different antibody preparations, one of which is commercially available. Using one of the antisera (986), the assay could detect as few as 200 × 10⁶ receptors. This is equal to 0.332 fmol. This sensitivity means that a minimum of 100 A-431 cells (human carcinoma) or 5,000 normal cells are needed to quantitate the number of EGF receptors. Two of the antisera (986 and 451) recognized EGF receptors from placental tissue. EGF receptors from as little as 667 ng of placental membrane protein were detectable. The assay is highly species specific, with the sensitivity for the EGF receptor from different species dependent on the antiserum used. The commercial antibody, 29.1, had especially strong reactivity against pig and dog EGF receptors. An ELISA using this antibody had the capacity to detect the number of EGF receptors in 10 μg of liver membrane protein. The assay is sensitive to receptor conformation. The binding of antisera 986 and 451 to 1% sodium dodecyl sulfate (SDS)-denatured receptor was reduced. The binding of antibody 29.1 was impaired by the presence of 1% Triton X-100 but not the same levels of Tween-20 or SDS. In addition to being a sensitive technique for the quantitation of the EGF receptor, this assay is very rapid, taking a total of 4 h. The microtiter dish format also allows hundreds of samples to be assayed at once. By using the appropriate antiserum and standards, the EGF receptor can be quantitated in tissues from humans, dogs, pigs, and mice.     

Conservation of epidermal growth factor receptor function in the human parasitic helminth Schistosoma mansoni

The epidermal growth factor receptor (EGF-R) plays an important role in development and cell differentiation, and homologues of EGF-R have been identified in a broad range of vertebrate and invertebrate organisms. This work concerns the functional characterization of SER, the EGF-R-like molecule previously identified in the helminth parasite Schistosoma mansoni. Transactivation assays performed in epithelial Madin-Darby canine kidney cells co-transfected with SER and a Ras-responsive reporter vector indicated that SER was able to trigger a Ras/ERK pathway in response to human epidermal growth factor (EGF). These results were confirmed in Xenopus oocytes showing that human EGF induced meiosis reinitiation characterized by germinal vesicle breakdown in SER-expressing oocytes. Germinal vesicle breakdown induced by EGF was dependent on receptor kinase activity and shown to be associated with phosphorylation of SER and of downstream ERK proteins. (125)I-EGF binding experiments performed on SER-expressing oocytes revealed high affinity (2.9 x 10(-9) M) of the schistosome receptor for human EGF. Phosphorylation of the native SER protein present in S. mansoni membranes was also shown to occur upon binding of human EGF. These data demonstrate the ability of the SER schistosome receptor to be activated by vertebrate EGF ligands as well as to activate the classical ERK pathway downstream, indicating the conservation of EGF-R function in S. mansoni. Moreover, human EGF was shown to increase protein and DNA synthesis as well as protein phosphorylation in parasites, supporting the hypothesis that host EGF could regulate schistosome development. The possible role of SER as a receptor for host EGF peptides and its implication in host-parasite signaling and parasite development are discussed.     

Characterization of a monoclonal antibody directed against the epidermal growth factor receptor binding site

Summary In this work a new monoclonal antibody (mAb), designated MGR1, which recognizes the epidermal growth factor receptor (EGF-R) binding site, is described. The main characteristic of this mAb is its ability to discriminate between cells that express normal levels of EGF-R from cells with overexpression, the detectability threshold by immunocytochemical tests being 5 × 10⁴ receptors/cell of 10 µm diameter. MGR1 was found to inhibit EGF binding on the relevant target cells, and vice versa its binding was inhibited by EGF, which indicated that MGR1 recognizes the EGF receptor binding site. MGR1 exerted an inhibitory effect on both the in vitro and in vivo growth of cells with EGF-R overexpression, but had no effect on cells with a normal expression of the receptor. Tumour growth inhibition in athymic mice was also obtained on already implanted tumours. MGR1 therefore seems to be an adequate reagent for the development of immunotherapeutical approaches suitable for the treatment of tumours with EGF-R overexpression.     

Epidermal growth factor induces tyrosine phosphorylation of epidermal growth factor receptors not occupied with ligand in intact A431 cells.

The mechanism of epidermal growth factor receptor (EGF-R) autophosphorylation in intact A431 cells was studied. We detected epidermal growth factor (EGF) induced tyrosine phosphorylation of EGF-R not occupied with ligand. Cell monolayers were subjected to irradiation after incubation with photoreactive derivative of EGF and uncoupled EGF was extracted by acidic treatment. Subsequent immunoprecipitation with antiphosphotyrosine antibodies resulted in precipitation of both EGF-R complexes with EGF and EGF-R with unoccupied ligand-binding site. The fact of precipitation of EGF-R with unoccupied ligand-binding site in conjunction with our finding of rapid dephosphorylation of EGF-R after EGF extraction by acidic treatment, strongly supports the interpretation that cross-phosphorylation of EGF-R may take place in intact cells.     

Transforming growth factor alpha production and epidermal growth factor receptor expression in normal and oncogene transformed human mammary epithelial cells

We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.     

Binding of Erythroagglutinating Phytohemagglutinin Lectin from Phaseolus vulgaris to the Epidermal Growth Factor Receptor Inhibits Receptor Function in the Human Glioma Cell Line, U373 MG

Abstract: Little is known about the role of the N -linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF-R). In a human glioma cell line, U373 MG, EGF-Rs contain the bisecting N -linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris (E-PHA). Incubation of E-PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF-induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF-R, phenotypic events that depend on EGF-R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin from P. vulgaris (L-PHA), an isolectin of E-PHA, had no effect on EGF-R activity or the biological functions of these cells even though L-PHA was able to bind to the EGF-R. These findings suggest the presence of an important bisecting N -linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E-PHA lectin binding may provide an additional approach to blocking EGF-dependent glioma cell growth.     

Epidermal growth factor, transforming growth factor-α, and epidermal growth factor receptor expression and localization in the canine endometrium during the estrous cycle and in bitches with pyometra

Gene expression and immunohistochemical localization of epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and epidermal growth factor receptor (EGF-R) were compared between the endometrium of bitches (Canis familiaris) with pyometra accompanied by cystic endometrial hyperplasia (CEH) and that of healthy bitches at similar stages of the estrous cycle. In normal bitches, endometrial TGF-α mRNA levels were highest at proestrus and gradually decreased as the cycle progressed to anestrus. Epidermal growth factor receptor mRNA levels were not significantly affected by the stage of the estrous cycle. Epidermal growth factor mRNA levels were higher at Day 35 of diestrus than at other stages of the estrous cycle (P < 0.05). In bitches with pyometra, endometrial TGF-α and EGF-R mRNA levels did not differ significantly from those at diestrus in normal bitches, but EGF mRNA levels were lower than those at Day 35 of diestrus in normal bitches (P < 0.05). In normal bitches, positive immunohistochemical staining for TGF-α, EGF, and EGF-R was mainly present in the glandular and luminal epithelial cells of the endometrium. In contrast, in bitches with pyometra, immunoreactivity for EGF was clearly present in endometrial stromal cells. Inflammatory cells that had infiltrated the endometrial stroma stained strongly for TGF-α and EGF-R. Luminal and glandular epithelial cells also stained positive for EGF-R. In conclusion, expression of TGF-α by inflammatory cells and a low level of expression and differential localization of EGF may be involved in aberrant growth of endometrial glands and development of CEH.     

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.     

Down-modulation of epidermal growth factor receptor affinity in fibroblasts treated with interleukin 1 or tumor necrosis factor is associated with phosphorylation at a site other than threonine 654.

Interleukin 1 or tumor necrosis factor alpha can cause a transient down-modulation of epidermal growth factor (EGF) binding to quiescent fibroblast monolayers; the effect results from a reduction in EGF receptor (EGF-R) affinity and appears to be mediated by a protein kinase C (PKC)-independent mechanism. Here we show transient increases in EGF-R serine/threonine phosphorylation which are temporally coordinated with the effects on EGF binding; we also demonstrate that the cytokine-mediated phosphorylations, unlike those caused by PKC activators, have little discernible effect upon intrinsic EGF-R-associated tyrosine kinase activity. Cytokine-mediated EGF-R phosphorylation is resistant to staurosporine, an extremely potent inhibitor of PKC. Analysis of tryptic 32P-phosphopeptides reveals that Thr654, the unique site of PKC-mediated phosphorylation, is not phosphorylated in cytokine-treated cells, but a different, relatively acidic, peptide containing phosphoserine can be detected instead.     

Ligand-Induced Translocation of Epidermal Growth Factor Receptor to the Nucleus of NR6/HER Fibroblasts Is Serum Dependent

Ligand-induced translocation of epidermal growth factor receptors (EGF-R) to the nucleus of NR6/HER fibroblasts has been studied by immunoelectron microscopy. Following treatment of NR6/HER cells with epidermal growth factor (EGF) for 1 h, there was a decrease in EGF-R labeling at the plasma membrane and a corresponding increase in EGF-R in the nucleus. This was preceded by a rapid and sustained increase in nuclear phosphotyrosine content, detectable within 2 min of EGF treatment. EGF-R translocation into the nucleus was completely prevented by 18 h serum starvation prior to treatment with EGF. These results indicate that translocation of EGF-R to the nucleus is a controlled process and they suggest theft EGF-R may directly influence nuclear function.     

Vascular endothelial growth factor (VEGF) and its receptors in tumor-bearing dogs

The molecular biology of the angiogenic growth factor, vascular endothelial growth factor (VEGF), has been studied in the dog. All major isoforms of VEGF are present in the dog. The amino acid sequences are identical between human and dog in the loop regions that are responsible for receptor binding. Accordingly, the VEGF receptors of dogs and humans are very similar and permit functional exchange of the growth factor. Here we show that canine VEGF activates human endothelial cells to the same extent as human VEGF. Similarly, the two proteins display identical cell binding properties. The VEGF receptor 1 (Flt-1) shows the same alternative splicing in humans and dogs and is overexpressed in the majority of tumors in both species. VEGF occurs also in canine tumors in similar relative quantities as in human malignancies. Based on the literature and our study we suggest that the molecular biology and the function of the VEGF signaling system are virtually identical in humans and canines and in healthy as well as in disease conditions.     

Prolactin inhibits epidermal growth factor (EGF)-stimulated signaling events in mouse mammary epithelial cells by altering EGF receptor function.

We have previously shown that lactogenic hormones stimulate epidermal growth factor (EGF) mRNA accumulation in mouse mammary glands in vivo and in mouse mammary epithelial cells (NMuMG line). However, our in vitro studies indicate that the lactogenic hormone prolactin (PRL) completely inhibits EGF-stimulated DNA synthesis. PRL does not alter cholera toxin or insulin-like growth factor-1-stimulated cell growth, thus the inhibition appears to be specific for EGF. Our current studies are designed to evaluate the effects of PRL on EGF-stimulated signaling events in the NMuMG cell line. Cells treated with PRL for 30 min demonstrated a loss of high affinity EGF-binding ability. After long-term PRL treatment (18 h) there was a decrease in EGF receptor (R) number, as determined by [125I]EGF binding. PRL treatment (8 h) also decreased EGF-R mRNA levels. An EGF-stimulated increase in EGF-R mRNA observed 2-4 h after treatment was decreased when PRL was added to the cultures. Furthermore, levels of EGF-stimulated tyrosine phosphorylation of the EGF-R (170 kDa) and phospholipase C gamma (145 kDa) are dramatically decreased in cells treated with PRL. Also of great interest was a decrease in EGF-stimulated c-myc mRNA in PRL-treated cells. We conclude that PRL is acting to down-regulate the EGF-R, thus limiting EGF-stimulated cell signaling in mammary tissue.     

Expression of epidermal growth factor and surfactant proteins during postnatal and compensatory lung growth

We examined whether lung growth after pneumonectomy (PNX) invokes normal signaling pathways of postnatal development. We qualitatively and quantitatively assessed the immunoexpression of epidermal growth factor (EGF), its receptor (EGFR), surfactant proteins (SP) [SP-A and -D and surfactant proproteins (proSP)-B and -C] and proliferating cell nuclear antigen (PCNA) in immature and mature dog lung. We also assayed these proteins in lungs of immature dogs 3 wk or 10 mo after they underwent right PNX compared with simultaneous matched sham controls. During maturation, alveolar cell proliferation is regionally regulated in parallel with EGF and EGFR levels and inversely correlated with SP-A and proSP-C levels. In contrast, post-PNX lung growth is not associated with EGF or EGFR upregulation but with markedly increased SP-A level and moderately increased SP-D level; proSP-B and proSP-C levels did not change. We conclude that 1) signaling of EGF axis and differential regulation of SPs persist during postnatal lung development, 2) post-PNX lung growth is not a simple recapitulation of maturational responses, and 3) SP-A and SP-D may modulate post-PNX lung growth.     

Role of epidermal growth factor receptor in basal and stimulated colonic epithelial cell migration in vitro.

Colonic mucosal wounds are repaired, in part, by epithelial migration. Signaling mechanisms regulating this migration are poorly characterized. This study aimed to examine the role that the epidermal growth factor (EGF) receptor (EGF-R) and its ligands, EGF and transforming growth factor-alpha (TGF-alpha), play in migration in wounded in vitro models of colonic epithelium. Migration was assessed over 24 h in circular wounds made in confluent monolayers of LIM1215 human colon cancer cells. EGF and TGF-alpha stimulated migration twofold from 4 h after wounding. Basal migration and the motogenic effects of short chain fatty acids and hepatocyte growth factor were mediated through enhanced binding of TGF-alpha to EGF-R, while trefoil peptide-mediated motogenesis required EGF-R activation independently of TGF-alpha binding. Activation of protein kinase C (PKC) stimulated migration, an effect more potent than, and independent of, EGF-R activation. However, neither inhibition of PKC by Ro 31-8220 nor depletion of PKC by pretreatement with phorbol myristate acetate attenuated EGF-R-mediated motogenesis. In conclusion, EGF-R activation via TGF-alpha binding, or intracellularly, mediates basal LIM1215 migration and the effects of several motogens, with the exception of PKC activators. Since EGF-R and PKC have physiological activators in vivo, they may control colonic mucosal repair processes following injury.     

Early events in the antiproliferative action of tumor necrosis factor are similar to the early events in epidermal growth factor growth stimulation

The process of TNF-induced cytotoxicity is complex but appears to be mediated through a TNF-specific cell surface receptor. Recent evidence suggests that TNF action on tumor cells may be antagonized by epidermal growth factor (EGF) and other EGF-receptor modulatory peptides implicating a role for EGF-R in the process of TNF-induced cytotoxicity. In the present report, we investigated the biochemical actions of TNF on several biochemical events known to occur in the process of EGF signal transduction in intact cells. The actions of TNF were compared directly to those of EGF in both TNF-sensitive and -resistant tumor cell lines. In TNF-sensitive ME-180 cervical carcinoma cells, TNF (20 ng/ml) stimulated the tyrosine protein kinase activity of the EGF-receptor (EGF-R) fivefold when measured by receptor autophosphorylation in an immune complex kinase assay. TNF activation of EGF-R kinase activity in ME-180 was measurable 10 min after TNF incubation and enzymatic activity remained elevated 20 min after TNF addition. Activation of the receptor by TNF correlated with increased 32P incorporation into EGF-R protein when receptor was immunoprecipitated from 32P-equilibrated cells following a 20 min incubation with TNF. Acid hydrolysis of EGF-R protein isolated from TNF-treated ME-180 cells demonstrates an increase in the phosphotyrosine content of EGF-R when compared to receptor isolated from untreated cells. The results suggest that TNF increased EGF-R tyrosine protein kinase activity and the state of EGF-receptor tyrosine phosphorylation in a manner similar to that reported for EGF. However, TNF does not appear to be structurally related to EGF since TNF was unable to directly activate EGF-R when incubated with extensively washed immunoprecipitates of EGF-R. In TNF-resistant T24 bladder carcinoma cells, TNF failed to alter EGF-R tyrosine protein kinase activity although both EGF and phorbol ester were shown to modulate the enzymatic activity of the receptor in these cells. These results indicate that the ability of TNF to modulate EGF-R kinase in target cells may correlate with its cytotoxic actions on TNF-sensitive tumor cells. Other biochemical activities associated with the induction or regulation of cellular growth were examined in TNF- or EGF-treated tumor cells. EGF stimulated a rapid 8-16-fold increase in the expression of the proto-oncogene c-myc when analyzed by dot-blot analysis of total cellular RNA or Northern blot hybridization of polyadenylated RNA. TNF treatment failed to alter c-myc expression in ME-180 cells when analyzed by either technique.(ABSTRACT TRUNCATED AT 400 WORDS)     

Egf binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3.

The epidermal growth factor receptor (EGF-R) and the erbB-2 proto-oncogene product protein are closely related by their structural homology and their shared enzymatic activity as autophosphorylating tyrosine kinases. We show that in mammary tumor cells (SK-BR-3) EGF causes a rapid increase in tyrosine phosphorylation of the erbB-2 protein. Phosphorylation of erbB-2 does not occur in cells lacking the EGF-R (MDA-MB-453). Phosphorylation of erbB-2 in SK-BR-3 cells is blocked if EGF is prevented from interacting with its receptor by specific monoclonal antibodies. While EGF induces the down-regulation of its receptor in SK-BR-3 cells, EGF has no effect on the stability of the erbB-2 protein. This result suggests that the erbB-2 protein is a substrate of the EGF-R and indicates the possibility of communication between these two proteins early in the signal transduction process.     

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.     

The effect of epidermal growth factor (EGF) on progestin secretion and cyclic AMP synthesis in cultured luteal cells from pregnant rats

It is reported that steroid synthesis in ovarian cells is affected by epidermal growth factor (EGF). We cultured luteal cells from pregnant rats for 2 days with or without EGF, followed by incubation of the cells with or without stimulants (hCG, forskolin and dibutyryl cyclic AMP) for 5 hours. The levels of progesterone, 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) and cyclic AMP (cAMP) in the media were assayed. EGF had no effect on the basal levels of progesterone, 20 alpha-dihydroprogesterone and cAMP, but it suppressed these levels which were increased by the stimulants. We investigated binding capacity of [125I]-EGF to ovarian tissue of pregnant rats. Ovarian tissue had specific binding sites for EGF. The maximum number of binding sites was 2.38 fmol/mg tissue and the Kd value was 0.547 nM. It was indicated that EGF modified the reactivity of luteal cells to stimulants; counteracting the tropic effect of gonadotropins. It was shown that this effect of EGF might be exerted through its receptor in luteal cells.