Characteristics of antibodies to the epidermal growth factor receptor-kinase

Polyclonal antibodies to different antigenic forms of the epidermal growth factor (EGF) receptor-kinase from human A-431 cells have been produced, and their properties have been characterized and compared. Biochemically active receptor-kinase purified by affinity chromatography was employed as one type of antigen. Denatured receptor-kinase prepared by sodium dodecyl sulfate-gel electrophoresis of the affinity-purified receptor was used as the second type of antigen. Animals immunized with either type of antigen produced antibody capable of immunoprecipitating the receptor-kinase molecule. Antibodies produced in response to the biochemically active antigenic form of the receptor-kinase are capable of blocking ¹²⁵I-EGF binding to the receptor and inhibited EGF-stimulated biological responses. These antisera are not species specific in their ability to inhibit growth-factor binding to the EGF receptor of various mammalian cells. However, these rabbit antisera were unable to inhibit ¹²⁵I-EGF binding to rabbit cells. Although antisera produced in response to the denatured receptor-kinase molecule are not able to block ¹²⁵I-EGF binding or EGF-stimulated biological responses, they are particularly efficient for the immunoprecipitation of solubilized ¹²⁵I-EGF:receptor complexes. None of the antisera contain antibodies capable of interfering with basal receptor-kinase phosphorylation activity. Although each of the antisera immunoprecipitated this kinase activity, none of the antisera contained antibody which served as a phosphorylation substrate for the EGF receptor-kinase in contrast to the immunoglobulins present antisera to the src gene product of the Rous sarcoma virus.     

Epidermal growth factor receptor kinase translocation and activation in vivo

The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity.     

Intrapeptide autophosphorylation of the epidermal growth factor receptor: regulation of kinase catalytic function by receptor dimerization

The epidermal growth factor (EGF) receptor is a transmembrane polypeptide of 170 000 daltons (Da) with a cytoplasmically facing protein kinase domain. The regulation of the tyrosine kinase activity of the EGF receptor by added EGF and by receptor association state was studied in an in vitro system. The rate of autophosphorylation of the solubilized and purified EGF receptor was found to be independent of receptor concentration. To determine whether the zero-order kinetics observed point to intrapeptide phosphorylation, we measured the sedimentation characteristics of the undenatured solubilized receptor. The receptor was found to exist in two association-dissociation states-a monomeric 7.7S form and a dimeric 12S form. The 7.7S form is an active tyrosine kinase; it has high basal activity, and the activity is not further stimulated by EGF; it appears to be an EGF-independent form of the receptor kinase. The 12S form is devoid of catalytic activity, but in the presence of EGF it dissociates into the active monomeric form. Freshly purified receptor preparations contain mainly the monomeric receptor, have high basal kinase activity, and show low EGF stimulatability (less than 1.3-fold). Aging of the receptor results in progressive dimerization and decay of EGF-independent kinase activity (and increase in EGF stimulatability). All of these processes are reversed in the presence of EGF or dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term, heterologous down-regulation of the epidermal growth factor receptor in PC12 cells by nerve growth factor

Cells of the rat pheochromocytoma clone PC12 possess receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), thus enabling the study of the interaction of these receptors in the regulation of proliferation and differentiation. Treatment of the cells with NGF induces a progressive and nearly total decrease in the specific binding of EGF beginning after 12 h and completed within 4 d. Three different measures of receptor show that the decreased binding capacity represents, in fact, a decreased amount of receptor: (a) affinity labeling of PC12 cell membranes by cross-linking of receptor-bound 125I-EGF showed a 60-90% decrease in the labeling of 170- and 150-kD receptor bands in cells treated with NGF for 1-4 d; (b) EGF-dependent phosphorylation of a src-related synthetic peptide or EGF receptor autophosphorylation with membranes from NGF-differentiated cells showed a decrease of 80 and 90% in the tyrosine kinase activity for the exogenous substrate and for receptor autophosphorylation, respectively; (c) analysis of 35S-labeled glycoproteins isolated by wheat germ agglutinin-Sepharose chromatography from detergent extracts of PC12 membranes showed a 70-90% decrease in the 170-kD band in NGF-differentiated cells. These findings permit the hypothesis that long-term heterologous down-regulation of EGF receptors by NGF in PC12 cells is mediated by an alteration in EGF receptor synthesis. It is suggested that this heterologous down-regulation is part of the mechanism by which differentiating cells become insensitive to mitogens.     

A specific insulin receptor and tyrosine kinase activity in the membranes of Neurospora crassa

Cells of the wall-less ("slime") strain of Neurospora crassa possess specific high affinity insulin binding sites on their cell surface. 125I-labeled bound insulin was not displaced from these cells by insulin-like growth factor II (IGF-II), and was only weakly displaced by IGF-I and proinsulin. Cross-linking of 125I-labeled insulin with N. crassa cells using disuccinimidyl suberate resulted in the labeling of a single band of ca. 67 kDa m.w. on a polyacrylamide gel. Two proteins of ca. 66 and 59 kDa m.w. were purified from detergent solubilized plasma membrane preparations by passage over an insulin-agarose affinity matrix. Antibodies against an autophosphorylation site on the human and Drosophila insulin receptors (anti P2) immunoprecipitated a single phosphoprotein of ca. 50 kDa m.w. from detergent solubilized plasma membranes, which possessed protein tyrosine kinase activity when histone H2 was used as substrate.     

Developments in the mechanism of growth factor action: activation of protein kinase by epidermal growth factor

The interaction between epidermal growth factor (EGF) and its target cells has been used as a model for studying the regulation of cell proliferation. Many of the details of binding and subsequent internalization and degradation of this growth factor have been elucidated by following the fate of [125I]EGF in the presence of responsive cells. To investigate the membrane-localized biochemical consequences of EGF-receptor complex formation, a subcellular membrane system has been developed. In this system, EGF enhances phosphorylation of its receptor as well as other endogenous proteins. This EGF-stimulable protein kinase activity is not separated from the EGF receptor activity either by detergent solubilization or by affinity purification of the solubilized membranes. The data suggest that the EGF-binding activity and EGF-sensitive protein kinase activity reside in a single membrane protein.     

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)     

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.     

The epidermal growth factor receptor tyrosine kinase in liver epithelial cells. The effect of ligand-dependent changes in cellular location

Epidermal growth factor (EGF) activates the intrinsic tyrosine-specific protein kinase of its receptor (EGF-R). We studied the effect of EGF-dependent EGF-R internalization on receptor autophosphorylation and on the appearance of tyrosine phosphoproteins in rat liver epithelial cells. Peak receptor autophosphorylation activity (3- to 6-fold over basal) was found in homogenates of EGF-treated cells at times when the majority of receptors (greater than 90%) had been internalized but not yet degraded (15 to 30 min). Stimulated activity persisted for at least 2 h if EGF-R degradation was blocked by methylamine or 18 degrees C incubation. Detection of stimulated autophosphorylation in homogenates of cells treated with EGF in culture required detergent in the assay. Detergent was not necessary to detect stimulated autophosphorylation when EGF was added directly to homogenates of untreated cells. Immunoblots using antibodies against phosphotyrosine (p-Tyr) demonstrated that EGF treatment of intact cells increased the p-Tyr content of at least seven proteins (EGF-R, 115, 100, 75, 66, 57, and 52 kDa) within 5 s. Incubation of intact cells with EGF at 0 degrees C to prevent endocytosis still resulted in tyrosine phosphorylation of these seven proteins. In contrast, several substrates (120, 78, and 38 kDa) showed delayed increases (45-90 s) in tyrosine phosphorylation at 37 degrees C; their phosphorylation was even slower at 18 degrees C and did not occur at 0 degrees C. In cells incubated with EGF at 18 degrees C or in the presence of methylamine, EGF-R p-Tyr in the intact cell was lost by 2 h even though receptor was not degraded and still exhibited enhanced autophosphorylation in the homogenate assay. These findings suggest that tyrosine phosphorylation in response to EGF occurs predominantly during the initial stages of endocytosis and is mediated for the most part by ligand-receptor complexes at the cell surface. A subset of phosphorylations may require intracellular movement.     

Solubilization of EGF receptor with Triton X-100 alters stimulation of tyrosine residue phosphorylation by EGF and dimethyl sulfoxide

In isolated hepatic membranes, epidermal growth factor (EGF) and the polar solvent dimethyl sulfoxide (Me2SO) selectively stimulated the phosphorylation of the 170,000-dalton EGF receptor (p170) by 13.6 +/- 2.0- and 10.9 +/- 1.1-fold, respectively. The stimulation by maximally effective concentrations of the two substances was similar in rapidity of onset (less than 30 s at 0 degree C), time course of phosphorylation, and tyrosine residue specificity. These maximal effects were not additive when the substances were combined, indicating that the same kinase/substrate combination is activated by each. The lectin concanavalin A, which inhibits EGF receptor binding, blocked the effect of EGF but not Me2SO. In membranes solubilized with Triton X-100, EGF stimulated p170 phosphorylation by 40- to 55-fold. Me2SO also stimulated phosphorylation, indicating that it acts directly on the protein. However, the effect of the solvent was reduced by half. Additionally, Me2SO blocks the effect of EGF in the solubilized preparation. A room temperature preincubation after addition of either substance was necessary for maximal stimulation of p170 phosphorylation in solubilized membranes. With EGF, 30-40 min was necessary; with Me2SO, only 10 min was required. Thus, a secondary process appears to be involved in EGF receptor/kinase activation.     

A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles

A method is presented for the preparation of a "native" epidermal growth factor (EGF) receptor-kinase complex of molecular weight 170,000 from A-431 cells. Although this receptor complex is capable of binding EGF, noncovalently, in quantities similar to the previously isolated 150,000 complex (Cohen, S., Carpenter, G., and King, L., Jr. (1980) J. Biol. Chem. 255, 4834-4842), the 170,000 preparation has 5 to 10 times the intrinsic kinase activity (autophosphorylation). However, the 170,000 kinase activity toward other proteins is lower than that of the 150,000 preparation. Both the 170,000 and 150,000 kinase activities are enhanced by EGF. The 170,000 and 150,000 proteins are also capable of forming covalent linkages to 125I-EGF, and each is precipitated by antisera directed against the 170,000 protein. We suggest the 150,000 protein is a proteolytic degradation product of the 170,000 protein. The EGF-enhanced kinase activity of the 170,000 preparation remains associated with the 125I-EGF-binding activity following EGF affinity chromatography, electrophoresis in nondenaturing gels, or immunoprecipitation with antisera directed against the sodium dodecyl sulfate (SDS) gel-purified 170,000 protein. These results indicate that the receptor, kinase, and substrate domains are linked, possibly covalently.     

EGF-dependent phosphorylation of the EGF receptor in plasma membranes isolated from young and senescent WI-38 cells

Tyrosine-specific phosphorylation of the receptor for epidermal growth factor (EGF) in plasma membranes isolated from WI-38 cells is EGF-dependent and occurs to an equivalent extent and on identical tryptic peptides in preparations from cells of various in vitro ages. There is a marked reduction, however, in phosphorylation of receptor molecules from senescent as compared with young WI-38 cells, if enzyme activity is assayed in an immune complex following solubilization of plasma membranes with Nonidet P-40 (NP-40). Differences in the level of receptor phosphorylation in young vs. senescent NP-40 extracts are not resolved by changing the temperature at which the assay is performed, or the length of incubation. Moreover, addition of NP-40 or chloroform-methanol extracts of young cells to assays measuring receptor phosphorylation in senescent cell NP-40 preparations does not augment the senescent enzyme activity. The immunopurified senescent receptor is, however, capable of catalyzing phosphorylation of exogenous substrates. These results indicate that the loss of receptor autophosphorylation in solubilized preparations may result from a differential sensitivity of the senescent cell receptor to the detergent. This finding provides a marker for senescence and suggests subtle changes in protein structure, conformation, or regulation of the EGF receptor in senescent cells.     

Post-Translational Acquisition of Ligand Binding-and Tyrosine Kinase-Domain Function by the Epidermal Growth Factor and Insulin Receptors

Abstract

The epidermal growth factor receptor (EGFR) and insulin receptor undergo slow post-translational modification by which they acquire hormone binding and tyrosine kinase (EGFR) function. The half-time for acquisition of EGF or insulin binding activity is 30-40 min and of tyrosine kinase activity (EGFR), is 10-15 min. Tunicamycin, an inhibitor of N-linked oligosaccharide addition, blocks acquisition of both EGF and insulin binding activity. With EGFR, activation precedes acquisition of resistance to endoglucos-aminidase H (t_1/2 75 min), a medial Golgi event. Treatment of active high mannose receptor with endo H generates fully active aglyco-receptor; thus, core oligosaccharide addition is a prerequisite for activation, but not for EGF binding per se. EGFR is activated in and translocated from the endoplasmic reticulum (ER) slowly (t_1/2 75 min). Since translocation rate equals the rate for acquisition of endo H resistance, transit from the ER is rate limiting for EGFR maturation. Tunicamycin inhibits exit from the ER parallel to its effect on acquisition of binding activity. Insulin proreceptor, a 210 kDa high-mannose glycopolypeptide, acquires insulin binding function (t_1/2 45 min) then is proteolytically cleaved (t_1/2 3 hr) into subunits of the mature α₂β₂ receptor. Modification giving rise to insulin binding activity is due to a conformational change in the binding domain, since human autoimmune antibody recognizes only the active species, while rabbit polyclonal antibody recognizes all forms. Newly-translated EGF proreceptor lacks a functional tyrosine domain capable of autophosphorylation; 30-40 min after translation, while still in     

Regulation of epidermal growth factor receptor number and phosphorylation by fasting in rat liver

The binding of 125I-epidermal growth factor (EGF) to microsomal membrane preparations from the livers of rats fasted for 72 h or fed control or high carbohydrate diets was examined to determine whether alterations in nutrient intake could affect the EGF receptor system. Fasted rats had 40-50% less membrane binding than did control or carbohydrate-fed rats. Scatchard analysis of the binding data indicated that the decrease in EGF binding in fasted rats was due to a decrease in receptor number with no change in receptor affinity. Cross-linking of 125I-EGF to EGF receptors with disuccinimidyl suberate revealed specific binding of a Mr 170,000 protein, which was diminished by approximately 75% in fasting, and a Mr = 150,000 protein, which accounted for 40-50% of the total labeling in the control and carbohydrate-fed rats and which was relatively unchanged by fasting. The sum of the labeling of the 2 bands was reduced by approximately 40% in fasting and is consistent with the reduction in EGF binding detected by Scatchard analysis. EGF stimulated a 1.5-3-fold increase in 32P incorporation into one major protein of 170 kDa in all 3 groups. Basal and EGF-stimulated autophosphorylation of 170 kDa, when normalized for protein, was 75% lower in membranes from fasted animals, compared to those from control or carbohydrate-fed rats. The comparable reduction of 125I-EGF binding to, and 32P incorporation into, the 170-kDa EGF receptor protein suggested that kinase activity/receptor was unaffected by fasting. Moreover, EGF receptor kinase activity in the 3 groups was comparable for an exogenous substrate, as judged by equal basal and EGF-stimulated phosphorylation of Val5-angiotensin II, when normalized for total EGF-binding capacity. These results suggest that fasting regulates EGF receptor kinase activity primarily by regulation of the number of hepatic EGF receptors. The possibility exists that some in vivo effects of fasting may be mediated by a reduction in EGF receptor levels.     

Cytoskeletal and Morphological Changes Associated with the Specific Suppression of the Epidermal Growth Factor Receptor Tyrosine Kinase Activity in A431 Human Epidermoid Carcinoma

The epidermal growth factor (EGF) receptor is well known as a mediator of mitogenic signaling and its tyrosine kinase activity has been suggested as a viable target in cancer chemotherapy. To explore the consequences of abolishing the kinase activity of this receptor, we have utilized a potent and specific inhibitor of the enzyme, PD 153035, to sustain a long-term suppression of its activity. This compound inhibits EGF receptor autophosphorylation in cells with an IC₅₀in the low nanomolar range and does not block PDGF or FGF receptor kinase until concentrations are greater than 10 μM.[1] Human epidermoid carcinoma A431 cells were grown in the presence of PD 153035 and were passed weekly until cells grew in the presence of 1 μMinhibitor. These cells, referred to as A431R, showed a remarkable change in morphology, becoming flattened and spread out. A comparison of the sensitivity of EGF receptor autophosphorylation to PD 153035 between A431 and A431R showed a similar dose response, indicating that the cells had not developed any defect in the kinase which might make it resistant to the inhibitor. Likewise, EGF receptor autophosphorylation in response to exogenously added EGF, as well as receptor internalization, was similar between the two cell lines. Furthermore, analysis of A431R cells by flow cytometry showed no significant change in DNA content or percentage of cells in any one phase of the cell cycle compared to the parent line.¹²⁵I-labeled EGF/receptor binding studies showed that receptor number in the A431R cells was equivalent to that of the parent line; however, the Scatchard plot was linear, in contrast to the typical biphasic plot obtained with the parent cells, implying a loss of high-affinity receptors. Cytoskeletal preparations from both cell lines indicated that the A431R had fourfold less EGF receptor associated with the cytoskeleton than A431. This was accompanied by a remarkable increase in polymerized actin stress fibers throughout the A431R cells, which most likely accounts for their flattened morphology. The A431R cells also exhibited a twofold increase in the expression of focal adhesion kinase, which is consistent with a greater contact area for their cell surface and increase in focal adhesions. Finally, although the A431R cells have a doubling time of 24 h, similar to that of the parent line, these cells stop growing as the monolayer approaches confluence, reminiscent of the contact inhibition seen in nontransformed cells. These data indicate that long-term suppression of the EGF receptor tyrosine kinase activity in A431 human epidermoid carcinoma results in certain cellular properties which are more consistent with a differentiated and nontransformed phenotype.     

Decreased autophosphorylation of the insulin receptor-kinase in streptozotocin-diabetic rats

It has been documented that streptozotocin-induced diabetes in rats is associated with diminished effects of insulin despite increased insulin binding to its receptor. This paradox led us to examine whether any alterations of insulin receptor-kinase activities occur in this type of insulin resistance. Insulin binding capacity/mg of protein of solubilized, wheat germ agglutinin-purified preparations from livers was increased by 1.8-fold in the streptozotocin (65 mg/kg) diabetic rats. This increase was associated with a parallel increase in receptor protein as measured by an immunoblotting method using anti-insulin receptor antibody. Moreover, no apparent change was observed in the stoichiometry of alpha and beta subunits of the insulin receptor between diabetic and control rats. Insulin-stimulated (10(-7) M) phosphorylation of the beta subunit of the insulin receptor was decreased by 40% in diabetic rats when equal quantities of insulin binding capacity were compared. Phosphorylation of an exogenously added synthetic peptide (similar in sequence to the tyrosine phosphorylation site in pp60src) by the insulin receptor-kinase was also decreased by 25% in diabetic rats. These abnormalities were partially restored by in vivo insulin treatment. These data suggest that diminished insulin receptor autophosphorylation and kinase activity could provide a possible mechanism for the "post-binding insulin resistance" in diabetic rats.     

Loss of cytotoxic effect of epidermal growth factor (EGF) on EGF receptor overexpressing cells is associated with attenuation of EGF receptor tyrosine kinase activity

The biological activity of epidermal growth factor (EGF) is mediated through the intrinsic tyrosine kinase activity of the EGF receptor (EGFR). In numerous cell types, binding of EGF to the EGFR stimulates the tyrosine kinase activity of the receptor eventually leading to cell proliferation. In tumor-derived cell lines, which overexpress the EGFR, however, growth inhibition is often seen in response to EGF. The mechanism for growth inhibition is unclear. To study the relationship between growth inhibition and EGFR kinase activity, we have used a cell line (PC-10) derived from a human squamous cell carcinoma that overexpresses EGFR. When exposed to 25 ng/ml EGF at low cell densities (1,300 cells/cm²), PC-10 cells exhibit cell death. In contrast, if EGF is added to high density cultures, no EGF mediated cell death is seen. When PC-10 cells were maintained at confluency in the presence of 25 ng/ml EGF for a period of 1 month, they were subsequently found competent to proliferate at low density in the presence of EGF. We designate these cells APC-10. The APC-10 cells exhibited a unique response to EGF, and no concentration of EGF tested could produce cell death. By ¹²⁵I-EGF binding analysis and [³⁵S]methionine labeling of EGFR, it was found that the total number of EGFR on the cell surface of APC-10 was not decreased relative to PC-10. No difference between PC-10 and APC-10 was seen in EGF binding affinity to the EGFR. Significantly, EGF stimulated autophosphorylation of the EGFR of APC-10 was 8–10-fold lower than that of PC-10. This reduced kinase activity was also seen in vitro in membrane preparations for EGFR autophosphorylation as well as phosphorylation of an exogenously added substrate. No difference between PC-10 and APC-10 in the overall pattern of EGFR phosphorylation in the presence or absence of EGF was detectable. However, the serine and threonine phosphorylation of the EGFR of APC-10 cells was consistently 2–3-fold lower than that seen in PC-10 cells. These results suggest a novel mechanism for EGFR overexpressing cells to survive EGF exposure, one that involves an attenuation of the tyrosine kinase activity of the EGFR in the absence of a change in receptor levels or receptor affinity. © 1994 Wiley-Liss, Inc.     

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.     

Quantitative dissociation between EGF effects on c-myc and c-fos gene expression, DNA synthesis, and epidermal growth factor receptor tyrosine kinase activity.

The exact relationship between EGF-stimulated tyrosine phosphorylation, induction of the cellular proto-oncogenes c-myc and c-fos, and DNA synthesis remains uncertain. Madin-Darby Canine Kidney (MDCK) cells possess EGF receptor sites with high binding capacity, and in contrast to A431 cells, respond to EGF by increasing DNA synthesis. Following EGF stimulation of intact MDCK cells, there was a rapid and marked increase in the autophosphorylation of the EGF receptor. This was associated with an increase in the tyrosine phosphorylation of a 120 kDa phosphoprotein believed to be an endogenous substrate of this receptor kinase. The ED50 for stimulation of phosphorylation of pp120 was approximately 0.05 nM versus 1.0 nM for receptor autophosphorylation, consistent with amplification of signalling at this step in EGF action. Stimulation of DNA synthesis occurred after 12 to 24 hours and revealed even further amplification with an ED50 of about 0.1 nM. Intermediate between these events was a time-dependent activation of c-fos and c-myc gene expression. However, the ED50 for these processes was approximately 10 nM, indicating a relatively lower sensitivity of EGF for stimulation of proto-oncogene expression. Tyrphostin (RG 50864), a compound reported to inhibit specifically the EGF receptor kinase, completely blocked EGF stimulation of proto-oncogene induction. Interestingly, under the same experimental conditions, EGF receptor autophosphorylation was decreased only 60%. These data, along with the dose-response studies, indicate that proto-oncogene induction requires near maximal stimulation of EGF receptor autophosphorylation. They also suggest that, in MDCK cells, the EGF dependent induction of the c-fos and c-myc genes is not strictly correlated to the extent of EGF receptor autophosphorylation or EGF-stimulated DNA synthesis, and that EGF stimulation of DNA synthesis likely involves additional rate-limiting intermediate steps.     

Substrate specificities of insulin and epidermal growth factor receptor kinases

The abilities of insulin and EGF stimulated protein kinases to phosphorylate a series of exogenous substrates were compared using wheat germ lectin purified preparations of solubilized rat liver membranes. Three different kinds of substrates were found: substrates phosphorylated primarily by insulin stimulated kinase, substrates phosphorylated primarily by EGF stimulated kinase and substrates phosphorylated by both kinases to a similar extent. These results indicate that the insulin and the EGF receptor kinase have different, but overlapping, substrate specificities. In vivo, phosphorylation of cellular proteins by various hormone receptor kinases may be part of the signal transmission process for actions of the hormones. Different substrate specificities of kinases of different hormone receptors may therefore represent an important mechanism to preserve the specificity of the hormonal signal at the post receptor level.