Mechanisms of Gurken-dependent pipe regulation and the robustness of dorsoventral patterning in Drosophila

The restriction of Pipe, a potential glycosaminoglycan-modifying enzyme, to ventral follicle cells of the egg chamber is essential for dorsoventral axis formation in the Drosophila embryo. pipe repression depends on the TGFalpha-like ligand Gurken, which activates the Drosophila EGF receptor in dorsal follicle cells. An analysis of Raf mutant clones shows that EGF signalling is required cell-autonomously in all dorsal follicle cells along the anteroposterior axis of the egg chamber to repress pipe. However, the autoactivation of EGF signalling important for dorsal follicle cell patterning has no influence on pipe expression. Clonal analysis shows that also the mirror-fringe cassette suggested to establish a secondary signalling centre in the follicular epithelium is not involved in pipe regulation. These findings support the view that the pipe domain is directly delimited by a long-range Gurken gradient. Pipe induces ventral cell fates in the embryo via activation of the Sp?tzle/Toll pathway. However, large dorsal patches of ectopic pipe expression induced by Raf clones rarely affect embryonic patterning if they are separated from the endogenous pipe domain. This indicates that potent inhibitory processes prevent pipe dependent Toll activation at the dorsal side of the egg.     

Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells. Evidence for protein kinase C-dependent and -independent pathways

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.     

Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent

We have used the soluble covalent cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) to examine the capacity of epidermal growth factor (EGF) to stimulate the dimerization of purified EGF receptor, of EGF receptor in membrane preparations and in intact A431 cells. The addition of EGF either to membranes from A431 cells or to EGF receptor which was purified from A431 cells by immunoaffinity chromatography caused the appearance of a cross-linked product of Mr 340,000 which was identified using EGF receptor-specific antibodies as an EGF receptor dimer. Three independent approaches including biosynthetic labeling, surface iodination, and immunoblotting experiments were utilized to follow EGF receptor dimerization in living A431 cells. These approaches provided consistent results indicating that EGF induced rapid dimerization of EGF receptor in living cells, suggesting that this process may play a role in transmembrane signalling mediated by EGF.     

Modeling the epidermal growth factor -- epidermal growth factor receptor l2 domain interaction: implications for the ligand binding process

Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of ligands such as EGF or transforming growth factor alpha (TGF-alpha) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10-16, 36-37, 40-47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-alpha which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor.     

Epidermal growth factor (EGF) and monoclonal antibody to cell surface EGF receptor bind to the same chromatin receptor

Cellular uptake, nuclear translocation, and chromatin binding of epidermal growth factor (EGF) and monoclonal antibodies (MAbs) against the protein domain of the EGF surface receptor (MAb 425) and against the carbohydrate Y determinant on the EGF receptor (MAb Br 15-6A) were analyzed in cell lines that express surface EGF receptor. Both EGF and MAb 425 were translocated to the nucleus and bound in nondegraded form to the chromatin of all cells tested. MAb Br 15-6A was taken up only by SW 948 colorectal carcinoma cells which express EGF receptor whereas neither EGF nor MAb 425 was taken up by SW 707 colorectal carcinoma cells which do not express EGF receptor. MAb 425 immunoprecipitated a 230- to 250-kDa chromatin protein, which appears to be the EGF chromatin receptor. EGF was localized in a single EcoRI DNA fragment suggesting that the chromatin binding was highly specific. Binding of EGF to primarily DNase II-sensitive chromatin regions protected these regions from nuclease action. The role of growth factor binding to chromatin in neoplastic transformation is discussed.     

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.     

Tumor necrosis factor regulates tyrosine phosphorylation on epidermal growth factor receptors in A431 carcinoma cells: evidence for a distinct mechanism.

Previous studies of tumor necrosis factor (TNF) action on tumor cells revealed a possible role for tyrosine phosphorylation of epidermal growth factor (EGF) receptor in the growth-regulatory activities of this cytokine (N. J. Donato, G. E. Gallick, P. A. Steck, and M. G. Rosenblum, J. Biol. Chem., 264: 20474-20481, 1989). EGF receptor immunoprecipitated from [32P] phosphate-equilibrated A431 cells demonstrated that TNF treatment resulted in both a time- and concentration-dependent stimulation of EGF receptor phosphorylation, which was maximal (approximately 3-fold) after 10-20 min of TNF exposure (10 nM). Incubation of A431 cells with an equivalent concentration of EGF resulted in similar stimulation of EGF receptor phosphorylation, albeit at different phosphotyrosine levels. Antiphosphotyrosine immunoblot analysis confirmed these results but suggested that the extent and kinetics of TNF-induced tyrosine phosphorylation were distinct from those obtained in EGF-treated cells. Resolution of tryptic phosphopeptides from EGF receptor demonstrated that TNF-induced phosphorylation of EGF receptor was similar, but not identical, to profiles obtained from EGF-treated cells and distinct when compared to the actions of phorbol ester. Unlike EGF, TNF was unable to directly stimulate EGF receptor tyrosine kinase activity in membranes prepared from A431 cells. In addition, TNF treatment had no significant effect on either the high- or low-affinity ligand-binding sites on EGF receptor and did not alter the kinetics or extent of ligand-induced internalization of EGF receptors. However, EGF receptor biosynthesis was consistently increased upon prolonged treatment with TNF (4-12 h). Our results suggest that TNF regulates both phosphorylation and biosynthesis of EGF receptor in a manner distinct from that of both EGF and phorbol ester, and studies of the differential phosphorylation of EGF receptor may aid in understanding the molecular mode of TNF action.     

Epidermal growth factor stimulates the synthesis of its own receptor in a human breast cancer cell line

The MDA 468 human breast carcinoma cell line was examined for changes in epidermal growth factor (EGF) receptor synthesis and degradation under the influence of EGF. This cell line was used because it overexpresses the EGF receptor such that each cell has 10(6) receptors, but unlike the well-studied A431 cell, its receptor gene is amplified but is not rearranged. On exposure to EGF, total cellular receptor protein, measured by immunoprecipitation with monoclonal antibody B1D8, is reduced. The half-life of receptor metabolically labeled with L-[35S]methionine is 24 h in the absence of EGF and is reduced to 12 h in the presence of 10(-9) M EGF. To measure the effect of EGF on synthesis of the receptor, pulse labeling conditions were selected in which the rate of synthesis of the receptor precursor were followed. EGF had no significant effect on the rate of general protein synthesis in these cells, yet stimulated the synthesis of the EGF receptor 1.8-fold over the unstimulated rate. This increase in receptor precursor synthesis showed time and dose dependence. Stimulation could be detected after 3 h exposure to EGF with a maximum at 6-8 h. A concentration of 10(-11) M EGF gave detectable stimulation with maximal stimulation occurring at 10(-9) M. Longer times and higher concentrations gave submaximal stimulation. A similar dose-response relationship was observed when the rate of mature 170-kDa receptor protein synthesis was measured. These studies demonstrate that EGF stimulates the synthesis of it own receptor. Downregulation of the receptor by EGF results from an increased rate of receptor degradation and not decreased synthesis.     

Two independent mechanisms for escaping epidermal growth factor- mediated growth inhibition in epidermal growth factor receptor- hyperproducing human tumor cells

Human squamous cell carcinoma cell lines often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGF. To investigate the mechanism of EGF-mediated inhibition of cell growth, variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGF receptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant isolated from Ca9-22 cells, CER-1, grew without being affected by EGF. CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicated an increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independent mechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.     

Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity

The effect of autophosphorylation and protein kinase C-catalyzed phosphorylation on the tyrosine-protein kinase activity and ligand binding affinity of the epidermal growth factor (EGF) receptor has been studied. Kinetic parameters for the phosphorylation by the receptor kinase of synthetic peptide substrates having sequences related to the 3 in vitro receptor autophosphorylation sites (tyrosine residues 1173 (P1), 1148 (P2), and 1068 (P3)) were measured. The Km of peptide P1 (residues 1164-1176) was significantly lower than that for peptides P2 (residues 1141-1151) or P3 (residues 1059-1072). The tyrosine residue 1173 was also the most rapidly autophosphorylated in purified receptor preparations, consistent with previous observations for the receptor in intact cells (Downward, J., Parker, P., and Waterfield, M. D. (1984) Nature 311, 483-485). Variation in the extent of receptor autophosphorylation from 0.1 to 2.8 mol of phosphate/mol of receptor did not influence kinase activity or EGF binding affinity either for purified receptor or receptor in membrane preparations. Phosphorylation of the EGF receptor by protein kinase C was shown to cause a 3-fold decrease in the affinity of purified EGF receptor for EGF and to reduce the receptor kinase activity. In membrane preparations, phosphorylation of the EGF receptor by protein kinase C resulted in conversion of high affinity EGF binding sites to a low affinity state. This suggests that activation of protein kinase C by certain growth promoting agents and tumor promoters is directly responsible for modulation of the affinity of the EGF receptor for its ligand EGF. The regulation of the EGF receptor function by protein kinase C is discussed.     

Variation in EGF-induced EGF receptor downregulation in human hepatoma-derived cell lines expressing different amounts of EGF receptor

The effect of epidermal growth factor (EGF) receptor overexpression on ligand-induced EGF receptor downregulation was examined using a hepatoma-derived cell line, PLC/PRF/5, which expresses normal amounts of the EGF receptor, and a subline, NPLC/PRF/5, which expresses 10-fold more receptors at its cell surface. PLC/PRF/5 cells efficiently downregulated surface receptor levels upon exposure to saturating and subsaturating concentrations of EGF; the rate of receptor downregulation corresponded to that of ligand-receptor internalization. Upon internalization, EGF receptors were degraded and receptor biosynthesis remained at basal levels. EGF surface receptor remained downregulated for as long as cells were exposed to EGF. By contrast, surface EGF receptor abundance in NPLC/PRF/5 cells decreased by only 5-15% after 1-4 h incubation with subsaturating doses of EGF and actually increased by 67% within 20 h. Exposure of these cells to saturating concentrations of EGF induced modest decreases in surface receptor abundance during the initial 12 h incubation, followed by a progressive decline to 30% of initial values by 24 h. Relative ligand-receptor internalization rates in NPLC/PRF/5 cells were lower than those in PLC/PRF/5, although their surface receptor population was even higher than that predicted by the decreased internalization rates. EGF receptor degradation in NPLC/PRF/5 cells was also inhibited; exposure to saturating levels of EGF for more than 16 h was necessary before significant degradation occurred. Receptor protein and mRNA biosynthesis in NPLC/PRF/5 were stimulated by 8 h exposure to EGF but when saturating concentrations of EGF were present for 16 h, receptor biosynthesis was inhibited. EGF receptor overexpression circumvents the downregulatory effect of EGF by decreasing the rate of receptor internalization, inhibiting degradation of the internalized receptor pool, and stimulating EGF receptor biosynthesis. Conversely, receptor downregulation becomes pronounced at late times when receptor degradation is high and biosynthesis is inhibited.     

Desensitization of the EGF receptor alters its ability to undergo EGF-induced dimerization

Treatment of A431 cells with EGF has been shown to induce the formation of EGF receptor dimers. Sucrose density gradient centrifugation as well as surface radio-iodination followed by crosslinking were used to study further the properties of EGF receptor monomers and dimers as well as the regulation of dimer formation. We have shown previously that treatment of A431 cells with high doses of EGF at 37 degrees C leads to the desensitization of the EGF receptor without a significant loss of cell surface 125I-EGF binding [Kuppuswamy and Pike (1989) J. biol. Chem. 264, 3357-3363; Cunningham et al. (1989) J. biol. Chem. 264, 15351-15356]. Desensitization of the EGF receptor led to a decrease in the ability of receptor monomers to be induced to form dimers by EGF both in vivo and in vitro. These data suggest that the sensitivity of a cell to EGF may be modulated by altering the capacity of the EGF receptor to form oligomers.     

Epidermal growth factor and transforming growth factor-alpha induce differential processing of the epidermal growth factor receptor

The capacity of epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) to induce internalization and degradation of the EGF receptor was compared in NIH-3T3 cells expressing the human EGF receptor. This study was initiated following the observation that TGF-alpha was much less efficient relative to EGF in generating a Mr = 125,000 amino-terminally truncated degradation product from the mature EGF receptor (EGF-dependent generation of this degradation product is described in S.J. Decker, J. Biol. Chem., 264:17641-17644). Pulse-chase experiments revealed that EGF generally stimulated EGF receptor degradation to a greater extent than TGF-alpha. Both ligands induced EGF receptor internalization to similar degrees. However, recovery of [125I]-EGF binding following incubation with EGF or TGF-alpha was much faster for TGF-alpha treated cells. Recovery of [125I]-EGF binding after TGF-alpha treatment did not appear to require protein synthesis. Tyrosine phosphorylation of EGF receptor from cells treated with TGF-alpha decreased more rapidly following removal of TGF-alpha compared to cells treated similarly with EGF. These data suggest that EGF routes the EGF receptor directly to a degradative pathway, whereas TGF-alpha allows receptor recycling prior to degradation, and that tyrosine phosphorylation could play a role in this differential receptor processing.     

Tumor necrosis factor increases the number of epidermal growth factor receptors on human fibroblasts

Tumor necrosis factor (TNF) was shown previously to stimulate the growth of human FS-4 fibroblasts. Here we show that human recombinant TNF can increase the binding of epidermal growth factor (EGF) to these cells. Incubation with TNF resulted in a 40-80% increase in the number of EGF-binding sites with no apparent change in receptor binding affinity. The increase in EGF binding was apparent 8-12 h after the addition of TNF. TNF also increased the amount of EGF receptor protein immunoprecipitated from cells labeled with [35S]methionine. Stimulation of EGF receptor protein synthesis was demonstrable 2-4 h following TNF treatment. TNF increased EGF binding with a dose-response relationship similar to that reported earlier for the mitogenic action. Increased expression of EGF receptors, due to enhanced synthesis of the EGF receptor protein, may be functionally related to the mitogenic action of TNF in human fibroblasts.     

Transient epidermal growth factor (EGF)-dependent suppression of EGF receptor autophosphorylation during internalization.

To study the activity of the epidermal growth factor (EGF) receptor during EGF-directed internalization, liver epithelial cells were exposed to EGF at 37 degrees C for various periods of time, washed, and homogenized at 0 degrees C. EGF receptor autophosphorylation was assessed in homogenates using [gamma-32P]ATP. Autophosphorylation was stimulated 3- to 6-fold in homogenates of cells incubated with EGF (100 ng/ml) for 15 min but was at or below basal levels in homogenates of cells treated with EGF for 2.5-5 min. This was surprising because immunoblotting revealed that EGF receptor phosphotyrosine (P-Tyr) content in intact cells was near maximal from 30 s to 5 min after EGF treatment. Excess EGF (1 microgram/ml), added after homogenization but prior to the assay, increased autophosphorylation in homogenates of cells that had not been treated with EGF, but failed to increase activity in homogenates of cells treated with EGF in culture for 2.5-5 min. Suppression of tyrosine phosphorylation of an exogenous kinase substrate was also observed at times paralleling the suppression of EGF receptor autophosphorylation. The transient suppression of receptor autophosphorylation in the cell-free assay was not explained by persistent occupation of autophosphorylation sites by phosphate added in the intact cells. The sites were greater than 80% dephosphorylated during the homogenization. Additionally phosphatase inhibition that prevented the normal loss of EGF receptor P-Tyr in intact cells at 15 min did not affect the pattern of early (2.5-5 min) suppression and later (15 min) stimulation of autophosphorylation measured in the cell-free assay. The suppression was not explained by activation of protein kinase C in that depletion of greater than 95% of cellular protein kinase C activity by an 18-h incubation of cells with 10 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the early suppression of autophosphorylation in EGF-treated cells. Moreover, under the conditions tested, activation of protein kinase C by short-term treatment (0.5-10 min) with TPA or angiotensin II did not appreciably alter subsequent autophosphorylation in the cell-free assay. In contrast, a 30 degrees C preincubation of homogenates from cells with suppressed EGF receptor autophosphorylation led to the recovery of the ability of EGF to stimulate EGF receptor autophosphorylation. These results suggest that a rapid reversible protein kinase C-independent process prevents detection of EGF receptor kinase activity during an early phase of EGF-dependent receptor internalization.     

Ectodomain shedding-dependent transactivation of epidermal growth factor receptors in response to insulin-like growth factor type I

Diverse extracellular stimuli activate the ERK1/2 MAPK cascade by transactivating epidermal growth factor (EGF) receptors. Here, we have examined the role of EGF receptors in IGF-I-stimulated ERK1/2 activation in several cultured cell lines. In human embryonic kidney 293 cells, IGF-I triggered proteolysis of heparin binding (HB)-EGF, increased tyrosine autophosphorylation of EGF receptors, stimulated EGF receptor inhibitor (AG1478)-sensitive ERK1/2 phosphorylation, and promoted EGF receptor endocytosis. In a mixed culture system that employed IGF-I receptor null murine embryo fibroblasts (MEFs) (R(-) cells) to detect paracrine signals produced by MEFs expressing the human IGF-I receptor (R(+) cells), stimulation of R(+) cells provoked rapid activation of green fluorescent protein-tagged ERK2 in cocultured R(-) cells. The R(-) cell response was abolished by either the broad-spectrum matrix metalloprotease inhibitor batimastat or by AG1478, indicating that it resulted from the proteolytic generation of an EGF receptor ligand from adjacent R(+) cells. These data suggest that the paracrine production of EGF receptor ligands leading to EGF receptor transactivation is a general property of IGF-I receptor signaling. In contrast, the contribution of transactivated EGF receptors to IGF-I-stimulated downstream events, such as ERK1/2 activation, varies in a cell type-dependent manner.     

Epidermal growth factor (EGF) stimulates EGF receptor synthesis

Epidermal growth factor (EGF) binds to the extracellular domain of a specific 170,000-dalton transmembrane glycoprotein; this results in rapid removal of both ligand and receptor from the cell surface. In WB cells, a rat hepatic epithelial cell line, ligand-directed receptor internalization leads to receptor degradation. We tested whether the EGF receptor was replenished at a constitutive or enhanced rate following EGF binding by immunoprecipitating biosynthetically labeled EGF receptor from cells cultured with [35S]methionine. EGF stimulated receptor synthesis within 2 h in a dose-dependent manner; this was particularly evident when examining the nascent form of the receptor. To determine the site of EGF action, total WB cell RNA was transferred to nitrocellulose paper after electrophoresis and was hybridized to cDNA probes from both the external and cytoplasmic coding regions of the human EGF receptor. EGF increased receptor mRNA by 3-5-fold. Therefore, at least in some cells, the surface action of EGF that leads to EGF receptor degradation is counterbalanced by a positive effect on receptor synthesis.     

Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells.

Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.     

Treatment of A431 cells with epidermal growth factor (EGF) induces desensitization of EGF-stimulated phosphatidylinositol turnover

Epidermal growth factor (EGF) stimulates the turnover of phosphoinositides in A431 cells. In cells that were pretreated with EGF for 30 min at 37 degrees C and then washed to remove surface-bound hormone, a 70-100% decrease in the EGF-stimulated production of inositol monophosphate, inositol bisphosphate, and inositol triphosphate was noted when the cells were exposed to the agonist a second time. Since only a 15% decrease in receptor number was observed in these pretreated cells, the loss of responsiveness to EGF for the production of inositol phosphates could not be attributed to a down-regulation of the EGF receptors. These data suggest that pretreatment of A431 cells with high concentrations of EGF leads to a desensitization of the EGF receptor. This desensitization of the receptor by EGF is apparent within 10-15 min of the addition of EGF and is maximal by 30 min. The desensitization appears to be homologous in nature since pretreatment of cells with EGF did not diminish their responsiveness to bradykinin; and conversely, pretreatment with bradykinin did not diminish the subsequent responsiveness of the cells to EGF. Desensitization to EGF was observed in cells in which protein kinase C had been down-regulated by prolonged treatment with 12-O-tetradecanoylphorbol-13-acetate, implying that EGF receptor desensitization is independent of protein kinase C. The desensitizing effects of EGF on growth factor-induced phosphatidylinositol turnover could be prevented by pretreatment of the cells with the calmodulin antagonist trifluoperazine, suggesting that calmodulin may be involved in the regulation of EGF receptor sensitivity.