Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two retroviral DNAs that encode the normal human epidermal growth factor (EGF) receptor hEGFR have been generated by inserting a hEGFR cDNA into two different retroviral vectors. One DNA (pCO11-EGFR-neo) also contained a linked selectable marker gene (neoR). The other (pCO12-EGFR) only expresses hEGFR. When introduced into NIH3T3 cells, the two DNAs and the viruses derived from them induced a fully transformed phenotype, including focal transformation and growth in agar or low serum, but transformation depended entirely upon EGF being present in the growth medium. Compared with pCO11-EGFR-neo, pCO12-EGFR induced EGF-dependent transformation 2-5 times more efficiently and expressed higher numbers of receptors (4 x 10(5) vs. 1 x 10(5) EGF receptors per cell). The results indicate that transforming potential is directly related to the number of EGF receptors. In defined, serum-free medium that contained only very low concentrations of insulin (0.6 microgram/ml) and transferrin (0.6 micrograms/ml), hEGFR-virus infected cells were able to grow with EGF as the only growth factor. Moreover, daily incubation of the cells with EGF for only 30 min was sufficient to induce growth. NR6 cells, which lack endogenous EGF receptors, were transformed as efficiently as NIH3T3 cells by the hEGFR virus. The dose-dependent growth response to EGF of infected NR6 cells grown in serum-free medium can be used as a highly sensitive bioassay for the quantitative assessment of EGF and transforming growth factor type alpha (TGF alpha). This bioassay is at least as sensitive as previously reported radioimmunoassays and can measure a much wider concentration range (10 pg-100 ng/ml). Uninfected NR6 cells or NR6 cells infected by helper virus alone can be used as controls for the EGF specificity of growth stimulation.
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PMID:Retroviruses expressing different levels of the normal epidermal growth factor receptor: biological properties and new bioassay. 256 8

The HER2/c-erbB-2 gene encodes the epidermal growth factor receptorlike human homolog of the rat neu oncogene. Amplification of this gene in primary breast carcinomas has been show to correlate with poor clinical prognosis for certain cancer patients. We show here that a monoclonal antibody directed against the extracellular domain of p185HER2 specifically inhibits the growth of breast tumor-derived cell lines overexpressing the HER2/c-erbB-2 gene product and prevents HER2/c-erbB-2-transformed NIH 3T3 cells from forming colonies in soft agar. Furthermore, resistance to the cytotoxic effect of tumor necrosis factor alpha, which has been shown to be a consequence of HER2/c-erbB-2 overexpression, is significantly reduced in the presence of this antibody.
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PMID:p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor. 256 7

Rat hepatocytes express large numbers of high and low affinity surface membrane receptors (EGFR) for epidermal growth factor (EGF) but the roles of EGF and EGFRs in hepatocyte proliferation in vivo are unclear. F344 rat hepatocytes in primary culture proliferated maximally in response to continuous serum-free culture with 3.3 nM (20 ng/ml) EGF, as quantified by cumulative [3H]thymidine labeling index. However, serum concentrations of EGF in rats with normal livers or induced hepatocyte proliferation due to partial hepatectomy, carbon tetrachloride-induced necrosis, or hepatic neoplasia were consistently below 0.1 nM. The 3- or 6-hour pulse exposures to EGF (1.7 nM) between 0 to 16 hours had minimal effect on labeling index at 48 hours, but these pulse exposures at 24 or 32 hours were equivalent to continuous exposure. At 24 and 32 hours, the total specific surface binding of [125I]EGF to hepatocytes cultured free of EGF decreased to 43 and 24% of the initial values, respectively. Scatchard analysis of EGF binding indicated that hepatocytes lost all high affinity EGFRs (Kd of 0.08 nM) by 24 hours. Low affinity [125I]EGF binding at 0 hour (Kd 0.8 nM) was further reduced at 24 hours (Kd = 3.9 nM) and corresponded more closely to mitogenic concentrations of EGF in culture. These studies demonstrate that proliferative responsiveness of hepatocytes to EGF increases during culture by a process that involves prior loss of constitutive high affinity EGFRs. These results suggest that constitutive high affinity EGFRs do not elicit the proliferative response to EGF.
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PMID:Inverse relationship between epidermal growth factor induced proliferation and expression of high affinity surface epidermal growth factor receptors in rat hepatocytes. 278 49

The tyrosine kinase activity of the epidermal growth factor (EGF) receptor is regulated by a truncated receptor of 100 kilodaltons (kDa) that contains the EGF-binding site but not the kinase domain. The inhibition of kinase is not due to competition for available EGF or for the kinase substrate-binding site. Chemical cross-linking studies suggest that the 100-kDa receptor may form a heterodimer with the intact EGF receptor. Structurally related receptor kinases, such as the platelet-derived growth factor receptor, the insulin receptor, and the Neu receptor, were not inhibited by the 100-kDa receptor. The results indicate that (i) the inhibition was specific for the EGF receptor, (ii) the kinase domain had little or no role in determining target specificity, and (iii) the regulation of kinase may be due to a specific interaction of the 100-kDa receptor with the ligand-binding domain of the EGF receptor kinase.
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PMID:Inhibition of tyrosine kinase activity of the epidermal growth factor (EGF) receptor by a truncated receptor form that binds to EGF: role for interreceptor interaction in kinase regulation. 278 40

The effects of epidermal growth factor (EGF) on membrane potential were investigated in suspensions of the following three cell types endowed with a large complement of specific receptors: EGFR-T17 (a clone of mouse NIH-3T3 fibroblasts overexpressing EGF receptors); A431 and KB (two human carcinoma lines). In all these lines EGF induced a rapid and marked hyperpolarization constituted by an initial peak (in all three cell lines) and a subsequent sustained plateau phase, concomitant with the well-known increase of [Ca2+]i. The time course and phorbol ester inhibitability of the membrane potential effects were the same as for the [Ca2+]i response. Experiments with Na+-free and chloride-free media excluded a major role of the latter ions in the EGF-induced hyperpolarization. In contrast, experiments with high K+ media, with the monovalent cation ionophore gramicidin and with Ca2+-free media together with either a Ca2+ ionophore (ionomycin, in A431 and EGFR-T17), or an agonist (bradykinin, in A431) addressed to a receptor coupled to phosphoinositide hydrolysis, were consistent with the involvement of Ca2+-activated K+ channels. The EGF-induced hyperpolarization was completely blocked by the K+ channel blocker, quinidine, and unaffected by a variety of other drugs. Patch clamping of individual EGFR-T17 cells confirmed the initial hyperpolarization (from approximately -30 mV, the resting potential, to -60, -80 mV) was due to activation of an outward current. This initial hyperpolarization was followed by fluctuations (period approximately 1 min) persisting as long as the cells could be analyzed. Thus, the changes of membrane potential appear to be not only novel members of the group of early events triggered by EGF in target cells but also long-lasting effects of the growth factor, which continue for unexpectedly long periods of time after EGF application.
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PMID:The effect of epidermal growth factor on membrane potential. Rapid hyperpolarization followed by persistent fluctuations. 278 95

The mouse monoclonal antibody (mAb) 225 IgG1 against the epidermal growth factor (EGF) receptor has been investigated for its capacity to localize in human tumor xenografts. The EGF receptor is the product of the c-erb-B proto-oncogene (also known as EGFR). Elevated expression of EGF receptors has been demonstrated in many human tumors and tumor cell lines. We studied A431 human vulvar squamous cell carcinoma cells, with 2 X 10(6) receptors per cell; MDA-MB-468 (MDA 468) human breast adenocarcinoma cells, with 3 X 10(5) receptors per cell; and MCF-7 human breast adenocarcinoma cells, with 5 X 10(3) receptors per cell. The 111In-labeled pentetic acid (DTPA), derivative of mAb 225 (111In-DTPA-225) was injected intraperitoneally into nude mice bearing subcutaneous tumor xenografts. We measured uptake by quantifying radioactivity in tumor and normal tissues and by obtaining gamma camera images. Uptake in A431 xenografts was 28% +/- 2.4% of the injected dose per gram of tumor on day 3 and 12.4% +/- 3.0% on day 7. Distribution ratios comparing uptake in the tumor with that in normal tissues were consistently greater than 4. In contrast, there was far less uptake of the control mAb KS1/4S-1 labeled with 111In. This conjugate, 111In-DTPA-KS1/4S-1, has an IgG1 isotype but does not bind to human or murine cells. Imaging of the tumor with mAb 225 was excellent, especially on days 3-7. MDA 468 xenografts exhibited reduced localization of mAb 225 in the tumor. For MCF-7 xenografts, the tumor uptake of mAb 225 after 7 days was only 0.70% +/- 0.10% of the injected dose per gram of tumor, which was comparable to the uptake of the KS1/4S-1 control mAb. The ratio of the concentration of radioactivity in the tumor to that in normal tissue (distribution ratio) showed poor selectivity of uptake, and imaging was not obtained. These observations suggest that labeled mAb can target the product of a proto-oncogene, the EGF receptor, when it is expressed at high levels in human tumor xenografts.
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PMID:Imaging of human tumor xenografts with an indium-111-labeled anti-epidermal growth factor receptor monoclonal antibody. 279 90

NIH 3T3 cells, which express a small number of EGF (epidermal growth factor) receptors, are poorly responsive to EGF. However, when the same cells overexpress the cloned human EGF receptor (EGFR T17 cells), they display EGF-dependent transformation. In EGFR T17 cells (but not in the parental NIH 3T3 cells), EGF is shown here to trigger polyphosphoinositide hydrolysis as well as the generation of the ensuing intracellular signals, the increase in the cytosolic Ca2+ concentration ([Ca2+]i) and pH. EGF induced a large accumulation of inositol 1,4,5-trisphosphate, with a peak at 15-30 s and a slow decline thereafter. Other inositol phosphates (1,3,4-trisphosphate and 1,3,4,5-tetrakisphosphate) increased less rapidly and to a lesser degree. [Ca2+]i increased after a short lag, reached a peak at 25 s and remained elevated for several minutes. By use of incubation media with and without Ca2+, the initial phase of the EGF-induced [Ca2+]i increase was shown to be due largely to Ca2+ release from intracellular stores. In contrast with previous observations in human A431 cells, the concentration-dependence of the EGF-triggered [Ca2+]i increase in EGFR T17 cells paralleled that of [3H]thymidine incorporation. It is concluded that polyphosphoinositide hydrolysis, [Ca2+]i increase and cytoplasmic alkalinization are part of the spectrum of intracellular signals generated by the activation of one single EGF receptor type. These processes might be triggered by the receptor via activation of the intrinsic tyrosine kinase activity. Large stimulation of DNA synthesis and proliferation by EGF in EGFR T17 cells could be due to a synergistic interplay between the two signal pathways initiated by tyrosine phosphorylation and polyphosphoinositide hydrolysis.
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PMID:Transmembrane signalling at epidermal growth factor receptors overexpressed in NIH 3T3 cells. Phosphoinositide hydrolysis, cytosolic Ca2+ increase and alkalinization correlate with epidermal-growth-factor-induced cell proliferation. 284 45

Interaction of epidermal growth factor (EGF) with its specific receptor (EGFR) was explored in the intact rat small intestine and in highly purified isolated enterocyte membrane preparations. Despite the fact that the EGF ligand is known to be present at physiological concentrations within the intestinal cavity, no significant binding of the ligand to the brush border surface was observed. Instead, binding of EGF to the EGFR was confined to other membrane populations, and correlation of ligand interaction with the laterobasal membranes (LBM) was nearly perfect (p less than 0.001) across a special equilibrium gradient enriched in brush border and LBM but devoid of intracellular membranes. Specific binding to another minor population of intracellular membranes that migrated to a position less dense than typical endoplasmic reticulum-Golgi vesicles on equilibrium gradients was also observed. Immunocytochemical exposure of intestine to EGFR antibody confirmed the localization of the EGFR to LBM and intracellular membranes. As estimated from the intensity of the staining, there may be immunologically active but nonbinding receptor species in the intracellular membrane compartment. Thus, despite the secretion of EGF into the intestinal lumen, the growth and maturational effects of EGF probably result from a specific interaction between EGF and EGFR solely at the laterobasal surface of the enterocyte. The functional role of the intracellular membrane species of EGFR, which remains to be established, may involve a source of inactive receptor that can be rapidly recruited and transferred to the LBM surface under changing environmental conditions.
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PMID:Epidermal growth factor receptor of the intestinal enterocyte. Localization to laterobasal but not brush border membrane. 291 82

A synthetic chimeric gene, coding for the human epidermal growth factor fused to the signal peptide of Escherichia coli alkaline phosphatase, was cloned into E. coli under the transcriptional control of the trp-lac (tac) promoter. Following induction with isopropylthiogalactoside, the secretion of the correctly processed protein product into the bacterial periplasm was detected and quantitated by its specific binding to the epidermal growth factor receptor. The purified protein was identical to authentic human epidermal growth factor in size, amino acid composition, primary sequence, receptor binding, and stimulation of receptor protein-tyrosine kinase activity. Based on interspecies homologies, structural considerations, and reported studies with peptide fragments, structure-function analysis was initiated with alterations of targeted amino acid residues by oligonucleotide-directed mutagenesis. The receptor binding affinity of each mutant, relative to the wild type, was measured by both radioreceptor competition and receptor tyrosine kinase stimulation assays. In general, the values obtained by the two methods were in agreement for each species of epidermal growth factor and followed the order: wild type greater than Glu24----Gly greater than Asp27----Gly much greater than Pro7----Thr greater than Tyr29----Gly greater than Leu47----His. The relatively low values obtained with the last two mutants suggest that Tyr29 and Leu47 may be important for the biological activity of human epidermal growth factor.
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PMID:Cloning of authentic human epidermal growth factor as a bacterial secretory protein and its initial structure-function analysis by site-directed mutagenesis. 304 17

In rat brain, distinct epidermal growth factor-receptor immunoreactivity (EGFR-IR) first appeared in astroglia at about day 16 postnatal, reached maximum intensity at 19 days and then became much weaker as the animals reached adulthood. EGFR-IR was also observed in cerebellar Purkinje cells as early as 11 days postnatal and was maintained into adulthood. In adult and aged animals the most prominent EGF receptor immunostaining occurred in cerebral cortex neurons (layers IV and V) that had the morphology of basket cells. Immunoreactive neurons were abundant in the cingulate, frontal, frontoparietal and striate cortices. In the frontoparietal cortex, EGFR positive neurons were most numerous in the motor area, diminishing laterally towards the somatosensory area. The localization and time of appearance of EGFR-IR did not appear consistent with a direct mitogenic role of the EGF domain in astroglia proliferation during development. However, the EGFR may be involved in neuronal survival and/or neuron-glia signalling.
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PMID:Epidermal growth factor receptor immunoreactivity in rat brain. Development and cellular localization. 334 47


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