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.     

Temperature-dependent lateral and transverse distribution of the epidermal growth factor receptor in A431 plasma membranes

Summary To elucidate further the structure and molecular dynamics of the epidermal growth factor receptor, temperature-dependent aggregation and extracellular protrusion of the epidermal growth factor receptor in isolated plasma membranes from A431 cells were examined by fluorescence energy-transfer techniques. Epidermal growth factor was labeled at the amino terminus with either fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate. A radionuclide receptor displacement assay demonstrated the bioactivity of these derivatives. Aggregation of the epidermal growth factor receptor was measured by determining the increase in fluorescence energy transfer between receptorbound fluorescein and tetramethylrhodamine-labeled epidermal growth factor. Energy transfer between receptor-bound fluorescent derivatives was reversibly greater at 37 than 4°C, indicating temperature-dependent aggregation of the receptor. The extracellular protrusion of the epidermal growth factor receptor was calculated from the magnitude of energy transfer between receptorbound fluorescein labeled epidermal growth factor and 5-(N-dodecanoylamino)-eosin partitioned into the lipid membrane at 4 and 37°C. No significant change in the distance of closest approach between the N-terminus of epidermal growth factor and the plasma membrane was observed at 4°C (69±2 Å) and 37°C (67±2 Å). Thus, the extracellular protrusion of the occupied epidermal growth factor receptor did not change detectably upon receptor aggregation.     

Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid.

Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.     

Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.     

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.     

Role of epidermal growth factor-stimulated protein kinase in control of proliferation of A431 cells

Epidermal growth factor (EGF), which stimulates tyrosine-specific protein kinase activity both in vivo and in vitro, inhibits proliferation of A431 human epidermoid carcinoma cells. After mutagenesis clonal cell lines that were resistant to the growth inhibitory effects of EGF were selected. All six variants examined contained decreased EGF-stimulated protein kinase. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in variant cell lines with up to tenfold reductions in both activities. This result suggests that both EGF binding and kinase activities reside in the same or closely coupled molecules. The effect of EGF on growth of two resistant variants was examined in detail. Clone 29 contains approximately 50% and clone 4 contains approximately 20% of the EGF-stimulated protein kinase activity of the parental A431 cell line. In serum-supplemented medium, EGF stimulated proliferation of clone 29 but did not affect growth of clone 4. In a 1:1 mixture of DME and F-12 medium without serum, EGF caused both clone 29 and clone 4 to grow as well as in 10% serum. These variants, which were selected for resistance to the growth inhibitory effects of EGF, thus exhibit a strong mitogenic response to EGF. This result suggests that resistance to the growth inhibitory effect of EGF may involve both a decrease in EGF-stimulated protein kinase and an alteration in the response pathway.     

Association of the epidermal growth factor receptor kinase with the detergent-insoluble cytoskeleton of A431 cells

The epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma cells, A431, was found to be predominantly associated with the detergent-insoluble cytoskeleton, where it retained both a functional ligand-binding domain and an intrinsic tyrosine kinase activity. The EGF-R was constitutively associated with the A431 cytoskeleton; this association was not a consequence of adventitious binding. The EGF-R was associated with cytoskeletal elements both at the cell surface, within intracellular vesicles mediating the internalization of the hormone-receptor complex, and within lysosomes. The EGF-R became more stably associated with cytoskeletal elements after its internalization. The cytoskeletal association of the EGF-R was partially disrupted on suspension of adherent cells, indicating that alteration of cellular morphology influences the structural association of the EGF-R, and that the EGF-R is not intrinsically insoluble. Cytoskeletons prepared from EGF-treated A431 cells, when incubated with gamma-32P-ATP, demonstrated enhanced autophosphorylation of the EGF-R in situ as well as the phosphorylation of several high molecular weight proteins. In this system, phosphorylation occurs between immobilized kinase and substrate. The EGF-R and several high molecular weight cytoskeletal proteins were phosphorylated on tyrosine residues; two of the latter proteins were phosphorylated transiently as a consequence of EGF action, suggesting that EGF caused the active redistribution of the protein substrates relative to protein kinases. The ability of EGF to stimulate protein phosphorylation in situ required treatment of intact cells at physiological temperatures; addition of EGF directly to cytoskeletons had no effect. These data suggest that the structural association of the EGF-R may play a role in cellular processing of the hormone, as well as in regulation of the EGF-R kinase activity and in specifying its cellular substrates.     

Regulation of the epidermal growth factor receptor by phosphorylation

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.     

Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)‐expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293‐EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (K_D, 8.49 ± 0.11 × 10^?7 M) > erlotinib (K_D, 1.07 ± 0.02 × 10^?6 M) > canertinib (K_D, 1.41 ± 0.07 × 10^?6 M) > afatinib (K_D, 1.80 ± 0.12 × 10^?6 M) > vandetanib (K_D, 1.99 ± 0.03 × 10^?6 M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.     

Epidermal growth factor induces changes of interaction between epidermal growth factor receptor and actin in intact cells

The epidermal growth factor receptor (EGFR) is a cyto-skeleton-binding protein. Although purified EGFR can interact with acting in vitro and normally at least 10% of EGFR exist in the insoluble cytoskeleton fraction of A431 cells, interaction of cytosolic EGFR with actin can only be visualized by fluorescence resonance energy transfer when epidermal growth factor presents in the cell medium. Results indicate that the correct orientation between EGFR and actin is important in the signal transduction process.     

Characterization of a comparative model of the extracellular domain of the epidermal growth factor receptor

The Epidermal Growth Factor (EGF) receptor is a tyrosine kinase that mediates the biological effects of ligands such as EGF and transforming growth factor alpha. An understanding of the molecular basis of its action has been hindered by a lack of structural and mutational data on the receptor. We have constructed comparative models of the four extracellular domains of the EGF receptor that are based on the structure of the first three domains of the insulin-like growth factor-1 (IGF-1) receptor. The first and third domains of the EGF receptor, L1 and L2, are right-handed beta helices. The second and fourth domains of the EGF receptor, S1 and S2, consist of the modules held together by disulfide bonds, which, except for the first module of the S1 domain, form rod-like structures. The arrangement of the L1 and S1 domains of the model are similar to that of the first two domains of the IGF-1 receptor, whereas that of the L2 and S2 domains appear to be significantly different. Using the EGF receptor model and limited information from the literature, we have proposed a number of regions that may be involved in the functioning of the receptor. In particular, the faces containing the large beta sheets in the L1 and L2 domains have been suggested to be involved with ligand binding of EGF to its receptor.     

Expression of epidermal growth factor receptor and associated glycoprotein on cultured human brain tumor cells

The expression of epidermal growth factor (EGF-R) in normal glial and glioma cells grown in culture was examined by using several independent assays. Immunoprecipitation with the monoclonal antibody R1 of extracts from metabolically labeled glial and glioma cells revealed a protein of Mr approximately 170,000, with a migration in sodium dodecyl sulfate-polyacrylamide gels identical to the EGR-R of A431 epidermal carcinoma cells. Furthermore, in the majority of glioma extracts, a protein of Mr approximately 190,000 was specifically immunoprecipitated by this antibody. Similar results were obtained by immunoblotting with a second antibody directed against a synthetic peptide in the sequence of the v-erb-B oncogene. In cell lines expressing both proteins, each was specifically phosphorylated on tyrosine in immune complex kinase assays. The majority of glioma cells bound between 40,000 to 80,000 125I-labeled epidermal growth factor molecules per cell. These results suggest that the expression of EGF-R is common in cultured human glioma cells. In addition, a structurally related protein, is expressed in some of these cells.     

Growth and secretion of erythropoietin of Chinese hamster ovary cells coexpressing epidermal growth factor receptor and erythropoietin genes: Design of cells for cell culture matrix

Chinese hamster ovary (CHO) cells were transfected withboth genes encoding erythropoietin (Epo) and epidermal growthfactor receptor (EGFR). The transfection of the Epo gene wasconfirmed by an enzyme-linked immunoassay. Overexpression ofEGFR was confirmed by Western blotting of EGFR. Thetransfected CHO cells were cultured in serum-free medium inthe presence of soluble epidermal growth factor (EGF) orimmobilized EGF. The CHO cells overexpressing EGFR grew in thepresence of less EGF than the cells not overexpressing EGFR.In addition, the growth of EGFR-overexpressing CHO cells wasenhanced in the presence of immobilized EGF more efficientlythan in the presence of soluble EGF. The amount of Eposecreted from the cells increased linearly with the increaseof growth rate. Consequently, culture of CHO cellscoexpressing Epo and EGFR on EGF-immobilized matrix was themost efficient for Epo production.     

Biosynthesis of the epidermal growth factor receptor in A431 cells.

A monoclonal antibody R1 against the human epidermal growth factor receptor has been used to study biosynthesis in the carcinoma cell line A431. Two glycoproteins of apparent mol. wts. 95 000 and 160 000 were immunoprecipitated from cells labelled for short times with [35S]methionine or [3H]mannose. Pulse-chase studies show the 160 000 mol. wt. glycoprotein to be a precursor of the 175 000 mol. wt. receptor, but do not establish a precursor role for the 95 000 mol. wt. glycoprotein. Limited proteolysis, peptide mapping, endoglycosidase digestion and the use of monensin and tunicamycin show that the 95 000 mol. wt. glycoprotein is structurally related to the 160 000 mol. wt. glycoprotein and that both glycoproteins have approximately 22 000 - 28 000 mol. wt. of oligosaccharide side chains. Monensin blocks conversion of the 160 000 to the 175 000 mol. wt. mature receptor, a process which involves complexing several of its N-linked oligosaccharide chains. Pulse-chase studies showed that an immunoprecipitable polypeptide of 115 000 mol. wt., or 95 000 mol. wt., in the presence of monensin, was secreted into the medium at late chase times. The possible mechanisms for the origins of all the receptor-related polypeptides are discussed.     

Nerve growth factor-induced reduction in epidermal growth factor responsiveness and epidermal growth factor receptors in PC12 cells: an aspect of cell differentiation.

The rat pheochromocytoma clone PC12 responds to nerve growth factor through the expression of a number of differentiated neuronal properties. One of the most rapid changes is a large, transient increase in the activity of ornithine decarboxylase. These cells also show an increase in ornithine decarboxylase activity in response to the mitogen, epidermal growth factor, but do not respond morphologically as they do to nerve growth factor. Specific, high-affinity epidermal growth factor receptors are present on the cells. When the cells are differentiated with nerve growth factor, the response to epidermal growth factor is markedly diminished and there is a marked reduction in the binding of epidermal growth factor to the cells.     

Topography of amphiregulin expression in cultured human keratinocytes: Colocalization with the epidermal growth factor receptor and CD44

Summary Much of the autonomous growth of cultured keratinocytes is attributable to the signaling of amphiregulin, a heparin-binding autocrine growth factor, through the epidermal growth factor receptor. Emerging evidence suggests, moreover, that the membrane proteoglycan, CD44, is a cofactor for the interaction of heparin-binding ligands with their receptors. This model was evaluated by characterizing the patterns of the immunolabeled molecules in cultured human neonatal keratinocytes, to test the hypothesis that involvement in a common function results in coordinate segregation within or on the cell. The molecules were localized by double immunofluorescence labeling to detect amphiregulin and either the epidermal growth factor receptor or CD44, and the immunostained products were imaged by scanning laser confocal microscopy. Both amphiregulin and the epidermal growth factor receptor segregated to a perinuclear distribution and to intercellular contacts. In addition, amphiregulin localized to the outer leading edge of colonies and focally to intranuclear sites. Metabolic blockade of proteoglycan sulfation with sodium chlorate inhibited growth of the cells and concurrently enhanced the nuclear, but decreased the outer leading edge, labeling for amphiregulin. There was no nuclear or perimeter labeling for the epidermal growth factor receptor. Cultures co-immunolabeled for CD44 and amphiregulin exhibited variable perinuclear staining for both, but otherwise CD44 was distributed to intercellular contacts. The intercellular localizations of CD44 with amphiregulin and of amphiregulin with the epidermal growth factor receptor were strongly concordant. These data are consistent with a concerted function at intercellular contacts, where cytokine signaling is mediated via receptor binding and possibly regulated by the CD44 proteoglycan as cofactor. The intranuclear and perimeter labeling of amphiregulin, however, suggests that this cytokine has additional functions, both in the nucleus and as a matrix receptor.     

表皮生长因子受体与肺脏发育的关系

表皮生长因子受体(Epidermal growth factor receptor,EGFR)是一种跨膜蛋白受体,是ErbB家族成员之一,具有酪氨酸激酶活性。EGFR与相应的配体结合引起EGFR形成同源或异源二聚体启动胞内信号转导,激活下游多种信号转导途径,产生生物学效应,RAS/RAF/MEK/ERK通路与细胞增殖、分化和凋亡有关;PI3K/PDK1/AKT通路与细胞的迁移和粘附有关。EGFR能促进肺泡II型上皮细胞的成熟和肺表面活性物质的合成、分泌。EGFR对哺乳动物肺脏的作用呈现时空效应及剂量依赖效应,EGFR的下调表达则会引起肺脏发育不成熟;而EGFR过度表达促进肺肿瘤细胞的增殖、侵袭和转移。文章综述了EGFR及其调节信号通路的研究进展,以及EGFR与动物肺脏发育不成熟和肺癌之间的关系。     

重组人表皮生长因子的稳定性

考察了不同条件下重组人表皮生长因子（rhEGF）的稳定性。结果表明,不同的保存条件（物理状态、温度、pH、浓度等）对rhEGF的稳定性有显影响,使用适当的添加剂（保护剂、抗氧剂、抑菌等）可以提高rhEGF的稳定性。上述结果为rhEGF的制剂学研究提供了依据。