Amiloride directly inhibits growth factor receptor tyrosine kinase activity

Addition of amiloride to A431 human epidermoid carcinoma cell membranes inhibited autophosphorylation of the epidermal growth factor (EGF) receptor. The tyrosine phosphorylation of histone H2B catalyzed by an affinity-purified preparation of EGF receptor was also inhibited by amiloride. The inhibition was noncompetitive with respect to histone but competitive with ATP, suggesting that amiloride may act as an ATP analogue which causes the formation of nonproductive enzyme-substrate complexes. The tyrosine phosphorylation of histone H2B catalyzed by the purified EGF receptor was inhibited by amiloride at concentrations identical to those previously reported to block EGF action on cell proliferation (Ki = 350 microM). Amiloride similarly inhibited the tyrosine phosphorylation of the human placental insulin receptor and the platelet-derived growth factor receptor of Swiss 3T3 cells. Immunoprecipitation of the EGF receptor from A431 cells labeled for 24 h with [32P]phosphate demonstrated that amiloride decreased the phosphorylation of the EGF receptor on serine and threonine residues and blocked the effect of EGF to cause phosphorylation of the receptor on tyrosine residues. Phosphoamino acid analysis of total cell proteins indicated that amiloride inhibited the increase in phosphotyrosine levels caused by EGF. We conclude that amiloride directly inhibits the tyrosine kinase activity of the receptors for EGF, insulin, and platelet-derived growth factor in in vitro and can mediate such actions in vivo. This effect of amiloride demonstrates that it is unsuitable as a drug to test the hypothesis that the stimulation of the Na+/H+ antiporter is essential for mitogenic signaling by growth factor receptors.     

Immunoaffinity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self-phosphorylation

Epidermal growth factor (EGF) receptor protein has been purified in a single high-yield step by immunoaffinity chromatography of extracts of A431 cells. A monoclonal antibody directed against the EGF binding site of the receptor was immobilized to Sepharose 4B as a specific immune absorbent and competitive elution with EGF was used to obtain purified EGF receptor protein with tyrosine kinase activity. The stoichiometry of EGF binding was determined by comparing 125I-EGF binding to A431 cells with the mass of EGF receptor protein in those cells as measured by immunoaffinity chromatography, radioimmunoassay, and immune precipitation. Each measurement indicated one EGF binding site/EGF receptor protein molecule. Study of the kinetics of autophosphorylation revealed rapid incorporation of 1 mol of phosphate/mol of enzyme followed by slower incorporation of additional phosphate groups. The autophosphorylation reaction has a Km for ATP (0.2 microM) which is about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation is an intramolecular reaction.     

Effects of gangliosides GM3 and De-N-acetyl GM3 on epidermal growth factor receptor kinase activity and cell growth.

Previously it was reported (Bremer, E.G., Schlessinger, J., and Hakomori, S.-I. (1986) J. Biol. Chem. 261, 2434-2440) that ganglioside GM3 inhibited epidermal growth factor (EGF)-stimulated phosphorylation of the EGF receptor in Triton X-100-treated preparations of human epidermoid carcinoma (A431) cell membranes. In addition, these authors reported that GM3 inhibited the growth of A431 cells. In contrast, a modified ganglioside, de-N-acetyl GM3, enhanced the EGF-dependent tyrosine kinase activity of the EGF receptor. In this work and in subsequent studies (Hanai, N., Dohi, T., Nores, G. A., and Hakomori, S.-I. (1988) J. Biol. Chem. 263, 6296-6301), the tyrosine kinase activity of the receptor from A431 cell membranes was assayed in the presence of Triton X-100. In this report, we confirm that GM3 inhibited and de-N-acetyl GM3 stimulated EGF receptor autophosphorylation in the presence of Triton X-100. However, in the absence of detergents, ganglioside GM3 inhibited EGF-stimulated receptor autophosphorylation, whereas de-N-acetyl GM3 had no effect on EGF-stimulated receptor autophosphorylation. The effects of these gangliosides on receptor autophosphorylation were measured in both A431 cell plasma membranes and in 3T3 cell membranes permeabilized to [32P]ATP by a freeze-thaw procedure, in intact A431 cells permeabilized with alamethicin, and in intact A431 cells grown in the presence of [32P]orthophosphate. Thus, the inhibitory effect of GM3 on receptor autophosphorylation was demonstrated in the presence and in the absence of detergent; the stimulatory effect of de-N-acetyl GM3 was observed only in the presence of detergent. We also demonstrate that ganglioside GM3 inhibited EGF-stimulated growth of transfected murine fibroblasts (3T3) that express the gene for human EGF receptor (Velu, T. J., Beguinot, L., Vass, W. C., Zhang, K., Pastan, I., and Lowy, D. R. (1989) J. Cell. Biochem. 39, 153-166). De-N-acetyl ganglioside GM3 had no effect on the growth of these cells. Growth of control fibroblasts, which lack endogenous EGF receptors (Pruss, R. M., and Herschman, H. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3918-3921), was not affected by the presence of either ganglioside. Similarly, ganglioside GM3, but not de-N-acetyl ganglioside GM3, inhibited the EGF-dependent incorporation of [3H]thymidine into DNA by transfected fibroblasts. Incorporation of labeled thymidine into DNA of control fibroblasts was not affected by the presence of either ganglioside. These studies indicate that ganglioside GM3, but not its deacetylated analogue, can affect EGF receptor kinase activity in intact membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that the tyrosine kinase domain of a small fraction of epidermal growth factor receptor molecules is exposed on the outer surface of A431 cells

Intact A431 cells were labeled with [gamma-32P]ATP. The major phosphorylation product of the ecto-kinase activity of A431 cells had the molecular mass of 170 kd and was identified as EGF receptor by specific immunoprecipitation. This phosphorylation was not stimulated by EGF added to the reaction buffer, but replacement of MgCl2 by MnCl2 in the buffer remarkably stimulated phosphorylation. An exogenous protein substrate, alpha-casein, was also phosphorylated by intact A431 cells. The analyses for phospho-amino acids of both EGF receptor and alpha-casein revealed that phosphorylation occurred mainly at phosphotyrosine residues. Tryptic phospho-peptides of the EGF receptor of intact A431 cells labeled with [gamma-32P]ATP were fractionated by HPLC. The elution patterns were essentially the same as that of the autophosphorylated EGF receptor, indicating that the phosphorylation sites of EGF receptor labeled in vivo with [gamma-32P]ATP are located in three tyrosine residues in the carboxyl terminus. These results indicate that the carboxyl-terminal tyrosine kinase domain of a small fraction of the EGF receptor molecules of an A431 cell is exposed on the outer surface of the cells.     

Effects of epidermal growth factor on transferrin receptor phosphorylation and surface expression in malignant epithelial cells

The transferrin (Tf) receptor is a major transmembrane protein which provides iron for normal and malignant cell growth. Epidermal growth factor (EGF) has been reported to rapidly and transiently alter the number of surface Tf receptors in normal and transformed epithelial cells. To investigate mechanisms of EGF-induced changes in surface Tf display, EGF effects on surface Tf receptors were compared in two cell lines which differ in their number of EGF receptors and growth responses to EGF. In cloned A431 cells with high receptor numbers which are growth-inhibited by EGF, EGF caused a 50% decrease in Tf receptor expression after 30 min. In contrast, EGF induced a rapid, transitory increase (within 5 min) in the number of surface Tf receptors on KB carcinoma cells which returned to basal levels by 15 min. The observed changes in Tf receptor display were due to altered receptor distribution and not changes in ligand affinity or total cellular transferrin receptor pools. Anti-EGF receptor monoclonal antibody blocked effects of EGF on transferrin receptor expression. Since the antibody is internalized and causes EGF receptor down-regulation, effects on transferrin receptor expression were independent of these events. EGF-induced alterations in Tf receptor display occurred even when cells were pretreated with colchicine, suggesting that changes in surface Tf binding were not mediated by cytoskeletal components. Na orthovanadate, which mimics some early cellular effects of EGF, duplicated EGF's effects on A431 Tf receptors, but had no effect on KB cells, suggesting these responses occur by differing mechanisms. To determine whether EGF caused changes in Tf receptor phosphorylation, 32P-labelled Tf receptors were immunoprecipitated after EGF treatment. After exposure to EGF, A431 cells showed no change in Tf phosphorylation, but KB cells showed a transient, 6-fold increase in transferrin receptor phosphorylation on serine residues. In both A431 and KB cells, phorbol ester (PMA) also increased phosphorylation on transferrin receptors, but had little effect on surface Tf receptor expression. In malignant cell lines, EGE induces rapid, variable changes in transferrin receptor expression and phosphorylation which differ from the effects of PMA. These early responses to EGF appear to differ with the cell type and correlate poorly with alterations in Tf receptor phosphorylation. These results suggest Tf receptor phosphorylation does not regulate Tf receptor display in all cells.     

Different forms of the epidermal growth factor receptor kinase have different autophosphorylation sites

Limited proteolysis converts the native (Mr 170 000) epidermal growth factor (EGF) receptor to the Mr 150 000 form of the receptor. Calcium-activated, neutral protease (purified to homogeneity from beef lung), chymotrypsin, and elastase were all similarly effective in generating the 150-kilodalton (150-kDa) form of the receptor in detergent-solubilized, membrane vesicles shed from A-431 cells. The rate of autophosphorylation with [gamma-32P]ATP of the 150-kDa form was only 10% of the rate with the native receptor. This decreased rate was not due to loss of kinase activity, since the phosphorylation of angiotensin was virtually unchanged after limited proteolysis of the native receptor kinase. However, maps of elastase-produced peptides from 170-kDa forms and elastase-generated 150-kDa forms of the EGF receptor showed that the major autophosphorylation sites in these two forms were totally different. Confirming this difference in autophosphorylation sites was the finding that the 32P label in the autophosphorylated native receptor could not be recovered in the 150-kDa form following proteolysis. This label was quantitatively recovered in 30-15-kDa peptide fragments generated simultaneously with the 150-kDa form of the receptor. Therefore, the decreased autophosphorylation of the 150-kDa form results from the loss of preferred autophosphorylation sites on the native receptor. Only 1-3% of the phosphate incorporated in the native receptor during autophosphorylation could be found on the 150-kDa autophosphorylation sites. Hence, autophosphorylation of the tyrosine sites in the 150-kDa form of the EGF receptor is markedly enhanced by removing the major sites autophosphorylated on the native form of the receptor.     

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of 110-130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of 70-100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c-src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies to the autophosphorylation sites of the epidermal growth factor receptor protein-tyrosine kinase as probes of structure and function.

Antisera were prepared against three synthetic peptides with amino acid sequences identical to those surrounding the three major autophosphorylation sites of the epidermal growth factor (EGF) receptor. The affinity-purified antibodies reacted strongly in an enzyme-linked immunosorbent assay against the immunizing peptide but showed little cross-reaction with the other two phosphorylation site peptides. EGF receptors labelled by autophosphorylation could be specifically precipitated by each of the phosphorylation site antibodies. The antibodies recognised EGF receptors labelled at each of the autophosphorylation sites, indicating that they could bind to the immunizing sequences irrespective of their states of phosphorylation. The antibodies were able to inhibit EGF receptor autophosphorylation without affecting EGF-stimulated tyrosine kinase activity towards exogenous peptide substrates, suggesting that the kinase and autophosphorylation sites were in distinct domains. Immunofluorescent staining of A431 cells showed that the autophosphorylation site sequences resided inside the cell. The autophosphorylation sites were shown to be within a domain of 20 000 mol. wt. which could be cleaved from the receptor through limited proteolysis by the calcium-dependent protease, calpain. The position of cleavage of the EGF receptor by the protease was mapped to lie between residues 996 and 1059. These results are discussed in the context of a model for the structure and function of the human EGF receptor.     

Epidermal growth factor receptor threonine and serine residues phosphorylated in vivo

The epidermal growth factor (EGF) receptor is regulated by EGF-stimulated autophosphorylation and by phorbol ester-stimulated, protein kinase C (Ca2+/phospholipid-dependent enzyme) mediated phosphorylation at identified sites. The EGF receptor contains additional phosphorylation sites including a prominent phosphothreonine and several phosphoserines which account for the majority of phosphate covalently bound to the receptor in vivo. We have identified three of these sites in EGF receptor purified from 32P-labeled A431 cells. The major phosphothreonine was identified as threonine 669 in the EGF receptor sequence. Phosphoserine residues were identified as serines 671 and 1046/1047 of the EGF receptor. Two other phosphoserine residues were localized to tryptic peptides containing multiple serine residues located carboxyl-terminal to the conserved protein kinase domain. The amino acid sequences surrounding the three identified phosphorylation sites are highly conserved in the EGF receptor and the protein products of the v-erb B and neu oncogenes. Analysis of predicted secondary structure of the EGF receptor reveals that all of the phosphorylation sites are located near beta turns. In A431 cells phosphorylation of the serine residues was dependent upon serum. In mouse B82 L cells transfected with a wild type human EGF receptor. EGF increased the 32P content in all tryptic phosphopeptides. A mutant EGF receptor lacking protein tyrosine kinase activity was phosphorylated only at threonine 669. Regulated phosphorylation of the EGF receptor at these threonine and serine residues may influence aspects of receptor function.     

A new monoclonal antibody for detection of EGF-receptors in western blots and paraffin-embedded tissue sections.

The prognostic significance of the epidermal growth factor receptor status (EGF-R-status) for certain human tumors requires the development of antibodies useful for clinical application. We used purified receptor preparations to generate monoclonal antibodies immunoreactive with the EGF-R purified from placenta membranes and A431 tumors. Four of the hybridomas contained antibodies (R2, R3, R5, and R9) which recognized both antigens. Antibody R3 was shown to display the following properties: it binds with a KD value of about 10(-9)-10(-10) M to the receptor, a half maximal inhibition of EGF-binding is achieved at 5 x 10(-8) M, and in Western blots of cell membranes R3 specifically detects the EGF-R at 0.1 micrograms/ml. R3 inhibits EGF-dependent clonogenic growth of NRK cells and completely blocks EGF stimulated autophosphorylation of the receptor. Moreover, R3 also detects EGF-R in paraffin-embedded tissue sections taken from human salivary gland, term placenta, and adult skin and mammary carcinomas. Thus, R3 can be used in retrospective diagnostic clinical studies and might help to develop new immunotherapeutic intervention.     

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.     

Detergent Solubilization is a Prerequisite for Aggregation-Induced Stimulation of Epidermal Growth Factor (EGF) Receptor Kinase

Abstract

Unlike EGF, concanavalin A and wheat germ agglutinin do not increase EGF receptor-kinase activity in intact A-431 membranes. However, they increase both autophosphorylation and phosphorylation of exogenous substrates about twice as much as EGF if the membranes are solubilized in detergent. Following solubilization, autophosphorylation due to the combined presence of a lectin and EGF is additive suggesting that each increases kinase activity by a different mechanism. These different mechanisms were studied by autophosphorylating membranes at increasing detergent concentrations after they had been permeabilized to [γ^?32P]ATP with alamethicin. As the detergent concentration increased, EGF stimulated autophosphorylation decreased 3–fold and 6–fold for the native 170 kDa receptor and for a protease-generated 150 kDa receptor form, respectively. However, in the presence of either lectin the same increase in detergent concentration only slightly altered the autophosphorylation rates which never exceeded the rate measured in the absence of EGF and detergent. Hence, the lectins increase kinase activity in solubilized membranes by preventing the adverse effects of detergent on the receptor-kinase and may not be useful models for how EGF activates its receptor.     

The epidermal growth factor receptor is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein in rat hepatocytes.

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.     

Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation

The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors.     

Transient epidermal growth factor (EGF)-dependent suppression of EGF receptor autophosphorylation during internalization.

To study the activity of the epidermal growth factor (EGF) receptor during EGF-directed internalization, liver epithelial cells were exposed to EGF at 37 degrees C for various periods of time, washed, and homogenized at 0 degrees C. EGF receptor autophosphorylation was assessed in homogenates using [gamma-32P]ATP. Autophosphorylation was stimulated 3- to 6-fold in homogenates of cells incubated with EGF (100 ng/ml) for 15 min but was at or below basal levels in homogenates of cells treated with EGF for 2.5-5 min. This was surprising because immunoblotting revealed that EGF receptor phosphotyrosine (P-Tyr) content in intact cells was near maximal from 30 s to 5 min after EGF treatment. Excess EGF (1 microgram/ml), added after homogenization but prior to the assay, increased autophosphorylation in homogenates of cells that had not been treated with EGF, but failed to increase activity in homogenates of cells treated with EGF in culture for 2.5-5 min. Suppression of tyrosine phosphorylation of an exogenous kinase substrate was also observed at times paralleling the suppression of EGF receptor autophosphorylation. The transient suppression of receptor autophosphorylation in the cell-free assay was not explained by persistent occupation of autophosphorylation sites by phosphate added in the intact cells. The sites were greater than 80% dephosphorylated during the homogenization. Additionally phosphatase inhibition that prevented the normal loss of EGF receptor P-Tyr in intact cells at 15 min did not affect the pattern of early (2.5-5 min) suppression and later (15 min) stimulation of autophosphorylation measured in the cell-free assay. The suppression was not explained by activation of protein kinase C in that depletion of greater than 95% of cellular protein kinase C activity by an 18-h incubation of cells with 10 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) did not affect the early suppression of autophosphorylation in EGF-treated cells. Moreover, under the conditions tested, activation of protein kinase C by short-term treatment (0.5-10 min) with TPA or angiotensin II did not appreciably alter subsequent autophosphorylation in the cell-free assay. In contrast, a 30 degrees C preincubation of homogenates from cells with suppressed EGF receptor autophosphorylation led to the recovery of the ability of EGF to stimulate EGF receptor autophosphorylation. These results suggest that a rapid reversible protein kinase C-independent process prevents detection of EGF receptor kinase activity during an early phase of EGF-dependent receptor internalization.     

The effect of phorbol esters and Ca2+ ions on the process of autophosphorylation of epidermal growth factor receptor in vivo

Monoclonal antibodies against phosphotyrosine were used to study tyrosine phosphorylation in human epidermal carcinoma A431 cells in vivo. Incubation of A431 cells with the epidermal growth factor (EGF) leads to tyrosine phosphorylation of the EGF receptor; the phosphotyrosine content in cellular EGF receptors increases 50-100-fold in the presence of the growth factor. The maximum level of the receptor autophosphorylation is reached on the 5th min and is then held constant during 90-min incubation with EGF. After preincubation of A431 cells with phorbol-12-myristoyl-13-acetate (PMA) or calcium ionophore A23187 the receptor autophosphorylation decreases significantly. After addition of A23187 and EGTA to the preincubation medium the phosphotyrosine content in cellular EGF receptors stimulated by the growth factor reaches the control level i.e., that observed in the absence of the ionophore. After preincubation of cells in the presence of phorbol ester and H-7 (protein kinase C inhibitor) the level of EGF receptor autophosphorylation does not practically differ from that of control.     

Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor

Glycosphingolipids added exogenously to 3T3 cells in culture were shown to inhibit cell growth, alter the membrane affinity to platelet-derived growth factor binding, and reduce platelet-derived growth factor-stimulated membrane phosphorylation (Bremer, E., Hakomori, S., Bowen-Pope, D. F., Raines, E., and Ross, R. (1984) J. Biol. Chem. 259, 6818-6825). This approach has been extended to the epidermal growth factor (EGF) receptor of human epidermoid carcinoma cell lines KB and A431. GM3 and GM1 gangliosides inhibited both KB cell and A431 cell growth, although GM3 was a much stronger inhibitor of both KB and A431 cell growth. Neither GM3 nor GM1 had any affect on the binding of 125I-EGF to its cell surface receptor. However, GM3 and, to a much lower extent, GM1 were capable of inhibiting EGF-stimulated phosphorylation of the EGF receptor in membrane preparations of both KB and A431 cells. Further characterization of GM3-sensitive receptor phosphorylation was performed in A431 cells, which had a higher content of the EGF receptor. The following results were of particular interest. (i) EGF-dependent tyrosine phosphorylation of the EGF receptor and its inhibition by GM3 were also demonstrated on isolated EGF receptor after adsorption on the anti-receptor antibody-Sepharose complex, and the receptor phosphorylation was enhanced on addition of phosphatidylethanolamine. (ii) Phosphoamino acid analysis of the EGF receptor indicated that the reduction of phosphorylation induced by GM3 was entirely in the phosphotyrosine and not in the phosphoserine nor phosphothreonine content. (iii) The inhibitory effect of GM3 on EGF-dependent receptor phosphorylation could be reproduced in membranes isolated from A431 cells that had been cultured in medium containing 50 nmol/ml GM3 to effect cell growth inhibition. The membrane fraction isolated from such growth-arrested cells was found to be less responsive to EGF-stimulated receptor phosphorylation. These results suggest that membrane lipids, especially GM3, can modulate EGF receptor phosphorylation in vitro as well as in situ.     

Modulation of EGF receptor activity by changes in the GM3 content in a human epidermoid carcinoma cell line, A431

Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP-glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 microM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype.     

Membrane vesicles of A431 cells contain one class of epidermal growth factor binding sites

Epidermoid carcinoma A431 cells exhibit two classes of epidermal growth factor (EGF) receptors as deduced from Scatchard analysis. Steady-state binding of EGF to isolated A431 membranes indicated, however, the presence of only one class of EGF binding sites. The apparent dissociation constant (Kd) of these sites was approx. 0.45 nM which is similar to that of the high-affinity receptor of intact A431 cells. These results suggest that the vesicle receptor population consists only of high-affinity receptors. However, further studies indicated that the binding sites were similar to the low-affinity class, since binding of EGF could be blocked entirely by 2E9, a monoclonal anti-EGF receptor antibody which is able to inhibit specifically EGF binding to low-affinity receptors in A431 cells. The difference in affinity of the receptors in membrane vesicles as compared to intact cells may be explained by differences in biophysical parameters such as diffusion-limited EGF binding and receptor distribution. Based upon these considerations, it is concluded that membrane vesicles of A431 cells contain one class of EGF receptors which are apparently identical to the low-affinity receptors of intact cells.     

Functional studies on the EGF receptor with an antibody that recognizes the intracellular portion of the receptor

An antibody against the human epidermal growth factor receptor (EGF), capable of activating its tyrosine kinase has been produced. Antibody 2913 recognizes only the cytoplasmic portion of the EGF receptor in A431 carcinoma cells, in normal human fibroblasts, and in a variety of other human tumor cell lines (Xu, Y.-A., Richert, N., Ito, S., Merlino, G. T., and Pastan, I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7308-7313). Indirect immunofluorescence and electron microscopy show that the antibody binds to intact cells only after membrane permeabilization. Moreover the antibody immunoprecipitates the v-erb-B gene product in avian myeloblastosis virus-infected cells but does not recognize the secreted form (105 kDa) of the A431 cell EGF receptor which lacks the cytoplasmic domain. Antibody 2913 activates the EGF receptor kinase in solubilized A431 membranes causing autophosphorylation on tyrosine residues only. Tryptic peptide maps suggest that antibody 2913 and EGF stimulate phosphorylation of the same amino acid residues. By electron microscopy, the cytoplasmic portion of the receptor was followed throughout its endocytotic pathway. The results show that the kinase domain is rapidly degraded in lysosomes with no accumulation in the cytoplasm or in the nucleus.