Estradiol-receptor Complex Research Articles

Effects of steroid hormones and antihormones in cultured cells.

Mouse L-929 fibroblasts contain androgen (AR), estrogen (ER) and glucocorticosteroid receptors (GR). The addition of androgens and estrogens (10 to 50 nM) to the culture medium stimulated cell growth and this growth stimulation was inhibited when the corresponding anti-hormones (cyproterone acetate or tamoxifen, 100 nM) were present at the same time. Glucocorticosteroids (10 to 100 nM) had negative effects on cell growth leading even to cell death over 1 microM concentrations. The addition of a new antiglucocorticosteroid RU 486, inhibited the negative effects of glucocorticosteroids. RU 486 displays also anti-progesterone activity, but as L-929 cells have no progesterone receptor, only the anti-glucocorticosteroid action of this new compound was studied. Cell adhesiveness to culture plates was increased by androgen or estrogen, but decreased by glucocorticosteroid and again, the corresponding anti-hormones abolished these positive or negative effects. All these observations can be made in serum-free culture medium supplemented with insulin but not with other hormones, proteins or growth factors. Cyproterone acetate, tamoxifen and RU 486 have specific affinity for AR, ER and GR, respectively. After incubation of whole cells with 3H-tamoxifen or 3H-RU 486, accumulation of either estradiol or glucocorticosteroid receptor complexes in the nuclear fraction of the cell was observed. A tamoxifen-resistant clone of L-929 cells was selected in serum-free medium. This variant had about 50% less ER than tam-sensitive L-929 cells and also less specific antiestrogen binding sites which were found in the particulate fraction of the cells. Shionogi SC-115 mammary cancer cells contain AR and ER. In the presence of testosterone (50 nM) a considerable difference of cell growth was observed. Testosterone increased cell proliferation, and the anti-androgen cyproterone acetate (100 nM) completely inhibited this growth stimulation. RU 486 (100 nM) showed also some antagonistic effect on androgen induced cell growth. The shape of cells cultured with or without androgens was completely different. In the presence of testosterone, clonal growth of the cells was observed with no contact inhibition, whereas in the absence of hormone, cells formed a monolayer. No clonal growth was observed when cyproterone acetate (100 nM) was present at the same time as testosterone in the culture medium. Both testosterone and cyproterone acetate were bound to AR. In the murine thymoma cell lines W7TB and T1M1b, RU 486 is a strong antagonist of the glucocorticosteroid induced cytolytic response.(ABSTRACT TRUNCATED AT 400 WORDS)

17beta-estradiol enhances the production of granulocyte-macrophage colony-stimulating factor in human keratinocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is effective for impaired wound repair. Estrogen is known to enhance wound repair. We examined if 17beta-estradiol (E2) may in vitro enhance GM-CSF production in human keratinocytes. E2 and membrane-impermeable bovine serum albumin-conjugated E2 increased GM-CSF secretion, mRNA stability, and promoter activity. The element homologous to activator protein-1 (AP-1) on the promoter was responsible for the activation. E2 enhanced transcriptional activity and DNA binding of AP-1. E2 transiently generated c-Fos protein, and shifted AP-1 composition from c-Jun homodimers to c-Fos/c-Jun heterodimers in keratinocytes. E2-induced enhancement of GM-CSF secretion, mRNA stability, and promoter activity were not suppressed by estrogen receptor antagonist ICI 182,780, however, suppressed by conventional protein kinase C inhibitor Gö6976 and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK). Gö6976 and PD98059 suppressed E2-induced c-Fos expression and enhancement of DNA-binding and transcriptional activity at AP-1. E2 induced membrane translocation of protein kinase Calpha, which was suppressed by phosphatidylinositol (PI)-specific phospholipase C (PLC) inhibitor U73122. E2 stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), which was suppressed by PD98059, Gö6976, and U73122. E2 transiently generated inositol 1,4,5-triphosphate in keratinocytes, which was suppressed by U73122 and guanine nucleotide-binding protein inhibitor. These results suggest that E2 may enhance GM-CSF production via guanine nucleotide-binding protein-coupled membrane receptors and signaling cascade of PI-specific PLC/protein kinase Calpha/MEK/ERK.

DNA-binding properties of the overexpressed recombinant estrogen receptor alpha.

Estrogen receptor alpha was overexpressed in COS-7 cells by transformation of an expression vector with the full length open reading frame of the receptor alpha. The nuclear estradiol-receptor complex and the soluble receptor from the COS-7 cells were cross-linked with an estrogen response element (ERE), which was substituted with 5-bromo-2'-deoxyuridine (BrdUVRE), as the receptor dimers by UV irradiation. In gel retardation analysis, the specific bindings of both nuclear and soluble receptors to ERE were decreased with increasing of KCl concentration compared with 0.1 M KCl. The ionic interactions of both receptors with ERE are thought to be similar.

Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells.

The mechanism by which estradiol acts on cell multiplication is still unclear. Under conditions of estradiol-dependent growth, estradiol treatment of human mammary cancer MCF-7 cells triggers rapid and transient activation of the mitogen-activated (MAP) kinases, erk-1 and erk-2, increases the active form of p21ras, tyrosine phosphorylation of Shc and p190 protein and induces association of p190 to p21ras-GAP. Both Shc and p190 are substrates of activated src and once phosphorylated, they interact with other proteins and upregulate p21ras. Estradiol activates the tyrosine kinase/p21ras/MAP-kinase pathway in MCF-7 cells with kinetics which are similar to those of peptide mitogens. It is only after introduction of the human wild-type 67 kDa estradiol receptor cDNA that Cos cells become estradiol-responsive in terms of erk-2 activity. This finding, together with the inhibition by the pure anti-estrogen ICI 182 780 of the stimulatory effect of estradiol on each step of the pathway in MCF-7 cells proves that the classic estradiol receptor is responsible for the transduction pathway activation. Transfection experiments of Cos cells with the estradiol receptor cDNA and in vitro experiments with c-src show that the estradiol receptor activates c-src and this activation requires occupancy of the receptor by hormone. Our experiments suggest that c-src is an initial and integral part of the signaling events mediated by the estradiol receptor.

A 67 kDa non-hormone binding estradiol receptor is present in human mammary cancers.

The presence of large amounts of a 67 kDa estradiol receptor that does not bind hormone was observed in 8 to 37 human mammary tumors (34 malignant and 3 benign). This form of receptor was detected by its conversion to hormone binding receptor by an endogenous tyrosine kinase in vitro. All 8 tumors were malignant. In these, the incubation of cytosol with ATP was seen to cause a 1- to 5-fold increase in estradiol-specific binding sites. These sites bound estradiol with physiological affinity, and their appearance was associated with tyrosine phosphorylation of estradiol receptor. The enzyme converting the non-hormone binding receptor into the hormone binding receptor is largely present in cytosol and scarce in membranes. It has been extensively purified. It is a 67 kDa protein under denaturating conditions, binds calmodulin-Sepharose in a Ca2+-dependent manner, is stimulated by Ca2+ and calmodulin, phosphorylates exogenous actin, is activated by the estradiol-receptor complex. The enzyme interacts with antibodies directed against the carboxy-terminal and catalytic domains of c-src. Therefore, it is a putative new member of the large c-src-related kinase family. Human mammary cancers with significant amounts of 67 kDa non-hormone binding receptor show relatively low levels of hormone binding estradiol receptor. The presence of non-hormone binding receptor that can be activated by in vitro tyrosine phosphorylation suggests that functional interaction of estradiol receptor with tyrosine kinases is altered in malignant tumors and has bearing on loss of hormone dependence and progression of the mammary cancer malignancy.

Properties of a purified estradiol-dependent calf uterus tyrosine kinase.

A uterus tyrosine kinase has been purified to a single 67-kDa protein when analyzed by SDS-PAGE. Under nondenaturing conditions the molecular weight of the enzyme ranges from 114 to 136 kDa, depending on the procedure employed. The kinase binds calmodulin in a Ca(2+)-dependent manner and the ATP analog [(fluorosulfonyl)benzoyl]adenosine. The purified enzyme phosphorylates the phosphatase-treated uterus estradiol receptor on tyrosine and activates its hormone binding. The kinase phosphorylates actin, calmodulin, and histone H2B. Whatever the substrate, the enzymic activity is dependent on purified estradiol-receptor complex and is activated by Ca(2+)-calmodulin. The kinase activates and phosphorylates the human estradiol receptor (HEO) within the hormone binding domain (HBD) [Migliaccio, et al. (1989) Mol Endocrinol. 3, 1061-1069] as well as four of the five mutants of the HEO obtained by substituting each of the five tyrosine residues present in the HBD of the receptor with phenylalanine by site-directed mutagenesis. The mutant substituted at tyrosine 537 is the only one that is neither phosphorylated nor activated by the kinase. This proves a causal relationship between the phosphorylation of estradiol receptor on tyrosine 537 and its hormone binding activity. A synthetic peptide corresponding to 11 out of 13 amino acids surrounding tyrosine at position 537 of the human estrogen receptor can be phosphorylated by the kinase. This and other findings indicate that this kinase, unlike other tyrosine kinases, phosphorylates tyrosyl residues with acidic amino acids close to the carboxyl side.

Effect of pesticides on oestradiol-receptor complex formation in rat uterus cytosol

The effect of γ-1,2,3,4,5,6-hexachlorocyclohexane (lindane), 2-chloro-4-ethylamino-6-isopropylamino- s-triazine (atrazine) and 2-methylthio-4,6-bis-isopropylamino- s-triazine (prometryne) on the formation of a specific oestradiol-receptor complex in the rat uterus cytosol has been examined in vitro and in vivo. Both in vitro and in vivo, the pesticides significantly ( P < 0.001) inhibited the formation of the complex in the rat uterus cytosol. The decrease in the number of free specific binding sites on the receptors was determined. The affinity of binding was not modified under the influence of pesticides, and the K d value was of the same order of magnitude (10 −9 m). The inhibition was found to be fully non-competitive.

Dissociation kinetics of the uterine estrogen receptor-estradiol complex are unaltered in aging C57BL/6 mice

Several, though not all, estrogen-dependent phenomena show reduced responsiveness to estradiol (E 2) during aging. One factor contributing to this reduced sensitivity could be an increase in the dissociation rate of the estrogen receptor-hormone complex. We therefore studied the effect of aging on the dissociation rate of 3HE 2 from the uterine cytosolic estrogen receptor (ER) of C57BL/6 mice that had been ovariectomized 48 h earlier. Measurements were made at 28°C at two concentrations of cytosol. In dilute cytosol ([ER] = 0.01 nM) the dissociation of ER- 3HE 2 displayed a single phase, first order profile which did not differ among young (4–6 month), middle-aged (15–18 month) and old (23–30 month) mice. In more concentrated cytosol ([ER] = 2 or 6 nM) the dissociation of ER- 3HE 2 displayed a biphasic first order profile that consisted of an initial rapidly dissociating phase followed by a more slowly dissociating phase. There was no effect of age on the dissociation rate constant ( K) of either the rapid ( K −1) or slow ( K −2) phase. Shortening the time alotted for the concentrated cytosol to equilibrate with 3HE 2 before measuring the dissociation rate reduced the fraction of the receptor hormone complex that dissociated in the slow phase, but, once again, the dissociation profiles did not differ between age groups. These results indicate that uterine ER dissociation kinetics remain unaltered in middle-aged and old mice and are therefore unlikely to play a role in the attenuated responsiveness to estrogen during aging.

Studies on the activation of the oestrogen receptor bound to the anti-oestrogens 4-hydroxytamoxifen and ICI 164,384 by using three monoclonal antibodies

Three monoclonal antibodies, H222, H226 and D547, which provided evidence of the structural transformation and change in exposure of the functional domains of the oestrogen receptor from fetal guinea-pig uterus upon activation, were used to study the receptor bound to the anti-oestrogens 4-hydroxytamoxifen and ICI 164,384. No differences in the structure of non-activated 4-hydroxytamoxifen- and ICI 164,384-receptor complexes, as compared with the oestradiol-receptor complex, were detected by the three monoclonal antibodies. When heated at 28 degrees C, both anti-oestrogen-receptor complexes became capable of binding the D547 antibody, which reacts selectively with the activated receptor; however, this binding was lower than that of the oestradiol-receptor complex. The interaction with the H226 antibody showed that anti-oestrogens can induce receptor dimerization, but to a lesser extent than oestradiol. In addition, both anti-oestrogen-receptor complexes can bind to DNA-cellulose and are retained in nuclei from intact cells at 28 degrees C, but less efficiently than the oestradiol-receptor complex. On the other hand, the nuclear receptor seems to have a similar dimeric structure when bound to either anti-oestrogens or oestradiol, as detected by the three monoclonal antibodies. The data suggest that 4-hydroxytamoxifen and ICI 164,384 induce and impaired activation of the oestrogen receptor; this difference, although quantitative rather than qualitative, might be related to the partial agonistic action of these anti-oestrogens in the fetal guinea-pig uterus.

Cell-Free Interaction of the Estrogen Receptor with Mouse Uterine Nuclear Matrix: Evidence of Saturability, Specificity, and Resistance to KC1 Extraction

An integral part of the mechanism of estrogen action is the interaction of estrogen receptor (ER) complexes with specific nuclear acceptor sites to effect alterations in genomic expression. The localization of nuclear acceptor sites has been in question, but an increasing body of indirect evidence implicates the nuclear matrix. To assess the binding characteristics of [3H]estradiol-receptor complexes (3HER) to nuclear matrix, ER from ovariectomized mice was partially purified by ammonium sulfate precipitation and incubated under cell-free conditions with mouse uterine nuclear matrix at 4 C. The binding capacity of the nuclear matrix was determined to be 36.4 +/- 5.7 fmol/100 micrograms DNA, with a Kd of 0.23 +/- 0.03 nM. Binding to nuclear matrix sites was specific, as determined by the ability of increasing concentrations of unlabeled ER complexes to inhibit binding of 3HER. Spleen, used as a nontarget tissue, contained fewer binding sites (n = 4.07 fmol/100 micrograms DNA) than matrix from liver (n = 14.2). The binding affinity was the same in all three tissues. Injection of animals with estradiol before death was associated with loss of assayable nuclear matrix binding sites, implying occupancy of sites by ER in vivo. Unbound receptor (R) also demonstrated the ability to bind to uterine matrix (n = 40.2 +/- 2.7 fmol/100 micrograms DNA; Kd = 0.26 +/- 0.05 nM) as well as to competitively inhibit the binding of 3HER complexes. However, heat-inactivated receptor displayed no binding or competing activity, nor did the progesterone receptor. The two forms of the receptor can be functionally distinguished by extraction with 0.6 M KCl; 43% of ER, but no R, were resistant to KCl extraction. These results indicate that nuclear acceptor sites are associated with the nuclear matrix. Furthermore, these sites demonstrate the criteria expected of specific binding sites, i.e. high affinity, limited capacity, hormone receptor, and relative tissue specificity. The apparent association of uncomplexed receptor to nuclear acceptor sites may explain the uterine tissue nuclear localization of ER in the absence of hormone.

Prolonged cultivation of rat uterine cells with preserved estrogen responsiveness

A rat uterine cell culture was prepared as an experimental system for investigation of mechanisms of steroid hormone actions. Cells frequently supplemented with fresh medium were successfully cultured for 4 weeks through 2 successive passages. Studies of estrogen responsiveness in the primary culture as well as in it's first subculture were performed by a small scale uptake assay for determination of specific steroid binding. Scatchard analysis of specific ovarian hormone binding confirmed that cultured uterine cells preserve both estradiol and progesterone receptors. Characteristics of specific [ 3H]estradiol binding detected in cells of the first subculture were comparable to those obtained in the initial primary culture. The number of specific estradiol binding sites was diminished to one third of the initial values only in cells of the second subculture, 22 days after isolation of cells from tissue. In the primary culture and in it's first subculture the cells responded to estradiol with a 2–3-fold increase in progesterone receptor level. The subcellular distribution of steroid receptors was also studied; estradiol receptor complexes were detected predominantly in the nuclei whereas progesterone receptors were nearly equally distributed between nuclei and cytosol.

Covalent immobilization of the estrogen receptor to an electrostatically neutral N-hydroxysuccinimide ester derivative of agarose

Immobilization of the estrogen receptor to the N-hydroxysuccinimide ester of succinylethylenediaminocarboxymethyl agarose (Reagent B) is described and compared with that to the charged N-hydroxysuccinimide ester derivative (Reagent A), previously described. The time course for immobilization was examined. Thirty-six percent of the input receptor was immobilized within 1 h. The optimum pH in immobilization is 7.0–7.4. The dissociation rate of [ 3H]estradiol(3,17β−1,3,5(10)-estratriene) from the [ 3H]estradiol-receptor complex immobilized to Reagent B was similar to that in Reagent A. The receptor immobilized to Reagent B was saturated with estradiol at 5 h. The [ 3H]estradiol concentration necessary for saturation was 10 nM. The dissociation constant (K D) for the receptor immobilized to Reagent B was 0.95 × 10 −9 M.

Mapping on the calf estrogen receptor of the binding domain for an antibody interfering with receptor activation

The localization on the calf estrogen receptor of the binding domain for B36 (an IgM antibody which prevents and reverses the effects of receptor activation) has been studied by means of controlled proteolysis of the receptor-estradiol complex using trypsin, chymotrypsin, and papain. We successively determined for intact and proteolyzed receptor-estradiol complex (i) the abilities of estradiol-binding species to aggregate in low salt medium, to bind to nonspecific DNA absorbed onto cellulose, and to interact with B36 antibody in sucrose gradients; (ii) the hydrodynamic properties of estradiol-binding species, by gel permeation chromatography and sucrose gradient centrifugation in high salt media and (iii) the molecular weights of B36-reactive species, by immunoblot analysis. Three tryptic receptor fragments of M r 36,000, 34,000, and 33,000 and two chymotryptic fragments of M r 36,000 and 33,000 included both the hormone- and B36-binding domains but did not interact with DNA, whereas at least two receptor fragments resulting from the action of chymotrypsin and papain bound estradiol with high affinity but interacted neither with DNA nor with B36. Taking into account these results and assuming that structure of the calf estrogen receptor is similar to those of sequenced estrogen receptors (which show a highly conserved organization with considerable homologies in the functional domains), we propose that the B36-binding domain is located either between the DNA- and hormone-binding domains (model I) or at the C-terminal end of the estrogen receptor (model II). The regions that include the main proteolytic cleavage sites of the receptor are also specified, and the abilities of the two models of the calf estrogen receptor to account for the effect of B36 on receptor activation are discussed.

Subunit composition of the estrogen receptor: Involvement of the hormone-binding domain in the dimeric state

The purified estrogen receptor (ER) whether in 9 or 5 S molecular form, binds more than one molecule of the monoclonal antibody JS 34/32 (Redeuilh, G., Moncharmont, B., Secco, C., and Baulieu, E.-E. (1987) J. Biol. Chem. 262, 6969-6975). We now have investigated the effects of controlled trypsin proteolysis and of a dissociating chaotropic salt (NaSCN) on the structure of the estrogen receptor covalently labeled with radioactive tamoxifen aziridine. When the DNA-binding transformed 5S ER was dialyzed against a buffer containing 0.5 M NaSCN, it was converted into a form sedimenting at 3.7 S +/- 0.1 (n = 3). It reverted to the 5 S molecular form when NaSCN was dialyzed away. Fluorographic analysis of both the 5 and 3.7 S ER following SDS-gel electrophoresis revealed one main band corresponding to Mr congruent to 66,000. After limited trypsin treatment of the 5 S ER, tamoxifen aziridine-binding protein sedimented at 4.3 S +/- 0.1 (n = 5), had a Stokes radius of 3.6 nm (calculated Mr = 65,000), and did not bind DNA. The same form was obtained after limited trypsin digestion of the ER bound to DNA-cellulose or to hsp 90 (the nontransformed 8-9 S ER molecular form). This 4.3 S trypsinized ER was reversibly dissociated by NaSCN into a congruent to 3 S +/- 0.1 (n = 3) molecular form. Fluorographic analysis of both the 4.3 and 3 S ER after SDS-gel electrophoresis showed one main radioactive band of Mr congruent to 30,000. Taken together our results suggest that 1) the 5 S ER is a homodimer of two Mr congruent to 66,000 hormone binding subunits which may be released as such from the nontransformed 8-9 S ER, 2) the trypsin digestion products yield two carboxyl-terminal fragments of Mr congruent to 30,000 that remain in the form of a dimer having lost their DNA-binding region, and 3) the trypsin cleavage would occur within the region located between the hormone-binding domain and the DNA-binding domain. These data indicate that the dimerization of the receptor occurs through hydrophobic interaction of its hormone-binding domain but cannot exclude that other part(s) of the receptor may also contribute to the dimer formation. The dimerization may be critically involved in the mechanism by which estradiol-receptor complexes promote change of gene transcription.

Estradiol receptor-nuclear interactions in aging mouse uteri: The role of DNA and nuclear matrix

We have studied the interactions of estradiol receptor (ER) with nuclear DNA or matrix (NM) in the uteri from young, middle-aged and old mice. Recombination studies using heatactivated ER and nuclear subfractions from various age groups suggested a 30% reduction for DNA binding ( P < 0.05) but not for NM with aging. Cross-incubation studies of heat-activated ER with nuclear subfractions from mice of all three age subgroups showed that this reduced DNA binding ability followed the onset of anestrous and did not result from proteolytic degradation of ER complexes.

Kinetics and in vitro origin of the temperature-dependent transition of the estrogen receptor monomer.

Partitioning of estrogen receptors in aqueous two-phase polymer systems has provided the basis for a detailed kinetic analysis of the effects of temperature on estrogen receptor (ER) structure in vitro. Exposure to temperatures of 0-30 degrees C increased the rate of change in ER partition coefficients by up to 100-fold but did not affect the final extent of the process. The temperature-dependent change in ER partition coefficients was characterized by a linear Arrhenius plot and an activation energy of 25 kcal/mol. The rate of the temperature-dependent ER transition (28 degrees C) was found to be unaffected by greater than 50-fold changes in receptor concentration, which indicates that the temperature-dependent change in partition coefficients reflects a first-order process. The partition coefficients of heated ER were unaffected by subsequent 18-h incubations at 0 degree C, indicating that the temperature-dependent ER transition is irreversible in vitro. Direct heating of the unoccupied ER resulted in both a change in ER partition coefficients and a loss of ER binding sites. The temperature-dependent change in unoccupied ER partition coefficients was complete within 30 min at 28 degrees C and yielded a first-order rate constant that was the same as that obtained for heating the receptor-estradiol complex at 28 degrees C. In contrast, the loss of unoccupied ER binding sites that occurred during 28 degrees C incubations did not reach completion after 150 min of heating and was found to behave as a second-order process.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro interaction of estradiol receptor with Ca2+-calmodulin.

Estradiol receptor (ER) activity requires interaction with hormone and specific DNA sequence. We now report that this receptor also interacts with calmodulin (CaM), the major intracellular mediator of Ca2+ action in eucaryotic cells. This interaction has been observed using both CaM-Sepharose and [125I]CaM. Crude and purified [3H]ER complex show high affinity interaction with CaM-Sepharose [dissociation constant (Kd) 0.12 and 0.16 nM, respectively]. Unoccupied receptor shows a similar high affinity interaction. Tamoxifen-ER complex also binds to CaM-Sepharose. Several findings show that this CaM-ER interaction is very specific: lack of this interaction has been observed in the presence of trifluoperazine, an inhibitor of protein binding to CaM; the receptor binds neither Sepharose, nor parvalbumin-Sepharose; competition of interaction of [3H]ER complex with CaM-Sepharose is observed by cold ER complex; rat liver glucocorticoid receptor does not bind to CaM-Sepharose. The interaction of purified receptor with 125I-labeled CaM has been detected by various techniques: centrifugation through sucrose gradient of CaM incubated with receptor shows that CaM binds to a protein forming a complex sedimenting at 5 S. This complex is shifted to the 7.5 S region by a monoclonal antireceptor antibody. Incubation of CaM with receptor followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography of the immunoprecipitated receptor shows that [125I]CaM coprecipitates with the receptor. Competition of this interaction by an excess of cold CaM is observed. Interaction of the receptor with CaM is also observed by the overlay technique.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological differences between the estradiol-, tamoxifen- and 4-hydroxy-tamoxifen-estrogen receptor complexes detected by two monoclonal antibodies

Two monoclonal antibodies (D547 and H222), obtained against the estrogen receptor from MCF-7 breast cancer cells, were used to study the estrogen receptor from fetal guinea-pig uterus bound to estradiol or to the antiestrogens tamoxifen and 4-hydroxytamoxifen. The estradiol-receptor complex binds partially to the monoclonal antibody D547, shifting its sedimentation coefficient in high salt sucrose density gradients from 4.5S to 7.5S. Recently, we demonstrated that the form selectively recognized by this monoclonal antibody is the activated form of the receptor. The estrogen receptor complexed with tamoxifen or 4-hydroxytamoxifen is also partially recognized by this monoclonal antibody but the fraction of total receptor bound to the antibody is significantly less than for the receptor complexed with estradiol. Another series of experiments showed that the monoclonal antibody H222, which recognizes a different antigenic site on the receptor molecule, binds all the estradiol-receptor complex (independently of the degree of activation), shifting its sedimentation coefficient to 7.5S. However, even if all the 4-hydroxytamoxifen-receptor complex is bound by this antibody, only a fraction of the receptor is recognized when it is complexed with tamoxifen. These data show different interactions between the estradiol-, tamoxifen- and 4-hydroxytamoxifen-receptor complexes and the two monoclonal antibodies tested and suggest that these compounds induce different conformational modifications of the estrogen receptor molecule.

Interaction of two nonhistone proteins with the estradiol response element of the avian vitellogenin gene modulates the binding of estradiol-receptor complex.

The DNA sequence corresponding to the estradiol response element has been synthesized and tested in vitro for the binding of specific proteins. Gel retardation experiments combined with dimethyl sulfate protection experiments revealed that this region binds two nonhistone proteins (NHPs). One of them, NHP-1, has a molecular weight of 70,000 and binds specifically to the dyad symmetry sequence GGTCAGCGTGACC. The NHP-1 can be separated from the estradiol receptor chromatographically; it does not bind estradiol and does not cross-react with an antibody directed against the estradiol receptor. A series of synthetic "mutant" oligonucleotides were tested in a protein-DNA binding competition assay. Deletion of the GCG in the center of the dyad symmetry sequence suppressed the binding of NHP-1 by 90%, and the conversion of any GC pair to an AT pair decreased the affinity of the binding site for NHP-1. Methylation of the two CpGs on both strands of the dyad symmetry sequence decreased the affinity of the binding site for NHP-1 by 60%, whereas hemimethylation of the same structure did not inhibit the binding of NHP-1. NHP-1 and NHP-2, the NHP binding to the DNA next to the dyad symmetry sequence, bind exclusively to double-stranded DNA. NHP-2 has a molecular weight of 60,000. NHP-1 and NHP-2 are neither tissue nor species specific. In vitro reconstitution experiments show that NHP-1 and NHP-2 increase the binding efficiency of the estradiol-receptor complex to the estradiol response element.

Immunological difference between ribonuclease and temperature, time and salt-induced forms of the estrogen receptor detected by a monoclonal antibody

The effect of RNAase A on the activation of the estrogen receptor from fetal guinea pig uterus was studied by DNA-cellulose binding assay and immunorecognition of the estradiol-receptor complex by the monoclonal antibody D547 raised against the human estrogen receptor. After RNAase treatment at 4°C or 25°C the binding of the receptor to DNA-cellulose doubled. This stimulation was partially prevented by sodium molybdate. RNAase treatment did not modify the interaction of the receptor with the monoclonal antibody D547; this antibody, as was demonstrated previously, selectively recognizes the activated form of the receptor when activation has been induced by temperature, time or high salt concentrations. In addition, RNAase had little or no effect on the transformation of the 8–9 S receptor to more slowly sedimenting forms under low salt concentrations. These observations suggest that even if RNAase induces receptor activation, which can be inferred from the increase in its binding to DNA-cellulose, the conformational modifications of the receptor molecule involved in this process are apparently different from those induced by factors such as temperature, time or high-salt concentrations.