Mechanism of biological synergy between cellular Src and epidermal growth factor receptor

Overexpression of both cellular Src (c-Src) and the epidermal growth factor receptor (EGFR) occurs in many of the same human tumors, suggesting that they may functionally interact and contribute to the progression of cancer. Indeed, in murine fibroblasts, overexpression of c-Src has been shown to potentiate the mitogenic and tumorigenic capacity of the overexpressed EGFR. Potentiation correlated with the ability of c-Src to physically associate with the activated EGFR and the appearance of two unique in vivo phosphorylations on the receptor (Tyr-845 and Tyr-1101). Using stable cell lines of C3H10T1/2 murine fibroblasts that contain kinase-deficient (K-) c-Src and overexpressed wild-type EGFR, we show that the kinase activity of c-Src is required for both the biological synergy with the receptor and the phosphorylations on the receptor, but not for the association of c-Src with the receptor. In transient transfection assays, not only epidermal growth factor but also serum- and lysophosphatidic acid-induced DNA synthesis was ablated in a dominant-negative fashion by a Y845F mutant of the EGFR, indicating that c-Src-induced phosphorylation of Y845 is critical for the mitogenic response to both the EGFR and a G protein-coupled receptor (lysophosphatidic acid receptor). Unexpectedly, the Y845F mutant EGFR was found to retain its full kinase activity and its ability to activate the adapter protein SHC and extracellular signal-regulated kinase ERK2 in response to EGF, demonstrating that the mitogenic pathway involving phosphorylation of Y845 is independent of ERK2-activation. The application of these findings to the development of novel therapeutics for human cancers that overexpress c-Src and EGFR is discussed.     

125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts

125I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37 degrees C, the cell-bound 125I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound 125I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0 degrees C, cell-bound 125I-hEGF is not degraded but slowly dissociates from the cell. The data are consistent with a mechanism in which 125I-hEGF initially is bound to the cell surface and subsequently is internlized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-CH2Cl, benzyl guanidobenzoate), a lysosomotropic agent (chloroquine) various local anesthetics (cocaine, lidocaine, procaine), and ammonium chloride. After the binding and degradation of 125I-hEGF the fibroblasts are no longer able to rebind fresh hormone. The binding capacity of these cells is restored by incubation in a serum-containing medium; this restoration is inhibited by cycloheximide or actinomycin D.     

Saturation of the endocytic pathway for the transferrin receptor does not affect the endocytosis of the epidermal growth factor receptor

Cell-surface receptors that undergo clathrin-mediated endocytosis contain short amino acid sequences in their cytoplasmic domain that serve as internalization signals. Interactions between these sequences and components of the endocytic machinery should become limiting upon overexpression of the constitutively recycling transferrin receptor (TfR). A tetracycline-responsive system was used to induce overexpression of the TfR up to 20-fold in HeLa cells. Internalization assays indicate the rate of 125I-transferrin uptake per surface TfR is reduced by a factor of 4 in induced cells. Consistent with endocytosis being the rate-limiting step, TfRs shift from an endosomal to more of a plasma membrane distribution with TfR overexpression. The clathrin-associated protein AP-2 has been proposed to interact directly with the cytoplasmic domain of many receptors, yet no changes in the amount or distribution of AP-2 were detected in induced cells. The internalization rate for the epidermal growth factor receptor was also measured, with or without induction of TfR expression. Even though endocytosis of the TfR is saturated in induced cells, 125I-labeled epidermal growth factor continues to be internalized at a rate identical to that seen in uninduced cells. We propose that there are different limiting steps for the endocytosis of these two receptors.     

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.     

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.     

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.     

Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligand-induced internalization of the growth hormone receptor

The major function of the ubiquitin-conjugating system is the targeting of cytosolic and nuclear proteins for degradation by the proteasome. Recently, ubiquitin conjugation has been implicated in the downregulation of signalling receptors such as the mammalian growth hormone receptor (GHR) and the alpha-factor receptor in yeast. By examining truncated receptors, the internalization-deficient receptor mutant F327A and conditions under which clathrin-mediated GHR endocytosis is inhibited, we show here that GHR ubiquitination and ligand-induced GHR internalization are coupled events. Previously, we had shown that GHR endocytosis is dependent on an intact ubiquitination system. Here we present evidence that GHR ubiquitination depends on an intact endocytic pathway. Our data indicate that the ubiquitin-conjugating system and the endocytic pathway interact at the cytoplasmic tail of the GHR at the plasma membrane, where they cooperate to regulate internalization of the GHR.     

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.     

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.     

Sialidase gene transfection enhances epidermal growth factor receptor activity in an epidermoid carcinoma cell line, A431

Glycosphingolipids expressed in cancer cells have been implicated in the modulation of tumor cell growth through their interaction with transmembrane signaling molecules such as growth factor receptors. For glycosphingolipids to interact with growth factor receptors, the presence of sialic acid seems to be essential. Stable transfection of a gene encoding a soluble Mr 42,000 sialidase into a human epidermoid carcinoma cell line (A431) provided an approach by which the level of terminal lipid-bound sialic acid on the cell surface could be altered. In the sialidase-positive clones, the level of ganglioside GM3 was diminished, and little change was observed in protein sialylation. Sialidase-transfected cells grew faster than control cells. Sialidase expression did not modify the binding of epidermal growth factor (EGF) to its receptor but enhanced EGF receptor (EGFR) tyrosine autophosphorylation as compared to that of parental cells or cells transfected with the vector (pcDNA3) alone. Moreover, the phosphorylation of the EGFR, as well as other protein substrates, was observed at low EGF concentrations, suggesting an increase in the receptor kinase sensitivity. These data provided evidence that changes in ganglioside expression in cancer cells by appropriate gene transfection can dramatically affect EGFR kinase activity. Hence, the modulation of ganglioside expression may represent an approach to alter tumor cell growth.     

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.     

Increased epidermal growth factor receptor expression in metaplastic bronchial epithelium

Epidermal growth factor receptor (EGFr) is expressed in human bronchial epithelial cells, and non-small cell lung cancers express increased EGFr. Squamous metaplasia of the bronchial epithelium occurs in chronic smokers and is considered an early premalignant change. In this study, EGFr expression was examined in biopsies of histologically normal and metaplastic bronchial tissues obtained from 69 smokers who were enrolled in a randomized placebo-controlled chemoprevention trial. This trial tested the effects of 6 months of treatment with 13-cis retinoic acid (13cRA) on bronchial metaplasia. EGFr expression was examined as a marker of bronchial metaplasia and response to 13cRA treatment. In bronchial biopsies obtained from patients in this study, EGFr expression was higher in metaplastic biopsies than in normal biopsies (P = 0.02). Smoking cessation during treatment correlated with reduced metaplasia (P < 0.001) and EGFr expression (P = 0.02), but multivariate analysis suggested that this effect of smoking cessation on EGFr expression was dependent upon reversal of bronchial metaplasia. 13cRA treatment did not alter EGFr expression (P = 0.23). Baseline EGFr expression levels in metaplastic biopsies did not predict metaplasia reversal. This study demonstrated that increased EGFr expression is a biomarker of bronchial metaplasia, but it did not support the hypothesis that EGFr is a biomarker of retinoid response in lung cancer chemoprevention trials.     

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.     

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.     

Small molecule inhibitors of the platelet-derived growth factor receptor, the fibroblast growth factor receptor, and Src family tyrosine kinases

The inhibition of tyrosine kinases involved in growth factor signal transduction pathways represents an attractive strategy for controlling aberrant cellular growth. Over the last 4-5 years, there have been numerous reports on the discovery of small molecule inhibitors for potential therapeutic applications to a number of proliferative diseases, principally cancer and restenosis, where the over-expression of certain tyrosine kinases has been demonstrated. These include, amongst others, the platelet-derived growth factor receptor, the fibroblast growth factor receptor, and the nonreceptor c-Src tyrosine kinase. This review compiles published reports and patent filings from 1995 to mid-1997 that include data directly related to inhibition of the platelet-derived growth factor receptor, fibroblast growth factor receptor, and Src family tyrosine kinases. Potential clinical applications for selected classes of tyrosine kinase inhibitors reviewed herein will likely depend on the demonstration of meaningful activity in a variety of therapeutic targets in animal models.     

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 that EGF-R may directly influence nuclear function.     

Characterization of the epidermal growth factor receptor in pig oviduct and endometrium

Two different plaque variants of Japanese encephalitis virus were selected from a wild-type Taiwanese isolate using Vero cells. One variant was found to exhibit small plaque morphology with retarded virus replication kinetics in Vero cells, and was demonstrated to be resistant to monoclonal antibody (mAb) E3.3 neutralization. The other variant showed large plaque morphology, was sensitive to mAb E3.3 neutralization, and manifested reduced virulence in mice on both intracranial and intraperitoneal inoculations. These two variants propagated in Vero cells retained high levels of infectivity but had relatively low HA titers as compared with the parent strain. The envelope sequences of these two variants showed four amino acid differences at residues E-85 (Glu/Arg), E-306 (Glu/Gly), E-331 (Ser/Arg), and E-387 (Met/Arg). Our results indicated the neutralizing epitope of Japanese encephalitis virus did not overlap with virus virulence determinant.     

The epidermal growth factor receptor tyrosine kinase phosphorylates connexin32

To elucidate the role of excessive nitric oxide (NO) via the inducible nitric oxide synthase (iNOS) in experimental allergic encephalomyelitis (EAE), the effect of a selective iNOS inhibitor, aminoguanidine, was investigated using mice with actively induced EAE. Administration of aminoguanidine by intraperitoneal or intracisternal injection from day 2 to day 12 after immunization produced a significant delay in the onset of EAE. On the other hand, administration of aminoguanidine by intraperitoneal or intracisternal injection for 10 days after the onset of EAE enhanced the clinical severity and mortality rate and hastened the onset of relapse significantly. The histological study at day 11 after the onset revealed that more inflammatory cells were present in the central nervous system of mice treated with aminoguanidine as compared with mice without aminoguanidine treatment. These results suggested that NO via iNOS was a pathogenetic factor in the induction phase of EAE, but had an inhibitory role in the progression phase of EAE. Although the effect of NO synthase inhibitors on EAE has been controversial, the present study suggested that the timing of administration might be an important consideration and might explain the previous contradictory reports.     

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.