Production and characterization of antibody blocking epidermal growth factor: Receptor interactions

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 · 10⁶ epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes.The immonoglobulin G fraction of this immune sera inhibited ¹²⁵I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of ¹²⁵I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5°C).Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A-431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.     

Epidermal growth factor: Characteristics of specific binding in membranes from liver,placenta, and other target tissues

Epidermal growth factor, a 6,400-dalton polypeptide from the mouse submaxillary gland, binds specifically to cells and membranes derived from a variety of human, rat, mouse, and bovine tissues. Liver, placenta, skin, cornea, and cultured chondrocytes, Hela cells, and Chang liver cells bind large amounts of epidermal growth factor, whereas fat cells, resting and lectin-stimulated human peripheral lymphocytes, mouse thymocytes, cultured rat hepatoma cells, and mammary cells from virgin and pregnant mice bind little or no epidermal growth factor. The binding site for epidermal growth factor is distinct from receptors for other anabolic peptides such as insulin, nerve growth factor, and growth hormone. The binding of epidermal growth factor is rapid and reversible. The rate constant of association is approximately 10⁶ mole^?1 sec^?1, the rate constant of dissociation is about 6 × 10^?4 sec^?1, and the apparent equilibrium dissociation constant is about 10^?9m. Trypsin at low concentrations (50–200 μg/ml) destroys the receptor site for epidermal growth factor. The binding of epidermal growth factor by membranes is not accompanied by appreciable degradation of the peptide present in the medium or of that bound to the membranes. Use can be made of the high affinity and specificity of membranes for epidermal growth factor to measure by a competitive binding assay as little as 200 pg of EGF per ml (3 × 10^?11m).     

Demonstration of protease activity for epidermal growth factor-binding protein

The epidermal growth factor can be isolated from the male mouse submaxillary gland as part of a high molecular weight complex. The complex is composed of two molecules of epidermal growth factor and two molecules of epidermal growth-factor binding protein (J.M. Taylor, W.M. Mitchell, and S. Cohen, 1974, J. Biol. Chem.249, 3198–3203). The proteolytic activity of epidermal growth-factor binding protein was demonstrated by its self-proteolysis in moderate (3–7 m) concentrations of urea, and, its inhibition by formation of a complex with pancreatic trypsin inhibitor. This complex was characterized by its pI and by its ability to yield pancreatic trypsin inhibitor and epidermal growth factor-binding protein in sodium dodecyl sulfate-urea gel electrophoresis. The association equilibrium constant was determined to be 3.6 × 10⁷m^?1 by inhibition studies of the esteropeptidase. These results, which indicate that epidermal growth factor-binding protein is capable of autodigestion and of forming a stable complex with a macromolecular inhibitor of trypsin, lend strong support to the hypothesis that epidermal growth factor-binding protein is capable of cleaving a larger precursor by its proteolytic action.     

Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.     

The effects of epidermal growth factor on cell proliferation and prolactin production by GH3 rat pituitary cells

The effects of epidermal growth factor (EGF) were studied in rat pituitary tumor cells, GH₃, grown in serum-supplemented and serum-free chemically defined media. EGF (1 nM) increased the cell number to 132% of the control cultured in the defined medium during a 6-day incubation period, while it decreased the cell number to 60% of the control in the serum-supplemented medium. EGF altered the morphology of the cells grown in the defined medium more markedly to an elongated conformation than that of cells grown in the serum-supplemented medium. EGF also stimulated prolactin (PRL) production by culture in the presence or absence of serum. The effects of the cell density of GH₃ on the action of EGF were shown to appear in two ways. The mitogenic influence of EGF was more effective on, and more responsive to, high-density cells, whereas the stimulatory action on PRL production was less effective on high-density cells. However, the inhibitory effects on cellular growth appeared independently of cell densities. The results obtained with ¹²⁵I-EGF binding experiments indicated that the number of binding sites, affinity, and internalization of EGF receptors were similar in either serum-supplemented or serum-free culture. At low cell density, the number of available ¹²⁵I-EGF binding sites per cell was larger than at high cell density. These results suggested that there was no apparent correlation between EGF binding and its differing effects on the growth of GH₃ cultured in the serum-supplemented and the defined medium.     

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

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

Regulation of hepatic growth hormone receptors by insulin.

Induction of diabetes in the rat with streptozotocin caused a decrease in the specific binding of human growth hormone to liver receptors. The decrease was due to a loss of binding sites, with no change in the affinity constant for growth hormone (5.6 × 10⁹M^?1). A highly significant correlation was seen between serum insulin levels and hepatic growth hormone binding. Specific insulin binding to hepatic receptors was increased in diabetes, with a highly significant negative correlation between serum insulin levels and insulin binding. The loss of growth hormone receptors was reversed by treating diabetic rats with insulin. Since hormones which bind to “lactogenic” binding sites in the liver are reported to regulate somatomedin levels, the insulin dependence of human growth hormone receptors might account for the decrease in serum somatomedin in diabetes.     

Amphiregulin-dependent proliferation of cultured human keratinocytes: Autocrine growth,the effects of exogenous recombinant cytokine,and apparent requirement for heparin-like glycosaminoglycans

Amphiregulin, a member of the epidermal growth factor family with heparin binding affinity, functions as a natural regulator of keratinocyte growth. Autocrine signaling by amphiregulin and the effects of exogenous recombinant cytokine were studied in serum-free cultures of human neonatal keratinocytes. A metabolic inhibitor of proteoglycan sulfation was used to assess the role of cellular heparin-like glycosaminoglycans in amphiregulin-dependent growth. Keratinocytes plated at >10³ cells/cm² grew in an autocrine manner in the absence of exogenous epidermal growth factor or amphiregulin. Incubation of keratinocytes with an amphiregulin-blocking antibody indicated that ～70% of autocrine growth is mediated by endogenous amphiregulin. Proliferation potential in the presence of recombinant human amphiregulin was dose dependent and saturable and above ～1 ng/ml was comparable to that achieved with similar concentrations of epidermal growth factor. Sodium chlorate, which blocks glycosaminoglycan sulfation, reversibly inhibited epidermal growth factor-dependent proliferation by 42%, exogenous amphiregulin-dependent proliferation by 75%, and autocrine growth by 95%; concurrent incubation with 1-100 μg/ml heparin partially reversed this inhibition. Exogenous heparin in the absence of chlorate, however, nearly completely inhibited growth under autocrine conditions and in the presence of recombinant amphiregulin. Structure-function studies indicate that the polymerization level, high sulfate group density, and possibly iduronic acid content of heparin-like moieties correlate with their inhibitory activity. Collectively, these observations indicate that amphiregulin is the major autocrine factor for keratinocytes and demonstrate that exogenous amphiregulin is an effective growth promoting factor with molar potency similar to that of epidermal growth factor. Autocrine and paracrine signaling by amphiregulin may require cellular heparin-like glycosaminoglycans, presumably as matrix or membrane proteoglycans, whereas soluble glycosaminoglycans inhibit signaling, possibly by competing for cytokine binding. © 1994 wiley-Liss, Inc.     

Variants of 3T3 cells lacking mitogenic response to the tumor promoter tetradecanoyl-phorbol-acetate

The potent tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) can stimulate quiescent, nonproliferating 3T3 cells to reenter the cell cycle and divide. We have previously used a slection technique developed in our laboratory to isolate variant cell lines which no longer divide in response to epidermal growth factor. We have now utilized the same selection procedure to isolate, from 3T3 cells, two variant cell lines, TNR-2 and TNR-9, which retain growth control and divide in response to elevated serum or fibroblast growth factor, but which do not respond to TPA. The variants do not incorporate precursors into DNA in response to TPA, demonstrating that the cells do not enter the S phase of the cell cycle. The TPA nonresponsive variant TNR-2 cannot respond to epidermal growth factor; TNR-9 responds to this mitogen. TNR-2 variant cells, which do not respond to EGF, do not bind ¹²⁵I-EGF. TPA can modulate ¹²⁵I-EGF binding to TNR-9 cells in a manner similar to its action on parental 3T3 cells. This TPA-induced alteration of EGF binding indicates that TNR-9 cells still interact with TPA, despite their inability to mount a mitogenic response.     

The effect of 1.25 dihydroxyvitamin D3 on binding and internalization of epidermal growth factor in cultured cells : Studies on BT-20 cells using quantitative electron microscope autoradiography

The biological effects of 1.25(OH)₂D₃ on epidermal growth factor receptor (EGF-R) and on EGF internalization were examined in human mammary carcinoma BT-20 cells. In this cell line, with known amplification of the epidermal growth factor receptor gene, EGF was not stimulatory for growth. Biological assay and quantitative EM autoradiography combined with iodinated ligand binding to specific receptors demonstrated that the number of binding sites per unit of length of plasma membrane was 2.48-fold higher in treated than in control cells. ¹²⁵I-EGF was progressively internalized in a time- and temperature-dependent manner after selective association with the membrane-coated pits. No modification of the time course of ¹²⁵I-EGF internalization was noted in the control and in the treated cells, but a different distribution of the labeling in the subcellular compartment was observed in treated cells. In 1.25(OH)₂D₃-treated batches, the grain density remained low in the receptosomes throughout the experiment, whereas it was high and occurred early in the lysosomes. On the other hand, in control cells, the grain density of the receptosomes was high, whereas it occurred late and was relatively low in the lysosomes. These data suggest that 1.25(OH)₂D₃ is a regulator of EGF-R level in BT-20 cell line, but it cannot be affirmed whether this effect is direct or mediated by other parameters.     

Decreased epidermal growth factor binding in cells growth arrested in G1 by nutrient deficiency

Previous studies have shown that the nontransformed AKR-2B mouse embryo derived cell line may growth arrest by two separate mechanisms in the G₁ phase of the cell cycle-growth factor deficiency arrest (G₀) and low molecular weight nutrient deficiency arrest. An examination of epidermal growth factor (EGF) receptors under the different resting or growth conditions has shown that rapidly growing cells or cells arrested due to growth factor deficiency have the expected amount of ¹²⁵I-EGF binding with approximately 10⁵ receptors per cell being present in G₀ arrested cells. In contrast, cells arrested due to nutrient deficiency show a reduction in ¹²⁵I-EGF binding to 10--20% of that observed under the other conditions. This effect appears to be due to decreased receptor number and not to a change in the affinity of the receptor. Stimulation of DNA synthesis by nutrient replenishment causes a tenfold increase in EGF binding 20 hours later, with some increase in binding being detectable as early as six hours. The increase in binding is inhibited by cycloheximide and actinomycin D. This suggests that new mRNA synthesis as well as increased protein synthesis is required for the increase in EGF binding.     

Binding and processing of epidermal growth factor in Panc-I human pancreatic carcinoma cells

The binding of ¹²⁵I-labeled epidermal growth factor (EGF) was studied in Panc-I human pancreatic carcinoma cells. At 37°C, binding was rapid and associated with marked endocytosis of the ligand. Bound EGF was sequentially converted to a number of more acidic species as follows: pI 4.55 to pI 4.2, to pI 4.35, to pI 4.0. EGF internalization and processing were blocked at 4°C. EGF did not alter cell growth when Panc-I cells were incubated in the presence of 2 to 10% serum. In contrast, when the serum concentration was lowered to 0.1%, EGF significantly enhanced cell replication after 6 days of culture.     

Effect of streptozotocin-induced diabetes on epidermal growth factor receptors in rat liver plasma membrane

The effect of streptozotocin-induced diabetes on 125I-labeled epidermal growth factor (EGF) binding was studied in microsomal membranes from rat liver. The binding of EGF in membranes from diabetic animals was significantly low, the value being about 60% of the control level. Scatchard analysis of the binding data clearly showed that the decrease in EGF binding was due to a decrease in the number of receptors. Treatment of diabetic animals with insulin restored EGF receptors to control levels, whereas the treatment with triiodothyronine had no effect. Serum EGF concentrations measured were almost the same among the control, diabetic, and insulin-treated diabetic groups. These results suggest that insulin deficiency in vivo causes a decrease in hepatic EGF receptors.     

Peptide binding to intestinal epithelium: distinct sites for insulin, EGF and VIP

Several peptides, including insulin, epidermal growth factor and vasoactive intestinal polypeptide bind to intestinal epithelial cells. However, it is unclear whether one binding site binds several peptides or whether separate sites exist for each peptide. These studies were designed to examine the specificity of peptide binding sites on intestinal epithelial cells. Peptide binding was measured directly with [125I]radiolabelled peptides to isolated enterocytes prepared from rabbit ileum. The characteristics of insulin and epidermal growth factor binding were similar. Both insulin and epidermal growth factor specific binding was saturable, directly correlated to cell concentration and temperature and pH dependent. The total number of insulin binding sites per cell was 4500, that for epidermal growth factor was 2280. Scatchard analysis for both peptides produced curvilinear plots. Dissociation of both peptides from the binding site was increased in the presence of their respective unlabelled peptide. However, insulin specific binding was not altered by epidermal growth factor, and epidermal growth factor specific binding was unaffected by insulin. Further, both insulin and epidermal growth factor failed to inhibit the specific binding of vasoactive intestinal polypeptide to ileal enterocytes, and vasoactive intestinal polypeptide did not inhibit insulin or epidermal growth factor specific binding. These studies demonstrate that insulin, epidermal growth factor and vasoactive intestinal polypeptide interact with three distinct membrane binding sites on the enterocyte.     

Epidermal growth factor (EGF) binding to membranes immobilized in microtiter wells and estimation of EGF-related transforming growth factor activity

The binding of radiolabeled epidermal growth factor (EGF) to immobilized A-431 target cell membranes coupled to polyvinyl chloride microtiter wells is described. Saturation curves and Scatchard analysis of the data indicate that the observed binding parameters are consistent with those previously reported. Binding capacity of the membranes are approx. 6.6 pmol EGF per mg membrane protein. Kinetics of ¹²⁵I-EGF binding were slower, however, than reported for binding to membranes in suspension, although binding constants were not greatly different. The high- and low-affinity binding constants for ¹²⁵I-EGF were calculated to be approximately 1 · 10¹² M^?1 and 2.5 · 10⁹ M^?1, respectively. Application of this technique in a competitive binding assay requires no more than 2.5 μg of membrane protein per assay, is essentially complete after 60 min, and facilitates screening of a large number of samples in a short time. Therefore, this will assist in the evaluation and quantitation of EGF and EGF-related transforming growth factor activity in physiological fluids. This technique may also be applied to analyses of other hormone-receptor systems.     

Adriamycin causes up regulation of epidermal growth factor receptors in actively growing cells

We have demonstrated a drug-dependent increase in the capacity of HeLa and 3T3 cells, grown in the presence of lethal and sublethal concentrations of adriamycin, to bind epidermal growth factor (EGF). Scatchard analysis ascribes this effect to an increase in the number of binding sites, with little change in affinity. The time course of binding of ¹²⁵I-EGF is unchanged by adriamycin treatment, in both 3T3 and HeLa cells, at both 0 and 37 °C. This increase appears gradually over 3 or 4 days' exposure to the drug and is reversible over a similar period. Although in HeLa cells the increase reaches a maximum of about 4-fold, regardless of cell density, the maximum observed in 3T3 cells, over 100-fold, is seen only at low cell densities. This could be related to the density-dependent growth regulation seen in 3T3 cells, but not in HeLa cells. We suggest that the ability of the anticancer agent adriamycin to alter the cellular response to a growth-regulatory substance may be related to the mechanism of its cytotoxic action.     

Enhanced binding of fibronectin-coated latex beads to quiescent 3T3-L1 cells is correlated with escape from growth arrest

The possible involvement of fibronectin receptors in growth stimulation was investigated by an analysis of fibronectin-coated latex bead binding to 3T3-L1 cells under various conditions. 3T3-L1 cells, growth-arrested in a medium with a low concentration of calf serum, bound few fibronectin-coated beads. After addition of serum at concentrations of 1.0% or higher, there was a rapid and transient increase in the number of cells with bound beads and a subsequent increase in the incorporation of bromodeoxyuridine (BrdU) into cell nuclei. Incorporation of BrdU was observed in about 60% of the cells with bound beads. Fibroblast growth factor and platelet-derived growth factor at concentrations of 5 ng/ml or higher also enhanced binding of fibronectin-coated beads to cells. Stimulation of bead binding by epidermal growth factor and insulin was weak. Fibroblast growth factor, but not epidermal growth factor, increased the incorporation of BrdU into nuclei. These results indicate a relationship between stimulation of cell proliferation in quiescent cells and increased binding by cells of fibronectin-coated latex beads.     

Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody

Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor‐alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB‐EGF (heparin‐binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C‐terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala⁴¹, Glu⁴⁴, and His⁴⁵. These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859.     

Effect of tunicamycin on the turnover of epidermal growth factor receptors in cultured calf aorta smooth muscle cells: Comparison with IMR-90 human lung fibroblasts

Treatment of cultured calf aorta smooth muscle cells with tunicamycin, a potent inhibitor of dolichol-mediated glycosylation, resulted in progressive loss of receptors for epidermal growth factor with 50% of receptors lost after 6 h. Receptor half-life was also 6 h with cycloheximide treatment but was 12 h with either actinomycin D or camptothesin treatment. The epidermal growth factor-induced processing (internalization and/or degradation) of residual receptors remaining after tunicamycin treatment appeared to be unaltered.50% decrease in ¹²⁵I-labeled epidermal growth factor binding was observed also with IMR-90 fibroblasts upon 6 h treatment with tunicamycin, although these cells were less sensitive to inhibition by tunicamycin of glycosylation and protein synthesis.