Effect of retinoic acid and 12-O-tetradecanoyl phorbol-13-acetate on the binding of epidermal growth factor to its cellular receptors

Binding of ¹²⁵I-labelled epidermal growth factor (EGF) to C3H/2K cells and the effect of a tumor promotor, 12-O-tetradecanoyl phorbol-13-acetate (TPA) and of a tumor promotor antagonist, retinoic acid, on the binding was studied. Scatchard plot analysis of the binding showed the presence of two type of binding sites with different affinity to EGF. Treatment of the cells with retinoic acid for 1 h resulted in elevation of the affinity of both sites without changing their number per cell. Prolonged exposure to retinoic acid abrogated this elevation of the affinity and caused cycloheximide-sensitive increase of the number of the binding sites of both types. TPA inhibited binding of EGF to the cells by abolishing the binding to the high affinity sites, whereas retinoic acid, in the presence of TPA, enhanced it by increasing the number of the low affinity sites.     

Effects of retinoic acid on the binding and mitogenic activity of epidermal growth factor

In this study the effects of retinoic acid on the binding and mitogenic activity of epidermal growth factor (EGF) in mouse fibroblast Balb/c 3T6 cells are further examined. Retinoic acid treatment of 3T6 cells results in a sixfold enhancement of 125I-labeled mouse EGF binding when assayed at 37 degrees C. In both retinoic acid-treated and control cells, cell-associated 125I-EGF is rapidly internalized, degraded, and secreted. Retinoic acid treatment does not seem to have a significant effect on the rate of internalization and degradation of EGF. At 0 degrees C, internalization of EGF is strongly inhibited in both retinoic acid-treated and control cells. Under these conditions retinoic acid-treated cells still exhibit a tenfold higher level of EGF binding compared to control cells. When exposed to high concentrations of EGF both retinoic acid-treated and control cells "down-regulate" their EGF receptors. And although the growth rate of retinoic acid-treated cells is about half that of control cells, the rate at which EGF binding capacity is restored after down-regulation is about three times as fast as in control cells. No direct antagonism on EGF binding was observed between the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and retinoic acid. EGF is a potent mitogen for 3T6 cells in serum-free medium; retinoic acid inhibits the mitogenic activity of EGF even though it increases EGF binding. Retinoic acid also inhibits cell proliferation induced by sarcoma growth factor (SGF) and insulin.     

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor.

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.     

Epidermal growth factor receptor synthesis is stimulated by phorbol ester and epidermal growth factor. Evidence for a common mechanism

Previously, we and others have shown that epidermal growth factor (EGF) stimulates the synthesis of its own receptor and the accumulation of EGF receptor mRNA. Here, we demonstrate that the tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA), like EGF, also stimulates receptor synthesis in the human breast carcinoma cell line, MDA468 cells. The receptor synthesis rate increased 5-fold with a peak at 8 h after exposure to TPA with half-maximal stimulation at a dose of 5 ng/ml TPA. This stimulation of receptor synthesis occurred despite a 30% decrease in general cellular protein synthesis. The increased receptor synthesis rate resulted in the accumulation of 60% more receptor protein as determined by quantitative immunoblotting using a newly developed monoclonal antibody, H9B4. Although TPA treatment resulted in an immediate loss of high affinity EGF-binding sites, the long-term effect was an increase in both the low and high affinity binding sites. The effects of EGF and TPA on receptor synthesis were not additive. Furthermore, down-regulation of protein kinase C (the Ca2+/phospholipid-dependent enzyme) by long-term TPA treatment resulted in cells unable to respond to the stimulatory effects of both TPA and EGF on receptor synthesis. Nevertheless, the TPA-pretreated cells were still growth-inhibited by EGF. These results suggest that the stimulatory effect of EGF on receptor synthesis requires protein kinase C, whereas the inhibitory effect of EGF on the proliferation of these cells does not. Although we confirmed that EGF stimulated the incorporation of phosphate into phosphatidylinositol in A431 cells, it failed to do so in the MDA468 cells. Thus, in MDA468 cells, EGF may require protein kinase C for part of its action, but we could not demonstrate an associated activation of phosphatidylinositol turnover by EGF. The exact mechanism of involvement of protein kinase C in EGF action is still not clear.     

Inhibition of epidermal growth factor binding to surface receptors by tumor promotors.

The tumor promotor, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), causes a marked (about 10-fold) decrease in the affinity of epidermal growth factor (EGF) receptors for [¹²⁵I]-EGF. The inhibition exhibits specificity, dependence on temperature, and persistence after removal of the promotor. TPA does not induce “down-regulation” of EGF receptors. Finally, the promotor interacts synergistically with EGF in stimulating glycolysis and DNA synthesis in 3T3 cells. The results suggest that TPA does not bind directly to EGF receptors.     

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.     

Phenobarbital decreases hepatocyte EGF receptor expression independent of protein kinase C activation

The liver tumor promoter, phenobarbital, directly applied to cultured, adult rat hepatocytes at concentrations of greater than 1 mM, decreases cellular surface binding of EGF. This effect of phenobarbital resembles that of 4 beta-phorbol-12 alpha-myristate-13 beta-acetate (TPA) in that both decrease EGF receptor number, but do not affect receptor affinity. The effects of the two tumor promoters differ however, in that only TPA reduces high affinity EGF binding by A431 cells. They also differ in that TPA, but not phenobarbital, causes redistribution of protein kinase C from a soluble to a membranous hepatocyte subcellular fraction. These data indicate that decreased EGF binding is a common hepatocyte response to the tumor promoters, TPA and phenobarbital, but that this response can be mediated by either a TPA-activated, protein kinase C-dependent pathway or by a phenobarbital-sensitive, protein kinase C-independent pathway.     

Specificity in the synergism between retinoic acid and EGF on the growth of adult human skin fibroblasts

Vitamin A (retinol) and five retinoids were tested for their ability to enhance epidermal growth factor (EGF) stimulation of adult human skin fibroblast growth in vitro. The retinoids utilized in this study were RO-1-5488 (all-trans-retinoic acid), RO-4-3780 (13-cis-retinoic acid), RO-10-9359, RO-10-1670, and RO-21-6583. Retinol and each retinoid were capable of stimulating fibroblast growth alone (0-86%), while 13-cis and all-trans-retinoic acid were the most potent in potentiating the EGF promotion of fibroblast growth. Other growth factors tested in addition to EGF were nerve growth factor (NGF), fibroblast growth factor (FGF), and thrombin. While EGF and FGF stimulated fibroblast growth to the same degree (2.3-fold), only growth stimulated by EGF was potentiated by retinoic acid. Since retinoic acid might enhance the EGF stimulation of cell growth by increasing either EGF receptor number or binding affinity, the binding of 125I-labeled EGF was carried out in the presence of retinoic acid and the data were subjected to a Scatchard-type analysis. No change in EGF receptor number or affinity was seen in the presence of retinoic acid. The data indicate a specific interaction between retinoid acid and EGF which results in the potentiation of the EGF-stimulated cell growth. Furthermore, the mechanism of this interaction does not seem to involve the initial binding of EGF to its plasma membrane receptor or the available number of EGF receptors located on the cell surface.     

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.     

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.     

Okadaic acid-induced hyperphosphorylation of the epidermal growth factor receptor. Comparison with receptor phosphorylation and functions affected by another tumor promoter, 12-O-tetradecanoylphorbol-13-acetate.

Okadaic acid, a potent tumor promoter and inhibitor of phosphoserine/threonine protein phosphatases 1 and 2A, produces a large increase in epidermal growth factor (EGF) receptor phosphorylation in several cell types. The increases are limited to phosphoserine and phosphothreonine residues. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a distinct tumor promoter and protein kinase C activator, also induces serine/threonine phosphorylation of the EGF receptor and is known to modulate receptor functions. Comparison of okadaic acid and TPA influences on the EGF receptor show significant differences. Okadaic acid did not promote phosphorylation of Thr-654, a major site of TPA-induced phosphorylation. However, other sites of phosphorylation were similar for the two tumor promoters. In vitro experiments with purified protein phosphatase 2A demonstrate the insensitivity of Thr-654 phosphorylation, which regulates EGF receptor function, to dephosphorylation by this okadaic acid-sensitive protein phosphatase. In contrast to TPA, okadaic acid did not attenuate the tyrosine kinase activity or ligand binding capacity of the EGF receptor. However, okadaic acid did produce a decrease in EGF-stimulated inositol phosphate formation in a manner distinct from that of TPA.     

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 modulation of epidermal growth factor- and nerve growth factor-induced adhesion and growth factor binding of PC-12 pheochromocytoma cells

Nerve growth factor (NGF) has previously been shown to increase the rate of adhesion of PC-12 pheochromocytoma cells to cell culture dishes. This increase in the rate of adhesion was postulated to be important in NGF-mediated neurite outgrowth. We now report that epidermal growth factor (EGF) is also able to increase the rate of adhesion of PC-12 cells to cell culture dishes, but does not elicit neurite outgrowth. The dose-response curve for EGF is bell-shaped, in contrast to the more classically shaped dose-response curve obtained with NGF. Tetradecanoyl-phorbol-acetate (TPA), a potent tumor promoter, blocks the EGF-induced increase in adhesion rate of PC-12 cells, but does not alter the NGF-induced increase in adhesion rate. TPA shifts the EGF binding curve to the right for PC-12 cells, but does not alter maximal EGF binding at saturating concentrations of EGF. The binding of NGF to PC-12 cells is not affected by TPA. NGF-induced neurite formation by PC-12 cells is unaffected by TPA, in contrast to the previously reported delay of neurite outgrowth of serum-deprived neuroblastoma cells and NGF-exposed chick embryonic ganglia cells. NGF and EGF both cause a decrease in the number of short microvilli and an increase in the number of long microvilli on PC-12 cells. TPA blocks the decrease in the number of short microvilli in EGF-treated cells, but not in NGF-treated cells. Long microvilli formation is blocked by TPA in both conditions, suggesting the latter are not involved in the increased adhesion rates.     

EGF-induced PGE2 release is synergistically enhanced in retinoic acid treated fetal rat lung cells

Retinoic acid has been shown to induce a 2.5-fold increase in 125I-EGF binding capacity through increased EGF receptor synthesis in a fetal rat lung (FRL) cell line (1). In FRL cells, incubation with either EGF or retinoic acid induces a modest increase in PGE2 secretion (80% or 40%, respectively). However, in the presence of both EGF and retinoic acid, FRL cells exhibit a 6.4-fold increase in PGE2 secretion. Retinoic acid and EGF dose-response curves demonstrate that the effect on PGE2 secretion correlates with the retinoic acid induced increase in EGF receptors. These data suggest a relationship between increased EGF receptor expression and increased EGF responsiveness. Furthermore, these data indicate a potential mechanism by which EGF and retinoic acid may interact in lung physiology.     

Heterologous regulation of EGF receptors in fibroblastic cells

Platelet-derived growth factor (PDGF) increases the mitogenic activity of epidermal growth factor (EGF) in several cells lines, including BALB/C-3T3. PDGF-treated BALB/C-3T3 cells manifest a reduced capacity to bind 125I-labeled EGF due to a loss of high affinity EGF receptors. Cholera toxin potentiates the ability of PDGF to both decrease EGF binding and initiate mitogenesis. Whether PDGF increases EGF sensitivity via its effects on EGF receptors is not known and requires a more complete understanding of the mechanism by which PDGF decreases EGF binding. 12-O-tetradecanoylphorbol 13-acetate (TPA) also reduces EGF binding in BALB/C-3T3 and other cells, presumably by activating protein kinase C and, consequently, inducing the phosphorylation of EGF receptors at threonine-654. PDGF indirectly activates protein kinase C, and EGF receptors in PDGF-treated WI-38 cells are phosphorylated at threonine-654. Thus, the effects of PDGF on EGF binding may also be mediated by protein kinase C. We investigated this hypothesis by comparing the actions of PDGF and TPA on EGF binding in density-arrested BALB/C-3T3 cells. Both PDGF and TPA caused a rapid, transient, cycloheximide-independent loss of 125I-EGF binding capacity. The actions of both agents were potentiated by cholera toxin. However, whereas TPA allowed EGF binding to recover, PDGF induced a secondary and cycloheximide-dependent loss of binding capacity. Most importantly, PDGF effectively reduced binding in cells refractory to TPA and devoid of detectable protein kinase C activity. These findings indicate that PDGF decreases EGF binding by a mechanism that involves protein synthesis and is distinct from that of TPA.     

Biphasic effects of type beta transforming growth factor on epidermal growth factor receptors in NRK fibroblasts. Functional consequences for epidermal growth factor-stimulated mitosis

Exposure of confluent NRK cells to transforming growth factor-beta (TGF-beta) results in distinct alterations in subpopulations of plasma membrane epidermal growth factor (EGF) receptors. The low affinity sites increase in number, whereas the high affinity sites undergo a transient decrease in affinity followed by a prolonged increase in number. Cycloheximide inhibits both of these effects. Functional assays measuring EGF-stimulated thymidine incorporation in the presence of TGF-beta show that the resulting long-term stimulation of EGF receptor binding is associated with an increased sensitivity to EGF. Similarly, the initial, transient decrease in EGF binding is associated with a temporary inhibition of EGF-stimulated thymidine incorporation. The results describe a bifunctional effect of TGF-beta at the biochemical level consistent with the action of this peptide on NRK cell growth.     

Effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the growth inhibitory and increased phosphatidylinositol (PI) responses induced by epidermal growth factor (EGF) in A431 cells

Epidermal growth factor (EGF) inhibited the growth of A431 human epidermoid carcinoma cells. The tumor promoting, phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also retarded A431 cell growth. Addition of both TPA and EGF inhibited cell growth in an additive or synergistic manner depending upon the initial plating density of the cultures. EGF increased the production of diacylglycerol (60-70%) and stimulated the synthesis of phosphatidylinositol (PI) from 3H-inositol (three- to fourfold increase). Both of these responses were attenuated in the presence of TPA. TPA alone stimulated the production of diacylglycerol (DG) but had little effect on PI synthesis. The biological effect of TPA appeared to be mediated by the presence of a high-affinity receptor for phorbol esters on A431 cells. Moreover, the binding of 125I-EGF to A431 cells was unaffected by TPA, suggesting that the antagonistic effects of TPA were occurring distal to the EGF receptor. These findings also indicated that although TPA and EGF both inhibited A431 cell growth, this effect could be dissociated from changes in PI synthesis but may be dependent upon transient changes in DG production.     

Effects of substitution of threonine 654 of the epidermal growth factor receptor on epidermal growth factor-mediated activation of phospholipase C

Addition of epidermal growth factor (EGF) to many cell types activates phospholipase C resulting in increased levels of diacylglycerol and intracellular Ca2+ which may lead to activation of protein kinase C. EGF treatment of cells can also lead to phosphorylation of the EGF receptor at threonine 654 (a protein kinase C phosphorylation site) which appears to attenuate some aspects of receptor signaling. Thus, a feedback loop involving the EGF receptor, phospholipase C, and protein kinase C may regulate EGF receptor function. In this report, the role of phosphorylation of threonine 654 of the EGF receptor in regulation of EGF-stimulated activation of phospholipase C was investigated. NIH-3T3 cells expressing the normal human EGF receptor or expressing EGF receptor in which an alanine residue had been substituted at residue 654 of the receptor were used. Addition of EGF to cells expressing wild-type receptor induced a rapid, but transient, increase in phosphorylation of threonine 654. EGF addition also caused the rapid accumulation of inositol phosphates in these cells. EGF-stimulated accumulation of inositol phosphates was significantly higher in cells expressing Ala-654 receptors compared to control cells. Treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulated phosphorylation of threonine 654 to a greater degree than EGF, completely inhibited EGF-dependent inositol phosphate accumulation in cells expressing wild-type receptor, but caused only a 20-30% inhibition in Ala-654 expressing cells. EGF stimulated phosphorylation of phospholipase C-gamma on serine and tyrosine residues in cells expressing wild-type of Ala-654 receptors. However, TPA treatment of cells inhibited EGF-induced tyrosine phosphorylation of phospholipase C-gamma only in cells expressing wild-type receptors. Similarly, TPA inhibited tyrosine-specific autophosphorylation of the EGF receptor and tyrosine phosphorylation of several other proteins in wild-type receptor cells, but not in Ala-654 cells. TPA treatment abolished high affinity binding of EGF to cells expressing wild-type receptors, while decreasing the number of high affinity binding sites 20-30% in Ala-654 cells. These data suggest that phosphorylation of threonine 654 can regulate early events in EGF receptor signal transduction such as phosphoinositide turnover, probably through a feedback mechanism involving protein kinase C. Subsequent dephosphorylation of threonine 654 could reactivate the EGF receptor for participation in later signaling events.     

Three classes of epidermal growth factor receptors on HeLa cells

The kinetics of 125I-labeled epidermal growth factor (EGF) binding to receptors on HeLa cells were investigated. Scatchard analysis revealed the presence of 22,000 high affinity receptors (Kd = 0.12 nM) and 25,000 low affinity receptors per cell (Kd = 9.2 nM). The kinetic analysis of EGF binding to high affinity receptors was performed with cells pretreated with the monoclonal antibody 2E9, which prevents specifically EGF binding to low affinity receptors. The study of EGF binding to only low affinity receptors was performed with cells pretreated with the phorbol ester phorbol 12-myristate 13-acetate, which induces a conversion of high affinity receptors to low affinity receptors. This kinetic analysis of EGF binding to HeLa cells revealed the presence of three types of receptors. High affinity receptors were found to consist of one receptor type (type I) with a kinetic association constant (kass) of 6.2 x 10(5) M-1.s-1 and a kinetic dissociation constant (kdis) of 3.5 x 10(-4) s-1. The low affinity receptors were found to consist of two kinetic distinguishable sites: type II or fast sites with kass = 3.3 x 10(6) M-1.s-1 and kdis = 8.1 x 10(-3) s-1 and the type III or slow sites with kass = 3.2 x 10(4) M-1.s-1 and kdis = 1.6 x 10(-4) s-1. The regulatory mechanism which may determine the EGF binding characteristics is discussed.