Heterodimerization of c-erbB2 with different epidermal growth factor receptor mutants elicits stimulatory or inhibitory responses.

Ligand-induced dimerization of growth factor receptors is crucial for stimulation of their intrinsic protein tyrosine kinase activity promoting receptor autophosphorylation by an intermolecular mechanism. Moreover, the suppressive and negative dominant action of defective epidermal growth factor receptor (EGFR) was shown to be caused by formation of inactive heterodimers with normal EGFR leading to diminished biological signaling. In this report we explore the structural requirements and functional significance of heterodimerization between EGFR and HER2. HER2 (also called c-erbB-2 or neu) is a member of the EGFR family whose natural ligand is still unknown. We show that in response to EGF, wild type EGFR and various EGFR mutants were able to undergo heterodimerization with HER2. Addition of EGF to transfected cells co-expressing HER2 with a kinase negative point mutant of EGFR (K721A) stimulated heterodimer formation, tyrosine phosphorylation of K721A and HER2, and tyrosine phosphorylation of one of their known substrates, phospholipase C gamma. However, the binding of EGF to transfected cells co-expressing HER2 together with another EGFR mutant CD533 (a deletion mutant lacking most of the cytoplasmic domain of EGFR) caused heterodimerization and inhibition of tyrosine kinase activity. It appears therefore that EGF-induced heterodimerization of EGFR and HER2 can promote either stimulatory or inhibitory influences on kinase activity. We propose that the nature of receptor interactions on the cell surface can either activate or inhibit the initiation of growth factor-controlled cellular signaling.     

Analysis of platelet-derived growth factor receptor domain function using a novel chimeric receptor approach.

A chimeric receptor consisting of an epidermal growth factor (EGF) receptor ligand-binding domain and platelet-derived growth factor (PDGF) receptor transmembrane and cytoplasmic signalling domains has been constructed and shown to be fully functional in phosphorylation, mitogenesis, transformation, Ca2+ release, and pH change assays. Expression of this receptor in EGF receptor-deficient, PDGF-responsive NIH 3T3 cells allows the activation of PDGF signalling pathways by EGF. This system was used to examine the function of kinase insertion sequences (KIS). While a mutant with a KIS deletion of 83 amino acids displayed a significant but reduced ability to induce mitogenic, transforming, and Ca2+ release responses in transfected cells, deletion of 20 additional amino acids resulted in abolishment of such activities. This differential loss of signalling potential correlated with the reduced or abolished potential of these receptor mutants to phosphorylate cellular substrates such as PLC gamma. Our results suggest an integral role for KIS in PDGF receptor cytoplasmic domain conformation and an involvement in substrate interaction, but provide no evidence for an exclusive role of KIS in the mediation of biological signals.     

H2O2-induced activation of transcription factors STAT1 and STAT3: the role of EGF receptor and tyrosine kinase JAK2

Reactive oxygen species initiate multiple signal transduction pathways including tyrosine kinase signaling. Here, we demonstrate tyrosine phosphorylation of EGF receptor, STAT3, and, to a lesser extent, STAT1 upon H2O2 treatment of HER14 cells (NIH3T3 fibroblasts transfected with full-length EGF receptor). Maximum phosphorylation levels were observed in 5 min of stimulation at 1-2 mM H2O2. It has been shown that the intrinsic EGF-receptor tyrosine kinase is responsible for the receptor phosphorylation upon H2O2 stimulation. STAT3 and STAT1 activation in HER14 cells was demonstrated to depend on EGF receptor kinase activity, rather than JAK2 activity, while in both K721A and CD126 cells (NIH3T3 transfected with kinase-dead EGF receptor, and EGF receptor lacking major autophosphorylation sites, respectively) STAT1 and STAT3 tyrosine phosphorylation requires JAK2 kinase activity. Furthermore, STAT3 is constitutively phosphorylated in K721A and CD126 cells, and STAT1 H2O2-stimulated activation in these cells is much more prominent than in HER14. In all the cell lines used, Src-kinase activity was demonstrated to be unnecessary for ROS-initiated phosphorylation of STATs. Herein, we postulate that EGF receptor plays a role in H2O2-induced STAT activation in HER14 cells. Our data also prompted a hypothesis of constitutive inhibition of JAK2-dependent STAT activation in this cell line.     

A chimeric EGF-R-neu proto-oncogene allows EGF to regulate neu tyrosine kinase and cell transformation.

The neu oncogene, characterized by Weinberg and colleagues, is a transforming gene found in ethylnitrosourea-induced rat neuro/glioblastomas; its human proto-oncogene homologue has been termed erbB2 or HER2 because of its close homology with the epidermal growth factor receptor (EGF-R) gene (c-erbB1). Expression of the rat neu oncogene is sufficient for transformation of mouse NIH 3T3 fibroblasts in culture and for the development of mammary carcinomas in transgenic mice, but the neu proto-oncogene has not been associated with cell transformation. We constructed a vector for expression of a chimeric cDNA and hybrid protein consisting of the EGF-R extracellular, transmembrane and protein kinase C-substrate domains linked to the intracellular tyrosine kinase and carboxyl terminal domain of the rat neu cDNA. Upon transfection with the construct, NIH 3T3 cells gave rise to EGF-R antigen-positive cell clones with varying amounts of specific EGF binding. Immunofluorescence and immunoprecipitation using neu- and EGF-receptor specific antibodies demonstrated a correctly oriented and positioned chimeric EGF-R-neu protein of the expected apparent mol. wt on the surface of these cells. EGF or TGF alpha induced tyrosine phosphorylation of the chimeric receptor protein, stimulated DNA synthesis of EGF-R-neu expressing cells and led to a transformed cell morphology and growth in soft agar. In contrast, the neu proto-oncogene did not show kinase activity or transforming properties when expressed at similar levels in NIH 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the influences of the E5 transforming protein on kinetic parameters of epidermal growth factor binding and metabolism.

The E5 protein of the bovine papillomavirus induces cellular transformation when transfected into NIH 3T3 cells, and the extent of focal transformation is enhanced by cotransfection with the epidermal growth factor (EGF) receptor (Martin et al., Cell 59:21-32, 1989). To determine whether E5 affects EGF:receptor interactions we analyzed the kinetics of 125I-EGF processing using a mathematical model that enabled us to evaluate rate constants for ligand association (ka), dissociation (kd), internalization (ke), recycling (kr), and degradation (kh). These rate constants were measured in NIH 3T3 cells transfected with the human EGF receptor (ER cells) and in cells transfected with both the EGF receptor and E5 (E5/ER cells). We found that the rate constant for 125I-EGF association ka was significantly decreased in E5/ER cells, but was apparently occupancy-independent in both cell lines. The 125I-EGF dissociation rate constant kd was significantly lower in E5 transformed cells, and increased with occupancy in both cell lines. This suggests that E5 alters the receptor before or during EGF binding so that ligand association is slower; however, once complexes are formed, EGF is bound more tightly to the receptor. Rate constants for internalization ke were also found to be occupancy-dependent, although at a given level of occupancy ke was similar for both cell lines. Also, there was no apparent effect of E5 on the recycling rate constant kr. The 125I-EGF degradation rate constant kh was 30% lower in E5 transformed cells, and was occupancy-independent. The overall effect of E5 is to stabilize intact EGF:receptor complexes which may alter mitogenic signaling of the receptor.     

Cell surface heparan sulfates mediate internalization of the PWWP/HATH domain of HDGF via macropinocytosis to fine-tune cell signalling processes involved in fibroblast cell migration

HDGF (hepatoma-derived growth factor) stimulates cell proliferation by functioning on both sides of the plasma membrane as a ligand for membrane receptor binding to trigger cell signalling and as a stimulator for DNA synthesis in the nucleus. Although HDGF was initially identified as a secretory heparin-binding protein, the biological significance of its heparin-binding ability remains to be determined. In the present study we demonstrate that cells devoid of surface HS (heparan sulfate) were unable to internalize HDGF, HATH (N-terminal domain of HDGF consisting of amino acid residues 1-100, including the PWWP motif) and HATH(K96A) (single-site mutant form of HATH devoid of receptor binding activity), suggesting that the binding of HATH to surface HS is important for HDGF internalization. We further demonstrate that both HATH and HATH(K96A) could be internalized through macropinocytosis after binding to the cell surface HS. Interestingly, HS-mediated HATH(K96A) internalization is found to exhibit an inhibitory effect on cell migration and proliferation in contrast with that observed for HATH action on NIH 3T3 cells, suggesting that HDGF exploits the innate properties of both cell surface HS and membrane receptor via the HATH domain to affect related cell signalling processes. The present study indicates that MAPK (mitogen-activated protein kinase) signalling pathways could be affected by the HS-mediated HATH internalization to regulate cell migration in NIH 3T3 fibroblasts, as judged from the differential effect of HATH and HATH(K96A) treatment on the expression level of matrix metalloproteases.     

Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells

The epidermal growth factor receptor (EGFR) gene is frequently amplified and/or overexpressed in human malignancies. To investigate the biological effects of its overexpression, we constructed a eukaryotic vector containing human EGFR cDNA. Introduction of this construct led to reconstitution of functional EGF receptors in NR6 mutant cells, which are normally devoid of this receptor. Transfection of NIH 3T3 resulted in no significant alterations in growth properties. However, EGF addition led to the formation of densely growing transformed foci in liquid culture and colonies in semisolid medium. NIH 3T3-EGFR clonal lines, which expressed the EGF at 500- to 1000-fold levels over control NIH 3T3 cells, demonstrated a marked increase in DNA synthesis in response to EGF. Thus EGF receptor overexpression appears to amplify normal EGF signal transduction. Finally, high levels of EGFR expression, which conferred a transformed phenotype to NIH 3T3 cells in the presence of ligand, were demonstrated in representative human tumor cell lines that contained amplified copies of the EGFR gene.     

Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing

Cultured NIH 3T3 cells devoid of endogenous EGF receptors were transfected with cDNA constructs encoding either the human EGF receptor or an EGF receptor mutant in which Lys721, a key residue in the ATP binding site, was replaced with an alanine residue. The mutant receptor was properly processed, and it displayed both high- and low-affinity surface binding sites. Unlike the wild-type receptor, the mutant receptor did not possess intrinsic protein-tyrosine kinase activity. The initial rate of EGF internalization was similar for wild-type and mutant EGF receptors. Surprisingly, the mutant receptors were not down regulated, but appeared to recycle in transfected cells. These data suggest that degradation of normal EGF receptors after endocytosis is due to the kinase activity endogenous to this receptor. A single amino acid substitution rendered a "down-regulated" receptor into a receptor that can recycle from cytoplasmic compartment back to the cell surface.     

Dimerization of internalized epidermal growth factor receptors.

Binding of epidermal growth factor (EGF) to cell surface EGF receptors initiates the formation of the receptor homodimers that can be detected by covalent cross-linking in intact cells or in detergent-solubilized cell extracts. Low pH dissociation of EGF from surface receptors results in immediate monomerization of receptor dimers. Using chemical cross-linking during mild permeabilization or cell solubilization, we have detected dimers of internalized EGF receptors in human carcinoma A-431 cells and transfected NIH 3T3 cells that express human EGF receptors. The percentage of internalized cross-linked receptor dimers was similar to that observed for surface EGF receptors. Furthermore, at the time of maximal accumulation of EGF-receptor complexes within the endosomal compartment (10-15 min of incubation at 37 degrees C), both the dimeric and monomeric forms of the EGF receptor are tyrosine-phosphorylated to the same extent as surface dimer and monomer species. In transfected NIH 3T3 cells, the level of dimerized and internalized kinase-negative EGF receptors was not different from that observed for wild-type receptors. These data suggest that for some time after internalization EGF does not dissociate from its receptor and indicate that a receptor conformation is preserved intracellularly that allows maintenance of receptor-receptor interactions and tyrosine kinase activity.     

Activation of HER4 by heparin-binding EGF-like growth factor stimulates chemotaxis but not proliferation.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent mitogen and chemotactic factor for fibroblasts, smooth muscle cells and keratinocytes. It is demonstrated that HB-EGF is not only a ligand for HER1, as previously reported, but for HER4 as well. HB-EGF binds to NIH 3T3 cells overexpressing either HER1 or HER4 alone, but not HER2 or HER3 alone. Binding to HER4 is independent of HER1. The ability of HB-EGF to bind to two different receptors is in contrast to EGF which binds to HER1, but not to HER4, and heregulin-beta1 which binds to HER4, but not to HER1. Besides binding, HB-EGF activates HER4. For example (i) it induces tyrosine phosphorylation of HER4 in cells overexpressing this receptor and of endogenous HER4 in MDA-MB-453 cells and astrocytes; (ii) it induces association of phosphatidylinositol 3-kinase (PI3-K) activity with HER4; and (iii) it is a potent chemotactic factor for cells overexpressing HER4. Chemotaxis is inhibited by wortmannin, a PI3-K inhibitor, suggesting a possible role for PI3-K in mediating HB-EGF-stimulated chemotaxis. On the other hand, HB-EGF is not a mitogen for cells expressing HER4, in contrast to its ability to stimulate both chemotaxis and proliferation in cells expressing HER1. It was concluded that HER4 is a newly described receptor for HB-EGF and that HB-EGF can activate two EGF receptor subtypes, HER1 and HER4, but with different biological responses.     

Cell transformation potential of a HER2 transmembrane domain deletion mutant retained in the endoplasmic reticulum.

The HER2 (c-erbB-2) gene encodes a protein, p185HER2, which possesses all of the structural characteristics and functional properties of a growth factor receptor, although its ligand has not yet been well characterized. HER2 is the human homolog of the rat proto-oncogene neu and is closely related to the gene encoding the epidermal growth factor receptor. Amplification of this gene and overexpression have been found to be a prognostic criterion for a 30% subpopulation of human breast cancer patients. In this study, we investigated the role of the transmembrane-spanning sequence in the biosynthesis and localization of p185HER2. A truncation mutant lacking the cytoplasmic and transmembrane domains was glycosylated and efficiently secreted. However, a mutant lacking only the transmembrane-spanning sequence was incompletely glycosylated and failed to reach the cell surface. Unexpectedly, although this deletion mutant was retained in the endoplasmic reticulum membrane, it was still able to transform NIH 3T3 cells when expressed at high levels.     

Herstatin, an autoinhibitor of the human epidermal growth factor receptor 2 tyrosine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.     

Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.     

High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses

We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAb108), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a cDNA encoding the full-length human EGF receptor gene, and fully responsive to EGF. The binding to saturation of mAb108 to the surface of these cells at 4 degrees C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGF. The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37 degrees C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 10(6) M-1.s-1) than did low-affinity receptor (2.5 x 10(5) M-1.s-1), whereas the off-rate constants, measured at 4 degrees C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors. At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAb108 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAb108 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.     

Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors.

The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing.     

Recombinant human epidermal growth factor precursor is a glycosylated membrane protein with biological activity.

NIH 3T3 cells were transfected with cDNA corresponding to human kidney prepro-epidermal growth factor (preproEGF) under control of the inducible mouse metallothionein promoter. The synthesis of recombinant human EGF precursor by these cells has provided us with a model system for analysis of the structure and activity of this precursor. In transfected cells, the precursor was present as an intrinsic 170-kilodalton membrane protein as well as a soluble protein in the extracellular medium; both forms were N glycosylated. Glycosylation of the EGF precursor was determined by (i) the direct incorporation of [3H]mannose and [3H]glucosamine, (ii) metabolic labeling in the presence or absence of glycosylation inhibitors, (iii) enzymatic cleavage of the precursor by N-glycanase or endoglycosidase II, and (iv) lectin chromatography. Recombinant human preproEGF was purified by affinity chromatography, using wheat germ lectin and antibodies to human EGF. The intact precursor was biologically active. Purified preparations of preproEGF (i) competed with 125I-labeled EGF for binding to the EGF receptor in intact fibroblast cells, (ii) activated the intrinsic tyrosine kinase activity of the EGF receptor in membrane preparations, and (iii) sustained the growth of a mouse keratinocyte cell line that is dependent on EGF for growth. These results suggest that proteolytic processing of the precursor may not be essential for its biological function.     

Enhancement of epidermal growth factor (EGF) and insulin-stimulated tyrosine phosphorylation of endogenous substrates by sodium selenate.

Sodium selenate stimulated tyrosine phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells and enhanced the tyrosine phosphorylation of endogenous proteins in response to EGF in A431 cells and insulin in NIH 3T3 HIR3.5 cells. These effects occurred without changes in ligand binding, were not abolished by mercaptoethanol in the case of the EGF receptor, and appeared distinct from the effects of vanadate. These results support a role for selenium or selenoproteins in regulating EGF and insulin receptor tyrosine kinase activity and suggest a mechanism whereby selenium-containing compounds contribute to cell growth.     

Transfer of functional EGF receptors to an IL3-dependent cell line

Epidermal growth factor (EGF) is a small protein that acts as a mitogen for various epidermal, epithelial, and fibroblastic cells that bear specific EGF receptors. The molecule that binds EGF is a 175-kD transmembrane protein, with an extracellular ligand binding domain and an intracellular domain that possesses tyrosine kinase activity, thought to be involved in the mitogenic signalling process. Here we have constructed a recombinant murine retrovirus that transduces a human cDNA encoding the 175-kD protein and used this retrovirus to infect BAF3, a murine, bone marrow-derived cell line, which is dependent on the haematopoietic factor interleukin-3 (IL3) for its growth in culture. The EGF receptors expressed in the infected cells exhibit two affinity states, as well as EGF-stimulated autophosphorylation. Furthermore, EGF can replace IL3 in supporting short-term proliferation of these cells. These data identify functional properties of the EGF receptor upon expression of the 175-kD EGF binding protein in a haemotopoietic cell that does not express endogenous receptors. They also suggest that gene transfer of growth factor receptors to heterologous cells may allow novel growth stimuli to be exploited.     

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.     

Kinetics of tyrosine phosphorylation and internalization of human EGF receptors overexpressed in NIH 3T3 fibroblasts

Binding of epidermal growth factor (EGF) to cells rapidly induces tyrosine phosphorylation of its receptor which is followed by its internalization and dephosphorylation. The kinetics of these processes differs widely in time from minutes to hours according to cell types. In this paper we analyzed EGF receptor phosphorylation and down-regulation in NIH 3T3 cells transfected with the recombinant hEGF-R cDNA which express 4 X 10(5) receptors/cell. In the presence of EGF receptor phosphorylation reached a maximum after 1 min and was then maintained for about 1 h, while during this time the number of EGF-binding sites was reduced to 40% of the initial number. Detailed analysis of the fate of a population of receptors previously activated and autophosphorylated at 4 degrees C, after warming to 37 degrees C in the absence of the ligand, showed that internalization of the cell surface-associated EGF and dephosphorylation of the receptor were rapid (t1/2 15 min) and followed a similar kinetics. Our data indicate that at any given time only a fraction of the total cell surface receptors is phosphorylated on tyrosine and that dephosphorylation occurs at the cell surface or very rapidly after internalization. In addition the data also suggest that a certain recycling of previously internalized receptors may occur in these cells during EGF treatment.