Disulfide bond structure of human epidermal growth factor receptor

The extracellular domain of the human epidermal growth factor receptor (sEGFR) consists of 621 amino acid residues, including 50 cysteines. The connections of the 25 disulfide bonds in the recombinant sEGFR protein, obtained from Chinese hamster ovary cells, have been determined using N-terminal sequencing and matrix-assisted laser desorption/ionization mass spectroscopy. We identified a basic repeat of eight cysteines with a 1-3, 2-4, 5-6, and 7-8 disulfide pairing pattern in the two cysteine-rich regions of sEGFR. By comparison to other cysteine-rich motifs, it was concluded that the cysteine-rich repeat of sEGFR belongs to the laminin-type EGR-like (LE) structural motif. Three-dimensional structure models of the two cysteine-rich regions have been built, based on the three-dimensional structures of the LE domains from the laminin gamma1 chain and secondary structure predictions for the EGF receptor.     

Expression of epidermal growth factors and epidermal growth factor receptor in normal cycling human ovaries

Immunolocalization of transforming growth factor-alpha (TGF alpha), epidermal growth factor (EGF), cripto-1, amphiregulin and epidermal growth factor receptor (EGFR) was studied in 51 premenopausal human ovaries at various phases of the menstrual cycle. Localization of mRNA for TGF alpha and EGF was also studied by in-situ hybridization. Immunoreactive TGF alpha was observed predominantly in theca cells in 12 of 33 antral follicles in the follicular phase (6/14 dominant follicles, and 6/19 non-dominant) but not in any of the 18 follicles in the luteal phase or in primordial and pre-antral follicles. TGF alpha immunoreactivity was present predominantly in the luteinized granulosa cells in 13 of 15 corpora lutea in the luteal phase, which are considered to be active in steroidogenesis, but not in any of the regressed corpora lutea. Accumulation of TGF alpha mRNA hybridization signal was observed only in the theca cells in the follicles and luteinized theca cells in the ovaries that were immunohistochemically positive for TGF alpha. EGFR immunoreactivity was detected in 24 of 33 antral follicles in the follicular phase and in two of 18 follicles in the luteal phase but not in any of the corpora lutea. Immunoreactive EGF, cripto-1 and amphiregulin or EGF mRNA was not detected in any follicles, corpora lutea, or the stroma cells examined. These results indicate that, of the epidermal growth factors examined in this study, TGF alpha is locally synthesized in normal cycling human ovaries and TGF alpha may be synthesized in theca cells and act on the granulosa cells in a paracrine fashion through the EGFR in ovarian follicles.     

Immunocytochemical localization of transforming growth factor alpha, epidermal growth factor and epidermal growth factor receptor in the cat endometrium and placenta

Hoxb-5 is one of the few homeobox genes strongly expressed in the developing mouse lung. To explore the hypothesis that Hoxb-5 acts to regulate epithelial cell fate and branching morphogenesis in the developing lung, we studied the temporal, spatial, and cell-specific expression of Hoxb-5 from gestational day (d) 13.5 to postnatal day (P) 2. Immunocytochemistry demonstrated regional localization of Hoxb-5 protein to developing conducting airways and surrounding mesenchyme. The cellular expression pattern changed from diffusely positive nuclei of mesenchymal cells on d13.5 to become more localized to nuclei of subepithelial fibroblasts and some adjacent columnar and cuboidal epithelial cells on d14.5. After d14.5, Hoxb-5 protein expression continued to decrease in mesenchymal cells distal from developing airways, but persisted in fibroblasts underlying conducting airways. Hoxb-5 protein expression persisted in nuclei of columnar and cuboidal epithelial cells on d16.5 and d17.5, with expression in low cuboidal epithelial cells as well from d17.5 to P2. Western blot analysis showed temporal and quantitative changes in Hoxb-5 protein expression with peak expression on d14.5-15.5. We conclude that Hoxb-5 protein is developmentally regulated in a temporal, spatial, and cell-specific manner throughout the pseudoglandular, canalicular, and terminal saccular periods of lung development in the mouse. This localization and expression pattern suggests that Hoxb-5 may influence branching morphogenesis, cell-cell communication, cell fate, and differentiation of conducting airway epithelia.     

Relation of epidermal growth factor receptor concentration to growth of human esophageal cancer cell lines

The relation between the concentration of epidermal growth factor (EGF) receptor and the effects of EGF on cell proliferation were studied using 16 newly established human esophageal cancer cell lines. According to 125I-EGF binding assay, the amount of EGF receptor was found to vary from 6 x 10(4) to 1.2 x 10(7) (sites/cell). Changes in EGF-stimulated tyrosine-specific protein kinase activity almost paralleled changes in the number of EGF receptors per cell. Amplification of EGF receptor gene was detected in only one cell line. Under monolayer culture conditions, we found three types of growth responses of esophageal cell lines to EGF; growth in 5 cell lines was inhibited and that in 4 cell lines was stimulated while that in the other 7 cell lines remained unaffected. Relation was observed between the number of EGF receptors per cell and the growth response to EGF. On the other hand, cell lines whose growth was inhibited by EGF in monolayer culture were stimulated by EGF in soft agar culture, though the opposite was not necessarily true.     

Effect of nocodazole on endocytosis of epidermal growth factor receptor

Although interferon-alpha (IFN-alpha) has proved beneficial in the treatment of some tumors, the basis for this is still uncertain. In this study, we examined the effects of IFN-alpha on the growth of tumor cells in vitro, using the Daudi line of B lymphoma cells as a model. There was a dose-dependent accumulation of these cells in the G1 phase of the cell cycle 24-48 h from the time of exposure to IFN-alpha. This was followed between 48 h and 96 h by an increasing degree of apoptosis, as assessed by cell survival, propidium iodine staining, and transmission electron microscopy. Concomitantly with the apoptosis, there was the appearance of pl8 Bax-alpha, an apparently novel variant low molecular weight form of the p21 Bax-alpha found in normal cells. There was also a slight diminution in Bcl-xL, with a resultant drop in the Bcl-xL:Bax-alpha ratio. Treatment of cells with CD40-L partially inhibited the development of apoptosis in response to IFN-alpha. At the same time, generation of p18 Bax-alpha was reduced, which suggests that this plays a part in the apoptotic process. These findings may throw light on the development of lymphomas and perhaps point to future ways of improving therapy with IFN-alpha.     

pH sensitivity of epidermal growth factor receptor complexes

The association/dissociation binding kinetics of 125I-labeled mouse epidermal growth factor (EGF) to receptors on human fibroblast cells in monolayer culture have been measured at 4 degrees C as a function of extracellular pH from pH 5-9. At pH 8, steady-state total binding is maximal. As pH is lowered to 6.5, total binding monotonically decreases dramatically. It changes further only slightly between pH 6.5 and 5 to about 20% of the maximum binding value. Scatchard binding plots at pH 7.5 and above show the commonly observed concave-upward, non-linear curve; as pH is lowered, this plot becomes much more linear, indicating that the "high affinity" bound receptor population is greatly diminished. Application of our ternary complex binding model [Mayo et al., J Biol Chem 264:17838-17844, 1989], which hypothesizes complexation of the EGF-bound receptor with a cell surface interaction molecule, indicates that pH may have some direct effects on ternary complex formation, but the major effect is on EGF-receptor dissociation.     

Phosphorylation of CrkII adaptor protein at tyrosine 221 by epidermal growth factor receptor

CrkII adaptor protein becomes tyrosine-phosphorylated upon various types of stimulation. We examined whether tyrosine 221, which has been shown to be phosphorylated by c-Abl, was phosphorylated also by other tyrosine kinases, such as epidermal growth factor (EGF) receptor. For this purpose, we developed an antibody that specifically recognizes Tyr221-phosphorylated CrkII, and we demonstrated that CrkII was phosphorylated on Tyr221 upon EGF stimulation. When NRK cells were stimulated with EGF, the tyrosine-phosphorylated CrkII was detected at the periphery of the cells, where ruffling is prominent, suggesting that signaling to CrkII may be involved in EGF-dependent cytoskeletal reorganization. The EGF-dependent phosphorylation of CrkII was also detected in a c-Abl-deficient cell line. Moreover, recombinant CrkII protein was phosphorylated in vitro by EGF receptor. These results strongly suggest that EGF receptor directly phosphorylates CrkII. Mutational analysis revealed that the src homology 2 domain was essential for the phosphorylation of CrkII by EGF receptor but not by c-Abl, arguing that these kinases phosphorylate CrkII by different phosphorylation mechanisms. Finally, we found that the CrkII protein phosphorylated upon EGF stimulation did not bind to the phosphotyrosine-containing peptide and that CrkII initiated dissociation from EGF receptor within 3 min even with the sustained tyrosine phosphorylation of EGF receptor. This result implicated phosphorylation of Tyr221 in the negative regulation of the src homology 2-mediated binding of CrkII to EGF receptor.     

Intestinal adaptation is enhanced by epidermal growth factor independent of increased ileal epidermal growth factor receptor expression

BACKGROUND/PURPOSE: Intestinal adaptation after massive small bowel resection (SBR) is augmented by epidermal growth factor (EGF) via an unknown mechanism. We recently have observed that EGF increases the expression of EGF receptor mRNA and protein content in the remnant ileum after SBR. The purpose of this study was to determine whether the magnitude of EGF-induced receptor expression correlates with intestinal adaptation. METHODS: A 50% proximal SBR or sham operation (bowel transection with reanastomosis) was performed on male ICR mice. Animals from each group were then selected randomly to receive either human recombinant EGF (150 microg/kg/d) or saline by twice daily intraperitoneal injections. The remnant ileum was harvested at 1 week, and parameters of adaptation measured as changes in protein content. Ileal EGF receptor mRNA was quantitated using a ribonuclease protection assay. Changes in the expression ileal EGF receptor protein were determined by Western blot after immunoprecipitation. Comparisons of mean values between groups was performed using analysis of variance (ANOVA) and a P value of less than .05 was considered significant. Values are presented as mean +/- SEM. RESULTS: EGF was mitogenic to the ileum after sham operation as monitored by increases in ileal protein content (2.21 +/- 0.002 mg/cm Sham v 2.97 +/- 0.25 mg/cm Sham +/- EGF; P < .05). After SBR, adaptation resulted in increased ileal protein content (4.45 +/- 0.27 mg/cm), which was substantially boosted by EGF (5.98 +/- 0.39 mg/cm; P < .05). No differences were detected in ileal EGF receptor mRNA or protein expression between Sham or SBR groups that did not receive EGF. However, EGF significantly enhanced the expression of ileal EGF receptor mRNA to an equal extent after both sham and SBR (approximately threefold). The magnitude of this increase in EGF receptor protein (four- to sixfold) was similar in both EGF groups as shown by Western blotting. CONCLUSIONS: Changes in ileal EGF receptor expression are not mandatory for adaptation to occur. EGF upregulates the expression of mRNA and protein for its own intestinal receptor in vivo. Because EGF-induced receptor expression was comparable after both SBR and Sham operation, the beneficial effect of EGF during adaptation is likely caused by other factors in addition to increased receptor expression.     

A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity

The development and neoplastic progression of human astrocytic tumors appears to result through an accumulation of genetic alterations occurring in a relatively defined order. One such alteration is amplification of the epidermal growth factor receptor (EGFR) gene. This episomal amplification occurs in 40-50% of glioblastomas, which also normally express endogenous receptors. Moreover, a significant fraction of amplified genes are rearranged to specifically eliminate a DNA fragment containing exons 2-7 of the gene, resulting in an in-frame deletion of 801 bp of the coding sequence of the extracellular domain. Here we used retroviral transfer of such a mutant receptor (de 2-7 EGFR) into glioblastoma cells expressing normal endogenous receptors to test whether the mutant receptor was able to augment their growth and malignancy. Western blotting analysis showed that these cells expressed endogenous EGFR of 170 kDa as well as the exogenous de 2-7 EGFR of 140-155 kDa. Although holo-EGFRs were phosphorylated on tyrosine residues only after exposure of the cells to ligand, de 2-7 EGFRs were constitutively phosphorylated. In tissue culture neither addition of EGF nor expression of the mutant EGFR affected the rate of cell growth. However, when cells expressing mutant EGFR were implanted into nude mice subcutaneously or intracerebrally, tumorigenic capacity was greatly enhanced. These results suggest that a tumor-specific alteration of the EGFR plays a significant role in tumor progression perhaps by influencing interactions of tumor cells with their microenvironment in ways not easily assayed in vitro.     

Transforming growth factor alpha, epidermal growth factor, and epidermal growth factor receptor expression in normal and diseased human adrenal cortex by immunohistochemistry and in situ hybridization

Because epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and epidermal growth factor receptor (EGFR) have been implicated in the regulation of adrenocortical function, we used immunohistochemistry and in situ hybridization of EGF and TGF-alpha to study 41 specimens of human adrenal cortex, including 10 normal specimens, 15 aldosteronomas, five Cushing's adenomas, six adrenocortical incidentalomas, and five carcinomas to determine what role these growth factors play in controlling human adrenocortical function. Neither immunoreactivity nor mRNA hybridization signals to EGF was detected in any specimens, and EGF therefore may exert its effects on adrenal function as an endocrine hormone. TGF-alpha expression was detected at both protein and mRNA levels in normal and neoplastic adrenal cortex, demonstrating that TGF-alpha is synthesized locally in human adrenal cortex. TGF-alpha expression was observed in the cells with increased steroidogenesis, including compact tumor cells and zona fasciculata cells with lipid depletion, but did not necessarily correlate with production sites of any specific steroid hormone. EGFR immunoreactivity was more widely distributed than TGF-alpha immunoreactivity. Both TGF-alpha and EGFR expression were markedly elevated in adrenocortical carcinomas. TGF-alpha and EGFR thus appear to be involved in biological function in both normal and neoplastic human adrenal cortex. In addition, TGF-alpha and EGFR may play important roles in some biological features of adrenocortical malignancy.     

Regulation of epidermal growth factor receptor by androgens in human endometrial cells in culture

Women with polycystic ovaries (PCO) have a thicker endometrium than women with normal ovaries. This cannot be due to unopposed oestrogen, as it occurs in ovulatory cycles. Androgens may be involved, as these are raised in women with PCO. The effects of steroids are partly mediated by growth factors and their receptors. The aim of this study was to investigate the effect of androgens on epidermal growth factor (EGF) receptor in human endometrium. Endometrium was enzymatically dispersed and glands and stromal cells separated. Cells were incubated in Ham's F10 medium supplemented with 5% charcoal-stripped fetal calf serum and either androgens or vehicle. Specific binding of [125I]-labelled EGF was measured. Testosterone and dihydrotestosterone (DHT) (10 micromol/l) increased EGF receptor concentration (control 100 +/- 9%, testosterone 196 +/- 23% control; control 100 +/- 1%, DHT 244 +/- 6% control) but did not alter receptor affinity. The effect of testosterone was inhibited by the anti-androgen hydroxyflutamide, but not by the antioestrogen ICI182780 nor the aromatase inhibitor 4-hydroxyandrostenedione. EGF receptor levels were increased by androstenediol (control 100 +/- 2%, androstenediol 120 +/- 10% control) but not by androstanediol, dehydroepiandrosterone (DHA), DHA sulphate or androstenedione. Testosterone and DHT increased EGF receptor concentrations in glandular epithelium (control 100 +/- 24%, testosterone 147 +/- 5%, DHT 185 +/- 30% control). These data suggest that androgens may have an effect on the endometrium via an increase in EGF receptor concentrations.     

Immunohistochemical localization of the epidermal growth factor, transforming growth factor alpha, and their receptor in the human mesonephros and metanephros

Nucleotide sequence analysis of a 3.3-kb genomic EcoRI fragment and of relevant subfragments of a genomic 13.2-kb SmaI fragment of Alcaligenes eutrophus, which were identified by using a dihydrolipoamide dehydrogenase-specific DNA probe, revealed the structural genes of the 2-oxoglutarate dehydrogenase complex in a 7.5-kb genomic region. The genes odhA (2850 bp), odhB (1248 bp), and odhL (1422 bp), encoding 2-oxoglutarate dehydrogenase (E1), dihydrolipoamide succinyltransferase (E2), and dihydrolipoamide dehydrogenase (E3), respectively, occur co-linearly in one gene cluster downstream of a putative -35/-10 promoter in the order odhA, odhB, and odhL. In comparison to other bacteria, the occurrence of genes for two E3 components for the pyruvate as well as for the 2-oxoglutarate dehydrogenase complexes is unique. Heterologous expression of the A. eutrophus odh genes in E. coli XL1-Blue and in the kgdA mutant Pseudomonas putida JS347 was demonstrated by the occurrence of protein bands in electropherograms, by spectrometric detection of enzyme activities, and by phenotypic complementation, respectively.     

Insufficiency of self-phosphorylation for the activation of epidermal growth factor receptor

Polyclonal immunoglobulins were produced against the carboxy terminus, -SEFIGA, of the receptor for epidermal growth factor (EGF). The addition of these immunoglobulins to a solution containing EGF receptor resulted in the activation of its protein tyrosine kinase. The levels of activation were assessed by measuring the initial velocities of the phosphorylation of the tyrosine in angiotensin II. The enzymatic activity induced by the immunoglobulins was significant, usually 50-70% of the maximum activity induced by EGF, and the induction occurred over a narrow range of concentration of the immunoglobulins. In order to achieve the activation, the immunoglobulins had to be bivalent; the addition of monovalent Fab fragments to EGF receptor did not produce any activation of the protein tyrosine kinase. The activation produced by the immunoglobulins was found to be reversible upon the addition of the synthetic peptide SEFIGA against which the immunoglobulins had been produced. Self-phosphorylation of the EGF receptor also occurred as the enzyme was activated by the immunoglobulins. Tryptic peptide maps demonstrated that the self-phosphorylation caused by the immunoglobulins had the same signature as that produced by EGF. When the synthetic peptide that had been used as the hapten was added to EGF receptor that had been self-phosphorylated in the presence of the immunoglobulins, the stimulated enzymatic activity was lost even though the protein remained phosphorylated. It follows from the results of deletion mutation [Walton, G. M., Chen, W. S., Rosenfeld, M. G., & Gill, G. N. (1990) J. Biol. Chem. 265, 1750-1754] and the results reported here that self-phosphorylation is neither necessary nor sufficient for the activation of EGF receptor.     

Rational design for the development of epidermal growth factor receptor antagonists

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF alpha) bind with similar high affinity to the human EGF receptor. Using a domain-exchange strategy we have shown that the C-terminal linear region of these molecules is involved in high affinity receptor binding. By further single amino acid substitution in this linear C-terminal region, a putative interaction site of these ligands with their receptor has been identified. This identification of a receptor binding domain in EGF/TGF alpha provides an important initial step in the development of EGF receptor antagonists with significant clinical potential.     

CrkII signals from epidermal growth factor receptor to Ras

A rat fibroblast mutant defective in oncogenic transformation and signaling from epidermal growth factor receptor to Ras has been isolated. The mutant contains dominant negative-type point mutations in the C-terminal SH3 domain of one crkII gene. Among the adapters tested, the mutant is complemented only by crkII cDNA. Expression of the mutated crkII in parent cells generates the phenotype indistinguishable from the mutant cell. Yet overexpression or reduced expression of Grb2 in the mutant before and after complementation with crkII have little effect on its phenotype. We conclude that adapter molecules are highly specific and that the oncogenic growth signal from epidermal growth factor receptor to Ras is predominantly mediated by CrkII in rat fibroblast.     

Human epidermal growth factor and the proliferation of human fibroblasts

The majority of the mRNA molecules in HeLa cells contain 1-2 residue(s) of m6Ap and one blocked, methylated 5' terminal "cap" structure. The hnRNA, which is longer than mRNA, contains both m6Ap and caps but 4-6 times as many m6Ap residues per chain. In addition, nuclear molecules contain T2 RNA ase-resistant, methyl-labeled oligonucleotides ("di-" and "tri-" nucleotides) which are not found in mRNA. Some of the dinucleotides may be precursors to the 2'-0-methylated nucleotides in the cap structures. These results are compatible with internal methylation of hnRNA molecules (both m6Ap and 2'-0-methyl) followed by hnRNA cleavage and the addition of the cap structure to generate at least some of the HeLa cell mRNA. It also appears that some hnRNA molecules, which are longer than most mRNA molecules, contain cap structures suggesting the derivation of some mRNA molecules from the 5' regions of hnRNA.     

Comparison of radioligand assay and immunostaining for epidermal growth factor receptor in human breast cancer

Epidermal growth factor (EGF) receptor status is a useful prognostic indicator in women with breast cancer. Lack of standardization and correlation of methodology for the detection of EGF receptor has hampered its further evaluation. EGF receptor status was ascertained by immunohistochemistry and radioligand assay in 120 breast cancers. Of 52 tumours negative for EGF receptor on radioligand assay, 47 were negative on immunohistochemistry and, of 68 tumours positive for the receptor on assay, 52 were positive on immunohistochemistry. If the more widely evaluated radioligand assay is assumed to be the 'gold standard', immunohistochemistry has a sensitivity of 81 per cent and a specificity of 91 per cent.     

Decorin is a biological ligand for the epidermal growth factor receptor

Ectopic expression of decorin induces profound cytostatic effects in transformed cells with diverse histogenetic backgrounds. The mechanism of action has only recently begun to be elucidated. Exogenous decorin activates the epidermal growth factor (EGF) receptor, thereby triggering a signaling cascade that leads to phosphorylation of mitogen-activated protein (MAP) kinase, induction of p21, and growth suppression. In this study we demonstrate a direct interaction of decorin with the EGF receptor. Binding of decorin induces dimerization of the EGF receptor and rapid and sustained phosphorylation of MAP kinase in squamous carcinoma cells. In a cell-free system, decorin induces autophosphorylation of purified EGF receptor by activating the receptor tyrosine kinase and can also act as a substrate for the EGF receptor kinase itself. Using radioligand binding assays we show that both immobilized and soluble decorin bind to the EGF receptor ectodomain or to purified EGF receptor. The binding is mediated by the protein core and has relatively low affinity (Kd approximately 87 nM). Thus, decorin should be considered as a novel biological ligand for the EGF receptor, an interaction that could regulate cell growth during remodeling and cancer growth.