Epidermal Growth Factor Binding Activity Research Articles

Isolation from phage display libraries of lysine-deficient human epidermal growth factor variants for directional conjugation as targeting ligands.

Ligand-targeted anticancer therapeutics represent an opportunity for the selective and efficient delivery of drugs to tumours. The chemical coupling of ligands to drugs or drug carrier systems is, however, often hampered by the presence of multiple reactive groups within the ligand, for example, epsilon-NH(2) groups in lysine side chains. In this paper, we describe the isolation by phage display of human epidermal growth factor (EGF) variants without lysine and a reduced number of arginine residues. The selection on A431 carcinoma cells also revealed that R41 is indispensable for EGF binding activity as all EGF variants contained an arginine residue at this position. One EGF variant (EGFm1) with K28Q, R45S, K48S and R53S mutations was expressed in bacteria and showed an identical binding activity as wild-type EGF. EGFm1 could be labelled with fluorescein isothiocyanate demonstrating the accessibility of the N-terminal amino group for coupling reagents. Furthermore, coupling of EGFm1 to PEGylated liposomes resulted in target cell-specific binding and internalization of the liposomes. These human EGF variants should be advantageous for the generation of anticancer therapeutics targeting the EGF receptor, which is overexpressed by a wide variety of different tumours.

Glycosylation-induced Conformational Modification Positively Regulates Receptor-Receptor Association: A STUDY WITH AN ABERRANT EPIDERMAL GROWTH FACTOR RECEPTOR (EGFRvIII/ΔEGFR) EXPRESSED IN CANCER CELLS

The epidermal growth factor receptor (EGFR) is a multisited and multifunctional transmembrane glycoprotein with intrinsic tyrosine kinase activity. Upon ligand binding, the monomeric receptor undergoes dimerization resulting in kinase activation. The consequences of kinase stimulation are the phosphorylation of its own tyrosine residues (autophosphorylation) followed by association with and activation of signal transducers. Deregulation of signaling resulting from aberrant expression of the EGFR has been implicated in a number of neoplasms including breast, brain, and skin tumors. A mutant epidermal growth factor (EGF) receptor missing 267 amino acids from the exoplasmic domain is common in human glioblastomas. The truncated receptor (EGFRvIII/DeltaEGFR) lacks EGF binding activity; however, the kinase is constitutively active, and cells expressing the receptor are tumorigenic. Our studies revealed that the high kinase activity of the DeltaEGFR is due to self-dimerization, and contrary to earlier reports, the kinase activity per molecule of the dimeric DeltaEGFR is comparable to that of the EGF-stimulated wild-type receptor. Furthermore, the phosphorylation patterns of both receptors are similar as determined by interaction with a conformation-specific antibody and by phosphopeptide analysis. This eliminates the possibility that the defective down-regulation of the DeltaEGFR is due to its altered phosphorylation pattern as has been suggested previously. Interestingly, the receptor-receptor self-association is highly dependent on a conformation induced by N-linked glycosylation. We have identified four potential sites that might participate in self-dimerization; these sites are located in a domain that plays an important role in EGFR functioning.

Regulation of the induction of ornithine decarboxylase in keratinocytes by retinoids

Treatment of SV40-transformed keratinocytes (Z114) with epidermal growth factor (EGF) resulted in an increase in ornithine decarboxylase (ODC) activity and a dose-dependent increase in ODC mRNA levels. Pretreatment of keratinocytes with all-trans-retinoic retinoic acid inhibited the EGF induction of ODC activity. In both quiescent and EGF-stimulated cells, all-trans-retinoic acid inhibited ODC gene transcription and lowered ODC mRNA levels, whereas glyceraldehyde phosphate dehydrogenase expression remained unaffected. Treatment with all-trans-retinoic acid for 24 h resulted in a dose- and time-dependent decrease of up to 52% in EGF binding to EGF receptors and a 30-75% decrease in EGF-receptor quantity. In addition, when cells were treated with both UV radiation and all-trans-retinoic acid, their effects were additive in causing a decrease in EGF binding. Blocking of EGF receptors with a neutralizing antibody for EGF receptors inhibited the induction of ODC activity by EGF. The effects of several other retinoids, including Ro15-0778, etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cis-retinoic acid and acitretin, were also studied to determine their effects on EGF binding and ODC activity. Two of these other retinoids, 13-cis-retinoic acid and Ro13-7410, inhibited EGF binding the most (35-46%, P < 0.001); several others (etarotene, Ro40-8757 and etretinate) were less effective (7-16%), but significantly decreased EGF binding (P < 0.05), and two retinoids (Ro15-0778 and acitretin) showed no significant effect on EGF binding. In contrast, all of the retinoids tested inhibited the induction of ODC activity by EGF, although etretinate and Ro15-0778 were less effective. EGF signal transduction is important in ODC gene regulation, and retinoids are significant modulators of this pathway.

Epidermal growth factor receptors in human corpora lutea during the menstrual cycle and pregnancy.

We have demonstrated the presence of epidermal growth factor (EGF) and its receptors in human non-gestational corpora lutea. To determine further the characteristics of EGF receptor binding, we examined 30 human corpora lutea throughout the luteal phase and during pregnancy. Scatchard plots of EGF binding in 29 of the 30 corpora lutea were curvilinear, suggesting negative co-operativity. The mean +/- SE of the association constant Ka was (0.9 +/- 0.2) x 10(9) l/mol, the dissociation constant Kd was (2.2 +/- 0.3) x 10(-9) mol/l and the number of binding sites (Rt) was (15.8 +/- 2.1) x 10(-19) mol/micrograms protein for non-gestational corpora lutea. The Kd increased significantly in late pregnancies compared to early pregnancies (P = < 0.005), while Rt was significantly higher in term pregnancies than in either early pregnancy (P < 0.01) or the menstrual cycle (P < 0.001). Corpora lutea atretica (n = 2) and ovarian stroma (n = 6) did not show any EGF binding activity. Our findings demonstrate the presence of specific EGF receptors in human corpora lutea of both the menstrual cycle and pregnancy. The changes in EGF binding parameters in early pregnancy suggest that there may be a relationship between the role of EGF and ovarian steroidogenesis.

Role of threonine residues in regulation of the epidermal growth factor receptor by protein kinase C and mitogen-activated protein kinase

Epidermal growth factor (EGF) receptor tyrosine kinase activity is down-regulated by a number of growth-modulating agents that activate protein kinase C and/or mitogen-activated protein (MAP) kinases. Although the mechanism is unclear, it has been hypothesized that phosphorylation of specific threonine residues leads to inhibition of the EGF receptor tyrosine kinase. Two sites phosphorylated on the EGF receptor in response to phorbol esters are possible mediators of this effect: threonine 654, the target of protein kinase C, and threonine 669, the target of MAP kinase and the major site of phosphorylation on the EGF receptor. In order to investigate the role of these residues in receptor regulation, we substituted glutamic acid to mimic the negative charge introduced by phosphorylation at these sites. The wild-type and mutant receptor cDNAs were then transfected into CHO cells that lack endogenous EGF receptor. The EGF binding properties of the mutant receptors were similar to those of the wild-type EGF receptors. EGF stimulated tyrosine kinase activity and DNA synthesis in cells expressing both mutant receptors, indicating that the mutant EGF receptors are biologically active. Treatment of cells with phorbol esters inhibited the high affinity EGF binding and tyrosine kinase activities of both mutant and wild-type EGF receptors. These results indicate that acidic residues at either the Thr-654 or Thr-669 site modulate but do not block EGF receptor signalling. Furthermore, this data demonstrates that the mutant EGF receptors are still a target for inhibition by phorbol esters. Thus, events other than phosphorylation of Thr-654 or Thr-669 appear to be required for receptor down-regulation by protein kinase C or MAP kinase.

Ontogenesis and distribution of epidermal growth factor immunoreactivity and binding activity in the mouse fetal and neonatal tissues.

Epidermal growth factor (EGF) immunoreactivity, EGF binding activity, and their ontogenic changes were investigated in placenta, amnion, and various organs of mouse fetus and neonate. EGF immunoreactivity was detected in all organs examined such as fetal and neonatal liver, brain, lung, intestine, placenta, and amnion. The fetal tissues having relatively higher EGF content were amnion, liver, and intestine. The tissue distribution of EGF immunoreactivity in the fetus did not differ from that in neonate. As for its ontogenesis, EGF immunoreactivity was almost constant during fetal development in liver, brain, and placenta, whereas it gradually increased in intestine. Scatchard plot analysis of EGF receptor, which was also demonstrated in all organs studied, revealed that EGF receptor in these tissues showed curvi-linear profile and its affinity was very high. Unlike EGF immunoreactivity, EGF binding activity showed tissue distributional difference between fetus and neonate except skin; EGF binding activity was relatively high in heart, kidney, lung, and intestine in the fetus, whereas it was high in liver, kidney, heart, and lung in the neonate. As for its ontogenesis, EGF receptor increased in liver as gestation proceeded, while it decreased in intestine. These results suggest that the biological importance of EGF in fetal development may be variable among fetal organs despite the ubiquitous existence of EGF and its receptor in many fetal tissues.

An EGF-pseudomonas exotoxin A recombinant protein with a deletion in toxin binding domain specifically kills EGF receptor bearing cells.

We constructed two chimeric toxins; one composed of epidermal growth factor (EGF) and pseudomonas exotoxin A (PE), designated EGF-PE and the other composed of EGF and PE with a deletion of the Ia domain (cell-binding domain), designated EGF-PE (delta Ia). Both chimeric toxins reacted with anti-EGF and anti-PE antibodies. The cell-killing experiments showed that EGF-PE, but not EGF-PE (delta Ia), was cytotoxic to the murine fibroblast cell line NR6, which carried the PE receptor, but not the EGF receptor. However, after NR6 was transfected with DNA for the expression of human EGF receptor, the transfected cell line, designated NRHER5, over-expressed human EGF receptors and became sensitive to EGF-PE(delta IA). The cytotoxicity of EGF-PE(delta Ia), but not EGF-PE, to NRHER5 can be completely blocked by an excess amount of EGF. To completely reverse the cytotoxicity of EGF-PE on NRHER5, both the EGF receptor pathway and the PE receptor pathway need to be blocked. These results suggest that EGF-PE exhibits both EGF and PE binding activities, while EGF-PE(delta IA) possesses only EGF binding activity. Thus, EGF-PE(delta Ia) may be a better chimeric toxin than EGF-PE in terms of target specificity to EGF receptor bearing cells. We, therefore, examined the cytotoxicity of EGF-PE(delta Ia) to various human cancer cell lines. We find that human cancer cells containing more EGF receptors are more sensitive to EGF-PE(delta Ia).

Polyoma middle T antigen or v- src desensitizes human epidermal growth factor receptor function and interference by a monensin-resistant mutation in mouse Balb/3T3 cells

Epidermal growth factor (EGF)-induced down-regulation of its receptor is an obligatory pathway for cellular regulation of EGF-specific receptor (EGF-R) in normal and malignant cells. BNER4 cells are mouse Balb/3T3 cells transfected with the human EGF-R complementary DNA (cDNA). Polyoma middle T antigen-transfectants of BNER4, B4/MT-2, B4/MT-13, B4/MT-23, and B4/MT-24, showed diminished down-regulation of cell surface human EGF-R in response to EGF relative to the parental BNER4 cells. Also, the v- src-transfectants B4/SRC-13 and B4/SRC-24 showed much less down-regulation than BNER4 cells, whereas H- ras-transfectants of BNER4, B4/RAS-24 and B4/RAS-25, showed EGF-induced down-regulation of the cell surface EGF-R similar to that of BNER4. EGF induced DNA synthesis more than 20-fold in BNER4, but induced only about a 1.5- to 6-fold increase in the middle T antigen- and v- src-transfectants. EGF-Rs of the middle T antigen-transfectants were metabolically stable in the presence of EGF in comparison with their parental BNER4 cells. EGF-Rs of BNER4 cells degraded with half-lives of about 2 h in the presence of EGF, but those of the middle T antigen transformants were found to be highly stabilized in the presence of EGF. On the other hand, transfection with polyoma middle T antigen (MTAg) cDNA causes malignant transformation of Balb/3T3 cells, but not its monensin (an ionophoric antibiotic)-resistant mutant MO-5 cells, which have no significant EGF binding activity. Transfection of human EGF-R cDNA into MO-5 leads to the expression of high levels of human EGF-R in MNER31. Unlike the polyoma MTAg transfectants of BNER4, EGF-R in polyoma MTAg cDNA-transfectants into MNER31, M31/MT-13 and M31/MT-14, were down-regulated to levels similar to those of their parental MNER31. Exposure to EGF induced a more than 10-fold increase in DNA synthesis of quiescent BNER4, MNER31, M31/MT-13, and M31/MT-14 cells. Polyoma middle T antigen or v- src appears to modulate EGF-induced down-regulation of EGF-R, possibly through interaction of the receptor with the viral oncogenes, and this interaction may be altered in the mutant.

Epidermal growth factor receptor in synaptic fractions of the rat central nervous system.

Functional relationships between epidermal growth factor (EGF) and neural tissues have of late attracted increasing interest. However, in spite of reported EGF effects on neurons, the expression of the EGF receptor (EGF-R) has not yet been unambiguously demonstrated in these cells. This 170-kDa protein bears an intracellular tyrosine kinase domain in which activity is ligand-dependent. We give definitive evidence here for its presence in neonatal and adult rat neurons showing also, for the first time, its binding and functional tyrosine kinase activities in the synaptic region. Immunohistochemistry using a polyclonal antibody prepared against the receptor purified from rat liver showed positive staining localized exclusively to neurons without regionalization to any particular brain zone. Binding studies made in Percoll-obtained synaptosomes revealed specific high affinity 125I-EGF binding sites (Kd, 1.42 x 10(-10) +/- 0.58 M) accounting for 17% of total binding and a great majority of low affinity (Kd, 2.55 x 10(-9) +/- 0.35 M) binding sites. Higher binding capacity was found in synaptosomal fractions obtained from newborn rats. The identity of the synaptosomal EGF binding activity with the 170-kDA EGF-R protein was demonstrated by cross-linking experiments. Furthermore, EGF-Affi-Prep affinity chromatography adsorbs a 170-kDa protein with EGF-R immunoreactivity from whole homogenates of adult rat brain. Phosphorylation assays made in freeze-thawed or intact synaptosomes showed EGF-induced tyrosine phosphorylation in the range of 170-, 126-150-, 124-, 113-, 98-, and 70-kDa proteins including the EGF-R. Thus, the EGF-R/EGF regulatory system could have a role in synaptic function that remains to be explored.

Inhibition by epidermal growth factor (EGF) of epidermal DNA synthesis in cultured chick embryonic skin pretreated with retinol and/or hydrocortisone: specific increment in EGF binding activity in both retinol- and hydrocortisone-pretreated epidermis without correlation to EGF-mediated inhibition of cell growth.

When tarsometatarsal skin of 13-day-old chick embryos that had been cultured in medium containing 5% delipidized FCS with or without retinol (20 microM) and/or hydrocortisone (20 nM) for 1 day was cultured in a chemically defined medium without either the hormone or retinol for 1 day, epidermal DNA synthesis of hydrocortisone- and/or retinol-pretreated skin was inhibited when compared to that of control skin. The addition of epidermal growth factor (EGF, 10 ng/ml) to retinol- or hydrocortisone-pretreated skin further inhibited the epidermal DNA synthesis. Epidermal DNA synthesis in retinol- and hydrocortisone-pretreated skin was more strongly inhibited than in retinol- or hydrocortisone-pretreated skin, but was not further inhibited by EGF. In epidermis which was induced to differentiation toward keratinization by hydrocortisone or mucous metaplasia by retinol, EGF inhibited DNA synthesis. The extent of [125I]-EGF binding to the epidermis of retinol- and hydrocortisone-pretreated skin was 160-180% that in control skin, with no change in affinity. Hence there is no correlation between EGF-binding and the mitogenic activity of EGF.

Potential Physiologic Roles for Epidermal Growth Factor in the Kidney

Epidermal growth factor (EGF) is a 53-amino acid polypeptide that is known to produce a number of biologic effects both in vitro and in vivo. High concentrations of EGF are found in urine, and high concentrations of prepro-EGF mRNA have been detected in kidney, localized to thick ascending limb of Henle (TALH) and distal convoluted tubule. Specific high-affinity EGF receptors have been demonstrated in mesangial cells, proximal tubule, and cortical and inner medullary collecting duct, as well as in medullary interstitial cells. In the proximal tubule, EGF binding and EGF receptor-associated tyrosine kinase activity are localized to basolateral membrane, and functional responses in collecting duct are observed only with basolateral administration of EGF. A number of renal responses to administration of EGF have recently been described, including modulation of glomerular hemodynamics, renal metabolism, tubular transport functions, and eicosanoid synthesis. In addition, EGF has been shown to be a potent mitogen in vitro for a variety of cell types in the kidney and may be an important mediator of renal repair following injury.

EGF receptor-binding activity in the urine of normal horses and horses affected by chronic laminitis

A heterologous radioreceptor binding assay (RRA) has been developed capable of detecting nanogram amounts of epidermal growth factor (EGF) receptor-binding activity in equine urine. The binding parameters of [ 125I]mEGF (murine EGF) to EGF receptors on equine plasma membranes are in good agreement with values from other EGF-RRA systems. The dissociation constant estimated from equilibrium methods (K D = 4 × 10 −10 M) is in reasonable agreement with that determined from the rate constants (K D = 6 × 10 −10 M) and is in good agreement with values determined in other species. The assay is specific for equine EGF (eEGF) receptor-binding activity and capable of detecting less than 0.34 nM eEGF receptor-binding activity in urine. Equine EGF receptor-binding activity in equine urine form adult horses varied widely between samples (8.5 ± 6.5 nM). This variability was somewhat reduced when values were adjusted for dilutional effects using urine creatinine as an indicator (3.6 ± 2.0 nanomoles/g creatinine). No significant differences were demonstrated between the means of EGF binding activity concentrations in clinically normal horses and horses affected by chronic laminitis.

Epidermal growth factor receptors and adenylate cyclase activity in human thyroid tissues

Thyroid stimulating hormone (TSH) and epidermal growth factor (EGF) are growth factors for some thyroid cells in cultures. We have previously found more EGF receptors in neoplastic human thyroid tissues than in normal thyroid tissues. We have also found a higher TSH-stimulated adenylate cyclase (AC) activity in neoplastic human thyroid tissues than in normal thyroid tissues. To clarify the relationship between the effect of EGF and TSH on thyroid tissue, we measured the binding of EGF and TSH and the basal, TSH-stimulated and forskolin-stimulated adenylate cyclase activity in 49 normal, hyperplastic and neoplastic human thyroid tissues (5 normal, 2 Hashimoto thyroiditis, 5 Graves' disease, 14 multinodular goiters, 9 follicular adenomas, 5 follicular carcinomas, 8 papillary carcinomas, and 1 undifferentiated carcinoma). Specific binding of EGF and TSH were measured by radioreceptor assays using competitive inhibition of radio-labeled ligand by unlabeled ligand. Basal, maximally (300 mU/ml) TSH-stimulated, and maximally (100 mM) forskolin-stimulated adenylate cyclase activities were also measured in the same membrane particulate fractions from the thyroid tissues. We found: neoplastic thyroid tissues bind more labeled EGF than nonneoplastic thyroid tissues; follicular adenomas and carcinomas have higher EGF binding than other thyroid tissues; a weak but significant correlation between specific EGF binding and specific TSH binding, and between specific EGF binding and TSH-stimulated adenylate cyclase activity of the thyroid membrane preparations. These findings are consistent with the hypothesis that TSH stimulates an increase in thyroid EGF receptors by increasing intracellular cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleocytoplasmic transport is enhanced concomitant with nuclear accumulation of epidermal growth factor (EGF) binding activity in both 3T3-1 and EGF receptor reconstituted NR-6 fibroblasts.

Measurements of nucleocytoplasmic transport of fluorescent-labeled macromolecules were performed in both an EGF-nonresponsive mutant fibroblast line (3T3-NR6) and in the same cell line reconstituted with active EGF receptors derived from rat hepatic membrane fraction. Immunolocalization studies of exogenously incorporated EGF receptors in reconstituted 3T3-NR6 fibroblasts demonstrated predominantly intracellular localization. The EGF receptor constructs also showed EGF-stimulated incorporation of [3H]thymidine, providing biochemical evidence for functional integration of the exogenously supplied EGF receptors into the reconstituted fibroblasts. Additional support for the functional incorporation of receptor may be inferred from the enhanced cellular accumulation of 125I-EGF in cells treated with chloroquine and leupeptin. 125I-EGF binding and transnuclear macromolecular transport measurements in mutant and reconstituted cells, in conjunction with such measurements on nuclei isolated from these cells, provide data consistent with a growth factor/nuclear signaling mechanism dependent on the nuclear acquisition of EGF binding activity from the plasma membrane.

Expression of epidermal growth factor receptors in four histologic cell types of lung cancer

Lung cancer tissues from 68 patients were examined for epidermal growth factor (EGF) receptor levels and EGF receptor gene copy numbers. Histologic cell types of these lung cancer tissues included squamous-cell carcinoma (n = 30), adenocarcinoma (n = 28), large-cell carcinoma (n = 4), and small-cell carcinoma (n = 6). Tissues of squamous-cell carcinoma exhibited exceptionally high 125I-EGF binding activity, and those of small-cell carcinoma showed no EGF binding activity. Southern blot hybridization analysis revealed EGF receptor gene amplification in the squamous-cell carcinomas with high EGF binding activity. The EGF receptor levels in squamous-cell carcinomas and adenocarcinomas were compared with their pathological staging grouping and pathological findings, including degree of differentiation, diameter of tumor, and lymph node metastasis. However, unlike previous reports on breast and bladder cancers, there was no obvious correlation between these pathological characteristics and the EGF receptor levels of lung cancer.

Subcellular localization of the EGF receptor maturation process

The glycosylation and the processing of the epidermal growth factor (EGF) receptor are suggested to play a crucial role(s) in the activation of ligand binding activity. To examine whether the receptor acquires EGF binding activity in the endoplasmic reticulum (ER) or in the Golgi complex, we carried out parallel kinetic analysis of the EGF binding activity and the intracellular transport of the newly synthesized receptor by immunoprecipitation with the anti-EGF receptor antibody B4G7 using the EGF receptor hyperproducing cell line NA. The kinetic analysis revealed that a receptor capable of binding EGF appeared after 30 to 60 min labeling with [ 35S]methionine. Pulse-chase experiments also indicated that the receptor capable of binding EGF appeared after a 30-min pulse with a 30-min chase. Subcellular fractionation analysis indicated that the newly synthesized receptor was present in the Golgi complex after labeling with [ 35S]methionine for 30 min. After a 30-min chase, the M r 170K receptor appeared in the Golgi complex and plasma membrane. Thus, these results together indicated that after a 30-min pulse incubation a fraction of the EGF receptors have been transported from the ER to the Golgi complex; however, the receptor is unable to bind EGF. Although the EGF receptor appeared on the cell surface after a 30-min pulse with a 30-min chase, only half of the receptors are capable of binding EGF. Therefore, the EGF receptor acquires ligand binding activity at a late stage of the maturation process, most likely in the Golgi complex.

Thapsigargin, a novel promoter, phosphorylates the epidermal growth factor receptor at threonine 669

Thapsigargin, a protein kinase C-independent tumor promoter, can negatively regulate the epidermal growth factor (EGF) receptor through inhibition of high affinity EGF binding and EGF-stimulated tyrosine kinase activity. In contrast to activators of protein kinase C, thapsigargin does not induce significant phosphorylation of threonine 654. However, thapsigargin does stimulate phosphorylation of the EGF receptor at other serine and threonine residues. We now identify threonine 669 as the major site of phosphorylation on the EGF receptor resulting from thapsigargin treatment. These results raise the possibility that phosphorylation of threonine 669 may mediate changes in the binding and kinase state of the EGF receptor.

Glycosylation of the epidermal growth factor receptor and its relationship to membrane transport and ligand binding.

Epidermal growth factor (EGF) receptor biosynthesis was examined in an oral squamous cell carcinoma line, NA, which overproduces the receptor to an even greater extent than the widely studied A431 cells. The EGF receptor of NA cells synthesized in the presence of tunicamycin had an apparent molecular weight of 130,000. The nascent protein in untreated cells was cotranslationally glycosylated to Mr 160,000 and further processed to Mr 170,000. The endo-beta-N-acetylglucosaminidase H (Endo H) digestion analysis revealed the presence of high mannose type oligosaccharide on the Mr 170,000 mature receptor. We extended the analysis by correlating the biosynthesis with the acquisition of binding activity. The unglycosylated Mr 130,000 receptor and the Mr 160,000 receptor seen after pulse-labeling had no EGF binding activity, whereas the Mr 160,000 receptor seen after chase-incubation and the Mr 170,000 receptor had binding activity. Thus, not only glycosylation but also some oligosaccharide processing is apparently necessary for the EGF binding. Treatment with processing inhibitors, such as monensin, swainsonine and 1-deoxynojirimycin, affected neither receptor transport to the plasma membrane nor binding activity. Inhibition by 1-deoxynojirimycin is thought to be incomplete since the surface receptor in treated cells had the same molecular weight as that in control cells. An Mr 160,000 receptor without binding activity accumulated in the intracellular fraction in the presence of brefeldin A, an inhibitor of intracellular transport. Thus, the EGF binding activity is thought to be acquired after the brefeldin A-sensitive process but prior to the swainsonine-sensitive mannose removal in NA cells.

Epidermal growth factor binding in intracranial neoplasms: preliminary biochemical and clinicopathological findings.

The binding capacity for epidermal growth factor (EGF) was determined in 34 intracranial neoplasms (14 glioblastoma, seven low-grade gliomas, six meningiomas, and seven others) and four specimens of normal brain by using [I125]EGF. EGF binding and binding affinity of the sites in the tumour and brain samples were compared to placenta and rat liver. All specimens of normal brain were negative. Ten of 14 glioblastoma specimens contained EGF binding (level range 10-39,660 fmol/mg protein), however, ligand binding affinity was high in only three tumours. Only one of nine low-grade gliomas contained EGF binding activity. Five of six meningiomas contained EGF binding sites (level range 49-776 fmol/mg protein) and binding affinity was high in two. When present EGF binding activity was found in all cellular fractions except the cytosol. There were no clinical or histopathological features within major tumour groups that were predictive of either high or specific EGF binding activity. These preliminary studies have confirmed that EGF receptor-like activity is present in the particulate fractions of intracranial neoplasms of both mesenchymal and neuroctodermal origin. In a large proportion of these tumours the EGF binding affinity is low, suggesting either a less specific or truncated EGF binding site.

Evidence that 2,3,7,8-tetrachlorodibenzo- p-dioxin and thyroid hormones act through different mechanisms in human keratinocytes

It has been proposed [ J. D. McKinney, J. Fawkes, S. Jordan, K. Chae, S. Oatley, R. E. Coleman, and W. Briner (1985). Environ. Health Perspect. 61, 41–53] that 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) produces toxic responses through persistent occupancy of nuclear thyroxine (T 4) receptors, and that maintenance of receptor occupancy by supraphysiologic concentrations of thyroid hormones mimics TCDD toxicity [ L. H. Hong, J. D. McKinney, and M. I. Luster (1987). Biochem. Pharmacol., 36, 1361–1365]. TCDD induces hyperkeratinization in cultured normal human epidermal cells and the human keratinocyte line, SCC-12F. This response is associated with a decrease in high-affinity epidermal growth factor (EGF) receptors. These cell systems were used as models to compare the actions of TCDD with those of triiodothyronine (T 3) and T 4 on human target cells. Keratinocytes were treated simultaneously with T 3 and T 4 in a 4:1 molar ratio ( T 3 T 4 ; Hong et al., 1987) and levels of EGF binding and 7-ethoxycoumarin O-deethylase activity (a marker for cytochrome P 1-450 induction) were measured. T 3 T 4 (at concentrations up to 10 μ m T 3/2.5 μ m T 4) and T 3 or T 4 alone (0.1 to 10 μ m) did not produce the hyperkeratinization, the decrease in EGF binding, or the increase in ECOD activity that are characteristic of TCDD exposure. Nonresponsiveness to T 3 T 4 was not due to metabolism of these hormones by the keratinocytes. T 3 and T 4 did not compete with [ 3H]TCDD for binding to cytosolic Ah receptor from C57BL6 mouse liver, SCC-12F, or normal human epidermal cells. TCDD and an active stereoisomer, 2,3,7,8-tetrachlorodibenzofuran, did not compete with [ 125I]T 3 or [ 125I]T 4 for binding to nuclear receptors from SCC-12F cells or C57BL6 mouse liver. Taken together, these data demonstrate that the actions of TCDD and thyroid hormones are mediated by distinct mechanisms in human keratinocytes.