Recycling of epidermal growth factor-receptor complexes in A431 cells: identification of dual pathways

The intracellular sorting of EGF-receptor complexes (EGF-RC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37 degrees C. The initial rate of EGF recycling was reduced at 18 degrees C. A significant pool of internalized EGF was incapable of recycling at 18 degrees C but began to recycle when cells were warmed to 37 degrees C. The relative rate of EGF outflow at 37 degrees C from cells exposed to an 18 degrees C temperature block was slower (t1/2 approximately 20 min) than the rate from cells not exposed to a temperature block (t1/2 approximately 5-7 min). These data suggest that there might be both short- and long-time cycles of EGF recycling in A431 cells. Examination of the intracellular EGF-RC dissociation and dynamics of short- and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125I-EGF recycled via the long-time pathway at a rate similar to that of 125I-EGF. Since EGF-RC degradation was also blocked at 18 degrees C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes.     

Recycling of epidermal growth factor in A431 cells

The fate of epidermal growth factor (EGF) after internalization by A431 cells was studied. First, cells containing 125I-EGF-receptor complexes in endosomes were obtained. Subsequent incubation of the cells at 37 degrees C resulted in the recycling of 125I-EGF from endosomes to the cell surface in the receptor-bound state and the gradual release of recycled ligand into the medium. The excess of unlabeled EGF blocked both rebinding and re-internalization of recycled 125I-EGF to produce enhanced accumulation of ligand in the medium. The rate of recycling was shown to be much higher than that of EGF degradation.     

The recycling of EGF-receptor complexes in A431 cells

As was demonstrated elsewhere (L. V. Teslenko et al., 1987), the epidermal growth factor (EGF) can recycle after internalization by A431 cells in membrane-bound state. In the present study, direct evidence on recycling of EGF-receptor complexes is presented using a covalently crosslinking reagent. The recycling was shown to occur via peripheral endosomes as well as through para-Golgi endosomes. It was found that among EGF degradation inhibitors tested only primaquine (300 microM) was able to decrease significantly the rate of recycling. The lowering of the temperature to 17 degrees C led to blocking the EGF degradation as well as to inhibiting the recycling. The data obtained suggested that the recycling of EGF-receptor complexes is relatively independent of their degradation.     

The endocytosis of epidermal growth factor in A431 cells: a pH of microenvironment and the dynamics of receptor complex dissociation

The endocytosis and intracellular fate of epidermal growth factor (EGF) were studied in A431 cells. After 15-20 min of internalization at 37 degrees C, rhodamine-labeled EGF (EGF-Rh) accumulated into large juxtanuclear compartment consisting of closely related vesicles. This structure was shown to be localized in the para-Golgi region. Fluorescein-labeled transferrin (Tr-FITC) was observed in the same region when added to the cells simultaneously with EGF-Rh. Using microscope spectrofluorometer, we determined that the Tr-FITC-containing para-Golgi structures have a pH of 6.1 +/- 0.3 while lysosomes containing dextran-fluorescein have a pH of 5.0 +/- 0.2. To study the dynamics of EGF-receptor dissociation during endocytosis a mild detergent treatment of living cells was used for extraction of an intracellular receptor-unbound EGF. During the first hour of internalization at 37 degrees C, neither significant dissociation of EGF-receptor complexes nor EGF degradation was observed. After 3 h of endocytosis, the percentage of unbound EGF increased to 55% of the total internalized EGF. These results suggest that EGF remains associated with receptors during endocytosis in A431 cells until it is transferred to lysosomes where the pH of the EGF microenvironment is dropped to 5. A prolonged presence of EGF-receptor complexes in the para-Golgi region might be of importance in mitotic signaling.     

Consumption of EGF by A431 cells: evidence for receptor recycling

We examined the extent of EGF consumption by EGFR in A431 cells. When 125I-EGF was added to A431 cell cultures at low or high density, at concentrations which corresponded to 10-fold excess of ligand over receptor on the cell surface, most of the 125I-EGF was consumed within 2 h. The amounts of 125I-EGF consumed were much greater than available EGFR on the A431 cells, by a factor of 6.5 in low-density cultures and 5.8 in high-density cultures. When the concentration of 125I-EGF was increased in low density cultures, further consumption of 125I-EGF by the A431 cells was greatly reduced, partially due to a rapid down regulation of EGFR. However, when higher concentrations of 125I-EGF were added to high density cultures, with reduced receptor down regulation, the cells continued to consume a large fraction of the EGF in the culture medium. The consumption of 125I-EGF by these cultures was in excellent agreement with the measured amount of ligand internalized into the cell. EGF consumption was far in excess of the number of EGFR down regulated or degraded. Only a minor portion of the EGFR could have been replaced during the assay period by synthesis of new EGFR or from the intracellular pool of EGFR, and the fluid-phase uptake of EGF is only temporarily increased by exposure to EGF. Our results demonstrate that EGFR in high density A431 cell cultures recycled many times. The apparent level of recycling was dependent upon the concentration of EGF and followed Michaelis-Menton kinetics for ligand concentrations as high as 215 nM. At this EGF concentration, high-density cultures consumed 45 EGF molecules per receptor over a period of 6 h.     

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.     

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.     

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.     

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.     

Endocytosis and intracellular transport of epidermal growth factor after destruction of the actin cytoskeleton by cytochalasin B

Endocytosis of the epidermal growth factor (EGF) was investigated in three cell lines--A431, 3T6 and Swiss 3T3--after their incubation with cytochalasin B (CB). CB was introduced into culture medium (10 mkg/ml) 1.5-2 hours before addition of 125I-EGF (20-40 ng/ml). The label uptake rate was measured after a 35-40 minutes incubation of cells with 125I-EGF. It appeared that disorganization of microfilamentous network caused by CB exerted no influence on the binding of EGF to the surface membrane receptors and its internalization. Nevertheless, the experiments performed on A431 cells using a fluorescent label--rhodamine--bound to EGF (EGF-R) indicate that CB, though not influencing the initial steps of endocytosis, inhibits the next step--the intracellular transport of EGF-receptor complexes from the trans-Golgi region to lysosomes. As was shown elsewhere (Barkan, Nikol'sky, 1986), CB inhibits the mitogenic effect of EGF on resting Swiss 3T3 cells. So, the process of EGF-receptor uptake and delivery to the trans-Golgi region is evidently not enough to stimulate the cell proliferation; next steps of transport and degradation of ligand-receptor complexes are presumably needed.     

Epidermal growth factor receptor. Characterization of a monoclonal antibody specific for the receptor of A431 cells

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells was obtained after fusion of immunized BALB/c mouse spleen cells with NS-1 myeloma cells. Specific binding of the antibody to the plasma membrane of A431 cells was demonstrated by indirect immunofluorescence and electron microscopy. The antibody did not react with human KB cells, normal rat kidney cells, or Swiss 3T3 cells. The antibody is an IgG3K; it specifically immunoprecipitated a Mr approximately 170,000 protein from radiolabeled A431 cell extracts. This protein is phosphorylated in a EGF-dependent manner in intact A431 cells and in Triton X-100-solubilized plasma membranes. The specificity of the interaction of the antibody with the Mr = 170,000 protein was confirmed by electrophoretic transfer of A431 cell proteins to nitrocellulose followed by incubation with the antibody and 125I-protein A. When 125I-EGF was covalently cross-linked to its receptor, the 125I-EGF-receptor complex was specifically precipitated by the antibody. The monoclonal antibody did not inhibit the binding of 125I-EGF to its receptor in intact A431 cells and also failed to stimulate the phosphorylation of the Triton X-100-solubilized EGF receptor. The results indicate that the antibody and EGF bind to different sites on the EGF receptor. The antibody will be useful for isolating the EGF receptor in an unactivated form.     

Chloroquine allows the secretion of internalized 125I-epidermal growth factor from fibroblasts

Incubation of cells with labelled hormone in the presence of the lysosomotropic agent chloroquine produces an enhanced intracellular accumulation of hormone and receptor. Using a pulse-chase paradigm in which cell surface receptors were labelled with 125I-EGF at 4 degrees C, it was found that when 100 microM chloroquine was present in the 37 degrees C chase medium intact hormone was accumulated in the medium. Without chloroquine, low molecular weight (mw) degradation products were found in the medium. The processes of receptor-mediated endocytosis and subcellular distribution of 125I-EGF-receptor complexes were unchanged by chloroquine. The source of the intact hormone accumulating in the medium was therefore an intracellular compartment(s). The 125I-EGF released from the cells could rebind to surface receptors and be re-internalized; rebinding was inhibited by unlabelled EGF or Concanavalin A in the incubation medium. The concentration of unlabelled EGF required to inhibit rebinding was more than three orders of magnitude greater than the amount of 125I-EGF whose rebinding was inhibited. Thus, the 125I-EGF released from intracellular sites was rebound preferentially over exogenous EGF. The possible pathways for secretion of intact 125I-EGF and mechanisms of its preferential rebinding are discussed.     

Tumor-promoting phorbol diesters mediate phosphorylation of the epidermal growth factor receptor

4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly inhibited the binding of low concentrations (less than 10(-9 m) of 125I-epidermal growth factor (EGF) to A431 human epidermoid carcinoma cells. However, very little change in the binding of 125-I-EGF at high concentrations (greater than 10(-8) M) was observed in response to PMA. Affinity labeling of the 170,000-dalton EGF receptor with 125I-EGF and disuccinimidyl suberate was also decreased by the tumor promoter at low, but not high, concentrations of 125I-EGF. In order to examine this action of PMA on the EGF receptor, the receptor phosphorylation state was evaluated in A431 cells that had been incubated with [32P]phosphate for 3 h prior to the addition of PMA. The 32P content of the EGF receptor purified with EGF-Sepharose was increased by 38% compared with the same amount of receptor isolated from control cells. The increase in EGF receptor phosphorylation was dose-dependent with a half-maximal effect between 0.1 and 1 nM PMA and was specific for tumor promoting analogues of phorbol diesters. Phosphoamino acid analysis indicated that the increase in the 32P content of the EGF receptor was mainly due to phosphoserine. These results demonstrate that the EGF receptor is a target for PMA action and suggest that the mechanism of PMA action on the response of cells to epidermal growth factor may be mediated in part by phosphorylation of the EGF receptor.     

Attenuated processing of epidermal growth factor in the face of marked degradation of transforming growth factor-alpha

T3M4 human pancreatic carcinoma cells avidly bound and internalized 125I-labeled epidermal growth factor (EGF) but did not readily degrade the ligand. Pulse-chase experiments in which the cell-bound radioactivity was allowed to dissociate into the incubation medium in the presence of unlabeled EGF indicated that the majority of the released 125I-EGF consisted of intact EGF and a slightly processed species that readily bound to the cell. Omission of unlabeled EGF during the chase period markedly decreased the amount of radioactivity in the incubation medium, mainly as a result of the rebinding of EGF to the cells. In contrast, T3M4 cells readily degraded 125I-labeled transforming growth factor-alpha (TGF-alpha), and the released radiolabeled products did not rebind to the cells. Both ligands were released from T3M4 cells under acidic conditions, complete dissociation occurring at a pH of 4.5 for EGF, and a pH of 6.5 for TGF-alpha. A 431 human epidermoid carcinoma cells and ASPC-1 human pancreatic carcinoma cells also failed to extensively degrade 125I-EGF, whereas Rat-1 fibroblasts markedly degraded the growth factor. As in the case of T3M4 cells, ASPC-1 cells extensively degraded 125I-TGF-alpha. Degradation of either ligand was blocked by the lysosomotropic compound methylamine in all the tested cell lines. Immunoprecipitation of the EGF receptor with specific polyclonal antibodies and Western blot analysis revealed the anticipated 170-kDa protein in T3M4 cells. Both EGF and TGF-alpha enhanced EGF receptor degradation, but TGF-alpha was less effective than EGF. These findings indicate that in certain cell types EGF and TGF-alpha may be differentially processed.     

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.     

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.     

The characteristics of the internalization of normal and mutant receptors for the growth factor released by thrombocytes]

The platelet-derived growth factor (PDGF) is a major mitogen in serum for connective tissue derived cells in culture. The influence of receptor structure on the ability of PDGF receptor to be internalized was studied using cell lines transfected with different PDGF-receptor constructions. CHO cell lines expressing either normal PDGF receptor (CHO wt cells) or truncated PDGF receptor, lacking all but 19 amino acids of the intracellular domain (CHO-ECTM cells) were stained according to the method of indirect immunofluorescence with monoclonal antibody B2 to PDGF receptor. It has been found that after a 30-min incubation of the CHO wt cells at 37 degrees C in the presence of PDGF-BB a typical process of endocytosis is observed, while after a 2 h incubation in the same conditions the staining of the cells is absent which suggested the existence of the down-regulation and the process of degradation of PDGF receptor in CHO wt cells. In contrast, in the case of CHO-ECTM cells after both 30-min and 2-h incubation of cells in the presence of PDGF-BB a bright staining of margins and a low staining of the cytoplasm are observed. When the cells where incubated in the presence of lysosomotropic drug chloroquine (0.1 mM), that inhibits degradation of the receptor, the immunostaining of CHO wt cells is changed, with bright compact spots appearing. But in CHO-ECTM cells the fluorescence pattern is not changed. To examine the rate of endocytosis of PDGF-BB, both the types of cells were incubated in the presence of 2 ng/ml of 125I-PDGF-BB.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.