Unlabeled Epidermal Growth Factor Research Articles

Characterization of epidermal growth factor receptors in cultured vascular smooth muscle cells of rat aorta.

Characteristics of specific receptors for epidermal growth factor (EGF) and its effect on cellular proliferation and synthesis of DNA and protein were studied in cultured vascular smooth muscle cells (VSMC) from rat aorta. Binding studies using 125I-EGF revealed the presence of high affinity binding sites for EGF on VSMC in culture: the apparent dissociation constant was approximately 2.5 X 10(-10)M and the maximal binding capacity was approximately 67,000 sites/cell. EGF stimulated cellular proliferation and incorporation of [3H]thymidine and [3H]leucine into the cells in a dose-dependent fashion; the approximate half-maximal stimulation was induced with 1.5 X 10(-10)M. Platelet-derived growth factor (PDGF) had an additive effect with EGF on DNA synthesis by VSMC. Preincubation of VSMC with unlabeled EGF resulted in a substantial reduction in the number of receptors without changing the affinity, suggesting receptor "down-regulation" mechanism. These data indicate that rat aortic VSMCs have specific receptors for EGF, and suggest that EGF, in addition to PDGF, is also involved in the cell growth of VSMC.

Identification of a novel receptor in Drosophila for both epidermal growth factor and insulin.

The notable amino acid homology among mammalian growth factor receptors with tyrosine-specific protein kinase activity has led to speculation that these receptors derived from a common evolutionary precursor. We report the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100-kDa protein is also related antigenically to the mammalian receptors for EGF and possibly insulin but may not correspond to the mammalian counterpart of either receptor in Drosophila. The Drosophila protein is recognized by antisera directed against the mammalian receptor for EGF in immunoblot hybridizations. It can be affinity labeled with either 125I-labeled insulin or 125I-labeled EGF after immunoprecipitation with anti-EGF receptor antiserum. Excess unlabeled EGF or insulin will block the affinity labeling with either growth factor, suggesting that both EGF and insulin share a common binding site on the 100-kDa Drosophila receptor. This Drosophila protein, therefore, may be closely related to an evolutionary precursor of the mammalian receptors for insulin and EGF.

Transport of 125I-EGF into milk and effect of sialoadenectomy on milk EGF in mice.

Epidermal growth factor (EGF) (urogastrone) is found in high concentrations in mouse and human milk. The origin of milk EGF is unknown. Milk samples were collected from lactating mice 2-6 h after the intravenous administration of a tracer dose of 125I-labeled EGF. The milk contained relatively high levels of radioactivity of which 35-46% was precipitated by trichloroacetic acid-phosphotungstic acid (TCA-PTA) and 26% by a specific antimouse EGF antiserum. Part of the radioactivity in milk was eluted from a Bio-Gel P-10 column at the point at which pure standard 125I-EGF was eluted. These data indicate that the mammary gland of the lactating mouse is capable of sequestering and transporting 125I-EGF into milk. Administration of a thousand-fold excess of unlabeled EGF caused no reduction in TCA-PTA-precipitable radioactivity in milk samples of mice given 125I-EGF. When mice were given 10 micrograms of unlabeled EGF and milk was collected 4 h later, compared with controls the EGF level in milk was doubled. Administration of EGF had no effect on lactose and protein concentrations in the milk, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no qualitative or quantitative changes in the major milk proteins. Milk collected from lactating mice that were sialoadenectomized 6 mo earlier contained about 50% less EGF compared with controls. After the administration of 125I-EGF high concentrations of radioactivity were also found in the mammary and submandibular glands and in the stomach. In the latter organs, however, 95-96% of the radioactivity was in the acid-soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of epidermal growth factor with receptors on human and porcine thyroid membranes

Recent evidence suggests that epidermal growth factor (EGF) may play an important role in the regulation of thyroid growth and function. We have examined the interaction of murine EGF (mEGF) with human and porcine thyroid membranes and compared this with the binding of bovine TSH (bTSH) using 125I-labelled hormones as tracers. The characteristics of the binding of mEGF were found to be similar for human and porcine thyroid membranes. Epidermal growth factor bound with high affinity (affinity constant = 1.4 X 10(9) l/mol); the density of binding sites was low compared with the TSH receptor. At 37 degrees C, the binding of 125I-labelled EGF was maximal at 1 h and was saturable in the presence of unlabelled EGF; half-maximal inhibition was at 1 ng EGF/tube (0.5 nmol/l) using 0.5 mg membrane protein/tube. Unlabelled bTSH had no effect on the binding of labelled EGF. Similarly, unlabelled EGF did not affect the binding of labelled TSH; hence it was concluded that mEGF and bTSH bound to independent sites. Epidermal growth factor had no effect on adenylate cyclase activity in membranes prepared from human non-toxic goitre; increasing concentrations of EGF did not affect basal, TSH-stimulated or fluoride-stimulated enzyme activity.

Binding sites for epidermal growth factor in human uterine tissues and leiomyomas.

There is no published data regarding whether human uterine tissues and leiomyomas contain binding sites for epidermal growth factor (EGF). The present study was undertaken with 33 myometria, 13 leiomyomas, and 4 endometria. All myometria (fundus) and leiomyomas and 3 of 4 endometria specifically bound 125I-labeled mouse EGF (myometria: mean, 1.1; range, 0.1-3.9 fmol/mg protein; leiomyomas: mean, 1.1; range, 0.2-2.6 fmol/mg protein; endometria: mean, 1.0; range, 0.0-3.1 fmol/mg protein). [125I]EGF binding to myometrium was ligand specific in that only unlabeled EGF, but not unlabeled insulin, hCG, human PRL, prostaglandin E1, or prostaglandin F2 alpha, competed with [125I]EGF for binding. The apparent dissociation constants and specific binding capacities for myometrium and leiomyoma were: 0.7 nM, 1.9 fmol/mg protein; and 0.1 and 3.7 nM, 0.1 and 4.4 fmol/mg protein, respectively. While smooth muscle content decreased from the fundus to the cervical end of uteri, [125I]EGF binding did not correspondingly decrease (r = 0.5; n = 4). The binding of [125I]EGF to myometrium did not vary with the phase of the menstrual cycle or the patients' diagnosis before hysterectomy (P greater than 0.1). In summary, these results demonstrate that human uteri and leiomyomas contain specific, high affinity binding sites for EGF, but the binding neither exhibited topographical changes nor varied with the phase of the menstrual cycle or benign pathological state of the tissue.

Binding and internalization of epidermal growth factor by rat pituitary tumor cells

Epidermal growth factor (EGF) regulates hormone synthesis and decreases the growth rate of clonal lines of rat pituitary tumor cells. [ 125I]iodo-EGF bound to specific receptors on GH 4C 1 rat pituitary cells with an apparent K d of 0.34 nM. At 0°C, 84% of specifically bound [ 125I]iodo-EGF appeared to be on the cell surface, while at 37°C, 84% of bound [ 125I]iodo-EGF appeared to be internalized and could not be removed by treatment with an acidic high-salt buffer. Internalization of surface-bound [ 125I]iodo-EGF required 10 min to reach completion and was markedly temperature-dependent. Monodansylcadaverine, bacitracin, ammonium chloride, chloroquine, quinacrine and vinblastin did not inhibit internalization of [ 125I]iodo-EGF, suggesting that transglutaminase is not involved; but these drugs did cause an increased accumulation of radioactivity, probably by inhibiting degradation. In 2 h at 37°C, approx. 35% of cell-associated [ 125I]iodo-EGF was degraded as analyzed by gel filtration, and 72% of the material that dissociated from the cells migrated as low molecular weight material or iodo-Tyr. Incubation of GH 4C 1 cells with unlabeled EGF caused a prolonged and dose-related decrease in the ability of the cells to bind [ 125I]iodo-EGF in a subsequent incubation, i.e. down-regulation. The pathway for internalization and degradation of the EGF-receptor complex in GH 4C 1 cells resembles that described for fibroblasts, in which the cellular response is different, but differs from the behavior of other hormone-receptor complexes in GH 4C 1 pituitary cells.

Epidermal growth factor-like transforming growth factor. II. Interaction with epidermal growth factor receptors in human placenta membranes and A431 cells.

A 125I-labeled derivative of rat epidermal growth factor-like transforming growth factor (125I-eTGF) that retains the chemical and biological properties of native eTGF has been prepared to characterize the interaction of eTGF with epidermal growth factor (EGF) membrane receptors. Binding of 125I-eTGF and 125I-labeled mouse EGF (125I-EGF) to membrane preparations from human placenta and the A431 human epidermoid carcinoma cell line was competed for to the same extent and with the same potency by either unlabeled eTGF or EGF. Components of placenta membranes and A431 membranes (145-170 kDa) were the major species affinity-labeled by cross-linking with membrane-bound 125I-eTGF or 125I-EGF. Affinity labeling of these receptor species was fully inhibited by both native eTGF and native EGF when present in excess during incubation of membranes with 125I-eTGF or 125I-EGF. The binding affinity and capacity of receptors in A431 membranes and placenta membranes were strikingly similar for eTGF and EGF. However, binding of 125I-eTGF to membrane receptors was highly sensitive to pH, being optimal only within the pH 8.0-8.5 range whereas EGF binding was optimal at pH 7.0-9.0. eTGF binding and EGF binding were modified in parallel by exposure of placental membranes to phytohemagglutinin, concanavalin A, or wheat germ agglutinin. Prolonged exposure of A431 cells to eTGF or EGF induced the same extent of down-regulation of both eTGF binding and EGF binding. eTGF-induced down-regulation of receptors in A431 cells followed the same kinetics as EGF-induced receptor down-regulation. The data indicate that despite the limited sequence homology between eTGF and EGF, these two ligands exhibit a remarkably similar mode of interaction with a common eTGF/EGF membrane receptor type.

Direct modulation of epidermal growth factor binding by cholecystokinin

The effects of cholecystokinin-octapeptide (CCK 8), the biologically active C-terminal moiety of cholecystokinin (CCK), on the binding of epidermal growth factor (EGF) were studied in isolated rat pancreatic acini. CCK 8 inhibited 125I-EGF binding in a dose-dependent manner. One-half maximal inhibition occurred at 5 × 10 −10M, and maximal inhibition at 10 −8M CCK 8. This inhibitory effect was detectable within 5 minutes of addition of CCK 8, and was not associated with enhanced degradation of 125I-EGF in incubation media. Unlabeled EGF exerted only a slightly greater inhibitory effect than CCK 8 on 125I-EGF binding at equivalent molar concentrations. In contrast to CCK 8, the gastrointestinal hormone vasoactive intestinal polypeptide (VIP) did not significantly alter EGF binding. CCK 8 also inhibited EGF binding in mouse pancreatic acini, but did not alter binding in A-431 human carcinoma cells. These findings suggest that physiological levels of CCK may regulate EGF binding in the pancreas and other tissues with receptors for both hormones. They thus point to a previously unrecognized mechanism for hormonal interaction.

Hepatic sequestration and biliary secretion of epidermal growth factor: evidence for a high-capacity uptake system.

Epidermal growth factor (EGF) promotes hepatocyte growth and is bound in the liver by specific receptors. We have determined hepatic uptake of EGF in intact rats after an intravenous or intraportal injection of a bolus of 125I-labeled EGF. Ninety-nine percent of the intraportal dose was taken up by the liver in 3 min, whereas only 58% of the intravenous dose appeared in the liver in 10 min. Uptake was inhibited by simultaneous treatment with an excess of unlabeled EGF. At time zero, uptake appeared to be complete. Disappearance from the liver followed first-order kinetics. Within 90 min of an intraportal injection, an average of 19% of the injected radioisotope appeared in bile, of which approximately one-fifth was shown to be immunoprecipitable with a specific anti-EGF antiserum. Light microscopic autoradiography demonstrated a very steep portal-to-central lobular concentration gradient consistent with a high-capacity uptake system. After intraportal injection or after incubation with cultured hepatocytes, labeled EGF was shown to be bound to its hepatic receptors. The main receptor-ligand complex had a Mr of approximately equal to 160,000-170,000, determined by NaDodSO4/polyacrylamide gel electrophoresis.

Affinity labeling of a transforming growth factor receptor that does not interact with epidermal growth factor.

Membrane components that interact with epidermal growth factor (EGF) and transforming growth factors (TGFs) have been identified by covalent crosslinking to their respective 125I-labeled ligands. Under appropriate conditions, disuccinimidyl suberate or hydroxysuccinimidyl p-azidobenzoate cross-link receptor-bound 125I-labeled EGF to a 140- to 170-kilodalton (kDal) receptor species in membranes from both A431 human carcinoma cells and normal rat kidney cells. 125I-Labeled sarcoma growth factor (SGF), a TGF from virally transformed mouse 3T3 cells, also can be affinity-crosslinked to the 140- to 170-kDal EGF receptor species in membranes from A431 and rat kidney cells. The labeling of this receptor is inhibited when either excess unlabeled EGF or SGF is present during incubation of membranes with either 125I-labeled EGF or 125I-labeled SGF. In contrast, a second receptor species of 60 kDal is affinity-labeled with 125I-labeled SGF but not with 125I-labeled EGF in membranes from both A431 and rat kidney cells. SGF and a TGF from virally transformed rat embryo cells inhibit the labeling of the 60-kDal species when present in excess during incubation of membranes with 125I-labeled SGF, whereas EGF is completely ineffective in inhibiting the labeling of this receptor. The data suggest that a specific 60-kDal receptor that displays high affinity for TGFs but not for EGF may mediate induction of the transformed phenotype. In addition, SGF and other TGFs interact with the 140- to 170-kDal EGF receptor that appears to mediate normal cell growth effects.

Receptor binding and processing of epidermal growth factor by human breast cancer cells.

Epidermal growth factor (EGF) stimulates the growth of human breast cancer cells (MCF-7). To better define the mechanisms for these effects, we studied receptor binding and subsequent processing of [l25I]EGF in cells growing in monolayer culture under conditions in which an effect on growth is observed. Binding was temperature dependent and was higher at reduced temperature. At 37 C, binding rapidly reached a maximum (20–60 min) and then gradually decreased. Binding was slower at a reduced temperature. High affinity was demonstrated by the ability of unlabeled EGF to compete for [125I]EGF binding; hah7 maximal displacement was observed at an EGF concentration that also resulted in half-maximal biological activity (1–2 ng/ml). Scatchard plots derived from these data or from those of studies using increasing concentrations of [125I]EGF were curilinear and superimposable. Plots from binding data obtained at 0 C were also curvilinear, suggesting that the processing and degradation of tracer alone cannot expla...

Epidermal growth factor: relationship between receptor down regulation in cultured NRK cells and epidermal growth factor enhancement of phosphorylation of a 170000 molecular weight membrane protein in vitro.

Incubation of confluent nondividing NRK cells in serum-free media with unlabeled epidermal growth factor (EGF) leads to a reduction in the specific binding capacity for 125I-labeled EGF. This modulation of the binding capacity for 125I-labeled EGF by unlabeled EGF, termed receptor down regulation, was dependent on EGF concentration and time. Membranes from untreated NRK cells have a phosphorylating system which catalyzed in vitro the phosphorylation of numerous membrane components; this phosphorylating system was stimulated by EGF. Although EGF enhanced the phosphorylation of many membrane proteins, one major component with Mr 170K and a minor band of Mr 150K were primarily affected. A comparison of the membrane phosphoproteins of untreated and down-regulated cells by in vitro phosphorylation and NaDodSO4 gel electrophoresis revealed that down regulation of EGF receptors results in a specific decrease in 32P phosphorylation of the 170K- and 150K-dalton components to subsequent stimulation with EGF in vitro. We further characterized the modulation of phosphorylation of the 170K protein by down regulation with EGF and found it to be dependent on EGF concentration and time. These studies demonstrated a correlation between the loss of 125I-labeled EGF binding activity by the cells and the loss of the vitro EGF-dependent 32P phosphorylation of the 170K-dalton membrane protein. In addition, the results suggest that the major 170K Mr phosphoprotein band is a component of the receptor for EGF which is a substrate of the phosphorylation reaction.

Regulation of metastatic ovarian cells in serum-free medium by epidermal growth factor and transferrin.

AbstractA rat ovarian tumorous cell line (OV1N) has been isolated and established in tissue culture whose growth can be controlled by the addition of epidermal growth factor (EGF) and transferrin to serum‐free medium. EGF at 1 ng/ml (1.5 × 10−10M) stimulates growth about 300% in a six‐day incubation assay. A combination of 1 ng/ml EGF plus 1 μg/ml (1.25 × 10−8M) transferrin stimulates growth 1700% above controls in six days. EGF and transferrin at these concentrations completely replaced fetal calf serum (FCS), which maximally stimulates the cells at 2%. At maximal stimulation (either by FCS or EGF and transferrin) a doubling time of from 16–20 hours is obtained. The growth of OV1N cells was also studied in vivo and compared to another rat ovarian cell line, 31A‐F2. Cells from both cell lines were injected intrasplenically into normal, ovariectomized and hypophysectomized female Fischer 344 rats. Animal data have indicated that 31A‐F2 cells behave more like “normal” ovarian cells in that they grew only in ovariectomized hosts. OV1N cells, on the other hand, grew metastatically in all hosts and killed 36% of those individuals.The binding of 125I‐EGF was also studied in OV1N and 31A‐F2 cells. OV1N cells bound about eightfold more 125I‐EGF than did 31A‐F2 cells at 37°C. The dose of 125I‐EGF required to specifically saturate 31A‐F2 sites was 10 ng/ml (1.5 × 10−9M) or half‐maximally at 4 ng/ml (6 × 10−10M). Scatchard plots indicate that 31A‐F2 cells contain roughly fourfold less EGF‐specific sites than OV1N cells. Incubation of either OV1N or 31A‐F2 cells with a 100‐fold excess addition of various proteins plus a maximal or a half‐maximal addition of 125I‐EGF demonstrated that unlabeled EGF was most effective in reducing 125I‐EGF binding to both OV1N and 31A‐F2 cell sites. Insulin, ovarian growth factor, transferrin, or their combinations, had minimal, if any effect on 125I‐EGF binding.

Sarcoma growth factor (SGF): specific binding to epidermal growth factor (EGF) membrane receptors.

Cells transformed by murine sarcoma viruses (MSV) produce and release into their tissue culture media several polypeptide growth stimulating factors. One of these has been partially purified using Bio-Gel P-60 column chromatography followed by DEAE-cellulose chromatography. This growth factor was assigned the name sarcoma growth factor (SGF), and is here shown to require the epidermal growth factor (EGF) receptor in order to function as a growth factor. DEAE-cellulose chromatography yielded a product that was several-fold purer than the material present in the Bio-Gel P-60 column pool II. The biologically active material from the DEAE-cellulose column, when labeled with 125I, showed specific binding to EGF membrane receptors. The specific binding could be prevented with the addition of either unlabeled EGF or SGF. Both radiolabeled SGF and EGF will bind to live or fixed cells. We were able to bind 125I-SGF as well as 125I-EGF to fixed cells and elute the bound material from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed cells. A 3T3 clone lacking EGF receptors was unable to respond to either EGF or SGF, whereas it responded well to serum and several other purified growth factors. The SGF isolated using DEAE-cellulose chromatography was unable to compete in a radioimmune assay using 125I-EGF and antibody to purified mouse submaxillary gland EGF; it also was not precipitated by anti-EGF antibody. From these studies it appears that the SGF produced and released by these MSV-transformed cells combines with and requires the EGF receptor in order to exert its biological effects. The peptide, however, is antigenically distinct from mouse submaxillary gland EGF.

Radioimmunoassay of epidermal growth factor.

Epidermal growth factor (EGF) is a biologically active polypeptide extracted from male mouse submaxillary glands. It exists either alone or complexed with an arginine esterase. It causes generalized epidermal growth and keratinization in the immature mouse and results in precocious tooth eruption and eye-opening that are the basis for an EGF bioassay. EGF is also active in a variety of epithelial tissues from several other species. A rapid and sensitive solid phase radioimmunoassay for EGF has been developed in which rabbit antibody to EGF was adsorbed onto the walls of nylon vessels. After 24 hr of incubation, 125I-labeled EGF was displaced by as little as 30 pg of unlabeled EGF. There was no cross-reaction with the arginine esterase; mouse nerve growth factor; rat growth hormone or prolactin; human growth hormone, ACTH, FSH, LH or TSH; beef or pork insulin; or angiotensin I. Immunoreactive EGF concentrations in submaxillary glands of immature, 15-dayold male mice were very low (0.016 ng/mg of wet tissue...