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.     

The effect of epidermal growth factor on neonatal incisor differentiation in the mouse

The effect of epidermal growth factor (EGF) on cellular differentiation of the neonatal mouse mandibular incisor was examined autoradiographically using tritiated thymidine ([3H]TDR) and tritiated proline ([3H]PRO). On days 0 (day of birth), 1, and 2, EGF was administered (3 micrograms/g body wt) sc to neonates. Mice were killed on Days 1, 4, 7, 10, and 13 after birth and were injected with either [3H]TDR or [3H]PRO 1 hr before death. [3H]TDR was used to analyze cell proliferation in eight cell types in the developing mouse incisor including upper (lingual) and lower (buccal) pulpal fibroblasts, preodontoblasts, inner and outer enamel epithelial cells (IEE and OEE), stratum intermedium (SI), stellate reticulum (SR), and periodontal ligament (PDL) fibroblasts. [3H]PRO was used to analyze protein synthesis in ameloblasts, and their secretion products (enamel and dentin), as well as PDL fibroblasts. The selected EGF injection scheme elicited acceleration of incisor eruption with minimal growth retardation. At Day 1, the upper and lower pulp, preodontoblasts, SI, and SR showed a significant decrease in labeling index (LI) 24 hr after a single EGF injection. After multiple injections (Days 0, 1, 2), two LI patterns were observed. In lower pulp, preodontoblasts, IEE, SI, SR, and OEE, a posteruptive change in LI was observed. In contrast, the upper pulp and PDL regions demonstrated a direct temporal relationship with eruption. Autoradiographic analysis with [3H]PRO indicated that EGF treatment caused significant increases in grain counts per unit area in ameloblast, odontoblast, and PDL regions studied. Significant differences were found in all four regions studied (ameloblasts, enamel, odontoblasts, dentin) at the 45-microns-tall ameloblast level as well as ameloblasts and odontoblasts at the 30-microns level at 13 days of age. The PDL demonstrated significant differences at all locations studied (base, 30 microns, 45 microns,) in 4-, 7-, and 13-day-old mice. Morphologically, EGF-treated groups demonstrated premature differentiation of ameloblasts and odontoblasts at the light microscopic level. The data indicate that EGF alters DNA and protein synthesis as well as differentiation patterns during the eruption process. While EGF affects both DNA and protein synthesis, the alteration of differentiation may be secondary to mitogenic effects on proliferative compartments. In order to determine the cellular target for EGF within the newborn mouse incisor, in vivo 125I-EGF binding was analyzed autoradiographically.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization and quantitation of 125I-epidermal growth factor binding in mouse embryonic tooth and other embryonic tissues at different developmental stages

We have shown earlier that epidermal growth factor (EGF) inhibits morphogenesis and cell differentiation in mouse embryonic teeth in organ culture. This inhibition depends on the stage of tooth development so that only teeth at early developmental stages respond to EGF (A-M. Partanen, P. Ekblom, and I. Thesleff (1985) Dev. Biol. 111, 84-94). We have now studied the quantity and pattern of EGF binding in teeth at various stages of development by incubating the dissected tooth germs with 125I-labeled EGF. Although the quantity of 125I-EGF binding per microgram DNA stays at the same level, localization of 125I-EGF binding by autoradiography reveals that the distribution of binding sites changes dramatically. In bud stage the epithelial tooth bud that is intruding into the underlying mesenchyme has binding sites for EGF, but the condensation of dental mesenchymal cells around the bud does not bind EGF. At the cap stage of development the dental mesenchyme binds EGF, but the dental epithelium shows no binding. This indicates that the dental mesenchyme is the primary target tissue for the inhibitory effect of EGF on tooth morphogenesis during early cap stage. During advanced morphogenesis the binding sites of EGF disappear also from the dental papilla mesenchyme, but the dental follicle which consists of condensed mesenchymal cells surrounding the tooth germ, binds EGF abundantly. We have also studied EGF binding during the development of other embryonic organs, kidney, salivary gland, lung, and skin, which are all formed by mesenchymal and epithelial components. The patterns of EGF binding in various tissues suggest that EGF may have a role in the organogenesis of epitheliomesenchymal organs as a stimulator of epithelial proliferation during initial epithelial bud formation and branching morphogenesis. The results of this study indicate that EGF stimulates or maintains proliferation of undifferentiated cells during embryonic development and that the expression of EGF receptors in different organs is not related to the age of the embryo, but is specific to the developmental stage of each organ.     

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.     

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.     

Accumulation of epidermal growth factor receptors in retinoic acid-treated fetal rat lung cells is due to enhanced receptor synthesis

125I-Epidermal growth factor (EGF) binding capacity in fetal rat lung (FRL) cells is increased approximately 2 to 3-fold within 18 h of retinoic acid addition. Analysis of 125I-EGF binding assays at 0 C reveals approximately 25,000 receptors per cell, while analysis of growth factor binding to retinoic acid-treated cells demonstrates an increase in receptor levels to approximately 70,000 receptors per cell with no detectable changes in receptor affinities. We show by immunoprecipitation of 35S-methionine labeled EGF receptors that retinoic acid addition produces an increase in the accumulation of EGF receptor protein. Using brief pulses of 35S-methionine, an increase in EGF receptor synthesis can be identified within 3 h after retinoic acid addition. These results are the first to demonstrate that a retinoic acid-induced increase in 125I-EGF binding capacity is due to increased EGF receptor protein synthesis. Also, we find that a transient decrease in the rate of EGF receptor turnover occurs when retinoic acid is initially added to FRL cells. On the basis of our data, we conclude that the retinoic acid-induced accumulation of EGF receptors in FRL cells is primarily due to increased receptor synthesis. The effect of retinoic acid on EGF receptor turnover may be a secondary factor, influencing the rate at which receptors accumulate.     

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.     

Inhibition of epidermal growth factor binding to mouse cultured cells by fibroblast-derived growth factor. Evidence for an indirect mechanism

Fibroblast-derived growth factor (FDGF), a basic, heat- and acid-stable polypeptide partially purified from the serum-free conditioned medium of BHK cells transformed by simian virus 40, is a potent mitogen for Swiss 3T3 cells and causes a marked reduction in 125I-labeled epidermal growth factor (125I-EGF) binding to these cells. The activity which inhibits EGF binding coelutes with the growth-stimulating activity after gel filtration, ion exchange chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both cellular responses are elicited by the same range of FDGF concentration in several murine cell types. The inhibition of EGF binding is rapid and results from a decrease in the apparent affinity of cellular receptors for 125I-EGF. FDGF does not affect the rate of cell-mediated 125I-EGF degradation. Several lines of evidence suggest that FDGF does not bind directly to EGF receptor. First, the effect of FDGF is dependent on the temperature of the assay; furthermore, treatment of cells with EGF results in loss of EGF receptors while exposure to FDGF for up to 24 h does not induce "down-regulation" of EGF receptors. Further, in A431 cells which display a large number of specific EGF receptors, 125I-EGF binding is not sensitive to FDGF. Finally, the effect of FDGF on 125I-EGF binding is not observed with isolated plasma membranes. Taken together, these findings suggest that FDGF binds to sites which are separate from EGF receptors. The results show a novel mechanism whereby a growth-promoting factor produced by a tumor cell line can rapidly modulate the affinity of the cellular receptors for EGF in an indirect manner.     

Protamine enhances epidermal growth factor (EGF)-stimulated mitogenesis by increasing cell surface EGF receptor number. Implications for existence of cryptic EGF receptors

Treatment of Swiss mouse 3T3 cells and human epidermoid carcinoma A431 cells with protamine at 37 degrees C increased the 125I-epidermal growth factor (EGF) binding activity at 4 degrees C. The effect of protamine on the increase of 125I-EGF binding activity appeared to be time, temperature, and dose dependent. This up-modulation of 125I-EGF binding by protamine correlated with protamine enhancement of EGF-stimulated mitogenesis, with respect to the magnitude of the effect and the dose response curves. Scatchard plot analyses indicated that protamine induced an increase in numbers of both high and low affinity EGF receptors without affecting their affinities. Protamine also increased functionally active EGF receptors in plasma membranes and solubilized membranes. This was evidenced by Scatchard plot analyses and by a protamine-induced increase of 125I-EGF-EGF receptor complex and an increase in EGF-stimulated phosphorylation of the EGF receptor. Combined with column chromatography of the solubilized EGF receptor on protamine-agarose gel, these results suggest that protamine may increase the EGF receptor number by directly activating cryptic EGF receptors in the plasma membrane.     

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.     

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.     

p-Azidophenylglyoxal-Epidermal Growth Factor: A Photoactivatable Affinity Crosslinking Reagent

Abstract

A photoaffinity derivative of highly purified ¹²⁵I-labelled epidermal growth factor (¹²⁵I-EGF) has been synthesized. The heterobifunctional crosslinking reagent p-azidophenylglyoxal (PAPG) was bound to arginine residues in ¹²⁵I-EGF. PAPG-¹²⁵I-EGF bound to EGF receptors on rat fibroblasts and human A431 epidermoid carcinoma cells in culture. An apparent decreased affinity of PAPG-¹²⁵I-EGF for the EGF receptor is in accord with at least one arginine being at or near the EGF receptor binding site. The PAPG-¹²⁵I-EGF:EGF receptor complexes on rat cells were internalized to the same extent as control EGF:receptor complexes. A431 cells treated with PAPG-¹²⁵I-EGF were irradiated with ultraviolet light and the labelled proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The 3 major labelled proteins had apparent molecular weights ranging from 75,000 to 200,000. Only the labelling of the 200,000-M_r protein was prevented by the addition of excess unlabelled EGF with the PAPG-¹²⁵I-EGF. This molecular weight is in agreement with the reported size of the EGF receptor plus EGF. A protein with apparent molecular weight of 100,000 was labelled by ¹²⁵I-EGF by an unknown mechanism which was dependent on the dose of UV light and blocked by the addition of excess unlabelled EGF.     

Inhibition of the apparent affinity of the epidermal growth factor receptor caused by phorbol diesters correlates with phosphorylation of threonine-654 but not other sites on the receptor.

Addition of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) to A431 human epidermoid carcinoma cells causes a marked increase in the phosphorylation state of the epidermal growth factor (EGF) receptor with a concomitant inhibition of both the high-affinity binding of 125I-EGF and the receptor tyrosine kinase activity. It was found in the present studies that the diuretic drug amiloride has no effect on the action of PMA to inhibit the binding of 125I-EGF. However, amiloride was observed to inhibit markedly the effect of PMA to cause a 3-fold increase in the phosphorylation state of the EGF receptors. In the presence of PMA and amiloride, the increase in the phosphorylation state of the EGF receptors was found to be only 1.2-fold over controls. Analysis of the EGF receptor phosphorylation sites by phosphopeptide mapping by reverse-phase h.p.l.c. demonstrated that PMA increases the phosphorylation state of the EGF receptor at many sites. One of these sites has been identified as a C-kinase substrate, threonine-654. In the presence of amiloride, PMA causes phosphorylation of threonine-654 to the same stoichiometry as that observed in the absence of amiloride. However, the marked increase in the phosphorylation state of the EGF receptor at other sites caused by PMA is abolished in the presence of amiloride. We conclude that the extensive phosphorylation of the EGF receptor at several sites caused by the addition of PMA to A431 cells is not required for the action of PMA to inhibit the high-affinity binding of 125I-EGF. The results indicate that the phosphorylation state of threonine-654 may play a role in this process.     

Protein kinase C inhibition of the epidermal growth factor receptor tyrosine protein kinase activity is independent of the oligomeric state of the receptor

Treatment of A431 human epidermoid carcinoma cells with 4-phorbol 12-myristate 13-acetate (PMA) causes an inhibition of the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an inhibition of the EGF receptor tyrosine protein kinase activity. The hypothesis that PMA controls EGF receptor function by regulating the oligomeric state of the receptor was tested. Dimeric EGF receptors bound to 125I-EGF were identified by covalent cross-linking analysis using disuccinimidyl suberimidate. Treatment of cells with PMA in the presence of 20 nM 125I-EGF caused no significant change in the level of labeled cross-linked monomeric and dimeric receptor species. Investigation of the in vitro autophosphorylation of receptor monomers and dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide demonstrated that the treatment of cells with PMA caused an inhibition of the tyrosine phosphorylation of both monomeric and dimeric EGF receptors. We conclude that the inhibition of the EGF receptor tyrosine protein kinase activity caused by PMA is not associated with the regulation of the oligomeric state of the EGF receptor.     

Characterization of binding and receptors for epidermal growth factor in smooth muscle

Summary The mitogenic and differentiation-inducing activities of epidermal growth factor (EGF) in epithelial tissues have been well described. Since non-mitogenic effects of EGF, especially in mesenchymal tissues such as smooth muscle are not well-known (Nanney et al. 1984), we have examined EGF-binding and receptors in smooth muscle from many sites. Specific EGF binding sites were detected by incubating small pieces of tissue with ¹²⁵I-EGF; immunoreactive EGF receptors were detected by immunohistochemistry. In-situ localization of ¹²⁵I-EGF binding sites and immunoreactive EGF receptors of smooth muscle cells in intact mammalian tissues were identical using either ¹²⁵I-EGF autoradiography or anti-EGF receptor antibody in an immunoperoxidase method. Cultured rat aortic smooth muscle also contained specific EGF receptors as detected by their biological response to EGF-binding and internalization of ¹²⁵I-EGF, as well as EGF-stimulated phosphorylation of a 170K protein. The presence of EGF receptors in a well-differentiated smooth muscle cell indicates that EGF may play a physiological, but non-mitogenic role in mammalian tissues in vivo.     

Glucocorticoid-mediated alteration in growth factor binding and action: Analysis of the binding change

The addition of the glucocorticoid analog dexamethasone (DX) to serum-free cultures of human fibroblasts caused a twofold enhancement of the mitogenic response to epidermal growth factor (EGF), although DX by itself was not mitogenic. A basis for this effect was suggested by studies showing that DX also increased the cellular binding of ¹²⁵I-EGF. DX increased the ability of the cells to bind ¹²⁵I-EGF only at low physiological concentrations of this polypeptide. Thus, data from ¹²⁵I-EGF binding to cells incubated without DX produced a linear Scatchard plot, whereas the data from ¹²⁵I-EGF binding to DX-treated cells led to an upwardly curvilinear Scatchard plot. Measurements of ¹²⁵I-EGF association with the cell surface and cytoplasm indicated that this binding change involved an alteration of cell surface EGF receptors. The binding change appeared not to involve negatively cooperative interactions between EGF receptors, nor a change in the number of receptors. The binding alteration could be explained by a model in which DX converted 25–30% of the cell surface EGF receptors to a form having a fourfold increased affinity.     

Regional ultrastructural and cytochemical comparisons of the epithelial-mesenchymal interface during rat incisor development

A major theme in understanding epithelial-mesenchymal interactions during development focuses upon regional mesenchyme specification of epithelial differentiation. One particularly useful epidermal organ system for studying this issue is the rodent continuously growing and erupting incisor tooth organ. One advantage of this particular system resides in the regional features of the rodent incisor tooth organ. Along the labial surface, inner dental epithelial cells differentiate into ameloblasts that produce enamel extracellular matrix, whereas the epithelia along the lingual surface do not become ameloblasts and do not produce enamel matrix. This study has been designed to compare ultrastructural features of labial versus lingual surfaces, with particular emphasis upon mesenchymal cell shape, the orientation of extracellular matrix collagen, the basal lamina, and the distribution of sulfated glycoconjugates. Critical analyses of the data indicated that different microenvironments exist between epithelia and mesenchyme in the labial versus the lingual surfaces of the developing rodent incisor tooth organ.     

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)     

甲状旁腺素、表皮生长因子及维生素A酸对UMR106细胞EGF受体的调节作用

本文研究了EGF、PTH和RA对UMR106细胞EGF受体的调节作用。结果显示PTH能上调EGF的受体,UMR106细胞经bPTH(1-34)处理3天,EGF受体的相对结合率与对照比较提高了40.3％,每个细胞的EGF受体数目从7.22×10~3增加到1.44×10~4,Kd从2.02×10~(-11)增加到3.68×10~(-11)mol/L。而RA则能下调EGF受体,以RA处理3天,EGF受体数目从7.22×10~3下降到4.28×10~3,Kd则从2.02×10~(-11)增加到4.17×10~(-11)mol/L。提示PTH和RA可能通过调变其EGF受体而分别起到正性和负性生长调节作用。     

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.