The epidermal growth factor receptor tyrosine kinase in liver epithelial cells. The effect of ligand-dependent changes in cellular location

Epidermal growth factor (EGF) activates the intrinsic tyrosine-specific protein kinase of its receptor (EGF-R). We studied the effect of EGF-dependent EGF-R internalization on receptor autophosphorylation and on the appearance of tyrosine phosphoproteins in rat liver epithelial cells. Peak receptor autophosphorylation activity (3- to 6-fold over basal) was found in homogenates of EGF-treated cells at times when the majority of receptors (greater than 90%) had been internalized but not yet degraded (15 to 30 min). Stimulated activity persisted for at least 2 h if EGF-R degradation was blocked by methylamine or 18 degrees C incubation. Detection of stimulated autophosphorylation in homogenates of cells treated with EGF in culture required detergent in the assay. Detergent was not necessary to detect stimulated autophosphorylation when EGF was added directly to homogenates of untreated cells. Immunoblots using antibodies against phosphotyrosine (p-Tyr) demonstrated that EGF treatment of intact cells increased the p-Tyr content of at least seven proteins (EGF-R, 115, 100, 75, 66, 57, and 52 kDa) within 5 s. Incubation of intact cells with EGF at 0 degrees C to prevent endocytosis still resulted in tyrosine phosphorylation of these seven proteins. In contrast, several substrates (120, 78, and 38 kDa) showed delayed increases (45-90 s) in tyrosine phosphorylation at 37 degrees C; their phosphorylation was even slower at 18 degrees C and did not occur at 0 degrees C. In cells incubated with EGF at 18 degrees C or in the presence of methylamine, EGF-R p-Tyr in the intact cell was lost by 2 h even though receptor was not degraded and still exhibited enhanced autophosphorylation in the homogenate assay. These findings suggest that tyrosine phosphorylation in response to EGF occurs predominantly during the initial stages of endocytosis and is mediated for the most part by ligand-receptor complexes at the cell surface. A subset of phosphorylations may require intracellular movement.     

IL-1 and TNF transmodulate epidermal growth factor receptors by a protein kinase C-independent mechanism

The effect of the human rIL-1 alpha and rTNF-alpha on the binding of 125I-labeled epidermal growth factor ([125I]EGF) to its receptor (EGF-R) has been studied in human gingival fibroblasts (HuGi). Incubation of these cells with recombinant cytokines at 37 degrees C caused a rapid, dose-dependent decrease in their ability to subsequently bind subsaturating levels of [125I]EGF at 4 degrees C. Inhibition was evident at 5 min after addition of cytokines, reached a maximal level (60-70% reduction) after 15 to 30 min, and declined thereafter. Normal EGF binding was attained by 2 h. Half-maximal inhibition of EGF binding occurred at 10 pM IL-1 and 50 pM TNF. The two cytokines were not additive in their effect. Competition experiments at 4 degrees C showed that the cytokines did not interact directly with EGF-R; Scatchard analysis of binding of [125I]EGF to HuGi after treatment with IL-1 and TNF revealed an increase in EGF-R Kd from 0.75 nM to 2.9 nM with no change in receptor number. The effect of IL-1 and TNF on EGF-R was compared with that of the tumor-promotor PMA which is known to "transmodulate" EGF-R affinity by activating protein kinase C which then phosphorylates EGF-R. PMA caused a greater inhibition of EGF binding to HuGi (80 to 85% inhibition; ED50 = 500 pM), and recovery of binding was much slower. Importantly, in HuGi made deficient in protein kinase C by prolonged incubation with PMA, addition of fresh PMA no longer affected EGF binding, while the response to IL-1 and TNF was intact. Cytokine- but not PMA-mediated EGF-R transmodulation was partially reversed by treatment of the cells with millimolar concentrations of the kinase inhibitor amiloride. HuGi were incubated with H3 32PO4, stimulated with PMA or cytokines, and EGF-R were immunoprecipitated; IL-1 and TNF, like PMA, caused a 2- to 5-fold increase in receptor phosphorylation. We conclude that occupation of IL-1 and TNF-R activates a protein kinase, distinct from kinase C, for which EGF-R is a substrate.     

Kinase activity controls the sorting of the epidermal growth factor receptor within the multivesicular body

We compared the internalization and intracellular sorting of epidermal growth factor receptor (EGF-R) and point mutant kinase-negative EGF-R separately expressed in NIH 3T3 cells lacking endogenous receptor. Both EGF-Rs internalized rapidly, but kinase-negative receptor was surface down-regulated only with monensin or at 20 degrees C. Furthermore, EGF internalized by mutant receptor alone was, in significant proportion, returned to the cell surface undegraded. Hence unlike wild-type receptor, kinase-negative EGF-R recycles. By electron microscopy the early pathways of endocytosis for the two receptors were identical; however, after 10-20 min the pathways diverged at the multivesicular body (MVB). Wild-type EGF-R, destined for degradation, localized to internal vesicles, while kinase-negative EGF-R, destined for recycling, localized to surface membranes of the MVBs and moved to small tubulovesicles. We conclude that sorting of internalized receptor for degradation or recycling can occur through spatial segregation within the MVB, and sorting of EGF-R is controlled by tyrosine kinase activity.     

Mobility of epidermal growth factor surface receptors and modulation by cytoplasmic actin.

Epidermal growth factor (EGF) receptors were demonstrated in cultured bovine odontogenic epithelial and ectomesenchymal cells and in rat embryonic and foetal lung fibroblasts by immunofluorescent reactivity with EGF and anti-EGF traced with a fluorescein-isothiocyanate-labelled goat anti-rabbit globulin. Mobility of EGF receptors, either alone or cross linked by anti-EGF, was manifest as sequential changes in staining patterns progressing from a random pattern to clusters, supranuclear caps and large perinuclear globules. Double fluorochrome studies showed that supranuclear caps of EGF receptors occurred in sites closely related to those occupied by cytoplasmic actin; the latter was demonstrated by anti-actin antibody traced with a rhodamine-labelled goat anti-human globulin. Prior treatment of cells with 2 x 10-5 M cytochalasin B for 10-120 min promoted capping of EGF receptors, while its presence throughout the staining procedure inhibited capping. The dual opposing effects of cytochalasin B on capping suggest that actin may have roles in the opposing functions of EGF receptor anchorage and mobility.     

Early events in the antiproliferative action of tumor necrosis factor are similar to the early events in epidermal growth factor growth stimulation

The process of TNF-induced cytotoxicity is complex but appears to be mediated through a TNF-specific cell surface receptor. Recent evidence suggests that TNF action on tumor cells may be antagonized by epidermal growth factor (EGF) and other EGF-receptor modulatory peptides implicating a role for EGF-R in the process of TNF-induced cytotoxicity. In the present report, we investigated the biochemical actions of TNF on several biochemical events known to occur in the process of EGF signal transduction in intact cells. The actions of TNF were compared directly to those of EGF in both TNF-sensitive and -resistant tumor cell lines. In TNF-sensitive ME-180 cervical carcinoma cells, TNF (20 ng/ml) stimulated the tyrosine protein kinase activity of the EGF-receptor (EGF-R) fivefold when measured by receptor autophosphorylation in an immune complex kinase assay. TNF activation of EGF-R kinase activity in ME-180 was measurable 10 min after TNF incubation and enzymatic activity remained elevated 20 min after TNF addition. Activation of the receptor by TNF correlated with increased 32P incorporation into EGF-R protein when receptor was immunoprecipitated from 32P-equilibrated cells following a 20 min incubation with TNF. Acid hydrolysis of EGF-R protein isolated from TNF-treated ME-180 cells demonstrates an increase in the phosphotyrosine content of EGF-R when compared to receptor isolated from untreated cells. The results suggest that TNF increased EGF-R tyrosine protein kinase activity and the state of EGF-receptor tyrosine phosphorylation in a manner similar to that reported for EGF. However, TNF does not appear to be structurally related to EGF since TNF was unable to directly activate EGF-R when incubated with extensively washed immunoprecipitates of EGF-R. In TNF-resistant T24 bladder carcinoma cells, TNF failed to alter EGF-R tyrosine protein kinase activity although both EGF and phorbol ester were shown to modulate the enzymatic activity of the receptor in these cells. These results indicate that the ability of TNF to modulate EGF-R kinase in target cells may correlate with its cytotoxic actions on TNF-sensitive tumor cells. Other biochemical activities associated with the induction or regulation of cellular growth were examined in TNF- or EGF-treated tumor cells. EGF stimulated a rapid 8-16-fold increase in the expression of the proto-oncogene c-myc when analyzed by dot-blot analysis of total cellular RNA or Northern blot hybridization of polyadenylated RNA. TNF treatment failed to alter c-myc expression in ME-180 cells when analyzed by either technique.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epidermal growth factor binding and steroid receptor content in human benign prostatic hyperplasia

The receptor for epidermal growth factor (EGF-R) was characterized on membrane fractions from human benign prostatic hyperplasia (BPH). Specific binding of [125I]EGF reached equilibrium after 40 min at 25 degrees C and was stable for up to 120 min. Saturation analysis of EGF-R, performed by incubating the membranes with 0.0156-15 nM [125I]EGF in the presence and in the absence of 100-fold excess of cold EGF for 60 min, revealed the presence of two classes of binding sites with high and low affinities (Kd = 0.35 +/- 0.23 and 9.60 +/- 2.87 nM respectively). Competition experiments revealed that FSH, insulin and calcitonin did not compete with [125I]EGF. The simultaneous determination of EGF-R and that of estradiol (ER), progesterone (PR) and androgen receptors (AR) was performed using the same buffer to homogenate the tissues and to obtain cellular membranes. The steroid receptors (SR) were determined by means of the dextran-coated charcoal method. There was a significant negative correlation between nuclear SR and binding capacity of EGF-R. The presence of specific and high affinity binding sites for EGF and the modulation of the level of these sites by steroid receptors suggest a possible role of EGF in prostatic hyperplasia.     

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.     

Transit of receptors for epidermal growth factor and transferrin through clathrin-coated pits. Analysis of the kinetics of receptor entry

Selective enrichment of clathrin-coated membranes by anticlathrin immunoadsorption was used to examine the internalization of receptor-ligand complexes through coated pits. Using Staphylococcus aureus-anticlathrin antibody and [35S]methionine-labeled KB cells, the kinetics of association of the epidermal growth factor (EGF-R) and transferrin receptors (TF-R) with coated membranes were directly examined. The accumulation of EGF-R in coated pits at the cell surface was dependent upon EGF binding. EGF-R then passed sequentially through a compartment which did not react with anticlathrin antibody and a second clathrin-coated compartment. The EGF-R was degraded in lysosomes with a half-life of approximately 41-55 min. The tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, appears to mimic the action of EGF in inducing EGF-R accumulation in coated pits at the cell surface and receptor internalization. In contrast to the results with EGF-R, the TF-R was found in clathrin-coated membranes in the presence or absence of TF, and the concentration of TF-R in clathrin-coated membranes did not significantly change with time. The method presented should be of great utility for examining the biochemical changes that occur during the receptor-mediated endocytosis and sorting of ligands and receptors.     

Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.     

Internalization and down-regulation of the human epidermal growth factor receptor are regulated by the carboxyl-terminal tyrosines

The C terminus of the epidermal growth factor receptor (EGF-R) contains three tyrosines (Y1068, Y1148, and Y1173) which correspond to the major autophosphorylation sites. To investigate the role of the tyrosines in internalization and down-regulation of the EGF-R, mutational analysis was performed with receptors in which 1, 2, or all 3 tyrosines were changed to phenylalanines. The triple point mutant EGF-R, expressed in NIH-3T3, exhibited low autophosphorylation in vivo, low biological and reduced kinase activities. Single and double point mutants were down-regulated, as well as wild type EGF-R in response to EGF showing a half-life of about 1 h. Degradation of the triple point mutant, however, was impaired and resulted in a half-life of 4 h in the presence of EGF. EGF-dependent down-regulation of surface receptors was decreased in the triple point mutant EGF-R as was internalization and degradation of EGF. The specific rate of internalization of the triple point mutant was reduced. By contrast, intracellular processing of ligand previously internalized at 20 degrees C was similar between wild type and mutant receptors. Taken together the data indicate that the delay in degradation observed in cells expressing the triple point mutant EGF-R can be attributed mainly to a slower removal from the cell surface. Our results show that in the full-length EGF-R all three C-terminal tyrosines are necessary for rapid internalization, suggesting that autophosphorylation is required for efficient EGF-dependent receptor endocytosis.     

Immunoelectron microscope analysis of epidermal growth factor receptor (EGFR) in isolated Mytilus galloprovincialis (Lam.) digestive gland cells: evidence for ligand-induced changes in EGFR intracellular distribution

In mammalian cells, the binding of epidermal growth factor (EGF) to its receptor (EGFR), a glycoprotein with intrinsic tyrosine kinase activity, leads to the pleiotropic responses to EGF. Among these, a negative feedback response by stimulation of receptor internalization and lysosomal degradation, this attenuating signal transduction. In this work, data are reported on the identification of specific EGFRs in isolated digestive gland cells from the marine mussel (Mytilus galloprovincialis Lam.) By immunoelectron microscopy. In control digestive cells, EGFR immunoreactivity was mainly associated with cytoplasmic membrane structures and, to a lesser extent, the cell membrane. The presence of EGFR-like receptors was confirmed by Western blotting of digestive gland cell extracts with two different monoclonal antibodies that recognize either intracellular or extracellular epitopes. The addition of mammalian EGF resulted in significant time and temperature-dependent changes in EGFR subcellular distribution in mussel cells. In cells exposed to EGF for 0-15 min at 4 degrees C, the distribution of EGFR was not significantly different from that of the control cells. On the other hand, at 18 degrees C, an increased labelling along the cell membrane was observed after 5-10 min after EGF addition, with a concomitant decrease in the cytoplasmic signal. Moreover, after 20 min of exposure to EGF, ligand binding apparently resulted in EGFR compartmentation within the lysosomes. These observations were confirmed by quantitative analysis of EGFR labelling at different times of EGF exposure. Similar results were obtained utilizing the two different monoclonal antibodies. The results indicate that, in mussel digestive cells, the binding of heterologous EGF to specific receptors induces a negative feedback response by stimulating the lysosomal degradation of EGFR, thus suggesting the presence of mechanisms responsible for receptor downregulation similar to those observed in mammalian cells.     

Localization of immunoreactive epidermal growth factor receptors in human nervous system

Epidermal growth factor is a well-defined peptide which stimulates cell growth and elicits cell responses in a variety of tissues by binding to specific receptors, EGF-R. A specific antiserum against the EGF receptor, which has previously been used to characterize EGF-R in human skin, fibroblasts, and smooth muscle, was used to survey the distribution of EGF-R in human nervous system. Portions of formalin-fixed, paraffin-embedded autopsy specimens were examined by use of immunohistochemical staining (PAP technique) with EGF-R antiserum. Many types of nerve cells, e.g., cerebral cortical pyramidal cells, hippocampal pyramidal cells, Purkinje cells, anterior horn cells, and dorsal root ganglion neurons, contained immunoreactive EGF-R. However, immunoreactive EGF-R were not detected in astrocytes, oligodendrogliocytes, and other small neurons such as granule cells. Intense immunostaining for EGF-R was also detected in ependymal cells from choroidal and extrachoroidal locations. Although immunoreactive EGF-R is widely distributed in human nervous system, the functional role of EGF and its receptor in the nervous system remains unknown.     

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.     

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.     

Prolactin inhibits epidermal growth factor (EGF)-stimulated signaling events in mouse mammary epithelial cells by altering EGF receptor function.

We have previously shown that lactogenic hormones stimulate epidermal growth factor (EGF) mRNA accumulation in mouse mammary glands in vivo and in mouse mammary epithelial cells (NMuMG line). However, our in vitro studies indicate that the lactogenic hormone prolactin (PRL) completely inhibits EGF-stimulated DNA synthesis. PRL does not alter cholera toxin or insulin-like growth factor-1-stimulated cell growth, thus the inhibition appears to be specific for EGF. Our current studies are designed to evaluate the effects of PRL on EGF-stimulated signaling events in the NMuMG cell line. Cells treated with PRL for 30 min demonstrated a loss of high affinity EGF-binding ability. After long-term PRL treatment (18 h) there was a decrease in EGF receptor (R) number, as determined by [125I]EGF binding. PRL treatment (8 h) also decreased EGF-R mRNA levels. An EGF-stimulated increase in EGF-R mRNA observed 2-4 h after treatment was decreased when PRL was added to the cultures. Furthermore, levels of EGF-stimulated tyrosine phosphorylation of the EGF-R (170 kDa) and phospholipase C gamma (145 kDa) are dramatically decreased in cells treated with PRL. Also of great interest was a decrease in EGF-stimulated c-myc mRNA in PRL-treated cells. We conclude that PRL is acting to down-regulate the EGF-R, thus limiting EGF-stimulated cell signaling in mammary tissue.     

The capping of receptors for the epidermal growth factor and the participation of the cytoskeleton in this process in A-431 cells

Epidermal growth factor (EGF) receptor capping results from the interaction between the receptors and polyvalent ligands in A-431 cells examined in suspension at 22 degrees C. Colocalization of actin and spectrin with the ligand-receptor complexes during the redistribution was shown using double immunofluorescence. The obtained data show that the cortical microfilaments are involved in capping. EGF receptors become associated with the Triton-insoluble cytoskeleton as a consequence of ligand binding. EGF-receptor capping is not sensitive to the action of cytochalasin B. Capping in A-431 cells is discussed as a new model for studying the redistribution of the ligand-receptor complex.     

Neuropeptide-induced transactivation of a neuronal epidermal growth factor receptor is mediated by metalloprotease-dependent formation of heparin-binding epidermal growth factor

Numerous external stimuli, including G protein-coupled receptor agonists, cytokines, growth factors, and steroids activate mitogen-activated protein kinases (MAPKs) through phosphorylation of the epidermal growth factor receptor (EGF-R). In immortalized hypothalamic neurons (GT1-7 cells), agonist binding to the gonadotropin-releasing hormone receptor (GnRH-R) causes phosphorylation of MAPKs that is mediated by protein kinase C (PKC)-dependent transactivation of the EGF-R. An analysis of the mechanisms involved in this process showed that GnRH stimulation of GT1-7 cells causes release/shedding of the soluble ligand, heparin binding epidermal growth factor (HB-EGF), as a consequence of metalloprotease activation. GnRH-induced phosphorylation of the EGF-R and, subsequently, of Shc, ERK1/2, and its dependent protein, p90RSK-1 (p90 ribosomal S6 kinase 1 or RSK-1), was abolished by metalloprotease inhibition. Similarly, blockade of the effect of HB-EGF with the selective inhibitor CRM197 or a neutralizing antibody attenuated signals generated by GnRH and phorbol 12-myristate 13-acetate, but not those stimulated by EGF. In contrast, phosphorylation of the EGF-R, Shc, and ERK1/2 by EGF and HB-EGF was independent of PKC and metalloprotease activity. The signaling characteristics of HB-EGF closely resembled those of GnRH and EGF in terms of the phosphorylation of EGF-R, Shc, ERK1/2, and RSK-1 as well as the nuclear translocation of RSK-1. However, neither the selective Src kinase inhibitor PP2 nor the overexpression of negative regulatory Src kinase and dominant negative Pyk2 had any effect on HB-EGF-induced responses. In contrast to GT1-7 cells, human embryonic kidney 293 cells expressing the GnRH-R did not exhibit metalloprotease induction and EGF-R transactivation during GnRH stimulation. These data indicate that the GnRH-induced transactivation of the EGF-R and the subsequent ERK1/2 phosphorylation result from ectodomain shedding of HBEGF through PKC-dependent activation of metalloprotease(s) in neuronal GT1-7 cells.     

Role of metalloproteinase-dependent EGF receptor activation in alpha-adrenoceptor-stimulated MAP kinase phosphorylation in GT1-7 neurons

Adrenoceptors (ARs) are involved in the regulation of gonadotropin-releasing hormone (GnRH) release from native and immortalized hypothalamic (GT1-7) neurons. However, the AR-mediated signaling mechanisms and their functional significance in these cells are not known. Stimulation of GT1-7 cells with the alpha1-AR agonist, phenylephrine (Phe), causes phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinases that is mediated by protein kinase C (PKC)-dependent transactivation of the epidermal growth factor receptor (EGF-R). Phe stimulation causes shedding of the soluble ligand, heparin-binding EGF (HB-EGF), as a consequence of matrix metalloproteinase (MMP) activation. Phe-induced phosphorylation of the EGF-R, and subsequently of Shc and ERK1/2, was attenuated by inhibition of MMP or HB-EGF with the selective inhibitor, CRM197, or by a neutralizing antibody. In contrast, phosphorylation of the EGF-R, Shc and ERK1/2 by EGF and HB-EGF was independent of PKC and MMP activity. Moreover, inhibition of Src attenuated ERK1/2 responses by Phe, but not by HB-EGF and EGF, indicating that Src acts upstream of the EGF-R. Consistent with a potential role of reactive oxygen species (ROS), Phe-induced phosphorylation of EGF-R was attenuated by the antioxidant, N-acetylcysteine. These data suggest that activation of the alpha1-AR causes phosphorylation of ERK1/2 through activation of PKC, ROS and Src, and shedding of HB-EGF, which binds to and activates the EGF-R.     

Direct visualization of the phosphorylated epidermal growth factor receptor during its internalization in A-431 cells

Epidermal growth factor (EGF) rapidly stimulates receptor autophosphorylation in A-431 cells. After 1 min the phosphorylated receptor can be identified at the plasma membrane using an anti- phosphotyrosine antibody. With further incubation at 37 degrees C, approximately 50% of the phosphorylated EGF receptor was internalized (t1/2 = 5 min) and associated with the tubulovesicular system and later with multivesicular bodies, but not the nucleus. During this period, there was no change in the extent or sites of phosphorylation. At all times the phosphotyrosine remained on the cytoplasmic side of the membrane, opposite to the EGF ligand identified by anti-EGF antibody. These data indicate that (a) the tyrosine-phosphorylated EGF receptor is internalized in its activated form providing a mechanism for translocation of the receptor kinase to substrates in the cell interior; (b) the internalized receptor remains intact for at least 60 min, does not associate with the nucleus, and does not generate any tyrosine-phosphorylated fragments; and (c) tyrosine phosphorylation alone is not the signal for receptor internalization.