Epidermal growth factor and epidermal growth factor receptor-dependent phosphorylation of a Mr = 34,000 protein substrate for pp60src

A Mr = 34,000 protein present in the 100,000 X g supernatant fraction from A431 human epidermoid carcinoma cells is the major radiolabeled phosphate acceptor from [gamma-32P]ATP in a cell-free system requiring epidermal growth factor (EGF) and EGF receptor kinase. This protein is immunoprecipitated by IgG directed against avian Mr = 34,000 cellular substrate for pp60src. Phosphoamino acid analysis of the Mr = 34,000 protein labeled with 32Pi from [gamma-32P]ATP in a cell-free system requiring EGF and EGF receptor kinase yielded radiolabeled phosphotyrosine with no detectable radioactivity in phosphoserine or phosphothreonine.     

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

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.     

Diacylglycerol modulates binding and phosphorylation of the epidermal growth factor receptor

Tumor promoters cause a variety of effects in cultured cells, at least some of which are thought to result from activation of the Ca2+-phospholipid-stimulated protein kinase C. One action of tumor promoters is the modulation of the binding and phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells. To determine if these compounds act on the EGF receptor by substituting for the endogenous activator of C kinase, diacylglycerol, we compared the effects of the potent tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) with those of the synthetic diacylglycerol analog 1-oleyl 2-acetyl diglycerol (OADG). When A431 cells were treated with TPA, the subcellular distribution of C kinase activity shifted from a predominantly cytosolic location to a membrane-associated state; OADG also caused the disappearance of cytosolic C kinase activity. The shift in the subcellular distribution of C kinase, caused by TPA or OADG, correlated with changes in binding and phosphorylation of the EGF receptor. OADG, like TPA, caused loss of binding to an apparent high affinity class of receptors, blocked EGF-induced tyrosine phosphorylation of the EGF receptor, and stimulated phosphorylation of the EGF receptor at both serine and threonine residues. No difference between the phosphopeptide maps of receptors from cells treated with OADG or TPA was observed. Thus, it appears that tumor promoters can exert their effects on the EGF receptors by substituting for diacylglycerol, presumably by activating protein kinase C. Further, these results suggest that endogenously produced diacylglycerol may have a role in normal growth regulatory pathways.     

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

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.     

Epidermal growth factor receptor threonine and serine residues phosphorylated in vivo

The epidermal growth factor (EGF) receptor is regulated by EGF-stimulated autophosphorylation and by phorbol ester-stimulated, protein kinase C (Ca2+/phospholipid-dependent enzyme) mediated phosphorylation at identified sites. The EGF receptor contains additional phosphorylation sites including a prominent phosphothreonine and several phosphoserines which account for the majority of phosphate covalently bound to the receptor in vivo. We have identified three of these sites in EGF receptor purified from 32P-labeled A431 cells. The major phosphothreonine was identified as threonine 669 in the EGF receptor sequence. Phosphoserine residues were identified as serines 671 and 1046/1047 of the EGF receptor. Two other phosphoserine residues were localized to tryptic peptides containing multiple serine residues located carboxyl-terminal to the conserved protein kinase domain. The amino acid sequences surrounding the three identified phosphorylation sites are highly conserved in the EGF receptor and the protein products of the v-erb B and neu oncogenes. Analysis of predicted secondary structure of the EGF receptor reveals that all of the phosphorylation sites are located near beta turns. In A431 cells phosphorylation of the serine residues was dependent upon serum. In mouse B82 L cells transfected with a wild type human EGF receptor. EGF increased the 32P content in all tryptic phosphopeptides. A mutant EGF receptor lacking protein tyrosine kinase activity was phosphorylated only at threonine 669. Regulated phosphorylation of the EGF receptor at these threonine and serine residues may influence aspects of receptor function.     

cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.     

Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases

Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with 32Pi. The 120 kDa 32P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the 32Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.     

Stimulation of epidermal growth factor receptor threonine 654 phosphorylation by platelet-derived growth factor in protein kinase C-deficient human fibroblasts

We have tested the hypothesis that the mechanism of platelet-derived growth factor (PDGF) and phorbol diester action to decrease the apparent affinity of the epidermal growth factor (EGF) receptor is the phosphorylation of the EGF receptor at the Ca2+/phospholipid-dependent protein kinase (protein kinase C) phosphorylation site, threonine 654. Protein kinase C-deficient cells were prepared by prolonged incubation of human fibroblasts with phorbol diester. Addition of phorbol diesters to these cells fails to regulate EGF receptor affinity or threonine 654 phosphorylation. In contrast, PDGF treatment of both control and protein kinase C-deficient fibroblasts causes a decrease in the apparent affinity of the EGF receptor and an increase in threonine 654 phosphorylation. Thus, the ability of PDGF or phorbol diester to modulate EGF receptor affinity occurs only when threonine 654 phosphorylation is increased. The stoichiometry of threonine 654 phosphorylation associated with a 50% decrease in the binding of 125I-EGF to high affinity sites was 0.15 versus 0.3 mol of phosphate per mole of EGF receptor when 32P-labeled fibroblasts are treated with PDGF or phorbol diester, respectively. It is concluded that EGF receptor phosphorylation at threonine 654 can be regulated by PDGF independently of protein kinase C, substoichiometric phosphorylation of the total EGF receptor pool at threonine 654 is caused by maximally effective concentrations of PDGF, and different extents of phosphorylation of EGF receptors at threonine 654 are observed for maximally effective concentrations of PDGF and phorbol diester, respectively. The data are consistent with the hypothesis that a specific subpopulation of EGF receptors that exhibit high affinity for EGF are regulated by threonine 654 phosphorylation.     

Amiloride inhibits constitutive internalization and increases the surface number of epidermal growth factor receptors in intact rat hepatocytes

In previous experiments the surface expression of epidermal growth factor (EGF) receptors in freshly isolated rat hepatocytes varied temperature- and time-dependently and was depleted by monensin and cycloheximide in a way suggesting that a subpopulation of these receptors are subject to constitutive cycling (Gladhaug and Christoffersen; 1988). We here report the finding that pretreatment of the hepatocytes with amiloride exerts marked effects on cellular EGF receptor movements. After 2 h incubation with 1 mM amiloride, the receptor level was approximately 270,000 sites/cell surface vs. 140,000 in the untreated cell, with no change in receptor affinity. Amiloride thus stabilized the surface EGF receptor pool at an elevated level. In cells pretreated with amiloride for 60 min, the relative endocytosis decreased from about 2.6 EGF molecules internalized per receptor during 15 min endocytosis in untreated cells to about 1.5 molecules/receptor in amiloride-treated cells. These results suggest that amiloride causes an accumulation of EGF receptors at the hepatocyte surface due to inhibition of constitutive receptor internalization. In addition, it was found that in amiloride-treated hepatocytes the phorbol ester TPA strongly inhibited high-affinity EGF binding without affecting the total surface receptor number. In control cells, TPA did not consistently affect binding. Pretreatment with amiloride prevented surface EGF receptor depletion induced by cycloheximide and puromycin, but it did not significantly inhibit surface receptor depletion caused by monensin. Although the underlying mechanism of the amiloride effect on intracellular receptor trafficking is not clear, the results provide further evidence for a continuous, ligand-independent EGF receptor cycling pathway in hepatocytes.     

Biosynthesis of the epidermal growth factor receptor in human squamous cell carcinoma lines: secretion of the truncated receptor is not common to epidermal growth factor receptor-hyperproducing cells

The biosynthesis of the EGF receptor was examined in the epidermoid carcinoma cell line A431 and five novel cell lines from human squamous cell carcinomas possessing high numbers of EGF receptors. Newly synthesized EGF receptors were visualized by labeling with [35S]methionine and immunoprecipitation with a monoclonal anti-EGF receptor antibody. In addition, the processing of the EGF receptor and its intracellular transport was analyzed by distinguishing cell surface receptors from intracellular receptors and by treating cells with inhibitors such as tunicamycin, monensin and brefeldin A. These analyses revealed that in all the tumor cell lines the EGF receptor is synthesized as a glycosylated protein of Mr 160,000 which is converted to the receptor of Mr 170,000 through posttranslational glycosylation. The receptors of Mr 160,000 and 170,000 appeared to possess high mannose type oligosaccharide chains because endoglycosidase H treatment reduced their molecular weights by approximately 30,000. A431 was the only tumor cell line studied that secreted the truncated EGF receptor of Mr 110,000. In A431 cells, the truncated EGF receptor was generated from a protein of Mr 60,000 through tunicamycin- and monensin-sensitive glycosylation. A431 cells treated with monensin secreted the truncated receptor as a Mr 95,000 form.     

Modulation of type alpha transforming growth factor receptors by a phorbol ester tumor promoter

Epidermal growth factor (EGF) and an EGF-like transforming growth factor (eTGF) from retrovirally transformed cells bind to a common receptor type in A431 cells. We have investigated the effects of the tumor promoter phorbol myristate acetate [PMA] on EGF/eTGF receptors in intact A431 cells. Treatment with PMA at 37 degrees C induces a complete loss of high-affinity (Kd = 35-50 pM) binding sites for eTGF and EGF on the cell surface of A431 cells. This effect is half-maximal at 0.1 nM PMA, exhibits rapid kinetics, and persists for at least 4 hr in the presence of PMA. eTGF and PMA added to intact A431 cells induce the phosphorylation of immunoprecipitable 170kd EGF/eTGF receptors. The EGF/eTGF receptor isolated from control cells was found to contain phosphoserine and phosphothreonine. PMA and eTGF caused a marked increase in the level of these two phosphoamino acids. In addition, eTGF but not PMA caused the appearance of phosphotyrosine in the EGF/eTGF receptor in vivo. We conclude that the tumor-promoting phorbol diester regulates both the affinity and phosphorylation state of the A431 cell receptor for the type alpha transforming growth factors, eTGF and EGF.     

Sarcoma growth factor (SGF): specific binding to epidermal growth factor (EGF) membrane receptors

Cells transformed by murine sarcoma viruses (MSV) produce and release into their tissue culture media several polypeptide growth stimulating factors. One of these has been partially purified using Bio-Gel P-60 column chromatography followed by DEAE-cellulose chromatography. This growth factor was assigned the name sarcoma growth factor (SGF), and is here shown to require the epidermal growth factor (EGF) receptor in order to function as a growth factor. DEAE-cellulose chromatography yielded a product that was several-fold purer than the material present in the Bio-Gel P-60 column pool II. The biologically active material from the DEAE-cellulose column, when labeled with 125I, showed specific binding to EGF membrane receptors. The specific binding could be prevented with the addition of either unlabeled EGF or SGF. Both radiolabeled SGF and EGF will bind to live or fixed cells. We were able to bind 125I-SGF as well as 125I-EGF to fixed cells and elute the bound material from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed cells. A 3T3 clone lacking EGF receptors was unable to respond to either EGF or SGF, whereas it responded well to serum and several other purified growth factors. The SGF isolated using DEAE-cellulose chromatography was unable to compete in a radioimmune assay using 125I-EGF and antibody to purified mouse submaxillary gland EGF; it also was not precipitated by anti-EGF antibody. From these studies it appears that the SGF produced and released by these MSV-transformed cells combines with and requires the EGF receptor in order to exert its biological effects. The peptide, however, is antigenically distinct from mouse submaxillary gland EGF.     

Insulin action inhibits insulin-like growth factor-II (IGF-II) receptor phosphorylation in H-35 hepatoma cells. IGF-II receptors isolated from insulin-treated cells exhibit enhanced in vitro phosphorylation by casein kinase II

Insulin caused a rapid, dose-dependent increase in the binding of 125I-insulin-like growth factor-II (IGF-II) to the surface of cultured H-35 hepatoma cells. The [32P]phosphate content of the IGF-II receptors, immunoprecipitated from extracts of H-35 cell monolayers previously incubated with [32P]phosphate for 24 h, was decreased after brief exposure of the cells to insulin. Analysis of tryptic digests of labeled IGF-II receptors by bidimensional peptide mapping revealed that the decrease in the content of [32P]phosphate occurred to varying degrees on three tryptic phosphopeptides. Thin layer electrophoresis of an acid hydrolysate of isolated IGF-II receptors revealed the presence of [32P] phosphoserine and [32P]phosphothreonine. Insulin treatment of cells caused a decrease in the labeled phosphoserine and phosphothreonine content of IGF-II receptors. The ability of a number of highly purified protein kinases (cAMP-dependent protein kinase, protein kinase C, phosphorylase kinase, and casein kinase II) to catalyze the phosphorylation of purified IGF-II receptors was examined. Casein kinase II was the only kinase capable of catalyzing the phosphorylation of the IGF-II receptor on serine and threonine residues under the conditions of our assay. Bidimensional peptide mapping revealed that the kinase catalyzed phosphorylation of the IGF-II receptor on a tryptic phosphopeptide which comigrated with the main tryptic phosphopeptide found in receptors obtained from cells labeled in vivo with [32P]phosphate. IGF-II receptors isolated by immunoadsorption from insulin-treated H-35 cells were phosphorylated in vitro by casein kinase II to a greater extent than the receptors isolated from control cells. Similarly, IGF-II receptors from plasma membranes obtained from insulin-treated adipocytes were phosphorylated by casein kinase II to a greater extent than the receptors from control adipocyte plasma membranes. Thus, the insulin-regulated phosphorylation sites on the IGF-II receptor appear to serve as substrates in vivo for casein kinase II or an enzyme with similar substrate specificity.     

Threonyl-tRNA synthetase from Chinese hamster ovary cells is phosphorylated on serine

A Chinese hamster ovary cell line, 2000A, in which threonyl-tRNA synthetase accounts for 1.5% of the total soluble protein, was used to demonstrate that this enzyme is a phosphoprotein. Threonyl-tRNA synthetase was isolated by immunoprecipitation from cells labeled with 32Pi for 18 h. Phosphoamino acid analysis of radiolabeled threonyl-tRNA synthetase showed that phosphorylation occurs on serine.     

Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

Protein phosphorylation was studied in primary cultures of thyroid epithelial cells after the addition of different mitogens: thyrotropin (TSH) acting through cyclic AMP, epidermal growth factor (EGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA). EGF or TPA increased the phosphorylation of five common polypeptides. Among these, two 42-kilodalton proteins contained phosphotyrosine and phosphoserine with or without phosphothreonine. Their characteristics suggested that they are similar to the two 42-kilodalton target proteins for tyrosine protein phosphorylation demonstrated in fibroblasts in response to mitogens. No common phosphorylated proteins were detected in TSH-treated cells and in EGF- or TPA-treated cells. The differences in the protein phosphorylation patterns in response to TSH, EGF, and TPA suggested that the newly emerging cyclic AMP-mediated mitogenic pathway is distinct from the better known growth factor- and tumor promoter-induced pathways.     

Effects of cell density and phorbol esters on the expression of epidermal growth factor receptors

Previously, it has been shown that the binding of epidermal growth factor (EGF) by a wide range of cells decreases as cell density increases. In this report, we demonstrate that KB cells treated chronically with phorbol esters continue to exhibit decreases in EGF receptor binding as cell density increases. This finding suggests that protein kinase-C may not be essential for density-induced down regulation of EGF receptors, since phorbol esters are known to down regulate protein kinase-C. We also report that short-term and long-term effects of phorbol esters on the binding of EGF are affected by density. As shown previously for several cell lines, the phorbol ester 12-0-tetradecanoylphorbol-13-acetate transiently reduces EGF binding. We now show that the magnitude of this reduction diminishes as cell density increases. In addition, we determined that long-term treatment of KB cells with phorbol ester increases EGF binding. Again, this effect is diminished at high cell densities. Finally, we report that the increases in EGF binding induced by long-term treatment with phorbol esters are due to increases in the number of EGF receptors.Abbreviations EGF epidermal growth factor - FGF fibroblast growth factor - PBS phosphate buffered saline - PDBu 4-phorbol-12,13-dibutyrate - PDGF platelet-derived growth factor - PK-C protein kinase-C - TGF- transforming growth factor- - TPA 12-0-tetradecanoylphorbol-13-acetate     

Presence of mannose phosphate on the epidermal growth factor receptor in A-431 cells

In this report, we demonstrate a novel post-translational modification of the epidermal growth factor (EGF) receptor. This modification involves the presence of phosphate, previously thought to exist only on amino acid residues in the EGF receptor, on oligosaccharides of the receptor. We have utilized several independent approaches to determine that mannose phosphate is present on the EGF receptor in A-431 cells. Following metabolic labeling with 32P, immunoisolation of the EGF receptor, and digestion with Pronase radioactivity was determined to be present on high mannose type oligosaccharides by concanavalin A chromatography. Also, after acid hydrolysis of in vivo 32P-labeled EGF receptor, radioactivity was detected that co-migrated with mannose 6-phosphate on two-dimensional thin layer electrophoresis. This radiolabeled material co-eluted with a mannose 6-phosphate standard from a high pressure liquid chromatography anion exchange column. Last, an acid hydrolysate of [3H]mannose-labeled EGF receptor contained two radiolabeled fractions, as analyzed by thin layer electrophoresis, and the radioactivity in one of these fractions was substantially reduced by alkaline phosphatase treatment prior to electrophoresis. These experiments indicate that the mature EGF receptor in A-431 cells contains mannose phosphate. This is a novel modification for membrane receptors and has only been reported previously for lysosomal enzymes and a few secreted proteins.     

sn-1,2-Dioctanoylglycerol. A cell-permeable diacylglycerol that mimics phorbol diester action on the epidermal growth factor receptor and mitogenesis

The cell-permeable diacylglycerol, sn-1,2-dioctanoylglycerol (DiC8), is shown to mimic the effect of tumor promoting phorbol diesters on epidermal growth factor (EGF) binding and action in intact cells. DiC8 inhibited the binding of [3H]phorbol dibutyrate to A431 cell monolayers indicating that the diacylglycerol interacts with the phorbol diester receptor. At 0.3 microM, DiC8 half-maximally inhibited the high affinity binding of 125I-EGF to A431 human epidermoid carcinoma cells. Scatchard analysis indicated that the inhibition of 125I-EGF binding was very similar to that observed in the presence of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). DiC8 also mimicked the action of PMA to increase the phosphorylation state of the EGF receptor in 32P-labeled cells. Phosphoamino acid analysis demonstrated that DiC8 and PMA caused an increase in the level of EGF-receptor phosphoserine and phosphothreonine, whereas EGF caused an increase in the level of phosphoserine, phosphothreonine, and phosphotyrosine. Phosphopeptide mapping of the EGF receptor showed that DiC8 and PMA enhanced the phosphorylation of the same tryptic peptides. DiC8 inhibited the EGF-dependent tyrosine phosphorylation of the EGF receptor in A431 cells in a similar manner to that observed with PMA. In further experiments with quiescent Swiss 3T3 fibroblasts, DiC8 mimicked the ability of PMA to stimulate the incorporation of [methyl-3H]thymidine synergistically with low concentrations of EGF. This result indicates that DiC8 will mimic the long-term effects of PMA to regulate mitogenesis and raises the possibility that it may be active in two stage carcinogenesis. As both DiC8 and PMA stimulate the Ca2+- and phospholipid-dependent protein kinase (C-kinase) in vitro, the results support the hypothesis that the activation of C-kinase is a critical component of phorbol diester action on EGF receptor modulation and cell proliferation.     

Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: Interactions with epidermal growth factor and nerve growth factor

The phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (¹²⁵I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent K_i of 11–26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of ³H-choline and ³²P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth factor (NGF) which, in contrast to the former agents, is a differentiating stimulus for the PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of ¹²⁵I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.     

Phorbol ester and interferon-gamma modulation of epidermal growth factor receptors on human amniotic (WISH) cells

In this study we report that pretreatment of human amniotic (WISH) cells with interferon gamma (IFN-gamma) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the down-modulation of epidermal growth factor (EGF) receptors with respect to both receptor number and affinity. Scatchard analysis of EGF binding in the absence of both IFN-gamma and TPA indicated biphasic binding whereas addition of TPA resulted in the loss of the higher affinity class of sites. Pretreatment with IFN-gamma for 24 h enhanced the TPA-induced inhibition of EGF binding whereas IFN-gamma alone had no effect on binding. Protein kinase C (Ca2+/phospholipid-dependent enzyme) was examined as a possible mediator of IFN-induced EGF-receptor modulation; pretreatment of cells with IFN-gamma affected neither the binding of [3H]phorbol 12,13-dibutyrate to membrane or cytosolic fractions nor the protein kinase C activity, suggesting that IFN-gamma pretreatment did not result in translocation or activation of protein kinase C.