Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanism whereby antiestrogens alter the ability of the estrogen receptor (ER) to enhance transcription of estrogen-regulated genes is largely unknown. The effect that selected estrogenic and antiestrogenic ligands have on binding of ER to specific DNA sequences, estrogen responsive elements (EREs) has been quantitated. No differences in purification properties of calf uterine ER liganded with 4-hydroxytamoxifen (4-OHT-ER), ICI 164,384 (ICI 164,384-ER) or estradiol (E2-ER) were detected. A microtiter well plate assay was employed in which liganded ER bound to plasmid DNA is preferentially retained compared to free liganded ER. Binding of E2-ER, 4-OHT-ER, or ICI 164,384-ER was measured to plasmids containing or lacking a 38bp consensus ERE in vitro. The EREs tested contain an inverted repeat (5'-CAGGTCAGAGTGACCTG-3'). Both E2-ER and 4-OHT-ER showed similar high affinity specific binding (Kd = 0.24 and 0.16 nM, respectively) to one copy of the ERE. ICI 164,384-ER did not bind to plasmids containing one ERE. At saturation, however, 4-OHT-ER binding was about 50% of that observed for E2-ER. When the plasmid contained 3 or 4 tandem copies of the ERE, binding of E2-ER, 4-OHT-ER, and ICI 164,384-ER binding was measurable. E2-ER bound in a cooperative manner as suggested by convex Scatchard plots and Hill coefficients > 1.5. In contrast, 4-OHT-ER binding displayed much reduced cooperativity, and ICI 164,384-ER did not display cooperative binding. From these results, we propose that the conformation of ER induced by 4-OHT reduces its binding capacity to this consensus ERE without altering its affinity of binding. Furthermore, higher order protein-protein interactions between antiestrogen-liganded ER bound to DNA differ from those of E2-ER bound to ERE.
J Steroid Biochem Mol Biol 1992 Oct
PMID:Antiestrogen-liganded estrogen receptor interaction with estrogen responsive element DNA in vitro. 139 Feb 77

The antiestrogen tamoxifen is used in the treatment of hormone-responsive breast cancer. However, therapeutic failure has frequently been observed in both patients and animal models after long term treatment. We have studied the effect of a point mutation that leads to the substitution of Val for Gly at codon 400 in the ligand-binding domain of the estrogen receptor (ER) on estrogenic and antiestrogenic activities of 4-hydroxytamoxifen (4-OHT) and its derivatives. Stable ER transfectants derived from MDA-MB-231 CL10A, an ER-negative breast cancer cell line, have been used in these studies. 4-OHT and its fixed ring derivatives showed more estrogen-like activity in ER transfectants than in MCF-7, an ER-positive breast cancer cell line. In this study, 4-OHT was a partial agonist of cell growth in the transfectant S30 cells, which express the wild-type ER. However, it was a full agonist in the mutant ER transfectant ML alpha 2H, which expressed ER with Val at codon 400. The increased estrogenic activity of 4-OHT in ML alpha 2H cells was not due to the preferential isomerization of trans 4-OHT to cis 4-OHT, since the nonisomerizable fixed ring trans 4-OHT was a partial agonist for cell growth in S30 cells and was a full agonist in ML alpha 2H cells. Transient transfection using a reporter plasmid containing an estrogen response element demonstrated that fixed ring trans 4-OHT had estrogenic activity in ML alpha 2H cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1992 Dec
PMID:Point mutation of estrogen receptor (ER) in the ligand-binding domain changes the pharmacology of antiestrogens in ER-negative breast cancer cells stably expressing complementary DNAs for ER. 149 96

The mechanism by which antiestrogens antagonize the ability of estrogen receptor (ER) to induce the transcription of estrogen-regulated genes is only partially understood. To examine the effect of estrogen responsive element (ERE) stereoalignment and flanking sequences on estradiol-liganded ER (E2-ER)-ERE and antiestrogen-liganded ER (4-hydroxytamoxifen-liganded ER or 4-OHT-ER)-ERE binding, several dimeric EREs, containing a perfect inverted repeat (5'-GGTCAgagTGACC-3') but lacking the AT-rich flanking sequences typical of highly estrogen-responsive promoters, were cloned into a plasmid vector. The ERE centers of symmetry were spaced 1.5, 2.0, 3.0, 6.4 and 6.7 helical turns apart. E2-ER and 4-OHT-ER binding to these constructs was specific and saturable, but orientation-independent and, in contrast to our earlier work with E2-ER binding to AT-rich EREs, not cooperative. The affinity of E2-ER binding decreased as the distance between adjacent EREs was increased, suggesting that E2-ER binding to closely spaced EREs is more stable (Kd = 0.38, 0.58, 0.83, 1.23, and 0.96 nM, respectively, for the above spacings). In contrast, the affinity of 4-OHT-ER binding increased with increased ERE spacing (Kd = 2.90, 4.79, 1.39, 1.77, and 0.92 nM, respectively). The presence of AT-rich sequences flanking the ERE increased the binding affinity of E2-ER and 4-OHT-ER, an increase reflected in slower dissociation rates of ER from these EREs. The AT-rich sequence also enhanced the binding capacity of E2-ER but not 4-OHT-ER. Since the binding capacity of 4-OHT-ER is identical with or without an AT-rich region, we suggest that flanking sequences are more important in stabilizing E2-ER binding and may be critical for cooperative binding to stereoaligned EREs.
J Steroid Biochem Mol Biol 1993 Dec
PMID:Differential impact of flanking sequences on estradiol- vs 4-hydroxytamoxifen-liganded estrogen receptor binding to estrogen responsive element DNA. 827 5

Our previous studies have shown that even in the absence of estrogen, the estrogen receptor (ER) is still involved in growth by way of its conversion to a transcriptionally active state by growth inducing cytokines. The following paper now provides evidence that under more physiological conditions, the ER within the GH3 cell line used for the previous investigations, not only controls growth, but that transcriptional activity of the receptor is required for cell survival. Therefore when GH3 cells, maintained under serum and steroid replete conditions, are exposed to the pure antiestrogen ZM 182780 (10 nM), marked cell death is observed 72-120 h after first exposure. Studies on the nature of this cell death suggested that it had some of the reported characteristics of apoptosis or programmed cell death. Removal of steroids from the culture medium also resulted in cell death and this was enhanced by the addition of the pure antiestrogen. Both steroid withdrawal and ZM 182780 induced cell death was completely reversed by the inclusion of estrogens in the steroid free culture medium. In contrast, the non-steroidal antiestrogen, 4-hydroxytamoxifen (4-OHT) was not able to enhance steroid withdrawal death and at 1 microM, this compound was shown to have marked ER agonist activity. Further studies on the addition of conditioned medium from high density GH3 cell cultures, to low density steroid free cells, strongly suggested that the ER within these cells was responsible for the production of autocrine/paracrine survival factors.
J Steroid Biochem Mol Biol 1995 Dec
PMID:Estrogen receptor blockade by the pure antiestrogen, ZM 182780, induces death of pituitary tumour cells. 854 Dec 29

Estradiol-liganded estrogen receptor (E2-ER) binds EREs with a stoichiometry of one E2-ER dimer per estrogen response element (ERE). In contrast, although 4-hydroxytamoxifen (4-OHT)-liganded ER (4-OHT-ER) binds EREs with high affinity, its saturation ERE binding capacity is consistently half that of E2-ER, giving an apparent stoichiometry of one 4-OHT-ER monomer per ERE. Here we show that one molecule of 4-OHT ligand dissociates from the ER dimer apparently during the process of binding to DNA. Under equilibrium conditions, the type I antiestrogen tamoxifen aziridine (TAz), covalently attached to ER (TAz-ER), binds a single ERE with high affinity (Kd = 0.27 nM), comparable to that of E2-ER and 4-OHT-ER. In contrast to 4-OHT-ER, the ERE binding stoichiometry of TAz-ER was identical to that of E2-ER: one dimeric receptor per ERE. By measuring [3H]ligand that was initially bound to ER, a significant loss of [3H]4-OHT from ER was detected after ERE binding, whereas all [3H]E2 or [3H]TAz remained ER-bound. These results confirm that one molecule of 4-OHT ligand dissociates from the ER dimer as a consequence of ERE binding. Binding of 4-OHT and TAz are likely to induce a conformation in ER dimers that alters their capacity for gene activation. Upon ER binding to DNA, this conformation reveals itself by allowing 4-OHT dissociation, and predictably would allow TAz dissociation were it not bound covalently.
J Steroid Biochem Mol Biol 1996 Jan
PMID:Dissociation of 4-hydroxytamoxifen, but not estradiol or tamoxifen aziridine, from the estrogen receptor as the receptor binds estrogen response element DNA. 864 17

Quantitative DNase I footprinting assays were employed to simultaneously measure the amount of estrogen receptor (ER) bound to each site in constructs containing multiple estrogen response elements (EREs). These assays revealed identical, high affinity ER-ERE binding, Kd of approximately 0.25 nM, for estradiol-liganded ER (E2-ER), 4-hydroxytamoxifen liganded ER (4-OHT-ER), tamoxifen aziridine liganded ER (TAz-ER), and unliganded dimeric ER, for each ERE in constructs containing up to four tandem EREs. Increasing concentrations of ER resulted in the same pattern of occupancy for each ERE, whether or not the site was located near other EREs. Similarly, the presence or absence of E2, 4-OHT, or TAz ligand did not change ER-ERE interaction. Since activated ER-ERE binding affinity is identical, whether ER is liganded or unliganded, ligand cannot regulate ER-ERE binding affinity. These results support the hypothesis that ligand-dependent conformational changes primarily determine how ER interacts with components of the transcription initiation complex that mediate gene transactivation. In addition, footprint assays revealed that, following ER binding, an AT-rich site adjacent to the ERE becomes hypersensitive to DNase I digestion. This sequence may be easily or intrinsically bent, assisting in recruiting ER to ERE sites.
J Steroid Biochem Mol Biol 1996 Apr
PMID:Footprint analysis of estrogen receptor binding to adjacent estrogen response elements. 880 85

To determine whether accessory proteins mediate the ligand- and DNA sequence-dependent specificity of estrogen receptor (ER) interaction with DNA, the binding of partly purified vs highly purified bovine ER to various estrogen response elements (EREs) was measured in the presence of different ER ligands. Partly purified estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned tandem EREs flanked by naturally occurring AT-rich sequences, with a stoichiometry of one E2-ER dimer per ERE. In contrast, highly purified E2-ER binds with a 10-fold lower affinity and non-cooperatively to EREs flanked by the AT-rich region. Moreover, the binding stoichiometry of highly purified E2-ER was 0.5 E2-ER dimer, or one monomer per ERE, independent of the ERE flanking sequence. Interestingly, the binding of ER liganded with the antiestrogen 4-hydroxytamoxifen (4-OHT-ER) was non-cooperative with an apparent stoichiometry of 0.5 4-OHT-ER dimer per ERE, regardless of ER purity or ERE flanking sequence. We recently showed that when 4-OHT-ER binds DNA, one molecule of 4-OHT dissociates from the dimeric 4-OHT-ER-ERE complex, accounting for the reduced apparent binding stoichiometry. In contrast, ER covalently bound by tamoxifen aziridine (TAz) gave an ERE binding stoichiometry of one TAz-ER dimer per ERE, and TAz-ER binds cooperatively to multiple AT-rich EREs, regardless of the purity of the receptor. We have obtained evidence that purification of ER removes an accessory protein(s) that interacts with ER in a sequence- and/or DNA conformational-dependent manner, resulting in stabilization of E2, but not 4-OHT, in the ligand binding domain when the receptor binds to DNA. We postulate that retention of ligand by ER maintains the receptor in a conformation necessary to achieve high-affinity, cooperative ERE binding.
J Steroid Biochem Mol Biol 1996 Dec
PMID:Stability of the ligand-estrogen receptor interaction depends on estrogen response element flanking sequences and cellular factors. 901 Mar 47

Bovine estrogen receptor (ER) was purified to near homogeneity by estrogen response element (ERE) affinity chromatography, and its ERE binding ability was measured in vitro. Highly purified ER bound EREs with reduced affinity compared to partially purified ER. Partially purified ER contained hsp70, but highly purified ER did not. We examined whether addition of purified recombinant human hsp70 or purified bovine hsp70 would restore the higher ERE binding affinity, stoichiometry, and ligand retention detected with partially purified receptor and how hsp70 affected the rate of ER-ERE association and dissociation. ER-ERE binding was not affected by antibodies to either constitutive or induced forms of hsp70, regardless of ER purity. Addition of purified hsp70, with or without ATP and Mg2+, did not affect the association or dissociation rates of highly purified liganded ER binding to ERE. hsp70 Did not alter the total amount of ER-ERE complex formed. Similarly, hsp70 did not affect the rate of [3H]estradiol (E2) or [3H]4-hydroxytamoxifen (4-OHT) ligand dissociation from ER in the presence or absence of EREs. These data contrast with a report showing that maximal ERE binding by highly purified recombinant human ER required hsp70. We conclude that ER, purified from a physiological source, i.e., calf uterus, does not require hsp70 for maximal ER-ERE binding in vitro. Additionally, once ER is activated and bound by ligand, the receptor assumes its proper tertiary structure, and hsp70 does not impact ER ligand binding domain conformation.
J Steroid Biochem Mol Biol
PMID:hsp70 is not required for high affinity binding of purified calf uterine estrogen receptor to estrogen response element DNA in vitro. 945 95

We have shown that 4-hydroxytamoxifen (4-OHT) has estrogen-like effects on induction of TGFalpha mRNA in estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells, transfected with either wildtype (S30 cells) or a codon 351asp-->tyr mutant ER (BC-2 cells). The mutant receptor used to produce the stable transfectants was identified in a tamoxifen-stimulated human breast tumor. We have also demonstrated that raloxifene exhibits a gene-specific estrogen-like effect with mutant ER (BC-2 cells) but not with wildtype ER (S30 cells) (Levenson, A.S., Catherino, W.H. and Jordan, V.C. (1997) Estrogenic activity is increased for an antiestrogen by a natural mutation of the estrogen receptor. J. Steroid Biochem. Mol. Biol., 60, 261-268). We now describe the regulation of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) endogenous gene expression by estradiol (E2) and different antiestrogens in BC-2 cells. Northern blot analyses revealed that 4-OHT and raloxifene have concentration-dependent agonistic (E2-like) effects on the regulation of these genes. In contrast, the pure antiestrogen ICI 182780 alone had no effect but could block the action of E2, 4-OHT and raloxifene. The E2-like effects of non-steroidal antiestrogens in this model system cannot be explained by the mutation in the ER alone because 4-OHT acts as an agonist with wildtype receptor as well. We propose that the clear cut biological expression of estrogen-like qualities with different antiestrogens will in the future serve as an important model to dissect the signal transduction pathway.
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PMID:Agonist activity of antiestrogen-receptor complexes to regulate urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) endogenous gene expression in breast cancer cells. 956 18

The estrogen receptor (ER) is a ligand-activated transcription factor that binds to specific DNA sequences, estrogen response elements (EREs). Estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned EREs that are surrounded by naturally-occurring AT-rich sequences with a stoichiometry of one E2-ER dimer per ERE. When ER is bound by 4-hydroxytamoxifen (4-OHT), the active metabolite of the widely used therapeutic antiestrogen tamoxifen (TAM), the receptor binds to EREs with high affinity. However, one molecule of 4-OHT ligand dissociates from the ER dimer apparently during the process of binding to DNA, yielding a stoichiometry of one [3H]4-OHT molecule per ERE. To determine whether DNA-binding induced ligand dissociation is a general property of type I antiestrogens that are not covalently attached to the ER, we examined the interaction of ER liganded by tamoxifen (TAM) with EREs. We demonstrate that TAM-ER binds EREs with lower affinity than E2-ER, 4-OHT-ER, or ER liganded by the covalent antiestrogen tamoxifen aziridine. Unlike E2-ER, both TAM and 4-OHT-ER bind EREs non-cooperatively. Like 4-OHT, TAM appears to dissociate from the liganded ER as the receptor binds EREs. Additionally, partial proteolysis of ERE-bound ER by trypsin revealed different cleavage patterns for E2 versus 4-OHT and TAM. These findings indicate that the behavior of the ER liganded by TAM is generally similar to that of the antiestrogen 4-OHT.
Mol Cell Endocrinol 1998 Aug 25
PMID:Comparison of tamoxifen ligands on estrogen receptor interaction with estrogen response elements. 980 52


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