A radioimmunoassay for human epidermal growth factor receptor

The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.     

The epidermal growth factor receptor tyrosine kinase phosphorylates connexin32

Oncoprotein 18 or stathmin was isolated from bovine brain, characterized and novel features of its function as a microtubule depolymerizing factor were tested.The effect of phosphorylation of stathmin on its function as a microtubule depolymerizing factor has been tested in vitro. Five different protein kinases, protein kinase A, MAP kinase, cdc2 kinase, glycogen synthase kinase 3 and casein kinase 2, were used to modify stathmin, since it is known that these kinases could phosphorylate several residues that are modified in vivo and could have important roles in stathmin function. The residues phosphorylated in vitro by the different protein kinases were identified and in some cases they correspond to those modified in vivo.Recombinant unphosphorylated stathmin and native stathmin, which was previously dephosphorylated with alkaline phosphatase, showed similar microtubule depolymerizing activity. This activity is higher than that of stathmin phosphorylated by protein kinase A, MAP kinase or cdc 2 kinase, whereas phosphorylation of the protein with casein kinase 2 or glycogen synthase kinase 3 resulted in a slight increase of the depolymerizing activity.     

Cellular localisation of human epidermal growth factor receptor

We show here, using immunohistological techniques and a monoclonal antibody to the receptor for epidermal growth factor (EGFR) (Waterfield et al. 1982) that EGFR is present on a wide range of normal epithelial tissues and tumours arising from those sites. The distribution of the receptor suggests that EGF may be involved in the control of proliferation and possibly differentiation of surface epithelia. The strong tumour cell staining suggests an increased expression of the receptor in certain carcinomas.     

Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts

The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1/2 = 10.1 h) was faster than the rate of degradation of total cell protein. When EGF was added to the nonradioactive medium, the half-life of prelabeled receptor was decreased to 1.2 h in human fibroblasts. These data demonstrate by direct analysis of receptor protein that during "down regulation" the EGF receptor is rapidly degraded. Enhanced receptor degradation was observed 5-10 min after the addition of EGF. The EGF-induced degradation of the receptor was blocked by methylamine, chloroquine, iodoacetate, or incubation at 25 degrees C. We have also shown that EGF-induced down regulation in human fibroblasts results in a decrease in the total amount of EGF receptor protein present. The amount of EGF receptor protein has been quantitated by radiolabeling cellular protein and immunoprecipitation of the receptor. The EGF receptor constitutes approximately 0.0035% of the cellular protein in human fibroblasts.     

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.     

Increased epidermal growth factor receptor in multidrug-resistant human neuroblastoma cells

Multidrug-resistant human neuroblastoma cell lines obtained by selection with vincristine or actinomycin D from two independent clonal lines, SH-SY5Y and MC-IXC, have 3- to 30-fold more cell surface epidermal growth factor (EGF) receptors than the drug-sensitive parental cells as indicated by EGF binding assays and immunoprecipitation, affinity-labeling, and phosphorylation studies. Reversion to drug sensitivity in one line was accompanied by a return to the parental level of EGF receptor. SH-EP cells, a clone derived from the same neuroblastoma cell line as SH-SY5Y but which displays melanocyte rather than neuronal lineage markers, also express significantly more EGF receptor than SH-SY5Y cells. By nucleic acid hybridization analysis with a molecularly cloned probe, increased receptor level in multidrug-resistant cells was shown to be the result of higher levels of EGF receptor mRNA in drug-resistant than in drug-sensitive cells. The increased steady state amount of specific RNA did not result from amplification of receptor-encoding genes. A small difference was observed in the electrophoretic mobility under denaturing conditions of EGF receptor immunoprecipitated from drug-resistant and drug-sensitive cells. Quantitative and qualitative modulation of the EGF receptor might reflect alterations in the transformation and/or differentiation phenotype of the resistant cells or might result from unknown selective pressures associated with the development of multidrug resistance.     

Structural basis for epidermal growth factor receptor function

The receptor for epidermal growth factor (EGF) has been the subject of intense study primarily as a consequence of the pioneering studies of Cohen on growth factors and also because of its homology to the transforming protein encoded by the avian oncogene v-erbB, which is a truncated receptor, and its consequent role in cancer. Although similar structural mutation of the EGF receptor has not yet been found in human tumours, aberrant overexpression of both EGF receptors and c-erbB2, a closely related putative receptor [1], have been found to occur in squamous cell carcinomas and glial tumours, and mammary carcinomas respectively [2–4]. In addition to EGF, the related polypeptides transforming growth factor α (TGFα) and vaccinia virus growth factor [5] are also ligands for the EGF receptor. Expression of TGFα occurs during embryonal development and in specific adult tissues; it may also play a role in cellular transformation (reviewed in Ref. 6). These important properties, as well as the potential roles of both TGFα and EGF in wound repair, have emphasized the need to understand EGF receptor structure, function and regulation. This review discusses the structural properties of the EGF receptor and how these can be related to receptor function and regulation.     

Western blot detection of epidermal growth factor receptor from plasmalemma of culture cells using 125I-labeled epidermal growth factor

We have developed a novel Western blot procedure for the detection of epidermal growth factor (EGF) receptors within a complex mixture of membrane proteins. Purified cell membranes from either human placenta or cultured A431 cells were solubilized, resolved by electrophoresis, and electroblotted onto nitrocellulose paper. With 5-15% gradient gels, electroblotting was completed in 2 h and both the high- and low-molecular-weight proteins were transferred evenly onto the nitrocellulose, as indicated by the radiolabeled protein markers. Upon hybridization with 125I-EGF, the membrane receptor was identified as two adjoining bands on the nitrocellulose of 150 and 170 kDa. Binding of 125I-EGF to the immobilized membrane receptor was specific and was displaced by excess unlabeled EGF. The receptor signal on the autoradiogram was optimized when 1% hemoglobin and 0.05% Tween 20 were present during the hybridization. The ligand-binding activity of the immobilized receptor was not affected by sodium dodecyl sulfate detergent or ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but was drastically reduced by either heat denaturation or the addition of dithiothreitol to the membrane samples. Using this method, we were able to demonstrate that no noticeable difference was observed between the pre- and postphosphorylated EGF receptors in their ability to bind to 125I-EGF. Because it allows both identification and purification of a receptor from a mixture of proteins, this protocol should have general application in characterizing various receptor-ligand systems.     

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.     

Analogs of human epidermal growth factor which partially inhibit the growth factor-dependent protein-tyrosine kinase activity of the epidermal growth factor receptor

Three site-directed mutants of human epidermal growth factor, Leu-26----Gly, Leu-47----Ala, and Ile-23----Thr, were examined for their ability to stimulate the protein-tyrosine kinase activity of the epidermal growth factor receptor. The receptor binding affinities of the mutant growth factors were 20- to 50-fold lower, as compared to wild-type growth factor. At saturating concentrations of growth factor, the velocities of the phosphorylation of exogenously added substrate and receptor autophosphorylation were significantly lower with the mutant analogs, suggesting a partial 'uncoupling' of signal transduction. The mutant analogs were shown to compete directly with the binding of wild-type, resulting in a decrease in growth factor-stimulated kinase activity.     

Calcineurin-mediated dephosphorylation of the human placental membrane receptor for epidermal growth factor urogastrone

The findings of our work were 2-fold: (1) calcineurin (from bovine brain) can catalyze the complete dephosphorylation of the phosphotyrosine and phosphoserine residues in the human placental receptor for epidermal growth factor urogastrone (EGF-URO), and (2) the major calmodulin-binding protein of human placental membranes is a calcineurin-related protein. In terms of its metal ion dependence (Ni2+ greater than Mn2+ greater than Co2+), its calmodulin dependence, and its sensitivity to inhibitors (Zn2+, fluoride, orthovanadate), the phosphotyrosyl protein phosphatase activity of calcineurin, using the EGF-URO receptor as substrate, paralleled the enzyme activity measured with p-nitrophenyl phosphate (PNPP) as a substrate. These characteristics distinguish calcineurin from other classes of protein phosphotyrosyl phosphatases. Calcineurin purified from placental membranes was similar to, if not identical with, bovine brain calcineurin in terms of enzymatic specific activity toward PNPP, subunit electrophoretic mobilities, and immunological cross-reactivity. The enzymatic properties and comparative abundance of calcineurin in the placenta membranes suggest that this enzyme may play an important role in regulating the phosphorylation state of those receptors (e.g., for EGF-URO or insulin) also known to be present in the membranes.     

A fully human recombinant IgG-like bispecific antibody to both the epidermal growth factor receptor and the insulin-like growth factor receptor for enhanced antitumor activity

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of cancers. Here we propose that simultaneous targeting of both receptors with a bispecific antibody would lead to enhanced antitumor activity. To this end, we produced a recombinant human IgG-like bispecific antibody, a Di-diabody, using the variable regions from two antagonistic antibodies: IMC-11F8 to EGFR and IMC-A12 to IGFR. The Di-diabody binds to both EGFR and IGFR and effectively blocked both EGF- and IGF-stimulated receptor activation and tumor cell proliferation. The Di-diabody also inherited the biological properties from both of its parent antibodies; it triggers rapid and significant IGFR internalization and degradation and mediates effective antibody-dependent cellular cytotoxicity in a variety of tumor cells. Finally, the Di-diabody strongly inhibited the growth of two different human tumor xenografts in vivo. Our results underscore the benefits of simultaneous targeting of two tumor targets with bispecific antibodies.     

Proteolytic domains of the epidermal growth factor receptor of human placenta

Microsomal membranes from human placenta, which bind 5–20 pmol of ¹²⁵I-epidermal growth factor (EGF) per mg protein, have been affinity-labeled with ¹²⁵I-EGF either spontaneously or with dimethylsuberimidate. Coomassie blue staining patterns on SDS polyacrylamide gels are minimally altered, and the EGF-receptor complex appears as a specifically labeled band of 180,000 daltons which is not removed by urea, neutral buffers, or chaotropic salts but is partially extracted by mild detergents. Limited proteolysis by alpha chymotrypsin and several other serine proteases yields labeled fragments of 170,000, 130,000, 85,000, and 48,000 daltons. More facile cleavage by papain or bromelain rapidly degrades the hormone-receptor complex to smaller labeled fragments of about 35,000 and 25,000 daltons. These fragments retain the binding site for EGF, are capable of binding EGF, and remain associated with the membrane. Alpha chymotryptic digestion of receptor solubilized by detergents yields the same fragments obtained with intact vesicles, suggesting that the fragments may represent intrinsic proteolytic domains of the receptor.     

Relation of epidermal growth factor receptor concentration to growth of human epidermoid carcinoma A431 cells

The relation between the concentration of epidermal growth factor (EGF) receptor/kinase and effects of EGF on cell proliferation has been studied using variant A431 cells and antagonist anti-EGF receptor monoclonal antibodies. Clonal A431 cell variants selected for escape from the EGF-mediated growth inhibition of parental A431 cells all have reduced concentrations of EGF receptor/kinase; Harvey sarcoma virus-transformed A431 cells, which have escaped from EGF-mediated growth inhibition, also have reduced EGF receptors. Three clonal variants which have reacquired EGF-mediated growth inhibition have 2- to 4-fold more EGF receptor than their respective parent variant. A biphasic response with stimulation at low and inhibition at high concentrations of EGF was especially evident in revertants of clone 29. Three separate antagonist monoclonal anti-EGF receptor antibodies block the growth inhibitory effects of EGF and uncover EGF-mediated growth stimulation. These studies indicate that in A431 cell variants a continuum of ligand-activated EGF receptors determines proliferative responses from low concentrations of active receptors under basal conditions to intermediate concentrations causing growth stimulation to high concentrations, causing inhibition of cell proliferation.     

Hydrogen peroxide inhibits epidermal growth factor receptor internalization in human fibroblasts

Several cellular signal transduction cascades are affected by oxidative stress. In this study, the effect of hydrogen peroxide (H2O2) on the endocytosis of the epidermal growth factor (EGF) receptor was investigated. Exposure of HER14 cells to H2O2 resulted in a concentration-dependent inhibition of EGF receptor internalization. Binding studies demonstrated that this H2O2-induced inhibition in internalization was not due to altered binding of EGF to its receptor. Addition of H2O2 at different time points during internalization showed that EGF receptor internalization was rapidly reduced, suggesting that one of the first steps in the internalization process is inhibited. In addition, H2O2 inhibited the internalization of a different receptor, the chicken hepatic lectin receptor, in a concentration-dependent manner as well. Treatment of cells with another inducer of oxidative stress, cumene hydroperoxide, also resulted in a decreased internalization. Finally, we showed that H2O2 inhibited EGF-induced mono-ubiquitination of the EGF receptor pathway substrate clone 15, a process that normally occurs during EGF receptor endocytosis. These results clearly show that oxidative stress interferes with EGF signaling.     

Ultrastructural localization of epidermal growth factor receptor in human parotid gland

The epidermal growth factor receptor (EGFR) is widely distributed in several organs in which, following interaction with its ligand, it can affect development and differentiation. The aim of this study was to define the distribution of EGFR in human parotid gland by means of a post-embedding immunogold staining method. Normal human parotid glands obtained at surgery were routinely prepared for electron microscopy. Semithin and ultrathin sections were treated for immunocytochemistry using a mouse monoclonal antibody specific for EGFR and a goat anti-mouse gold conjugated secondary antiserum. At the light microscope level, EGFR reactivity was revealed by a specific dark staining in both acinar and ductal cells. At the electron microscope level, EGFR was strongly stained in the cytoplasmic compartments and occasionally labeled on cell surfaces. In acinar cells, it appeared to be associated with small vesicles of uncertain nature that were scattered among the secretory granules. EGFR-positive vesicles were also observed in the ductal cells, with the most intense labeling being localized in striated ducts. Since cytoplasmic vesicles were previously found to be EGF-positive, these results may be due to the presence of the EGF-EGFR complex that is internalized after binding of EGF to the surface EGFR.     

Biosynthesis of the epidermal growth factor receptor in human epidermoid carcinoma-derived A431 cells

Using human-specific antibody reagents, we have examined the biosynthesis of the epidermal growth factor receptor in human epidermoid carcinoma-derived A431 cells. Four Mr species (Mr = 70,000, 95,000, 135,000, and 145,000) are detected when cells are subjected to a brief pulse of L-[35S]methionine; an Mr = 165,000 species is detected after 45-60 min of exposure of cells to radiolabel. In pulse-chase experiments, the four lower Mr species appear to bear a precursor relation to the Mr = 165,000 protein. The molecule acquires N-linked oligosaccharide cotranslationally, and two of the species (Mr = 95,000 and 145,000) are susceptible to digestion with endo-beta-N-acetylglucosaminidase H. The Mr = 145,000 and Mr = 165,000 proteins, which become labeled with 125I-epidermal growth factor after treatment of intact cells with a bifunctional cross-linking reagent, are phosphorylated at serine and threonine on identical tryptic peptides.     

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.