Inhibition of the binding of 125I-labelled epidermal growth factor to mouse cells by a mitogen in goat mammary secretions.

Pre-colostrum and colostrum from goats cause a marked inhibition of the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The ability of these secretions to inhibit 125I-EGF binding is closely correlated with the ability to stimulate DNA synthesis in quiescent 3T3 cell cultures, suggesting that goat mammary secretions may contain an EGF-related mitogen. However, the material in colostrum which inhibits 125I-EGF binding to Swiss 3T3 cells is a basic protein with Mr greater than 20000 and is thus quite different from mouse and human EGF. Furthermore, the colostral-mediated inhibition of 125I-EGF binding, although rapid and apparently competitive, differs from the inhibition of binding induced by native, unlabelled EGF. Thus, the inhibitory effect of colostrum is markedly decreased when the assay temperature is shifted from 37 degrees C to 4 degrees C whereas unlabelled EGF is an effective competitive inhibitor at both 37 degrees C and 4 degrees C. Incubation of cells with EGF causes a reduction in cell surface EGF receptors whereas exposure to colostrum does not induce down-regulation of the EGF receptor. Our results suggest that the colostral factor does not bind directly to EGF receptors but inhibits 125I-EGF binding by an indirect mechanism which involves a temperature-sensitive step.     

Inhibition of epidermal growth factor binding to surface receptors by tumor promotors.

The tumor promotor, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), causes a marked (about 10-fold) decrease in the affinity of epidermal growth factor (EGF) receptors for [¹²⁵I]-EGF. The inhibition exhibits specificity, dependence on temperature, and persistence after removal of the promotor. TPA does not induce “down-regulation” of EGF receptors. Finally, the promotor interacts synergistically with EGF in stimulating glycolysis and DNA synthesis in 3T3 cells. The results suggest that TPA does not bind directly to EGF receptors.     

Insulin-like growth factor 1 and insulin reduce epidermal growth factor binding to Swiss 3T3 cells by an indirect mechanism that is apparently independent of protein kinase C

Insulin-like growth factor 1 and insulin reduced the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells by 15-20% at 37 degrees C, but not at 4 degrees C. Scatchard analysis indicated that IGF-1 and insulin affected the higher-affinity component of EGF binding, an effect previously associated with the activation of protein kinase C. However, the inhibition of 125I-EGF binding by IGF-1 and insulin was increased, not reduced, when the cells were treated with high concentrations of phorbol esters to down-modulate protein kinase C. We suggest that IGF-1 and insulin activate a protein kinase with similar or overlapping specificity to that of protein kinase C.     

Inhibition of epidermal growth factor binding to Swiss 3T3 cells by human platelet release-products

Human platelet ionophore release-products (IRP) inhibit the binding of 125I-labelled epidermal growth factor (125I-EGF) to its receptors on Swiss 3T3 cells. The inhibition appears to be caused by platelet-derived growth factor (PDGF) in the IRP and results from a decrease in the apparent affinity of cellular receptors for 125I-EGF. However, our results indicate that PDGF does not bind directly to EGF receptors, since (1) PDGF does not down-regulate EGF receptors; (2) the PDGF-mediated inhibition of 125I-EGF binding is temperature-dependent; (3) cells which possess EGF receptors but lack PDGF receptors do not exhibit a PDGF-mediated inhibition of 125I-EGF binding.     

Platelet-derived growth factor. II. Specific binding to cultured cells

We have prepared radioiodinated purified platelet-derived growth factor (125I-PDGF) which retains full mitogenic activity. The binding of 125I-PDGF to Swiss 3T3 cells is saturable and highly competed by whole blood serum, purified unlabeled PDGF, and by material from each stage in the purification of PDGF from platelet-rich plasma. Other purified mitogens and substances tested do not compete. 125I-PDGF binding to fibroblasts, 3T3 cells, and arterial smooth muscle cells shows an apparent dissociation constant of 10(-11) M, comparable to the range in which PDGF is mitogenic. A clone of Swiss 3T3 cells obtained from a population selected repeatedly against mitogenic response to PDGF shows a greatly reduced mitogenic response to PDGF and binds only 5% as much 125I-PDGF/cell. The binding capacity of the different cell types tested ranges from 2,500 binding sites/cell on the poorly responding variant to 390,000 binding sites/cell on one strain of Swiss 3T3 cells. Cell types that do not respond to PDGF do not show specific high affinity binding of 125I-PDGF. At 4 degrees C, 125I-PDGF binding to monolayer cultures is relatively slow. Equilibrium binding of low concentrations of 125I-PDGF is not achieved during 7 h unless the binding medium is constantly mixed. 125I-PDGF binding at 4 degrees C shows a broad pH optimum between 6.3 and 8.0. Binding does not seem to require Ca2+ or Mg2+ but is reduced more than 6-fold if both monovalent and divalent salts are omitted. The initial rate of 125I-PDGF binding is greater at 37 degrees C than at 4 degrees C but cell-associated 125I begins to decline soon after reaching a peak value at 30-60 min. Coincident with this decline, trichloroacetic acid-soluble 125I appears in the medium and the binding capacity of the cells declines. These phenomena suggest that PDGF and its receptor may be internalized and degraded.     

Epidermal growth factor receptor synthesis is stimulated by phorbol ester and epidermal growth factor. Evidence for a common mechanism

Previously, we and others have shown that epidermal growth factor (EGF) stimulates the synthesis of its own receptor and the accumulation of EGF receptor mRNA. Here, we demonstrate that the tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA), like EGF, also stimulates receptor synthesis in the human breast carcinoma cell line, MDA468 cells. The receptor synthesis rate increased 5-fold with a peak at 8 h after exposure to TPA with half-maximal stimulation at a dose of 5 ng/ml TPA. This stimulation of receptor synthesis occurred despite a 30% decrease in general cellular protein synthesis. The increased receptor synthesis rate resulted in the accumulation of 60% more receptor protein as determined by quantitative immunoblotting using a newly developed monoclonal antibody, H9B4. Although TPA treatment resulted in an immediate loss of high affinity EGF-binding sites, the long-term effect was an increase in both the low and high affinity binding sites. The effects of EGF and TPA on receptor synthesis were not additive. Furthermore, down-regulation of protein kinase C (the Ca2+/phospholipid-dependent enzyme) by long-term TPA treatment resulted in cells unable to respond to the stimulatory effects of both TPA and EGF on receptor synthesis. Nevertheless, the TPA-pretreated cells were still growth-inhibited by EGF. These results suggest that the stimulatory effect of EGF on receptor synthesis requires protein kinase C, whereas the inhibitory effect of EGF on the proliferation of these cells does not. Although we confirmed that EGF stimulated the incorporation of phosphate into phosphatidylinositol in A431 cells, it failed to do so in the MDA468 cells. Thus, in MDA468 cells, EGF may require protein kinase C for part of its action, but we could not demonstrate an associated activation of phosphatidylinositol turnover by EGF. The exact mechanism of involvement of protein kinase C in EGF action is still not clear.     

Secretion of an epidermal growth factor-like growth factor by epidermal growth factor-independent rat mammary carcinoma cells.

Rat mammary carcinoma (RMC) cells derived from serially transplantable mammary tumors are independent of epidermal growth factor (EGF) for long-term growth in serum-free medium. This phenotype is in contrast to that of normal mammary epithelial cells or cells derived from nontransplantable tumors that express an absolute requirement for EGF for growth in culture. The results of the experiments reported here indicate that EGF-independent RMC cells secrete a growth factor with potent EGF-like mitogenic activity. Conditioned media obtained from these cells can substitute for EGF for the growth of the EGF-dependent cell line MCF-10. This growth factor is neither EGF nor transforming growth factor alpha and does not compete with 125I-EGF for binding to EGF receptors. Phosphotyrosine Western blot analysis of lysates obtained from EGF-independent RMC cells revealed the presence of a 190 kilodalton (kDa) protein that was distinct from the EGF receptor. Similarly, growth of MCF-10 cells to confluence in serum-free medium supplemented with conditioned medium growth factor in place of EGF resulted in the disappearance of the EGF receptor band and appearance of the 190 kDa band in phosphotyrosine Western blots. The 190 kDa tyrosine-phosphorylated protein detected in cells stimulated by the conditioned medium factor is unlikely to be the c-erbB-2 protein, as indicated by negative results in immunoprecipitation experiments and in vitro kinase assays. In summary, EGF-independent RMC cells secrete a factor with potent EGF-like mitogenic activity. This suggests that an autocrine loop involving this growth factor mediates EGF independence in these cells.     

Heterogeneity of epidermal growth factor binding kinetics on individual cells.

Binding of fluorescein-conjugated epidermal growth factor (EGF) to individual A431 cells at 4 degrees C is measured by a quantitative fluorescence imaging technique. After background fluorescence and cell autofluorescence photobleaching corrections, the kinetic data are fit to simple models of one monovalent site and two independent monovalent sites, both of which include a first-order dye photobleaching process. Model simulations and the results from data analysis indicate that the one-monovalent-site model does not describe EGF binding kinetics at the single-cell level, whereas the two-site model is consistent with, but not proved by, the single-cell binding data. In addition, the kinetics of binding of fluorescein-EGF to different cells from the same coverslip often differ significantly from each other, indicating cell-to-cell variations in the binding properties of the EGF receptor.     

Evidence for isoaspartyl (deamidated) forms of mouse epidermal growth factor

A variant form of mouse submaxillary gland epidermal growth factor (EGF) was identified by isocratic reversed-phase HPLC of EGF obtained by Bio-Gel P-10 column chromatography ("culture grade"). The variant form was essentially absent in preparations of EGF further purified by chromatography on DEAE-cellulose ("receptor-grade" EGF). The spectral properties and amino acid composition of the variant form (EGF-I) could not be distinguished from those of the intact polypeptide isolated by HPLC (alpha-EGF). Receptor-binding and mitogenic properties of EGF-I were also equivalent to those of alpha-EGF. These data suggested that EGF-I was structurally very similar to EGF. However, the very low yield (less than 4%) obtained by Edman degradation indicated that the N-terminal (Asn1) of the polypeptide was modified. Isoelectric focusing of EGF-I revealed two major immunoreactive bands: one with a pI equivalent to that of alpha-EGF (pI 4.6) and another at pI 4.1. Alkaline treatment of alpha-EGF (0.1 M NH4OH) yielded peak material by HPLC that coeluted with EGF-I; the alkaline-generated EGF-I yielded bands that also focused at pH 4.6 and 4.1. Ammonium hydroxide treatment of [des-Asn1]-EGF (beta-EGF) did not produce conversion to EGF-I. On the basis of these data, we propose that EGF-I was formed by selective deamidation of the N-terminal Asn of intact EGF. This notion is also supported by liquid secondary ion mass spectrometry, which showed that EGF-I was approximately 1.5 mass units greater than alpha-EGF. The heterogeneity observed by isoelectric focusing supports previous studies which have shown that, following deamidation of N-terminal asparagine, a beta-aspartyl shift can occur, which in the present study might yield succinimido-aspartyl1-EGF and beta-aspartyl1-EGF. Low yields observed during Edman degradation indicate that negligible amounts occur as the alpha-aspartyl1-EGF isomer.     

Insulin-like growth factor II binding to cultured human chondrosarcoma cells

Cultured cells originally derived from a human chondrosarcoma (A1684) were used to investigate somatomedin binding in terms of kinetics and specificity. In this study, the rat somatomedin, multiplication-stimulation activity (MSA) was utilized. While the human chondrosarcoma cells did not exhibit a mitogenic response to MSA, the rate of transport of glucose and amino acids was significantly increased. In competitive binding experiments a specific insulin-insensitive MSA receptor was identified which showed half maximal displacement of tracer at a concentration of 250 ng/ml of MSA using whole cells. This receptor had an affinity constant of 4.8 X 10(7) M-1. Kinetic analysis of MSA binding to membrane preparations and to Triton X-100 solubilized membranes revealed an increase in the binding affinity to 1.28 X 10(8) M-1 and 2.8 X 10(8) M-1, respectively. Of particular significance is the observation that these cells have especially high levels of MSA receptors. Determination of binding capacity revealed that these cells contain approximately 1.9 X 10(6) MSA receptors per cell and therefore are an excellent model system for the characterization and purification of somatomedin receptors. Affinity labeling of the MSA receptor using the chemical crosslinking reagent, disuccinimidyl suberate, confirmed that this receptor was of the type II class of somatomedin receptors and exhibited a molecular weight of 218,000 under nonreducing conditions.     

Inhibition of autophosphorylation of epidermal growth factor receptor by a small peptide not employing an ATP-competitive mechanism

Previously we found that short peptides surrounding major autophosphorylation sites of EGFR (VPEY(1068)INQ, DY(1148)QQD, and ENAEY(1173)LR) suppress phosphorylation of purified EGFR to 30-50% at 4000 microM. In an attempt to improve potencies of the peptides, we modified the sequences by substituting various amino acids for tyrosine or by substituting Gln and Asn for Glu and Asp, respectively. Among the modified peptides, Asp/Asn- and Glu/Gln-substitution in DYQQD (NYQQN) and ENAEYLR (QNAQYLR), respectively, improved inhibitory potencies. The inhibitory potency of NYQQN was not affected by the concentration of ATP, while that of QNAQYLR was affected. Docking simulations showed different mechanisms of inhibition for the peptides: inhibition by binding to the ATP-binding site (QNAQYLR) and inhibition by binding to a region surrounded by alphaC, the activation loop, and the catalytic loop and interfering with the catalytic reaction (NYQQN). The inhibitory potency of NYQQN for insulin receptor drastically decreased, whereas QNAQYLR inhibited autophosphorylation of insulin receptor as well as EGFR. In conclusion, NYQQN is not an ATP-competitive inhibitor and the binding site of this peptide appears to be novel as a tyrosine kinase inhibitor. NYQQN could be a promising seed for the development of anti-cancer drugs having specificity for EGFR.     

Multidomain binding of transforming growth factor alpha to the epidermal growth factor receptor.

Solubilized epidermal growth factor receptor (EGF-R) has been used in an extension of the Geysen epitope mapping protocol in order to provide additional insight into the amino acid residues in human transforming growth factor alpha (hTGF alpha) which are critical to recognition and binding. Overlapping heptapeptides which encompassed the 50 amino acid primary sequence of hTGF alpha were synthesized on a polyethylene solid phase, and the amount of detergent-solubilized EGF-R bound to each peptide was measured using ELISA. EGF-R appeared to bind reproducibly to four heptapeptides cognate to sequences in both the N- and C-domains of hTGF alpha (residues 22-28, 28-34, 36-42, and 44-50). Visualization of these four regions on three-dimensional solution phase structures of hTGF alpha, derived from 1H NMR measurements [Kline, T.-P., Brown, F.K., Brown, S.C., Jeffs, P.W., Kopple, K.D., & Mueller, L. (1990) Biochemistry 29, 7805-7813], indicated that the peptide segments are located on a single face of the protein and suggested the presence of a potential receptor binding cavity. If peptide segments within both the N- and C-domains of hTGF alpha are involved in binding to EGF-R, then this has direct consequences for possible molecular mechanisms by which receptor activation might take place. For example, the observed conformational flexibility in the six NMR-derived hTGF alpha structures due to variations in the main-chain torsion angles of Val-33, in combination with the involvement of residues from both domains in the proposed binding cavity, may imply that receptor activation results from interdomain reorientation in the protein ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduced binding of epidermal growth factor by avian sarcoma virus-transformed rat cells

Rat cells transformed by Rous sarcoma virus and Fujinami sarcoma virus bound 5-10% of the amount of epidermal growth factor (EGF) bound by normal cells. Scatchard plot analysis indicated that the reduction in binding by transformed cells was due to a decreased number of receptors rather than to altered binding affinity. In experiments with temperature sensitive mutants of Rous sarcoma virus and Fujinami sarcoma virus significant loss of EGF binding occurred within one hour of shift from non-permissive to permissive temperature. Conditioned media from various normal and transformed cell lines were examined for the ability to inhibit EGF binding to normal cells or to cause "down regulation" of EGF receptors. No activity of either type was found. EGF-dependent phosphorylation in isolated membrane preparations was also examined. Membranes from normal cells displayed EGF-dependent phosphorylation of a Mr 180,000 protein presumed to be the EGF receptor. This activity was absent in membranes from transformed cells. The data suggest a close correlation between activation of avian sarcoma virus transforming gene products and modulation of the EGF growth regulatory system.     

Identification of insulin-like growth factor binding proteins from cultured human epidermal keratinocytes

The role and mechanisms of action of insulin-like growth factors (IGFs) in skin remain unclear. Epidermal keratinocytes possess IGF-I receptors and are responsive to IGF-I, which is primarily derived from underlying dermal fibroblasts. IGF binding proteins (IGFBPs), also synthesized by fibroblasts, may be involved in paracrine targeting of IGF-I to its receptors. We therefore examined whether human keratinocytes synthesize IGFBPs and their mRNAs. Following culture in complete medium (containing bovine pituitary extract and epidermal growth factor) Western ligand blotting (WLB) of cell conditioned medium revealed a major band of 32 kD, a less abundant IGFBP of 24 kD at all passages, and a 37–42 kD IGFBP which increased in abundance in late passage. Immunoprecipitation followed by WLB confirmed that the predominant 32 kD band was IGFBP-2. Radioimmunoassay of IGFBP-1, -3, and -6 revealed detectable levels of IGFBP-3 and significant levels of IGFBP-6, but not IGFBP-1. Northern analysis following culture in complete medium revealed that at early passage IGFBP-1, -2, -4, and -6 mRNAs were detectable. IGFBP-3 and -5 mRNAs were not detectable. Following culture in growth factor-free medium a 37–42 kD band, consistent with IGFBP-3, was predominant and a 24 kD band consistent with IGFBP-4 was also present. These data demonstrate the expression of a distinct pattern of IGFBPs by cultured human keratinocytes dependent on culture conditions. Keratinocyte-derived IGFBPs are likely to play a role in the transport and targeting of IGF-I from dermally derived fibroblasts to the epidermis. © 1995 Wiley-Liss, Inc.