scholarly journals Effects of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on metabolism of the epidermal growth factor receptor in normal human fibroblasts.

1984 ◽  
Vol 4 (9) ◽  
pp. 1718-1724 ◽  
Author(s):  
S J Decker
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Half Life ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Tumor Promoter ◽  
Minor Amount ◽  
Detectable Effect ◽  
Normal Human ◽  
Epidermal Growth

The biosynthesis, phosphorylation, and degradation of the epidermal growth factor (EGF) receptor were examined in normal human fibroblasts. The receptor was initially synthesized as an Mr = 160,000 immature form which matured to an Mr = 170,000 form in a monensin-sensitive manner. Tunicamycin treatment led to the accumulation of an Mr = 130,000 protein. The receptor was phosphorylated on serine and threonine residues in normally growing and quiescent cells, and treatment with EGF or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a two- to threefold increase in receptor-bound phosphate. EGF increased the amount of phosphoserine and phosphothreonine and caused the appearance of a minor amount of phosphotyrosine. TPA increased the levels of phosphoserine and phosphothreonine exclusively. Prior treatment with TPA inhibited the EGF-dependent appearance of phosphotyrosine in the receptor. Analysis of tryptic phosphopeptides revealed that six of the seven major peptides were common to the receptor from cells treated with EGF or TPA. EGF strongly stimulated [3H]thymidine incorporation in confluent cells, increased final saturation density three to fourfold, and increased whole-cell levels of phosphotyrosine about threefold. Treatment of cells with TPA before addition of EGF inhibited all three of these EGF-dependent responses. EGF also decreased the receptor half-life from 15 h to 1 h, but this was not inhibited by TPA. TPA alone had no detectable effect on the receptor half-life.

Effects of epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate on metabolism of the epidermal growth factor receptor in normal human fibroblasts

1984 ◽  
Vol 4 (9) ◽  
pp. 1718-1724
Author(s):  
S J Decker
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Half Life ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Tumor Promoter ◽  
Minor Amount ◽  
Detectable Effect ◽  
Normal Human ◽  
Epidermal Growth

The biosynthesis, phosphorylation, and degradation of the epidermal growth factor (EGF) receptor were examined in normal human fibroblasts. The receptor was initially synthesized as an Mr = 160,000 immature form which matured to an Mr = 170,000 form in a monensin-sensitive manner. Tunicamycin treatment led to the accumulation of an Mr = 130,000 protein. The receptor was phosphorylated on serine and threonine residues in normally growing and quiescent cells, and treatment with EGF or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a two- to threefold increase in receptor-bound phosphate. EGF increased the amount of phosphoserine and phosphothreonine and caused the appearance of a minor amount of phosphotyrosine. TPA increased the levels of phosphoserine and phosphothreonine exclusively. Prior treatment with TPA inhibited the EGF-dependent appearance of phosphotyrosine in the receptor. Analysis of tryptic phosphopeptides revealed that six of the seven major peptides were common to the receptor from cells treated with EGF or TPA. EGF strongly stimulated [3H]thymidine incorporation in confluent cells, increased final saturation density three to fourfold, and increased whole-cell levels of phosphotyrosine about threefold. Treatment of cells with TPA before addition of EGF inhibited all three of these EGF-dependent responses. EGF also decreased the receptor half-life from 15 h to 1 h, but this was not inhibited by TPA. TPA alone had no detectable effect on the receptor half-life.

scholarly journals Calmodulin antagonists decrease the binding of epidermal growth factor to transformed, but not to normal, human fibroblasts

1984 ◽  
Vol 218 (2) ◽  
pp. 629-632 ◽  
Author(s):  
P V Bodine ◽  
J T Tupper
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dependent Manner ◽  
Calmodulin Antagonists ◽  
Normal Human ◽  
Epidermal Growth

Four psychoactive agents which inhibit calmodulin activity were used to study their effect on the binding of epidermal growth factor (EGF) to normal and simian-virus-40-transformed human fibroblasts (WI38). These calmodulin antagonists decreased the binding of 125I-labelled EGF to the transformed, but not to the normal, cell in a dose-dependent manner. The mechanism of this effect appears to be due to a decrease in the apparent affinity of the plasma-membrane EGF receptor for the EGF molecule.

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

1991 ◽  
Vol 2 (8) ◽  
pp. 663-673 ◽  
Author(s):  
R Campos-González ◽  
J R Glenney
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
C Terminus ◽  
Epidermal Growth

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.

scholarly journals Tumor promoter and epidermal growth factor stimulate phosphorylation of the c-erbB-2 gene product in MKN-7 human adenocarcinoma cells.

1988 ◽  
Vol 8 (3) ◽  
pp. 1019-1026 ◽  
Author(s):  
T Akiyama ◽  
T Saito ◽  
H Ogawara ◽  
K Toyoshima ◽  
T Yamamoto
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Gene Product ◽  
Egf Receptor ◽  
Binding Activity ◽  
Tumor Promoter ◽  
Adenocarcinoma Cells ◽  
Epidermal Growth ◽  
Phosphopeptide Mapping

Treatment of human adenocarcinoma MKN-7 cells with epidermal growth factor (EGF) or phorbol tetradecanoate acetate (TPA) stimulated phosphorylation of the c-erbB-2 gene product. EGF induced a rapid increase in phosphotyrosine followed by relatively gradual increases in phosphoserine and phosphothreonine. On the other hand, the TPA-induced increase in phosphorylations occurred exclusively on serine and threonine residues. Tryptic phosphopeptide mapping analysis suggested that treatments with EGF and TPA induced phosphorylation of many common sites in the c-erbB-2 gene product. However, in contrast to TPA, EGF increased the phosphorylation of the c-erbB-2 protein in cells whose protein kinase C had been down-regulated by long-term pretreatment with TPA, suggesting that EGF and TPA induce phosphorylation by different mechanisms. Since the c-erbB-2 gene product did not show detectable EGF-binding activity, phosphorylation of tyrosine of the c-erbB-2 gene product might be catalyzed directly by the EGF receptor kinase that was activated by EGF.

scholarly journals Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

1988 ◽  
Vol 8 (3) ◽  
pp. 1345-1351 ◽  
Author(s):  
E Sturani ◽  
R Zippel ◽  
L Toschi ◽  
L Morello ◽  
P M Comoglio ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Atp Depletion ◽  
Egf Receptors ◽  
A431 Cells ◽  
Intact Cells ◽  
Receptor Phosphorylation ◽  
Epidermal Growth

We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986). In this report, the time course of receptor phosphorylation is investigated. In normal human fibroblasts, ligand-induced phosphorylation of PDGF and EGF receptors is followed by rapid dephosphorylation. However, in A431 cells the tyrosine-phosphorylated form of EGF receptor persists for many hours after EGF stimulation, allowing a detailed analysis of the conditions affecting receptor phosphorylation and dephosphorylation. In A431 cells, the number of receptor molecules phosphorylated on tyrosine was quantitated and found to be about 10% of total EGF receptors. The phosphorylated receptor molecules are localized on the cell surface, and they are rapidly dephosphorylated upon removal of EGF from binding sites by a short acid wash of intact cells and upon a mild treatment with trypsin. ATP depletion also results in rapid dephosphorylation, indicating that continuous phosphorylation-dephosphorylation reactions occur in the ligand-receptor complex at steady state. Phorbol 12-myristate 13-acetate added shortly before EGF reduces the rate and the final extent of receptor phosphorylation. Moreover, it also reduces the amount of phosphorylated receptors if it is added after EGF. Down-regulation of protein kinase C by chronic treatment with phorbol dibutyrate increases the receptor phosphorylation induced by EGF, suggesting a homologous feedback regulation of EGF receptor functions.

Tumor promoter and epidermal growth factor stimulate phosphorylation of the c-erbB-2 gene product in MKN-7 human adenocarcinoma cells

1988 ◽  
Vol 8 (3) ◽  
pp. 1019-1026
Author(s):  
T Akiyama ◽  
T Saito ◽  
H Ogawara ◽  
K Toyoshima ◽  
T Yamamoto
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Gene Product ◽  
Egf Receptor ◽  
Binding Activity ◽  
Tumor Promoter ◽  
Adenocarcinoma Cells ◽  
Epidermal Growth ◽  
Phosphopeptide Mapping

Treatment of human adenocarcinoma MKN-7 cells with epidermal growth factor (EGF) or phorbol tetradecanoate acetate (TPA) stimulated phosphorylation of the c-erbB-2 gene product. EGF induced a rapid increase in phosphotyrosine followed by relatively gradual increases in phosphoserine and phosphothreonine. On the other hand, the TPA-induced increase in phosphorylations occurred exclusively on serine and threonine residues. Tryptic phosphopeptide mapping analysis suggested that treatments with EGF and TPA induced phosphorylation of many common sites in the c-erbB-2 gene product. However, in contrast to TPA, EGF increased the phosphorylation of the c-erbB-2 protein in cells whose protein kinase C had been down-regulated by long-term pretreatment with TPA, suggesting that EGF and TPA induce phosphorylation by different mechanisms. Since the c-erbB-2 gene product did not show detectable EGF-binding activity, phosphorylation of tyrosine of the c-erbB-2 gene product might be catalyzed directly by the EGF receptor kinase that was activated by EGF.

scholarly journals Activity of the epidermal-growth-factor receptor and phospholipase C-γ 1 in heat-stressed fibroblasts and A-431 cells

1992 ◽  
Vol 286 (2) ◽  
pp. 541-547 ◽  
Author(s):  
S M Liu ◽  
G Carpenter
Keyword(s):  
Growth Factor ◽  
Heat Shock ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Egf Receptor ◽  
Inositol Phosphates ◽  
Human Fibroblasts ◽  
Egf Receptors ◽  
Epidermal Growth ◽  
Phospholipase C Gamma

A variety of changes in the functions of specific plasma-membrane components have been reported in cells exposed to a heat shock. In this study, we examined the consequences of heat stress on epidermal-growth-factor (EGF)-induced receptor autophosphorylation and receptor-mediated tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), a cellular substrate. Although the tyrosine kinase activity of the EGF receptor is rapidly inactivated at 45 degrees C in vitro [Carpenter, King & Cohen (1979) J. Biol. Chem. 254, 4884-4891], EGF stimulates autophosphorylation of its receptor in both A-431 cells and human fibroblasts after a prolonged heat shock. Phosphoamino acid analysis of the receptor reveals an EGF-induced increase in phosphotyrosine and phosphoserine at 46 degrees C. EGF also stimulates the phosphorylation of phospholipase C-gamma 1 and induces the formation of inositol phosphates under heat-shock conditions. 125I-EGF binding and internalization in A-431 cells is not decreased during incubations at 46 degrees C for up to 90 min. EGF-induced dimerization of EGF receptors on the cell surface is preserved during heat shock. Though EGF-receptor-mediated endocytosis is not inhibited by elevated temperature, the degradation of internalized 125I-EGF is dramatically decreased. These results indicate that, aside from ligand degradation, the EGF-mediated pathway of signal transduction through phospholipase C-gamma 1 remains remarkably intact during conditions of extreme cellular stress.

scholarly journals Interactions between the receptors for platelet-derived growth factor and epidermal growth factor.

1983 ◽  
Vol 96 (3) ◽  
pp. 679-683 ◽  
Author(s):  
D F Bowen-Pope ◽  
P E Dicorleto ◽  
R Ross
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Insulin Binding ◽  
Platelet Derived Growth Factor ◽  
Egf Receptors ◽  
3T3 Cells ◽  
Low Concentrations ◽  
Epidermal Growth

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.

scholarly journals Large-Scale Screening Assay to Measure Epidermal Growth Factor Internalization

2000 ◽  
Vol 5 (3) ◽  
pp. 133-139 ◽  
Author(s):  
Renate De Wit ◽  
Carolien M.J. Hendrix ◽  
Johannes Boonstra ◽  
Arie J. Verkleij ◽  
Jan Andries Post
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Large Scale ◽  
Egf Receptor ◽  
Human Fibroblasts ◽  
Receptor Internalization ◽  
Inexpensive Method ◽  
Screening Assay ◽  
Epidermal Growth ◽  
Large Scale Screening

Recently, we showed that the internalization of the epidermal growth factor (EGF) receptor is inhibited by hydrogen peroxide (H202) in human fibroblasts. In order to test the effect of various stress conditions on receptor internalization and to test a variety of antioxidants in their capacity to prevent or reduce the H202-induced inhibition of internalization, a screening assay was developed to measure the internalization in 96-well plates. In this assay, cells are exposed to biotin-conjugated EGF and the amount of internalized EGF is detected with horseradish peroxidase-conjugated streptavidin. We show that the results obtained by this new assay are comparable with those from internalization studies performed with radioactive labeled EGF. Therefore, the cellular internalization assay as presented here is a reliable method to measure EGF receptor internalization. Moreover, because elaborate processing of the cells is not required, the assay is a relatively fast and inexpensive method to study ligand-induced internalization in 96-well plates and thereby is suitable for large-scale screening of compounds or conditions interfering with this internalization.

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