Diacylglycerol treatment rapidly decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells

The synthetic diacylglycerol 1-oleoyl-2-acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37 degrees C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I-EGF binding to Swiss 3T3 cells at 4 degrees C after a 1 h incubation with a saturating dose of OAG at 37 degrees C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I-EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells.     

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

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.     

Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor

Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity. The present study examined the effect of TRH on EGF binding to intact GH4C1 rat pituitary tumor cells to test whether TRH activates protein kinase C. Cells were incubated with TRH at 37 degrees C and specific 125I-EGF binding was then measured at 4 degrees C. 125I-EGF binding was decreased by a 10-min treatment with 0.1-100 nM TRH to 30-40% of control in a dose-dependent manner. 125I-EGF binding was not altered if cells were incubated at 4 degrees C, although TRH receptors were saturated or in a variant pituitary cell line without TRH receptors. TRH (10 min at 37 degrees C) decreased EGF receptor affinity but caused little change in receptor density, 125I-EGF internalization, or degradation. When cells were incubated continuously with TRH, there was a recovery of 125I-EGF binding after 24 h. Incubation with the protein kinase C activating phorbol ester TPA caused an immediate (less than 10 min) profound (greater than 85%) decrease in 125I-EGF binding followed by partial recovery at 24 h. Maximally effective doses of TRH and TPA decreased EGF receptor affinity with half-times of 3 min. EGF treatment (5 min) caused an increase in the tyrosine phosphate content of several proteins; prior incubation with TRH resulted in a small decline in the EGF response. GH4C1 cells were incubated with 500 nM TPA for 24 h in order to down-regulate protein kinase C. Protein kinase C depletion was confirmed by immunoblots and the effects of TRH and TPA on 125I-EGF binding were tested. TRH and TPA were both much less effective in cells pretreated with phorbol esters. TRH increased cytoplasmic pH measured with an intracellularly trapped pH sensitive dye after mild acidification with nigericin. This TRH response is presumed to be the result of protein kinase C-mediated activation of the amiloride-sensitive Na+/H+ exchanger and was blunted in protein kinase C-depleted cells. All of these results are consistent with the view that TRH acts rapidly in the intact cell to activate protein kinase C and that a consequence of this activation is EGF receptor phosphorylation and Na+/H+ exchanger activation.     

Ligand-receptor interactions involved in the stimulation of Swiss 3T3 fibroblasts by insulin-like growth factors and insulin.

1. The binding of 125I-labelled insulin-like growth factor 1 (125I-IGF-1) to Swiss mouse 3T3 fibroblasts was time- and concentration-dependent. Unlabelled IGF-1 had a slightly higher potency than multiplication-stimulating activity (MSA) in inhibiting the binding of 125I-IGF-1, and insulin gave a parallel inhibition curve at 300-1000-fold lower potency. Chemical cross-linking of bound 125I-IGF-1 to its receptors, followed by polyacrylamide-gel electrophoresis under reducing conditions, revealed a major band of Mr 130,000, the labelling of which was inhibited by IGF-1 or high concentrations of insulin. 2. The binding of 125I-IGF-1 was not affected by either co-incubation or preincubation of the cells with a range of heterologous growth factors and mitogens. However, IGF-1 and MSA each induced down-regulation of 125I-IGF-1 binding sites. 3. The maximal stimulations of DNA synthesis induced by IGF-1, MSA and insulin, in the presence of a synergizing mitogen, were similar. The dose-response curve for insulin was not parallel to those for IGF-1 and MSA; in particular, low concentrations of insulin induced a greater stimulation than expected on the basis of its potency in the inhibition or down-regulation of 125I-IGF-1 binding. 4. The preincubation of 125I-IGF-1 with Swiss 3T3 cells at 37 degrees C decreased its ability to bind to a second batch of cells. This inactivation did not occur when the preincubation was performed at 4 degrees C or in the presence of cycloheximide. Chemical cross-linking revealed that the cells released an IGF-binding protein, giving a complex of Mr about 48,000. 5. It is concluded that type I IGF receptors mediate the stimulation of Swiss 3T3 cells by insulin-like mitogens, but that insulin probably stimulates the cells through insulin receptors. The cells can modulate the amount of ligand binding, both by down-regulation of the receptors and by the secretion of an IGF-binding protein.     

Inhibition of the binding of 125I-labelled epidermal growth factor to mouse cells by a mitogen in goat mammary secretions.

Pre-colostrum and colostrum from goats cause a marked inhibition of the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The ability of these secretions to inhibit 125I-EGF binding is closely correlated with the ability to stimulate DNA synthesis in quiescent 3T3 cell cultures, suggesting that goat mammary secretions may contain an EGF-related mitogen. However, the material in colostrum which inhibits 125I-EGF binding to Swiss 3T3 cells is a basic protein with Mr greater than 20000 and is thus quite different from mouse and human EGF. Furthermore, the colostral-mediated inhibition of 125I-EGF binding, although rapid and apparently competitive, differs from the inhibition of binding induced by native, unlabelled EGF. Thus, the inhibitory effect of colostrum is markedly decreased when the assay temperature is shifted from 37 degrees C to 4 degrees C whereas unlabelled EGF is an effective competitive inhibitor at both 37 degrees C and 4 degrees C. Incubation of cells with EGF causes a reduction in cell surface EGF receptors whereas exposure to colostrum does not induce down-regulation of the EGF receptor. Our results suggest that the colostral factor does not bind directly to EGF receptors but inhibits 125I-EGF binding by an indirect mechanism which involves a temperature-sensitive step.     

Intracellular Ca2+ and phorbol esters synergistically inhibit internalization of epidermal growth factor in pancreatic acini.

The association of 125I-labelled epidermal growth factor (125I-EGF) with mouse pancreatic acinar cells was inhibited by secretagogues which increase intracellular free Ca2+ concentrations. These agents included cholecystokinin-octapeptide (CCK8) and the Ca2+ ionophore A23187. Inhibition by CCK8 was blocked by lowering the incubation temperature from 37 degrees C to 15 degrees C. Moreover, in contrast with studies of intact acini, the binding of 125I-EGF to isolated acinar membrane particles was not affected either by CCK8, or by varying the level of Ca2+ in the incubation medium. These results indicated, therefore, that the inhibition of 125I-EGF association with acinar cells required intact cells that are metabolically active. Since intact cells at 37 degrees C are known to internalize bound EGF rapidly, acid washing was used to distinguish membrane-associated hormone from internalized hormone. Under steady-state conditions 86% of the 125I-EGF associated with the acini was found to be internalized by this technique. When agents that increased intracellular Ca2+ were tested they all markedly reduced the amount of internalized hormone, whereas surface binding was only minimally affected. The phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA), which is known to activate protein kinase C, a Ca2+-regulated enzyme, also inhibited the association of EGF with acini. This inhibition was similar to that induced by elevated intracellular Ca2+. To test whether these two inhibitory phenomena were related, the effects of TPA in combination with the Ca2+ ionophore A23187 were examined. At low concentrations the effects were synergistic, whereas at high concentrations the maximal level of inhibition was not changed. We suggest therefore that elevated intracellular Ca2+ and phorbol esters may inhibit EGF internalization by a mechanism involving activation of protein kinase C.     

beta-Adrenergic regulation of insulin and epidermal growth factor receptors in rat adipocytes

Incubation of intact rat adipocytes with physiological concentrations of catecholamines inhibits the specific binding of 125I-insulin and 125I-epidermal growth factor (EGF) by 40 to 70%. Affinity labeling of the alpha subunit of the insulin receptor demonstrates that the inhibition of hormone binding is directly reflective of a specific decrease in the degree of receptor occupancy. The stereospecificity and dose dependency of the binding inhibitions are typical of a classic beta 1-adrenergic receptor response with half-maximal inhibition occurring at 10 nM R-(-)-isoproterenol. Specific alpha-adrenergic receptor agonists and beta-adrenergic receptor antagonists have no effect, while beta-adrenergic receptor antagonists block the inhibition of 125I-insulin and 125I-EGF binding to receptors induced by beta-adrenergic receptor agonists. Further, these effects are mimicked by incubation of adipocytes with dibutyryl cyclic AMP or with 3-isobutyl-1-methylxanthine. The beta-adrenergic inhibition of both 125I-insulin and 125I-EGF binding is very rapid, requiring only 10 min of isoproterenol pretreatment at 37 degrees C for a maximal effect. Removal of isoproterenol by washing the cells in the presence of alprenolol leads to complete reversal of these effects. The inhibition of 125I-EGF binding is temperature dependent whereas the inhibition of 125I-insulin binding is relatively insensitive to the temperature of isoproterenol pretreatment. Scatchard analysis of 125I-insulin and 125I-EGF binding demonstrated that the decrease of insulin receptor-binding activity may be due to a decrease in the apparent number of insulin receptors while the inhibition of EGF receptor binding can be accounted for by a decrease in apparent EGF receptor affinity. The decrease in the insulin receptor-binding activity is physiologically expressed as a dose-dependent decrease of insulin responsiveness in the adipocyte with respect to two known responses, stimulation of insulin-like growth factor II receptor binding and activation of the glucose-transport system. These results demonstrate a beta-adrenergic receptor-mediated cyclic AMP-dependent mechanism for the regulation of insulin and EGF receptors in the rat adipocyte.     

Inhibition of epidermal growth factor binding to Swiss 3T3 cells by human platelet release-products

Human platelet ionophore release-products (IRP) inhibit the binding of 125I-labelled epidermal growth factor (125I-EGF) to its receptors on Swiss 3T3 cells. The inhibition appears to be caused by platelet-derived growth factor (PDGF) in the IRP and results from a decrease in the apparent affinity of cellular receptors for 125I-EGF. However, our results indicate that PDGF does not bind directly to EGF receptors, since (1) PDGF does not down-regulate EGF receptors; (2) the PDGF-mediated inhibition of 125I-EGF binding is temperature-dependent; (3) cells which possess EGF receptors but lack PDGF receptors do not exhibit a PDGF-mediated inhibition of 125I-EGF binding.     

Transmodulation of epidermal growth factor binding by platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate is not sodium-dependent in Balb/c/3T3 cells

The addition of platelet-derived growth factor (PDGF) to many types of cells causes a rapid decrease in high affinity binding of 125I-epidermal growth factor (EGF), a process which has been termed transmodulation. Treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) also results in the transmodulation of the EGF receptor in many cell types. PDGF can transmodulate EGF binding through a mechanism that is not dependent on protein kinase C activity. A recent report (Wattenberg, E. V., McNeil, P. L., Fujiki, H., and Rosner, M. R. (1989) J. Biol. Chem. 264, 213-219) described the requirement for a sodium ion influx in the down-modulation of the EGF receptor stimulated by a non-TPA-type tumor promoter, palytoxin, in Swiss 3T3 cells. We tested for a similar sodium requirement in Balb/c/3T3 and Swiss 3T3 cells stimulated by PDGF or TPA in Balb cells treated with TPA for prolonged periods to down-regulate protein kinase C activity. Our results clearly show that the PDGF- and TPA-stimulated transmodulation of the EGF receptor does not require external sodium nor is the process affected by amiloride. In each of these experiments, the loss of 125I-EGF binding occurred to a similar extent and at a similar rate in the presence or absence of sodium. Intracellular pH also did not appear to have a role in the response. The sodium ionophore, monensin, was previously shown to bring about the down-modulation of 125I-EGF binding in Swiss cells. However, our results indicate that monensin-induced transmodulation of the EGF receptor occurs with or without external sodium, suggesting that the loss of binding is not the result of a sodium ion influx. These findings demonstrate that an increase in intracellular sodium does not cause nor is it required for PDGF- or TPA-stimulated EGF receptor transmodulation.     

Regulation of thyroidal inducibility of Na,K-ATPase and binding of epidermal growth factor in wild-type and cold-sensitive E1a mutant type 5 adenovirus-transformed CREF cells

We have analyzed the relationship between expression of the transformed phenotype and thyroid hormone (triiodothyronine, T3) inducibility of Na,K-ATPase and binding of 125I-epidermal growth factor (EGF) to cell membrane receptors in wild-type (wt) and mutant type 5 adenovirus (Ad5)-transformed CREF cells displaying a cold-sensitive (cs) expression of the transformed phenotype. CREF cells respond to thyroid hormone treatment with increased Na,K-ATPase activity and bind similar levels of 125I-EGF at 32 degrees C, 37 degrees C and 39.5 degrees C. In contrast, CREF cells transformed by wt Ad5 or the E1a plus E1b-transforming genes of wt Ad5 are refractile to T3 treatment and bind lower levels of 125I-EGF than CREF cells at all three temperatures. By employing a series of cloned CREF cell lines transformed by a host-range cold-sensitive mutant virus, H5hr1 or H5dl101, or the E1a or E1a plus E1b genes from these viruses, we have investigated expression of the transformed state and its relationship with hormone inducibility and EGF binding. When cs virus, cs E1a- or cs E1a plus E1b-transformed CREF clones were grown at 32 degrees C, a nonpermissive transforming temperature in which cs-transformed cells exhibit properties similar to untransformed CREF cells, T3 induced Na,K-ATPase activity and these cells bound similar levels of 125I-EGF as CREF cells. However, when cs virus- and cs Ela plus E1b-transformed CREF clones were incubated at 37 degrees C or 39.5 degrees C, temperatures at which cs-transformed cells exhibit properties similar to wt Ad5-transformed CREF cells, they did not respond to T3 and bound lower levels of 125I-EGF than CREF cells. In the case of cs E1a-transformed CREF clones, thyroid hormone responsiveness was observed at both 32 degrees C and 37 degrees C, but not at 39.5 degrees C. By performing temperature shift experiments--i.e. 32 degrees C to 37 degrees C, 32 degrees C to 39.5 degrees C, 37 degrees C to 32 degrees C, and 39.5 degrees C to 32 degrees C, it was demonstrated that after a shift from lower to higher temperature a 24-hr lag period was required for cs-transformed CREF cells to lose T3 inducibility and exhibit reduced EGF binding, whereas 96 hr after a shift from higher to lower temperature a 96-hr lag period was required for cs-transformed cells to regain T3 inducibility and increased 125I-EGF binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Receptors for epidermal growth factor and insulin-like growth factor-I on preimplantation trophoderm of the pig.

125I-labelled epidermal growth factor (125I-EGF) and 125I-labelled insulin-like growth factor-I (125I-IGF-I) bound to trophoderm cells from pig blastocysts obtained on days 15-19 of pregnancy. Specific binding was detected on freshly isolated cell suspensions and on cells cultured for several days. The binding of 125I-EGF was inhibited by increasing concentrations of EGF, but not by various other growth factors and hormones. Chemical cross-linking of 125I-EGF to its receptors using disuccinimidyl suberate (DSS) revealed a radiolabelled band of relative molecular mass 160,000, similar to that identified as the EGF receptor in other cell types. The binding of 125I-IGF-I was inhibited by both IGF-I and insulin, indicating that the receptors were either type I IGF receptors or insulin receptors. Cross-linking of 125I-IGF-I to serum-free supernatants from trophoderm cultures showed that the cells secreted an IGF-binding protein, giving a complex of relative molecular mass about 45,000. The presence of receptors for EGF and IGF/insulin suggests that these factors could be involved in regulating the growth and development of the early blastocyst.     

Receptor-bound somatostatin and epidermal growth factor are processed differently in GH4C1 rat pituitary cells

GH4C1 cells, a clonal strain of rat pituitary tumor cells, have high-affinity, functional receptors for the inhibitory hypothalamic peptide somatostatin (SRIF) and for epidermal growth factor (EGF). In this study we have examined the events that follow the initial binding of SRIF to its specific plasma membrane receptors in GH4C1 cells and have compared the processing of receptor-bound SRIF with that of EGF. When cells were incubated with [125I-Tyr1]SRIF at temperatures ranging from 4 to 37 degrees C, greater than 80% of the specifically bound peptide was removed by extraction with 0.2 M acetic acid, 0.5 M NaCl, pH 2.5. In contrast, the subcellular distribution of receptor-bound 125I-EGF was temperature dependent. Whereas greater than 95% of specifically bound 125I-EGF was removed by acid treatment after a 4 degrees C binding incubation, less than 10% was removed when the binding reaction was performed at 22 or 37 degrees C. In pulse-chase experiments, receptor-bound 125I-EGF was transferred from an acid-sensitive to an acid-resistant compartment with a half-time of 2 min at 37 degrees C. In contrast, the small amount of [125I-Tyr1]SRIF that was resistant to acid treatment did not increase during a 2-h chase incubation at 37 degrees C. Chromatographic analysis of the radioactivity released from cells during dissociation incubations at 37 degrees C showed that greater than 90% of prebound 125I-EGF was released as 125I-tyrosine, whereas prebound [125I-Tyr1]SRIF was released as a mixture of intact peptide (55%) and 125I-tyrosine (45%). Neither chloroquine (0.1 mM), ammonium chloride (20 mM), nor leupeptin (0.1 mg/ml) increased the amount of [125I-Tyr1]SRIF bound to cells at 37 degrees C. Furthermore, chloroquine and leupeptin did not alter the rate of dissociation or degradation of prebound [125I-Tyr1]SRIF. In contrast, these inhibitors increased the amount of cell-associated 125I-EGF during 37 degrees C binding incubations and decreased the subsequent rate of release of 125I-tyrosine. The results presented indicate that, as in other cell types, EGF underwent rapid receptor-mediated endocytosis in GH4C1 cells and was subsequently degraded in lysosomes. In contrast, SRIF remained at the cell surface for several hours although it elicits its biological effects within minutes. Furthermore, a constant fraction of the receptor-bound [125I-Tyr1]SRIF was degraded at the cell surface before dissociation. Therefore, after initial binding of [125I-Tyr1]SRIF and 125I-EGF to their specific membrane receptors, these peptides are processed very differently in GH4C1 cells.     

Specific desensitization of the epidermal growth factor receptor by pp60v-src

BALB/c 3T3 cells infected with a temperature-sensitive mutant (LA90) of RSV have been used to investigate possible heterologous interactions between the pp60v-src tyrosyl kinase and the epidermal growth factor (EGF) and bradykinin receptors. The LA90 pp60v-src exhibits a very rapid activation t1/2 (less than 5 min) of protein kinase activity on decreasing the temperature from 40 degrees C to 35 degrees C. This change in temperature was also found to induce a very rapid decrease in the affinity for 125I-EGF of receptors on the RSV-LA90-infected cells but not of those on control parental cells. However, no significant changes were detected in the binding of 3H-bradykinin to either cell line. Two separable processes control the desensitization of the EGF receptor by pp60v-src, both of which are independent of protein kinase C. The first is rapid and transient, while the second is sensitive to cycloheximide and persists long after inactivation of pp60v-src.     

Ligand-induced desensitization of 125I-epidermal growth factor internalization

The internalization of 125I-epidermal growth factor (EGF) by A431 cells was investigated. Control cells were able to internalize over 80% of receptor-bound 125I-EGF. By contrast, cells treated with EGF before incubation with 125I-EGF internalized only 50% of the surface-bound radioligand. The ligand-induced decrease in 125I-EGF internalization showed a dose response to EGF with half-maximal effect occurring at 3 nM. The alteration in the extent of 125I-EGF internalization did not require extended treatment with high concentrations of the hormone. When the internalization of picomolar versus nanomolar concentrations of EGF were compared, the lower concentrations of 125I-EGF were more completely internalized than the higher concentrations of radioligand. These data are consistent with the hypothesis that occupation of the EGF receptor by hormone rapidly leads to the activation of cellular processes which effectively desensitize the system to further ligand-induced internalization. The decrease in the extent of ligand internalization occurred in cells in which the protein kinase C (Ca2+/phospholipid-dependent enzyme) activity had been down-regulated by prolonged treatment with 12-O-tetradecanoyl-phorbol-13-acetate implying that the desensitization process is independent of protein kinase C. However, the effects of EGF on the extent of hormone internalization could be mimicked by the addition of A23187 and could be prevented by pretreatment of the cells with calmodulin antagonists suggesting the possibility that Ca2+-calmodulin is involved in the regulation of EGF receptor internalization in A431 cells.     

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding

Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an Mr 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent; enhancement in 80k phosphorylation could be detected as early as 10 s after the addition of peptide. Recently, a rapid increase in the phosphorylation of an 80k cellular protein after treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts (Rozengurt, E., A. Rodriguez-Pena, and K. A. Smith, 1983, Proc. Natl. Acad. Sci. USA., 80:7244-7248; Rozengurt, E., A. Rodriguez-Pena, M. Coombs, and J. Sinnett-Smith, 1984, Proc. Natl. Acad. Sci. USA., 81:5748-5752). The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125I-labeled epidermal growth factor (125I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. Peptides structurally related to bombesin, including gastrin-releasing peptide, also stimulated 80k phosphorylation and inhibited 125I-EGF binding; both effects were selectively blocked by a novel bombesin antagonist. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca2+ concentration does not mediate the bombesin inhibition of 125I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125I-EGF to its cellular receptor.     

Effects of platelet-derived growth factor on phosphorylation of the epidermal growth factor receptor in human skin fibroblasts

Heterologous regulation of the epidermal growth factor (EGF) receptor by platelet-derived growth factor (PDGF) was studied in FS4 human skin fibroblasts. The addition of PDGF to FS4 cells inhibited high affinity binding of 125I-EGF and stimulated phosphorylation of the EGF receptor. Phosphopeptide analysis by high performance liquid chromatography revealed that PDGF treatment of cells increased phosphorylation at several distinct sites of the EGF receptor. However, PDGF did not stimulate phosphorylation of threonine 654, a residue previously shown to be phosphorylated when protein kinase C is activated. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated phosphorylation of the same peptides from the EGF receptor as PDGF, and, in addition, induced phosphorylation of threonine 654. TPA inhibited both high and low affinity 125I-EGF binding by these cells. PDGF treatment of cells had no effect on EGF-dependent, tyrosine-specific autophosphorylation of the receptor, whereas TPA treatment was inhibitory. TPA, but not PDGF, stimulated phosphorylation of a Mr = 80,000 protein, known to be a substrate for protein kinase C, even though PDGF appeared to mediate breakdown of phosphoinositides. These data suggest that regulation of EGF receptor function by PDGF and TPA are distinct in these cells, even though some elements of regulation are shared. The results differ from those previously reported for a human lung fibroblast isolate, indicating that cell type-specific differences may exist in metabolism of the EGF receptor.     

Partial purification and characterization of a growth factor present in goat's colostrum. Similarities with platelet-derived growth factor.

A factor in goat's colostrum which stimulates DNA synthesis and cell proliferation in Swiss 3T3 fibroblasts has been purified approx. 350-fold by a sequence of acid precipitation, cation-exchange chromatography and gel filtration. The growth factor is a highly basic, heat stable (100 degrees C for 5 min) polypeptide with Mr approx. 35000. The polypeptide resists denaturation by guanidinium chloride or urea but is totally inactivated by treatment with reducing agents. The factor, which we have termed colostric basic growth factor ( CBGF ), inhibits the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 fibroblasts but does not inhibit 125I-EGF binding to epidermoid A431 cells. CBGF interacts synergistically with plasma in stimulating DNA synthesis in quiescent Swiss 3T3 cells. The chemical and biological properties of CBGF are thus very similar to the properties reported for the human platelet-derived growth factor. Although high concentrations of CBGF are present in the colostrum of goats, cows, and sheep, the milk of these species contains little or no factor. The origin and possible functions of CBGF are unknown.     

Characterization of the rebinding of 125I-epidermal growth factor released from BALB/c-3T3 cells following accumulation in the presence of chloroquine

Lysosomotropic amines, such as chloroquine and methylamine, increase the intracellular accumulation of 125I-EGF by inhibiting lysosomal degradation. It has been shown previously that BALB/c-3T3 cells, prelabeled at 4 degrees C with 125I-EGF for 3 h and subsequently chased at 37 degrees C in the presence of chloroquine, internalized the surface bound 125I-EGF which was subsequently released into the extracellular medium in a high molecular weight form which co-migrated with native 125I-EGF. The secreted 125I-EGF rebound to the cells from which it was released more efficiently than does peptide in the extracellular media. We now show that when the BALB/c-3T3 cells were prelabeled at 37 degrees C for 2 h in the presence of chloroquine, the internalized 125I-EGF released into the medium was in a high molecular weight form which co-migrated with native 125I-EGF and did not rebind anymore efficiently than did peptide in the extracellular media. This lack of rebinding was not due to an alteration in the 125I-EGF molecule since it was still capable of rebinding to naive A431 cells, nor was it due to the exhaustion of EGF receptors on the BALB/c-3T3 cells. The inhibition of rebinding was observed only when the cells were treated with EGF in the presence of chloroquine, and was not due to a general down-regulation of membrane receptors. The differences between the rebinding of 125I-EGF at 4 degrees C and 37 degrees C suggest that EGF may be processed via different pathways in the cell.     

Characterization of epidermal growth factor receptor induction by retinoic acid in a chemically transformed rat liver cell line

Levels of epidermal growth factor (EGF) receptor expression vary widely among cell lines derived clonally from a chemically transformed population of rat liver epithelial cells. Retinoic acid (RA), a derivative of vitamin A that stimulates differentiation in a number of embryonal cell lines, increases the level of 125I-EGF binding in several clones of the transformed cell lines. One such cell line, GP6ac, which reverts to a less transformed phenotype when treated with RA, exhibited a 3-4-fold increase in surface EGF receptors with prolonged (2-5-day) RA exposure. The increase persisted as long as the cells were treated with RA. The increase in surface EGF receptors was due to induction of receptor biosynthesis, which occurred within 4 h at both the mRNA and protein levels and persisted until the RA was withdrawn. Paradoxically, the RA response was accompanied by an initial 40-50% decrease in 125I-EGF binding during the first 12 h of RA treatment. The decrease was due primarily to a reduction of receptor affinity. Since the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also decreases 125I-EGF binding and increases EGF receptor biosynthesis in GP6ac cells, we tested the effect of RA in cells depleted of protein kinase C by prolonged treatment (18 h) with 10 microM 12-O-tetradecanoylphorbol-13-acetate. The absence of protein kinase C did not affect the induction of receptor mRNA and protein or the decrease in binding during the early period of RA exposure. This indicates that RA induction of EGF receptor synthesis in GP6ac cells involves signaling pathways distinct from those utilized by phorbol esters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross talk among tyrosine kinase receptors in PC12 cells: desensitization of mitogenic epidermal growth factor receptors by the neurotrophic factors, nerve growth factor and basic fibroblast growth factor.

We have studied the effects of nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) on epidermal growth factor (EGF) binding to PC12 cells. We show that NGF and bFGF rapidly induce a reduction in 125I-EGF binding to PC12 cells in a dose-dependent manner. This decrease amounts to 50% for NGF and 35% for bFGF. Both factors appear to act through a protein kinase C(PKC)-independent pathway, because their effect persists in PKC-downregulated PC12 cells. Scatchard analysis indicates that NGF and bFGF decrease the number of high affinity EGF binding sites. In addition to their effect on EGF binding, NGF and bFGF activate in intact PC12 cells one or several serine/threonine kinases leading to EGF receptor threonine phosphorylation. Using an in vitro phosphorylation system, we show that NGF- or bFGF-activated extracellular regulated kinase 1 (ERK1) is able to phosphorylate a kinase-deficient EGF receptor. Phosphoamino acid analysis indicates that this phosphorylation occurs mainly on threonine residues. Furthermore, two comparable phosphopeptides are observed in the EGF receptor, phosphorylated either in vivo after NGF treatment or in a cell-free system by NGF-activated ERK1. Finally, a good correlation was found between the time courses of ERK1 activation and 125I-EGF binding inhibition after NGF or bFGF treatment. In conclusion, in PC12 cells the NGF- and bFGF-stimulated ERK1 appears to be involved in the induction of the threonine phosphorylation of the EGF receptor and the decrease in the number of high affinity EGF binding sites.