PubMed Health⌕ Search

Biomedical subjects

A C Notides

Publications and source records attributed to A C Notides.

At least 37 records · Page 2Linked to original sources
PubMed1992

Estrogen receptor phosphorylation. Hormonal dependence and consequence on specific DNA binding.

We have shown that the 32P-phosphorylation of the nuclear estrogen receptor from human MCF-7 cells or the calf uterus is estrogen-dependent. Within 2 min of estradiol treatment the phosphorylation of the estrogen receptor from MCF-7 cells doubled, and increased 4-fold within 20-40 min of estradiol treatment. Progesterone was ineffective in stimulating the phosphorylation of the estrogen receptor. Phosphoamino acid analysis indicated that the estrogen-stimulated phosphorylation of the human or calf estrogen receptor occurred only on serine residue(s). Phosphotryptic peptide analysis of the human estrogen receptor by two-dimensional peptide mapping or reverse-phase high pressure liquid chromatography revealed that only a single tryptic peptide (site) was phosphorylated. Treatment of the estrogen receptor with potato acid phosphatase resulted in the dephosphorylation of the 32P-labeled estrogen receptor and a decrease of the receptor's affinity for specific DNA sequences. These data suggest that transcriptional activation by the estrogen receptor involves an estrogen-dependent phosphorylation of the receptor resulting in its increased affinity for specific DNA sequences.

Adenocarcinoma↗
PubMed1991

Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene.

This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.

Base Sequence↗
PubMed1991

Estradiol inhibits transcription of the human glycoprotein hormone alpha-subunit gene despite the absence of a high affinity binding site for estrogen receptor.

Chronic administration of estradiol inhibits transcription of the gene encoding the alpha-subunit of pituitary glycoprotein hormones. Here, we show, using transfection analyses and a filter binding assay, that 1500 basepairs of proximal 5' flanking sequence of the human alpha-subunit gene lack a functional estrogen response element when transfected into heterologous cell lines, and fail to bind estrogen receptor purified from calf uterus. Yet, this same region of the alpha-subunit gene confers estradiol responsiveness (transcriptional suppression) to the bacterial chloramphenicol acetyltransferase gene in transgenic mice. A smaller promoter fragment of the bovine alpha-subunit gene also confers responsiveness to estradiol in transgenic mice, suggesting that the same element may mediate the steroid responsiveness of both promoters. Furthermore, regulation by estradiol of the chimeric human or bovine alpha-chloramphenicol acetyltransferase genes is pituitary specific, underscoring the physiological significance of these studies. Based on these results, we conclude that estradiol regulates expression of the alpha-subunit gene in vivo through a mechanism that does not involve high affinity binding of estrogen receptor to the alpha-subunit gene. Whether this mechanism is manifest at the level of the pituitary or hypothalamus remains to be determined.

Animals↗
PubMed1991

Phosphate-sensitive binding of the estrogen receptor to its response elements.

Although the nucleotide bases that constitute the consensus DNA sequence of the estrogen response element (ERE) have been identified, the involvement of electrostatic contacts between the sugar-phosphate backbone of the ERE and the estrogen receptor (ER) is not known. Moreover, the contribution of these contacts to sequence-specific DNA binding has not been determined. Therefore, the interactions of highly purified ER with the phosphate residues of the ERE derived from the chick vitellogenin (cVit)-II gene were examined by phosphate ethylation interference. Specific ER-DNA complexes were evident in electrophoretic gel mobility shift assays using DNA fragments containing either the perfect ERE (-625 relative to gene start site; 5'-GGTCAGCGTGACC) or the imperfect ERE (-353; 5'-GGTCAACATAACC). The phosphate ethylation interference footprint identified a 2-fold, symmetrical exclusion of phosphate residues essential for specific binding to the perfect ERE with a 5' stagger, indicating that each monomer of the ER dimer is bound in the major groove of the DNA. The interference footprint of the imperfect ERE did not detect interactions between the receptor and the phosphate residues in the 3' half of the response element on the noncoding strand. In contrast, the corresponding footprint of the perfect ERE displayed strong interactions between the ER and the phosphate backbone of the DNA. Consequently, the absence of these electrostatic contacts very likely accounts for the reduced binding affinity of the ER for the imperfect ERE. These results indicate that specific contacts between the ER and the sugar-phosphate backbone of its cognate response elements are an important aspect of DNA sequence recognition and high affinity binding.

Base Sequence↗
PubMed1989

An upstream region of the rat luteinizing hormone beta gene binds estrogen receptor and confers estrogen responsiveness.

Regulation of gonadotropin gene expression by sex steroids may occur via direct effects on the pituitary and/or by indirect effects of steroid on the hypothalamus. To study direct estrogen regulation of the rat luteinizing hormone beta (LH beta) gene, we performed estrogen receptor-DNA binding studies and transient expression gene transfer experiments. Nitrocellulose filter binding studies were performed with purified estrogen receptor from calf uterus and labeled fragments of the LH beta gene. Dose-dependent specific binding to receptor occurred only with LH beta gene fragments containing a common 284-base region from -1388 to -1105 bases upstream from the transcriptional start site. This DNA region contained a 15-base imperfect palindromic region (GGACACCATCTGTCC) with sequence similarity to other estrogen-responsive elements. Biological function was tested by inserting portions of the 5'-flanking region of the gene next to the herpes simplex virus thymidine kinase promoter fused to the chloramphenicol acetyltransferase gene (LH beta-tkCAT) and performing gene transfer experiments with the pituitary GH3 cell line. Promoter activity in LH beta-tkCAT constructs containing LH beta gene sequences from bases -2013 to -613, or from bases -1388 to -613 in either orientation, exhibited stimulation with 17 beta-estradiol (E2) treatment; in contrast, constructs containing bases -885 to -613 were not regulated by E2. Positive regulation by E2 exhibited dose- and time-dependent stimulation, with a maximum 2- to 6-fold effect achieved after 48 h of treatment with 10(-8) M E2. The estrogen receptor appeared to be required for this biological response. Stimulation of LH beta-tkCAT constructs did not occur in L cells with undetectable levels of E2 receptor, but did occur after cotransfection of an LH beta-tkCAT construct and an expression vector containing the human estrogen receptor cDNA. These studies demonstrate that a 5'-flanking region of the rat LH beta gene can bind to the estrogen receptor and that this region can confer hormonal responsiveness to a heterologous promoter. Thus, positive steroid regulation of luteinizing hormone may occur directly on the pituitary at the level of the LH beta gene.

Animals↗
PubMed1989

Estrogen receptor selectively binds the "coding strand" of an estrogen responsive element.

An initial step in the transcriptional activation of the prolactin gene by estrogen is the binding of the estrogen-receptor complex to a specific nucleotide sequence [estrogen responsive element (ERE)]. Using the gel mobility assay, we examined the binding mechanism of purified estrogen receptor to the ERE contained on a 255-base-pair fragment from the upstream region between nucleotides -1784 to -1531 of the rat prolactin gene. Remarkably, specific high-affinity binding was detected to the dissociated "coding strand" but not to the "noncoding strand" of the ERE-containing fragment. The dissociated strands of this fragment possess unusual secondary structure, as indicated by their anomalous migration in the gel mobility assay. The estrogen receptor binds to the coding strand of the ERE with a 60-fold higher affinity than to the double-stranded ERE. Furthermore, the receptor binds with a 1000-fold greater affinity to the coding strand of the ERE than to a double-stranded nonspecific DNA fragment. We propose that, in vivo, the estrogen receptor initially binds to the double-stranded ERE. Subsequently, the DNA strands separate due to transitory strand separation and supercoiling, allowing folding of the coding strand of the ERE into a structure that is then bound more tightly by the receptor. The formation of this receptor-ERE coding strand complex may be a crucial step in the mechanism of estrogen-stimulated transcription.

Animals↗
PubMed1988

A chemical kinetic model for ligand binding to identical and independent binding sites in vivo.

In living systems, hormones bind to receptor proteins that are continuously synthesized and degraded. Since these systems cannot be described by equilibrium binding equations, we present a chemical kinetic model for the binding of a hormone to a receptor with identical and independent binding sites in which synthesis and degradation occur. We have derived, from the model, equations that can be used to calculate the bound ligand concentration and the total protein concentration as a function of the free ligand concentration and time. These results show that the methods for experimental measurements, parameter estimation, interpretation of the parameters, and error analysis that are commonly used for equilibrium binding to identical and independent binding sites can be adapted for the analysis of steady-state binding data. The equations we derived for the steady-state ligand binding permit determination of the total receptor protein concentration and the binding affinity from experimental data. In contrast, if an equilibrium model were used, the values of these parameters would not necessarily approximate the true values. A useful approximation to the total receptor concentration as a function of time was found that requires only three of the five rate constants required for exact description by the model. This approximation is shown to be accurate in the biologically relevant range by using previously published parameters estimated from steroid binding data, and adding perturbations to include experimental error and variations among biological systems. When complexities exist, such that the model does not describe the data, these analyses aid in assessing the types of additional components and interactions that may exist.

Binding Sites↗
PubMed1988

The effects of dimethylformamide on the interaction of the estrogen receptor with estradiol.

The study of the mechanism of interaction of antiestrogens with the estrogen receptor is complicated by the limited solubility of these compounds and their nonspecific, hydrophobic interactions with proteins in estrogen receptor preparations and with the glass or plastic wall of the test tube. The organic solvent dimethylformamide increases the solubility of these compounds in aqueous solution and interferes with hydrophobic interactions with solid phases and thereby reduces their loss from the solution. For these reasons some investigators use dimethylformamide in the antiestrogen-estrogen receptor binding assay. In this study we report that dimethylformamide interferes with the estrogen receptor's binding kinetics and the estradiol-induced activation of the estrogen receptors, and inactivates the receptor.

Allosteric Regulation↗
PubMed1988

Mechanism of the estrogen receptor interaction with 4-hydroxytamoxifen.

The binding mechanism of the estrogen receptor with 4-[3H]hydroxytamoxifen was investigated. The equilibrium binding analysis with 4-[3H]hydroxytamoxifen indicated a positive cooperative interaction: the Scatchard plot was convex and the Hill coefficient was 1.4-1.5. This binding appears similar to the positively cooperative interaction of the estrogen receptor with [3H]estradiol. However, a competitive binding assay with a saturating concentration of [3H] estradiol and variable concentrations of 4-hydroxytamoxifen produced nonparallel displacement curves indicating that the binding mechanism of the receptor with these two ligands is different. The competitive binding assay with [3H]estradiol and 4-hydroxytamoxifen at constant molar ratios demonstrated that the receptor's affinity for estradiol was reduced and the receptor preferentially bound 4-hydroxytamoxifen. These data suggest that 4-hydroxytamoxifen interacts with the receptor differently than estradiol; it antagonizes the binding of estradiol when these two ligands are simultaneously present.

Animals↗
PubMed1987

Reduction of irreversible binding of diethylstilbestrol in hamster renal cortex by inhibitors of cytochrome P-450.

Hamster renal cortical slices metabolized [3H]diethylstilbestrol (DES) to reactive intermediates that irreversibly bound to macromolecules. Protein isolated from cortical slices following incubation with 50 nM [3H]DES for 90 min at 37 degrees C had 0.160 pmol [3H]DES eq/mg protein irreversibly bound. Samples of protein analyzed by gel electrophoresis revealed several radioactive peaks, indicating that specific adduct formation had occurred. No radioactivity was associated with DNA isolated from the same tissue slices. Incubation of the slices with [3H]DES under an atmosphere enriched in carbon monoxide decreased the nonextractable binding of [3H]DES metabolites to protein. The cytochrome P-450 inhibitors diethylaminoethyl 2,2-diphenylpentanoate HCl (SKF 525-A), metyrapone, butylated hydroxytoluene (BHT), and dicumarol decreased the irreversible binding of [3H]DES by 38 to 72%. Analysis of metabolites isolated from the incubation medium by high-pressure liquid chromatography indicated that carbon monoxide, BHT, and dicumarol inhibited the hydroxylation of [3H]DES. Arachidonic acid and indomethacin did not alter the irreversible binding of [3H]DES, indicating a lack of involvement of prostaglandin H synthetase in the metabolism of DES to reactive intermediates. These findings suggest that cytochrome P-450 isozymes in the hamster renal cortex metabolize DES to reactive species that covalently bind to macromolecules.

Animals↗
PubMed1987

Estrogen receptor purification by affinity chromatography using an orange triazine dye.

A rapid two-step procedure was devised for the purification of the estrogen receptor from the calf uterus. A 900- to 1700-fold purification of the estrogen receptor was obtained using ammonium sulfate precipitation followed by dye affinity chromatography with Reactive Orange 14 immobilized to Sepharose. The Reactive Orange 14-Sepharose was used to purify the estrogen receptor in the presence or absence of estradiol as well as to purify the progesterone receptor. The purified estrogen receptor retained its estradiol- and DNA-binding properties and sedimented into sucrose gradients as the 5 S receptor dimer. The Reactive Orange 14-Sepharose is easily prepared and offers a higher yield and purity of the estrogen receptor than that afforded by estrogen- or heparin-Sepharose chromatography.

Animals↗
PubMed1987

Glutathione protection against irreversible binding of diethylstilbestrol in the hamster renal cortex.

Hamster renal cortical slices bioactivated the synthetic estrogen, diethylstilbestrol (DES), to reactive metabolites that bound irreversibly to cellular proteins. Incubation of the slices for 30 min at 37 degrees C with 5 mM diethyl maleate prior to the addition of 50 nM [3H]DES for 60 min increased the nonextractable binding of [3H]DES metabolites to cellular protein by 150%, whereas addition of 5 mM glutathione (GSH) decreased the irreversible binding of [3H]DES by 17%. The addition of the 5 mM GSH to the incubation medium caused a 24% increase in tissue nonprotein sulfhydryl (NP-SH) content as estimated by reaction with Ellman's reagent after 30 min at 37 degrees C, while the addition of diethyl maleate for the same time period depleted tissue NP-SH levels by 54%. Kidneys from hamsters treated with the GSH synthesis inhibitor L-buthionine-(S,R)-sulfoximine at a dosage of 1 mmol/kg body wt for 2 hr had 45% of the NP-SH content as compared with kidneys of saline-treated controls. The irreversible binding of [3H]DES metabolites was increased by 60% in renal cortical slices from L-buthionine sulfoximine-treated hamsters. Non-extractable binding of [3H]DES metabolites to renal DNA was not observed. These results suggest that GSH, the predominate NP-SH in the cell, protects against the irreversible binding of DES metabolites to cellular macromolecules.

Animals↗
PubMed1987

Identification of an estrogen-responsive element from the 5'-flanking region of the rat prolactin gene.

The DNA sequences which interact with the estrogen receptor and which mediate the estrogenic regulation of prolactin gene transcription have been investigated by the use of receptor-DNA-binding experiments and gene transfer studies. Nitrocellulose filter binding assays using highly purified estrogen receptor and cloned fragments of the 5'-flanking region of the rat prolactin gene demonstrate that the receptor selectively binds to DNA sequences located between nucleotides -1713 and -1532 with respect to the transcription initiation site. The binding of the estrogen receptor to this region of the prolactin gene was strongly dependent on receptor concentration, suggesting that receptor dimers may be important in DNA binding. These data demonstrate that the selective binding of purified estrogen receptor to specific sequences of the rat prolactin gene is an intrinsic property of the receptor and is not due to the interaction of receptor with other proteins. The role of specific prolactin gene sequences in mediating the estrogenic regulation of prolactin gene transcription was confirmed by the use of prolactin-chloramphenicol acetyltransferase fusion genes. These studies demonstrated that sequences upstream of position -1532 are required for estrogen responsiveness. Furthermore, the region of the prolactin gene at -1713 to -1495 was able to confer estrogen responsiveness on the thymidine kinase promoter. Exonuclease III protection experiments further localized the receptor-binding sequences to positions -1587 to -1563. Comparison of the nucleotide sequence of the region of the prolactin gene which binds the estrogen receptor with the sequence of other estrogen-responsive genes suggested the presence of the conserved sequence [sequence in text], which shows similarity to sequences thought to mediate glucocorticoid receptor effects on transcription.

Acetyltransferases↗
PubMed1986

Metabolism and irreversible binding of diethylstilbestrol in the kidney of the Syrian golden hamster.

Oxidative metabolism of [3H]diethylstilbestrol (DES) and the irreversible binding of reactive [3H]DES metabolites to the macromolecules in kidney slices of Syrian golden hamster were investigated. Non-extractable binding of [3H]DES to kidney macromolecules was observed after incubating hamster kidney slices under aerobic conditions (95% O2/5% CO2), but not under anaerobic conditions (100% nitrogen + 2 mM KCN). A number of oxidative metabolites of [3H]DES were detected in the incubation medium of kidney slices incubated under aerobic, but not anaerobic, conditions. The amount of radiolabeled macromolecules formed in male cortical slices under aerobic conditions increased with time of incubation. At a medium concentration of 50 nM [3H]DES, 0.08 pmole [3H]DES equiv./mg dry weight at 30 min and 0.19 pmole [3H]DES equiv./mg dry weight at 120 min were observed. The amount of irreversible [3H]DES-macromolecular complexes also increased with the concentration of [3H]DES in the incubation medium: 1.59 pmole [3H]DES equiv./mg dry weight was formed with 0.5 microM [3H]DES and 21.89 pmole [3H]DES equiv./mg dry weight was formed with 10 microM [3H]DES. Non-extractable [3H]DES binding was detected in all the subcellular fractions of hamster kidney with the highest amount in the microsomal and soluble fractions, followed by the mitochondrial and nuclear fractions. The macromolecular-[3H]DES complexes were solubilized by proteases but not nucleases, suggesting that [3H]DES irreversible binding is principally to the proteins and not the nucleic acids. The cortex as compared with the medulla of the male hamster kidney displayed a 5-fold greater capacity to irreversibly bind [3H]DES metabolites. The male hamster renal cortex showed a 2- to 3-fold greater capacity to form irreversible macromolecular-[3H]DES complexes than the female hamster renal cortex. These data demonstrate that: (1) renal oxidative metabolism of DES results in [3H]DES metabolites binding irreversibly to macromolecules; and (2) the sex and organ site specificity of the [3H]DES-macromolecular binding corresponds with the sex and organ site specificity of renal tumors of the hamster.

Aerobiosis↗
PubMed1986

Computer modeling of estradiol interactions with the estrogen receptor.

Two computer models for the binding of estradiol to estrogen receptors were constructed, based solely upon the thermodynamic constraints of the most likely equilibria involved and known equilibrium constants. Previous data had suggested that the positive cooperativity of the system was dependent upon a monomer-dimer equilibrium (Notides et al., Proc. natn. Acad. Sci., U.S.A. 78 (1981) 4926-4930). Using computer modeling, we confirmed that the thermodynamic constraints of a monomer-dimer equilibrium system result in convex Scatchard plots in agreement with experimental data, including the progression to linearity at low receptor concentrations. This technique yielded estimates of the equilibrium constant for dimerization (approx. 10(10) to 10(14) M-1). The dose-response characteristics of the monomer-dimer equilibrium system revealed steep dose-response curves that were sensitive to the receptor concentration. In contrast, the dose-response curves that did not undergo a monomer-dimer equilibrium system and had a single step equilibrium process were more gradual.

Computers↗