help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2002-0027
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
144/7/2967    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by García Pedrero, J. M.
Right arrow Articles by Ramos, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by García Pedrero, J. M.
Right arrow Articles by Ramos, S.
Endocrinology Vol. 144, No. 7 2967-2976
Copyright © 2003 by The Endocrine Society

The Naturally Occurring Variant of Estrogen Receptor (ER) ER{Delta}E7 Suppresses Estrogen-Dependent Transcriptional Activation by Both Wild-Type ER{alpha} and ERß

Juana M. García Pedrero, Pedro Zuazua, Carlos Martínez-Campa, Pedro S. Lazo and Sofía Ramos

Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología Principado de Asturias, Universidad de Oviedo, 33071 Oviedo, Spain

Address all correspondence and requests for reprints to: S. Ramos, Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología Principado de Asturias, Universidad de Oviedo, 33071 Oviedo, Spain. E-mail: sramos{at}correo.uniovi.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have isolated and functionally characterized the exon 7-skipped variant (ER{Delta}E7) of estrogen receptor (ER){alpha}, which has emerged as the predominant variant expressed in multiple normal and tumoral tissues. However, to date no function has been established for this variant in mammalian cells. ER{Delta}E7 exhibits a negligible ability to bind ligands, insensitivity to allosteric modulation by estrogen and antiestrogens, and loss of estrogen-dependent interaction with p160 coactivators such as SRC-1 and AIB1. ER{Delta}E7 is able to form heterodimers with both ER{alpha} and ERß in a ligand-independent manner. Transient expression experiments in HeLa cells show that increasing amounts of ER{Delta}E7 result in a progressive inhibition of the estrogen-dependent transcriptional activation by both wild-type ER{alpha} and ERß on estrogen response element-driven promoters. The inhibitory effect of ER{Delta}E7 is due to the inhibition of binding of wild-type receptors to their responsive elements. Surprisingly, the activation function (AF)-1-dependent transactivation triggered by epithelial growth factor and phorbol-12-myristate-13-acetate is also abolished in ER{Delta}E7 despite AF1 integrity, suggesting a cross-talk between AF1 and AF2 regions of the receptor. These results indicate that the naturally occurring variant ER{Delta}E7 is a dominant negative receptor that, when expressed at high levels relative to wild-type ERs, might have profound effects on several estrogen-dependent functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN (E2) has long been known to promote the growth of certain human neoplasms, notably tumors of the breast, endometrium, and pituitary. It also modulates the development and function of normal tissues, such as the mammary gland and bone. It also has influence on the cardiovascular and central nervous systems (1, 2). The mitogenic and regulatory effects of E2 are mediated through two closely related nuclear receptors, estrogen receptor (ER)-{alpha} and the more recently described ERß (3, 4), which are encoded by separate genes. Both receptors are members of the steroid receptor superfamily that act principally as ligand-activated DNA-binding dimers (5, 6). It is well documented the existence of multiple ER{alpha} variants generated by alternative splicing (exon skipping) of the single ER{alpha} pre-mRNA (7, 8, 9).

ER{alpha} is a protein composed of discrete functional domains. The DNA-binding domain consists of exons 2 and 3, each of which encodes a single zinc-finger motif. This domain is essential for sequence-specific DNA binding and transcriptional activation through canonical estrogen response elements (EREs) (10). The N-terminal transactivation function (AF)1 encoded by exon 1 and a portion of exon 2 operates in a ligand-independent manner and may be activated by a variety of agents (11, 12). A ligand-binding domain (LBD) confers regulatory function to the receptor and is encoded by exons 4–8. This region is the most complex functionally and includes determinants for 1) heat-shock protein association in the cytoplasm, 2) ligand-dependent receptor dimerization, 3) a ligand-dependent activation function (AF2), which promotes gene transcription by recruiting coactivators on ligand binding, and 4) estrogen and antiestrogen ligand binding (13, 14, 15). Both AF1 and AF2 domains are required for optimal stimulation of transcription, but their relative contribution varies in a promoter- and cell type-specific manner (16, 17).

Evidences for the function of ER{alpha} variants have been elusive. Thus, it has been reported that ER{Delta}E5 can support weak, cell type-dependent activity (18, 19). Alternatively, both ER{Delta}E5 and ER{Delta}E3 have been reported as dominant negative receptor forms in the presence of wild-type (wt) ER{alpha} (20, 21, 22). With regard to ER{Delta}E7, contrasting results have been obtained. Thus, it has been reported to repress 60% of the action of equimolar wt ER{alpha} in yeast (23, 24) and be ineffective as a dominant negative of ER{alpha} in mammalian cells (20, 21). Moreover, no studies exist in literature assessing the possible role of ER{Delta}E7 on transactivation mediated by the more recently described ERß.

This work focuses on the functional characterization of the exon 7-skipped variant of ER{alpha} (ER{Delta}E7) isolated from MCF-7 cells. This is the most abundant splicing form of ER{alpha} expressed in this ER (+) mammary carcinoma cell line. We have examined the ability of the ER{Delta}E7 to bind ligand, the interaction with coactivators, its heterodimerization with both wt ER{alpha} and ERß, and its role in complex formation with DNA as well as on AF1- and AF2-dependent transcriptional activation. Our results indicate that ER{Delta}E7 acts as a dominant negative receptor with ability to suppress the E2-dependent transcriptional activation by both wt ER{alpha} and ERß. Therefore, ER{Delta}E7 that had been labeled as transcriptionally inert should be really considered an important receptor isoform in controlling E2-dependent functions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
17ß-Estradiol, 4-hydroxytamoxifen, epidermal growth factor, and other chemicals were purchased from Sigma (St. Louis, MO). ICI 182,780 was provided by Dr. A. E. Wakeling (Zeneca Pharmaceuticals, Macckesfield, Cheshire, UK). [35S]-methionine (Pro-mix; 14.3 mCi/ml; >1000 Ci/mmol) was from Amersham Pharmacia Biotech (Little Chalfont, UK).

Plasmids
Recombinant plasmids allowing expression of chimeric proteins containing ER sequences were constructed as follows. The cDNA fragments encoding LBD regions (exons 4–8) of the wt human (h) ER{alpha} and the exon 7-skipped variant were amplified by RT-PCR from MCF-7 cells using the following primers: 5'-CGGGATCCGGGTCTGCTGGAGAC-3' (at positions 1133–1147) and 5'-GCGAATTCTCAGACTGTGGCAGGG-3' (at positions 2065–2082). The amplification products of 964 and 780 bp were subcloned downstream of the glutathione-S-transferase (GST) gene into the BamHI/EcoRI sites of pGEX-2TK vector (Amersham Pharmacia Biotech) to generate the recombinant plasmids pGTK-LBD and pGTK-LBD{Delta}E7, respectively. Both constructions were verified to be free of mutations and in frame with GST by sequencing.

The expression vector pcDNA-ER{alpha} was constructed by ligating the full-length hER{alpha} cDNA into the BamHI/EcoRI sites of the eukaryotic expression vector pcDNA 3 (Invitrogen, San Diego, CA), as we previously described (25). For pcDNA-ER{Delta}E7 construction, the 518-bp BglII/EcoRI portion of the wt ER{alpha} from pcDNA-ER{alpha} plasmid was replaced with the corresponding exon 7-deleted ER fragment of 334 bp from pGTK-LBD{Delta}E7 plasmid.

Expression and purification of recombinant proteins
The resulting GST fusion proteins were expressed in Escherichia coli and purified by adsorption onto glutathione sepharose essentially as described by Frangioni and Neel (26). SDS-PAGE analysis showed a molecular mass of about 65 kDa for GST-LBD (which contains residues 280–595 of wt hER{alpha}) and about 50 kDa for the truncated protein GST-LBD{Delta}E7 (residues 280–466). The precise deletion of exon 7 results in a reading frame shift, causing premature termination of translation immediately downstream of the novel splice junction with the inclusion of 10 non-ER residues after codon 457 (23). Both wt and variant hybrid proteins were expressed at similar levels in E. coli as monitored by Coomasie-stained SDS-PAGE analysis.

Cell culture, transient transfections, and luciferase assay
HeLa cells were propagated as we previously described (27). Before transfection, HeLa cells were seeded in 12-well plates Linbro (ICN Biomedicals, Inc., Aurora, OH) and incubated 12–18 h at 37 C. Then cells were transferred to phenol-red free DMEM containing 0.5% charcoal/dextran-treated fetal calf serum (sFCS) and maintained for 3 d at 60–80% confluency. Cells were transfected with 0.5 µg of an ERE-driven reporter plasmid, 0.05–1.5 µg ER expression vectors and 50 ng of an internal control Renilla luciferase plasmid, pRL-TK (Promega Corp., Madison, WI) using FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer’s protocols. After 18–24 h, the medium was renewed and cells were stimulated during 24 h with different chemicals, as indicated.

Luciferase assays were performed as recommended by dual luciferase system (Promega Corp.). To correct for differences in transfection efficiency, the experimental values were normalized to Renilla luciferase activity.

To generate [{Delta}7ER{alpha}]MCF-7 cell lines, MCF-7 cells plated in a 10-cm culture dish (80% confluency) were stably transfected with 10 µg ER{Delta}E7 expression vector using 75 µl lipofectamine and 50 µl Plus reagent (Invitrogen) following the manufacturer’s instructions. After 24 h, medium was replaced with RPMI 1640 medium (BioWhittaker, Inc., Walkersville, MD) containing 10% heat-inactivated FCS and 500 µg/ml geneticin G418 (Invitrogen) for selection. The medium was renewed every 3–4 d. In 4 wk, visible colony foci were isolated and propagated in medium containing G418.

EMSA
Binding of the E2-ER complex to ERE was performed as we previously described (27). Five to ten microliters of cellular lysates of transient transfections were mixed with buffer B (20 mM HEPES-KOH, pH 7.9; 10 mM MgCl2; 1 mM EDTA; 10% (vol/vol) glycerol; 100 mM KCl; 0.2 mM phenylmethylsulfonyl fluoride; 0.2 mM dithiothreitol (DTT); 0.5% Nonidet P-40; and protease inhibitors) and incubated with 1 µg poly (deoxyinosine-deoxycytidine) in a total volume of 40 µl. Mixtures were preincubated at 0 C for 15 min, followed by incubation with the indicated hormones at 0 C for 10 min. [32P]-labeled probe (10 fmol containing 3–5 x 104 dpm) was added to the reaction and allowed to proceed for 1 h at 0 C, followed by 30 min at room temperature. The samples were loaded onto a preelectrophoresed (10 mA) 5% polyacrylamide gel (acrylamide to bisacrylamide ratio of 40:1) in TBE (45 mM Tris Borate, 1 mM EDTA) at 11 mV/cm. For specificity assays, 100-fold excess of unlabeled oligonucleotide was used as competitor before adding the probe to the binding reaction.

In vitro protein-protein interaction assays
GST pull-down experiments were performed as previously described by Cavailles et al. (28). [35S]-labeled proteins of wt hER{alpha}, hERß, ER{Delta}E7, SRC-1a, or AIB1 coactivators were synthesized by in vitro transcription-translation (Promega Corp.) using pcDNA-ER{alpha}, pCXN2-hERß (29), pcDNA-ER{Delta}E7, pCR-SRC-1a, or pcDNA-3.1AIB1, respectively, as templates. The fusion proteins loaded on glutathione-sepharose beads (25 µl) were preincubated with 1-µM concentrations of ligands [E2, 4-hydroxytamoxifen (OHT), or ICI] for 30 min at 4 C, followed by incubation with [35S]-labeled proteins for 1.5 h at 4 C in a total volume of 150 µl IPAB buffer [20 mM HEPES-KOH, pH 7.9; 5 mM MgCl2; 150 mM KCl; 0.02 mg/ml BSA; 0.1% (vol/vol) Triton X-100; 0.1% Nonidet P-40; and protease inhibitors]. Beads were washed four to five times with IPAB without BSA, collected by centrifugation, and resuspended in 20 µl loading buffer for SDS-PAGE analysis. The gel was vacuum dried, and the radiolabeled products were visualized by autoradiography.

Far-Western blot experiments were carried out essentially as described by Cavailles et al. (28). Purified GST-proteins were subjected to SDS-PAGE and electroblotted onto nitrocellulose. After denaturation/renaturation in 6 M to 0.187 M guanidine hydrochloride in HB buffer (25 mM HEPES-KOH, pH 7.9; 25 mM NaCl; 5 mM MgCl2; 1 mM DTT), filters were saturated at 4 C in blocking buffer and incubated with [32P]-labeled GST-LBD probe (28) in H buffer (20 mM HEPES-KOH, pH 7.9; 75 mM KCl; 0.1 mM EDTA; 2.5 mM MgCl2; 0.05% Nonidet P-40; 1% milk; 1 mM DTT) using 200,000 cpm of probe per milliliter in the presence of 1 µM E2 and cold GST to block nonspecific binding. After washes with H buffer, filters were dried and exposed for autoradiography at -80 C.

Immunoprecipitation
Five microliters of in vitro translated [35S]-labeled wt hER{alpha} or ERß were mixed with equal amounts of [35S]-labeled ER{Delta}E7 posttranslationally and incubated with 30 µl buffer B [20 mM HEPES-KOH, pH 7.9; 10 mM MgCl2; 1 mM EDTA; 10% (vol/vol) glycerol; 100 mM KCl; 0.2 mM phenylmethylsulfonyl fluoride; 0.2 mM DTT; 0.5% Nonidet P-40; and protease inhibitors] with the indicated hormones at 0 C for 10 min. Then ER{alpha}-ER{Delta}E7 and ERß-ER{Delta}E7 heterodimers were immunoprecipitated with monoclonal anti-ER{alpha} antibodies NCL-ER-LH1 (Novocastra Laboratories, Newcastle upon Tyne, UK), C-314 (SC-786) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal anti-hemagglutinin (HA), or rabbit polyclonal anti-ER antibodies (raised in our laboratory against C-terminal amino acid residues 280–595 of hER{alpha}) (25) on ice for 1 h, followed by incubation with 50 µl of 50% protein G sepharose slurry in buffer B at room temperature for 1 h by rocking. The immunoprecipitates were pelleted by centrifugation, washed, and analyzed by SDS-PAGE. After suitable separation, the gel was vacuum dried, quantified with an Instantimager (Packard, Downers Grove, IL) and exposed for autoradiography.

Western blot analysis
Western blot analysis was carried out as described (25) using monoclonal anti-ER{alpha} antibodies NCL-ER-6F11 (Novocastra Laboratories). Goat antimouse IgG antibodies coupled to horseradish peroxidase (Sigma) were used as secondary antibodies. Immunoreactive bands were visualized with the ECL detection system (Amersham Pharmacia Biotech).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ER{Delta}E7 binds neither estrogens nor antiestrogens
To identify and isolate ER{Delta}E7, we performed RT-PCR assays using total RNA isolated from MCF-7 cells actively growing with 10% FCS and cells arrested at G0-G1 phase by FCS and estrogen depletion. Reverse transcription was carried out priming with oligo-dT. The synthesized cDNAs were then amplified by PCR using oligonucleotide primers that flank the LBD of wt hER{alpha}. Two forms of ER{alpha} (964 and 780 bp) were detected by hybridization with an internal probe, corresponding to wt ER{alpha} and ER{Delta}E7, respectively (Fig. 1AGo). Thus, ER{Delta}E7 appears as the predominant spliced variant of ER{alpha} in MCF-7 cells. This was confirmed when measurements of the protein levels were carried out (Fig. 1BGo). When reverse transcription reactions were performed by priming with a 3'-specific oligonucleotide complementary to sequences downstream of the termination codon of ER{alpha} ORF, additional PCR products corresponding to multiple ER{alpha} variants were detected, as previously described (30).



View larger version (18K):
[in this window]
[in a new window]
  		Figure 1. ER{Delta}E7 represents the most abundant of the ER{alpha} splicing variants in MCF-7 cells. A, Amplification of LBD region of ER{alpha} was performed by RT-PCR using total RNA extracted from proliferating MCF-7 cells (+) or G0-G1 arrested cells (-). PCR products were separated on agarose gels and hybridized with an internal 309-bp probe (positions 1454–1763). B, ER{Delta}E7 protein expression was determined by Western blot analysis. Whole-protein extracts (100 µg) from HeLa (lane 1), MCF-7 (lane 2), and [{Delta}7ER{alpha}]MCF-7 cells (lane 3) were resolved on 10% SDS-PAGE and analyzed using the monoclonal anti-ER{alpha} antibody NCL-ER-6F11. C, Schematic description of GST fusion proteins, corresponding to GST fragment (lane 1), GST fused to the LBD of wtER{alpha} (residues 280–595) named as GST-LBD (lane 2), and GST fused to the LBD of ER{Delta}E7 variant (residues 280–466) named as GST-LBD{Delta}E7 (lane 3). The figure also includes sequence details of the exon 7 deletion, and black triangle points to the novel splice junction.

 
We investigated the ability of the LBDs of wt ER{alpha} and ER{Delta}E7 to bind [3H]-estradiol. Increasing amounts (0.75–3 pmol) of GST-LBD or GST-LBD{Delta}E7 hybrid proteins purified and immobilized onto glutathione sepharose were incubated with [3H]-estradiol and the hormone-receptor complex was determined by measuring the radioactivity retained on sepharose affinity matrices. Only wt ER{alpha} was able to bind [3H]-estradiol, whereas ER{Delta}E7 showed no specific ligand binding (Fig. 2Go, A and B).



View larger version (26K):
[in this window]
[in a new window]
  		Figure 2. Binding of [3H]-estradiol by the fusion proteins containing LBD regions of the wt hER{alpha} and ER{Delta}E7 variant. The fusion proteins were expressed in E. coli and purified by adsorption onto glutathione sepharose. A, Equal amounts of each protein (0.75–3 pmol, as indicated) were incubated with 10 nM [3H]-estradiol or [3H]-estradiol plus 1000-fold excess of unlabeled estradiol for 30 min at 4 C. The matrix was washed and pelleted to remove unbound ligand and the radioactivity determined. B, Binding to 10 pmol GST-proteins was determined varying the concentration of [3H]-estradiol from 0.1 to 1 nM in the absence and presence of 1000-fold excess of unlabeled estradiol. Values correspond to specifically bound [3H]-estradiol, and bars represent the mean ± SD of triplicates in two separate experiments.

 
Additional evidence for the lack of interaction of ER{Delta}E7 with E2 was obtained analyzing the specific effects of ligands on receptor sensitivity to trypsin digestion, compared with that of wt ER{alpha}. This was accomplished by using in vitro [35S]-labeled wt ER{alpha} and ER{Delta}E7 and preincubating these receptors with E2. The resulting complexes were then subjected to limited digestion with trypsin, and the products were analyzed by SDS-PAGE. The wtER{alpha} was highly sensitive to trypsin degradation in the absence of ligand (Fig. 3Go, lane 3). In the presence of E2, however, a trypsin-resistant 32-kDa receptor fragment was observed (Fig. 3Go, lane 6). Incubation of the labeled receptor with the antiestrogens OHT or ICI also rendered a fragment that was resistant to further digestion with trypsin, as previously reported (31). ER{Delta}E7 was also highly sensitive to protease digestion, but the presence of E2 failed to protect this receptor from degradation by trypsin, as expected from its inability to bind ligand (Fig. 3Go, lanes 9 and 12). Moreover, neither OHT nor ICI protected ER{Delta}E7 from digestion, indicating that ER{Delta}E7 was also unable to interact with antiestrogens (data not shown). This suggests that this variant lacks allosteric modulation by both estrogen and antiestrogens. The deletion of exon 7 eliminates a significant portion of the LBD, and thus the loss of ligand binding should be expected.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 3. Trypsin digestion of E2-ligated and unligated wtER{alpha} and ER{Delta}E7. Radiolabeled hER{alpha} and ER{Delta}E7 were synthesized in vitro and subjected to digestion with different concentrations of trypsin (as indicated) in the absence or the presence of 1 µM E2. The product of the digestion reactions were resolved by SDS-PAGE and visualized by autoradiography. Lanes 1–6 represent ER{alpha} digestions performed in the presence of ligand E2 (lanes 4–6) or vehicle alone (ethanol) (C, lanes 1–3). Lanes 7–12 correspond to the products of identical reactions performed with ER{Delta}E7 in absence (C, lanes 7–9) or presence of 1 µM E2 (lanes 10–12).

 
ER{Delta}E7 forms heterodimers with both wtER{alpha} and ERß
We performed GST pull-down experiments with [35S]-methionine-labeled ER{Delta}E7 and GST-LBD of wtER{alpha} to assess the interaction between the two proteins in vitro. As shown in Fig. 4AGo, ER{Delta}E7 protein was successfully coprecipitated with the GST-LBD fusion protein, and this interaction was unaffected by the absence (C) or presence of E2 or OHT at 1 µM. This indicates that ER{Delta}E7 heterodimerizes with wtER{alpha} in a ligand-independent manner.



View larger version (34K):
[in this window]
[in a new window]
  		Figure 4. ER{Delta}E7 forms heterodimers with both wtER{alpha} and ERß in vitro. A, GST pull-down experiment performed using in vitro translated [35S]-methionine-labeled ER{Delta}E7 incubated with GST alone (lanes 2 and 3) or GST fusion protein containing LBD of the wt hER{alpha} (GST-LBD, lanes 4–6) immobilized on GSH-sepharose in the absence of ligand (C) or presence of 1 µM E2 or 1 µM OHT. Below the panel, the percentage of the input pulled down (counts per minute) is shown. The input lane represents 10% of the total amount of labeled ER{Delta}E7 used in the binding reactions (lane 1). B (left panel), Far-Western analysis of GST fragment (lane 1), GST-LBD (lane 2), and GST-LBD{Delta}E7 (lane 3) immobilized onto nitrocellulose and incubated with 200,000 cpm/ml [32P]-labeled GST-LBD probe in the presence of 1 µM E2. B (right panel), Coomasie-blue stained SDS-PAGE analysis of GST proteins used for Far-Western experiments. C, Equivalent aliquots of [35S]-labeled wtER{alpha} and ER{Delta}E7 were immunoprecipitated with the antibody NCL-ER-LH1 (which binds only to the wtER{alpha}) in the absence of ligand (C, lane 1) or presence of 1 µM E2 (lane 2) or 1 µM OHT (lane 3). In parallel reactions, aliquots of [35S]-labeled wtERß and ER{Delta}E7 were immunoprecipitated with the antibody C-314 (which binds only to the ER{Delta}E7 variant) in the absence of ligand (C, lane 5) or presence of 1 µM E2 (lane 6) or 1 µM OHT (lane 7). Lanes 4, 8, and 12 correspond to the immunoprecipitation of total ERs (ERT) using polyclonal anti-ER antibodies, which bind wtER{alpha}, ERß, and ER{Delta}E7. Lanes 9–11 correspond to immunoprecipitation using anti-HA antibodies, as negative control. The lower level of ERß in the immunoprecipitates is due to a low efficiency in the synthesis of this protein; nevertheless, ERß fractions immunoprecipitated with C-314 antibody quantitatively represent about 50% of the total protein (compare lanes 5–7 with lane 8).

 
To investigate dimer formation with ER{Delta}E7, we used Far-Western blotting. This technique restricts the detection to direct interactions only between proteins. Thus, equal amounts of GST and the purified hybrid proteins GST-LBD and GST-LBD{Delta}E7 (Fig. 4BGo, right panel) were immobilized onto nitrocellulose and, after denaturation/renaturation, proteins on filters were incubated with an in vitro [32P]-labeled GST-LBD probe in the presence of 1 µM E2 (Fig. 4BGo). The ER{alpha} probe was found to bind to both LBDs of wtER{alpha} and ER{Delta}E7, whereas no interaction was detected with GST alone (Fig. 4BGo, left panel, lane 1). This experiment demonstrates the direct interaction between wtER{alpha} and ER{Delta}E7 and reveals that the capacity of wtER{alpha} to heterodimerize with ER{Delta}E7 variant appears to be comparable with its homodimerization ability.

Finally, we analyzed the interaction of ER{Delta}E7 with both full-length wt hER{alpha} and ERß, using in this case proteins in solution (Fig. 4CGo). For this purpose, aliquots of [35S]-labeled wtER{alpha} or ERß were mixed with an equal volume of [35S]-labeled ER{Delta}E7 and incubated in either the absence of hormone (C) or in the presence of 1 µM E2 or 1 µM OHT, as indicated. ER{alpha}-ER{Delta}E7 heterodimers formation was determined by immunoprecipitation with the monoclonal anti-ER{alpha} antibody NCL-ER-LH1, which recognizes an epitope located within the C-terminal of LBD (lanes 1–3). Because the truncated receptor ER{Delta}E7 lacks this epitope, only ER{alpha} homodimers and heterodimers containing ER{alpha} and ER{Delta}E7 will be immunoprecipitated by this antibody. Similarly, ERß-ER{Delta}E7 heterodimerization was detected by immunoprecipitation assays using the ER{alpha}-specific monoclonal antibody C-314 raised against the N-terminal of ER{alpha} that therefore allows visualization of both ER{Delta}E7 homodimers and ERß-ER{Delta}E7 heterodimers (lanes 5–7). Additionally, total ERs were immunoprecipitated with polyclonal anti-ER antibodies as a control (lanes 4, 8, and 12). No immunoprecipitation was observed when an unrelated antibody (anti-HA) was used (lanes 9–11). These results imply that the ER{Delta}E7 variant is able to form heterodimers with both wtER{alpha} and ERß and these interactions are not subjected to hormonal regulation.

Coactivator-binding properties of ER{Delta}E7
The LBD also includes a well-characterized C-terminal transactivation function (AF2), which promotes gene transcription by recruiting coactivator proteins in a ligand-dependent manner (32, 33). We tested whether exon 7 deletion might affect the binding of coactivators to ER{Delta}E7. Thus, we determined the interaction of ER{Delta}E7 with p160 coactivators in vitro by GST pull-down experiments using [35S]-labeled SRC-1a (Fig. 5AGo) or AIB1 (also named RAC3/ACTR/pCIP/SRC-3) (Fig. 5BGo) and GST-LBD or GST-LBD{Delta}E7 hybrid proteins as affinity reagents. As expected, the binding of both p160 coactivators to LBD of wtER{alpha} was greatly stimulated in the presence of E2, whereas the estrogenic antagonist OHT failed to induce their binding to the wtAF2 domain. On the other hand, using the GST-LBD{Delta}E7 hybrid protein, no induction of coactivators binding was observed in the presence of either E2 or OHT.



View larger version (40K):
[in this window]
[in a new window]
  		Figure 5. Estrogen-dependent interaction of p160 coactivators with the AF2 domains of wtER{alpha} and ER{Delta}E7. A, In vitro translated [35S]-methionine-labeled SRC-1a was incubated with GST alone (lanes 2 and 3) or GST fusion proteins containing LBDs of the wtER{alpha} (GST-LBD, lanes 4–6) or the ER{Delta}E7 variant (GST-LBD{Delta}E7, lanes 7–9) immobilized on GSH-sepharose in the absence of ligand (C) or presence of 1 µM E2 or 1 µM OHT. B, In vitro translated [35S]-methionine-labeled AIB1 was treated as in A. Below each panel, the percentage of the input pulled down (counts per minute) for each assay is shown. In each panel, the input lane represents 10% of the total volume of lysate used in each reaction (lane 1).

 
ER{Delta}E7 inhibits E2-dependent transcriptional activation by both wtER{alpha} and ERß on ERE-driven promoters
To investigate the biological activity of ER{Delta}E7, we constructed the expression plasmids pcDNA-ER{alpha} and pcDNA-ER{Delta}E7 that contain the full-length cDNAs of wtER{alpha} and ER{Delta}E7, respectively. Using in vitro transcription/translation systems, we determined that both constructs directed the expression of the corresponding [35S]-labeled receptors with the expected sizes of 66 and 52 kDa (see Fig. 3Go, lanes 1 and 7). In agreement with previously published results (20, 23), we confirmed that ER{Delta}E7 is an isoform of ER{alpha} that failed to stimulate transcription of ERE-driven reporter genes (not shown).

Because alternatively spliced forms of the ER{alpha} are present in MCF-7 cells along with the intact receptor, it was of interest to determine whether ER{Delta}E7 interferes with the activity of both wtER{alpha} and ERß. To address this question, we performed titration experiments in which we varied the ratio of wtER to ER{Delta}E7 (Fig. 6Go). Thus, we transiently transfected HeLa cells with ER{alpha} or ERß expression vectors and increasing amounts of ER{Delta}E7 plasmid along with the ERE-driven reporter plasmids pEREtkLuc (Fig. 6AGo) or pS2Luc (Fig. 6BGo). A saturating concentration of hormone (100 nM E2) was used. In both experiments we observed that increasing amounts of ER{Delta}E7 resulted in a progressive inhibition of the E2-dependent induction of luciferase activity by both ER{alpha} and ERß.



View larger version (36K):
[in this window]
[in a new window]
  		Figure 6. ER{Delta}E7 inhibits estrogen-dependent transactivation of ERE-driven reporters by ER{alpha} and ERß in a dose-related fashion. HeLa cells were cotransfected with 50 ng expression vectors encoding hER{alpha} or mERß; 0.5 µg ERE-driven reporter plasmids pEREtkLuc (A) or pS2Luc (B); 50 ng of internal control plasmid pRL-TK; and increasing amounts of ER{Delta}E7 expression vector (0–1.5 µg as indicated). The total amount of DNA was held constant to 2.1 µg by addition of empty expression vector, pcDNA3. C, This experiment was carried out with a constant amount of ER expression vectors (0.1 µg total DNA per well) and varying the ratios of wtERs to ER{Delta}E7 as indicated. After transfection, cells were treated with 100 nM E2 for 24 h and then harvested. Luciferase activities were normalized to the Renilla luciferase activities. The data are expressed as the percentage of wtER ({alpha} or ß) luciferase activity remaining; 100% was assigned to the response obtained with wtER ({alpha} or ß) plus estradiol. The activation varied from 3- to 5-fold in different experiments. The bars represent the mean ± SD of three independent experiments.

 
To rule out the possibility that the potent transcriptional suppression observed with high amounts of ER{Delta}E7 was due to high levels of this variant that might poison the transcription apparatus, we performed HeLa transfections using a constant amount of ER expression vectors (0.1 µg per well) and varying the relative amounts of wtERs and ER{Delta}E7 (Fig. 6CGo). In this case, ER{Delta}E7 also inhibited ERE-driven transcription reaching values to below basal activity. Thus, ER{Delta}E7 appears as a genuine dominant negative inhibitor that did not act by simply saturating the transcription machinery.

ER{Delta}E7 blocks the binding of wtER{alpha} and ERß to their responsive element
To investigate whether inhibition of transcription by ER{Delta}E7 is exerted at level of DNA binding, we compared the ability of ER{Delta}E7 and wild-type receptors to bind to the ERE in vitro. For this purpose we conducted EMSAs using whole extracts from HeLa cells that were transfected with wtER{alpha} or ER{Delta}E7 independently or with different ratios of both receptors (Fig. 7AGo). The wtER{alpha} formed a complex with the ERE probe that increased in the presence of E2 (lane 2). The specificity of the retarded band was demonstrated by supershift induction with anti-ER antibodies (lane 3). In contrast, ER{Delta}E7 showed no binding to the ERE in this in vitro EMSA (lanes 8 and 9). Interestingly, when both receptors were coexpressed the ER{alpha}-ERE complex was attenuated, and this reduction was proportional to the increase of ER{Delta}E7 (lanes 4–7). Similar experiments were performed with extracts from cells expressing ERß or different ERß:ER{Delta}E7 ratios (Fig. 7BGo), and identical results were obtained, suggesting that the transcriptional inhibition by ER{Delta}E7 is arisen from the inhibitory effect of this receptor on the binding of both wtER{alpha} and ERß to ERE.



View larger version (45K):
[in this window]
[in a new window]
  		Figure 7. ER{Delta}E7 inhibits estrogen-dependent binding of both wtER{alpha} and ERß to ERE. A, Whole extracts prepared from transfected HeLa cells expressing ER{alpha}, ER{Delta}E7 or 1:1 and 1:3 proportions of both receptors (as indicated) were assayed for ERE-binding activity by EMSA in the absence (-) or presence of 100 nM E2 (+). Specific ER{alpha}-ERE complexes were determined by supershift induction with polyclonal anti-ER antibodies (lanes 3, 5, 7, and 9). B, Whole extracts prepared from transfected HeLa cells expressing ERß or the indicated proportions of ERß/ER{Delta}E7 were assayed as above. Specific ERß-ERE complexes were determined by supershift induction with polyclonal anti-ER antibodies (lanes 3 and 6). C, Three microliters of in vitro synthesized wtER{alpha} were mixed with increasing amounts of ER{Delta}E7 as indicated. In lane 2, an approximately 100-fold excess of unlabeled ERE was added.

 
We also performed a series of EMSA in which wtER{alpha} and ER{Delta}E7 were mixed after in vitro translation (Fig. 7CGo). The translational efficiencies of the two synthetic mRNAs were determined to be equivalent by performing parallel reactions in the presence of [35S]-methionine (data not shown). As expected, the wtER{alpha} was able to form specific ER-ERE complexes (lane 1), which could be competed out by addition of an excess of unlabeled ERE (lane 2). When ER{Delta}E7 was mixed with wtER{alpha}, as the amount of ER{Delta}E7 increased, the binding of wtER{alpha} to its response element was progressively inhibited (lanes 3 and 4), even though ER{Delta}E7 was by itself unable to bind to the ERE (lane 5).

AF1-dependent transcriptional activation by wtER{alpha} and ER{Delta}E7
The ER{Delta}E7 variant is unable to bind ligand and devoid of ligand-dependent activity and modulation by estrogens and antiestrogens. In spite of all these properties, this variant might activate transcription through steroid-independent mechanisms that involve the AF1 domain, which remains intact. It has been described that the activity of the N-terminal AF1 of ER{alpha} is modulated by the phosphorylation of Ser (118) through the Ras-MAPK pathway (11). Thus, epithelial growth factor (EGF) and phorbol-12-myristate-13-acetate (PMA) have been shown to activate ER{alpha} (11, 12). On the other hand, it has been shown that estradiol contributes with these agents to the phosphorylation of Ser (118) by a MAPK-independent mechanism (34).

To investigate the E2-independent activation, we examined the ability of EGF and PMA to activate the transcription mediated by ER{Delta}E7. For this purpose, we conducted transfection experiments in HeLa cells with either wtER{alpha} or ER{Delta}E7 to evaluate the transcriptional activity of these receptors on luciferase reporter plasmids containing ERE sites into a synthetic promoter (Fig. 8AGo) or the natural promoter (Fig. 8BGo). In the presence of wtER{alpha}, EGF and PMA activated transcription 8- to 10-fold, and this effect was potentiated in the presence of E2. We performed two types of control experiments that clearly demonstrate that the activation with EGF and PMA was mediated by ER{alpha}: 1) in the absence of the ER, the expression of luciferase was significantly reduced; and 2) the response was blocked by the antiestrogen ICI. ER{Delta}E7 was not activated by EGF or PMA. This variant showed only a partial activation by PMA on pS2 promoter, which was neither stimulated with E2 nor abolished by ICI treatment. Although the relative activation of ER{Delta}E7 by PMA appears to be similar to that of wtER{alpha}, the level of transcription obtained with ER{Delta}E7 represented about 50% of that of wtER{alpha}. These findings suggest that the integrity of the AF1 region of ER{Delta}E7 is not sufficient to support full activation by EGF and PMA. This is in fact not too surprising because ER{Delta}E7 fails to bind ERE.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 8. Ligand-independent transcriptional activation of wtER{alpha} and ER{Delta}E7. HeLa cells were cotransfected with 100 ng expression vectors encoding ER{alpha} or ER{Delta}E7 or empty expression vector, pcDNA3 (indicated as -ER); 0.5 µg ERE-driven reporter plasmids, pEREtkLuc (A) or pS2Luc (B); and 50 ng of internal control plasmid pRL-TK. When indicated, the transfected cells were treated with 100 ng/ml EGF, 100 nM PMA, 100 nM E2, 1 µM ICI, or combinations of them. Luciferase activities were normalized to the Renilla luciferase activities. The data are reported as fold induction with respect to untreated cells (C), to which it was assigned the value 1. The bars represent the mean ± SD of three separate experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presence of ER variants has been shown in human breast cancer tissues (7), breast cancer cell lines (8), and a number of human normal and neoplasmic tissues (7, 9, 35, 36). Recently, using a splice-targeted primer approach, 20 alternatively spliced ER{alpha} mRNAs that are present in both breast cancer cell lines and tumors have been identified (8). Information on these variants, however, is limited to in vitro analysis at the mRNA levels. The biological significance of the alternatively spliced messengers remains an enigma. It is possible that variant receptor proteins are defective in folding, dimerization, or interaction with heat shock proteins (37) or other cellular factors (38) that may lead to rapid degradation. Thus, Dauvois et al. (39) have reported that impaired dimerization leads to a decreased half-life of the receptor protein. Characterization of variant receptor proteins is just beginning to emerge. Translation into protein has been shown for only ER{Delta}E4 (40) and ER{Delta}E5 (19, 41). Recently, a 52-kDa protein corresponding to ER{Delta}E7 was detected by Western analysis and shown to be the major variant ER protein expressed in various ER (+) breast cancer cell lines and extracts from ER (+) breast and uterine tumors (42). In addition, ER{Delta}E7 mRNA has been reported to be the major alternatively spliced form in most human breast tumors and cancer cell lines as well as in human uterus and endometrial tumors (43, 44).

Up to now, no function has been established for the ER{Delta}E7 variant in mammalian cells. Two reports indicated that ER{Delta}E7 is a dominant inhibitor of wtER{alpha} function in yeast (23, 24). In this work we have approached the functional characterization of this variant receptor obtained from MCF-7 cells. ER{Delta}E7 represents the prevalent spliced form of ER{alpha} expressed in this breast carcinoma-derived cell line, as determined at both mRNA and protein levels. Our studies provide evidence for the first time that ER{Delta}E7 suppresses the estrogen-dependent transcriptional activation by both wtER{alpha} and ERß. At a 1:1 ratio, the suppression of estrogen-induced transcription varied from 20–40%, depending on the wtER and ERE promoter used. Increasing amounts of ER{Delta}E7 resulted in the progressive inhibition of E2-dependent response, achieving a complete inhibition at a 10-fold molar excess of ER{Delta}E7. This observation may have physiological significance in breast cancer cells that predominantly express this ER{alpha} variant.

Powerful dominant negative mutants generated by chemical mutagenesis of the ER{alpha} LBD have been described (45). ER{Delta}E7 has the additional interest that it is a naturally occurring ER variant that may have profound effects on several estrogen-dependent functions if expressed at high levels relative to wtERs.

With regard to the selective modulation observed using two different ERE promoters, it has been reported that the EREs may act as allosteric modulators of ER conformation. Thus, the Xenopus vitellogenin A2 ERE, (GGTCAnnnTGACC) and the human pS2 ERE (GGTCAnnnTGGCC) induce changes in receptor conformation that could lead to association of the receptor with different transcription factors and assist in the differential modulation of estrogen-responsive genes in target cells (46).

The dominant negative character of ER{Delta}E7 suggests that this variant is able to interact with at least one component of the ERE-directed transcription complex in a manner that disrupts positive gene regulation mediated by both ER{alpha} and ERß. Based on gel mobility shift assays, ER{Delta}E7 is unable to bind to ERE by itself, and it prevents both wtER{alpha} and ERß from binding to DNA. This refutes the results of Fuqua et al. (23), who claimed to identify ER{Delta}E7 by complex formation with ERE and upshift induction with anti-ER antibody H222. It is unlikely that the protein detected was ER{Delta}E7, which is now known not to react with H222 (42). We cannot rule out a weak protein-DNA interaction, which would not be detected in a gel shift assay. In any case, it is clear that the ability of ER{Delta}E7 to suppress the activity of wtER is not due to the high affinity of the former for the ERE. Additionally, ER{Delta}E7 might form inactive heterodimers with wtERs. These heterodimers could be unable to either bind to the ERE or activate transcription when bound to the ERE. We found that ER{Delta}E7 can form a stable complex with ER{alpha} in a ligand-independent manner, as expected by the inability of ER{Delta}E7 to bind ligands. This is consistent with the absence of allosteric modulation of ER{Delta}E7 by estrogens and antiestrogens but disagrees with other observations using the two-hybrid system in yeast in which ER{Delta}E7 could form neither homodimers nor heterodimers with wtER{alpha} (24). Identical conclusions can be drawn from immunoprecipitation studies using in vitro translated ER{alpha} and ERß. Although immunoprecipitation assays are extremely difficult to use to quantitate the percentages of the various heterodimers that are immunoprecipitated, our data clearly demonstrate that heterodimers with and without ligand are immunoprecipitated to a similar extent. Thus, ligand binding is not a prerequisite for receptor dimerization, as indicated also by Zhuang et al. (47). Finally, Far-Western analysis also showed that wtER{alpha} can form heterodimers with ER{Delta}E7 with the same efficiency that it can form homodimers. Altogether, these studies show that ER{Delta}E7 is able to form mixed dimers with both wtER{alpha} and ERß and these heterodimers are unable to bind stably to DNA.

The ER{Delta}E7 variant might also interfere with associated transcription factors required for ER activity. Using pull-down experiments, we determined that the estrogen-dependent association of both SRC-1a and AIB1 coactivators with the AF2 domain of wtER{alpha} is prevented in the truncated AF2 of the ER{Delta}E7 variant. This result is in agreement with those reported by Heery et al. (48), who showed that the ability of SRC-1 to bind the ER and enhance its transcriptional activity is dependent on the integrity of the LXXLL motifs and on key hydrophobic residues in the conserved helix 12 of the ER. ER{Delta}E7 lacks this essential region.

Like other nuclear receptors, ER{alpha} is a modular protein in which individual domains are capable of demonstrating autonomous functions (10, 32). It can reasonably be assumed that the exclusion of a particular exon will result in a protein lacking the function ascribed to that exon. Alternatively, it is possible that the loss of a particular exon will result in unpredictable functional deficits or perhaps even bestow a novel function on the variant receptor. Some properties determined for ER{Delta}E7 are consistent with these predictions. Thus, the inability of ER{Delta}E7 to bind ligands, its insensitivity to estrogens and antiestrogens, and the lack of association with coactivators is not surprising because the loss of exon 7 implies the elimination of a significant portion of HBD/AF2 domain including helix 12. Less predictably, although ER{Delta}E7 contains both the DNA-binding domain and AF1 domains, this receptor shows a strong defect in ERE recognition and DNA binding and therefore the loss of AF1-dependent activation by EGF and PMA. Our results indicate that AF1 and AF2 exert mutual influence because the loss of AF2 in ER{Delta}E7 affects transactivation through AF1. It has been indicated that mutations in or near the AF2 transactivation region or elimination of the AF2 region are responsible for the dominant negative phenotype of the C-terminal ER mutants, whereas ER mutants made inactive by mutations in the hormone-binding region did not possess the capacity to act as effective blockers of ER action (45). These observations reinforce the idea that it is the disruption of the transactivation domain, and not the loss of ligand binding, that leads to the dominant negative phenotype exhibited by ER{Delta}E7.

Variant forms of the ER that function as dominant negative may play an important role in the loss of hormone responsiveness and the progression to hormone independence. The existence of different variants generated by alternative splicing of ER{alpha} and ERß that function as dominant negative has been interpreted as a physiological protective mechanism of regulating the E2-dependent growth of responsive tissues (49) and, alternatively, as a deleterious mechanism that render the ER+ cancer cells resistant to antiestrogen therapy (23, 50).

Because the growth of nearly 50% of all human breast cancers is dependent on the presence of an active estradiol-ER complex, it is interesting to explore ways to functionally inactivate ERs (51). In this regard, elevated levels of a dominant negative receptor that interferes with normal ER function could render a tumor unresponsive to estrogen and antiestrogens (23). Thus, the ER{Delta}E7 variant is significantly more abundant in ER+/PgR- tumors, compared with ER+/PgR+ tumors (23), being the ER+/PgR- phenotype more aggressive tumors, growing much faster and with a lower response to antihormone therapy (52). Also, it has been shown that the estrogen-independent LCC2 cells express significantly higher levels of ER{Delta}E7 transcripts, compared with the estrogen-dependent MCF-7 cells (53).

The studies of ER variants that have been published thus far been aimed at the identification of possible causes of the hormone-independent and antihormone-resistant growth of human breast cancers. Perhaps too little attention has been paid to establish the relative wtER/variant ratio of expression in both normal and tumoral tissues, a circumstance that in our view severely conditions the possible involvement of ER variants in physiological and/or pathological processes.


   Acknowledgments
 
We thank Ana Corao Trueba for technical assistance; Dr. V. Giguère for providing pERE-TKLuc, pS2Luc, and pCMX-mERß; Dr. M. Muramatsu for providing pCXN2-hERß; Dr. M. J. Tsai for pCR-SRC-1a; and Dr. Ana Aranda for pcDNA-3.1AIB1.


   Footnotes
 
This work was supported by grants from Fondo de Investigaciones Sanitarias 00/1086, CICYT SAF 2001-2750, MCT-02 SAF00136, and Plan de I+D+I del Principado de Asturias (PC-SPV-10). J.M.G.P. and C.M.C. were recipients of a fellowship from IUOPA, Obra Social Cajastur; and P.Z. was recipient of a fellowship from Fondo de Investigaciones Sanitarias.

Abbreviations: AF, Activation function; DTT, dithiothreitol; E2, estrogen; EGF, epithelial growth factor; ER, estrogen receptor; ERE, estrogen response element; FCS, fetal calf serum; GST, glutathione-S-transferase; h, human; HA, hemagglutinin; LBD, ligand-binding domain; OHT, 4-hydroxytamoxifen; PMA, phorbol-12-myristate-13-acetate; wt, wild-type.

Received November 11, 2002.

Accepted for publication March 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Horowitz MC 1993 Cytokines and estrogen in bone: antiosteoporotic effects. Science 260:626–627[Free Full Text]
  2. Nabulsi AA, Folsorn AR, White A, Patsch W, Heiss G, Wu KK, Szklo M 1993 Association of hormone-replacement therapy with various cardiovascular risk factors in postmenopausal women. The Atherosclerosis Risk in Communities Study Investigators. N Engl J Med 328:1069–1075[Abstract/Free Full Text]
  3. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
  4. Mosselman S, Polman J, Dijkema R 1996 ER-ß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  5. Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320:134–139[CrossRef][Medline]
  6. Katzenellenbogen BS, Korach KS 1997 Editorial: a new actor in the estrogen receptor drama-enter ER-ß. Endocrinology 138:861–862[Free Full Text]
  7. Pfeffer U, Fecarotta E, Vidali G 1995 Coexpression of multiple estrogen receptor variant messenger RNAs in normal and neoplastic breast tissues and in MCF-7 cells. Cancer Res 55:2158–2165[Abstract/Free Full Text]
  8. Poola I, Koduri S, Chatra S, Clarke R 2000 Identification of twenty alternatively spliced estrogen receptor {alpha} mRNAs in breast cancer cell lines and tumors using splice targeted primer approach. J Steroid Biochem Mol Biol 72:249–258[CrossRef][Medline]
  9. Rice LW, Jazaeri AA, Shupnik MA 1997 Estrogen receptor mRNA splice variants in pre- and postmenopausal human endometrium and endometrial carcinoma. Gynecol Oncol 65:149–157[CrossRef][Medline]
  10. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[CrossRef][Medline]
  11. Kato S, Endoh H, Masuhiro Y, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494[Abstract/Free Full Text]
  12. Bunone G, Briand PA, Miksicek R, Picard D 1996 Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 15:2174–2183[Medline]
  13. Pierrat B, Heery DM, Chambon P, Losson R 1994 A highly conserved region in the hormone-binding domain of the human estrogen receptor functions as an efficient transactivation domain in yeast. Gene 143:193–200[CrossRef][Medline]
  14. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  15. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[CrossRef][Medline]
  16. Tora L, White J, Brou C, Tasset D, Webster N, Scheer E, Chambon P 1989 The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell 59:477–487[CrossRef][Medline]
  17. Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike WJ, McDonnell DP 1994 Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8:21–30[Abstract]
  18. Castles CG, Fuqua SA, Klotz DM, Hill SM 1993 Expression of a constitutively active estrogen receptor variant in the estrogen receptor-negative BT-20 human breast cancer cell line. Cancer Res 53:5934–5939[Abstract/Free Full Text]
  19. Chaidarun SS, Alexander JM 1998 A tumor-specific truncated estrogen receptor splice variant enhances estrogen-stimulated gene expression. Mol Endocrinol 12:1355–1366[Abstract/Free Full Text]
  20. Bollig A, Miksicek RJ 2000 An estrogen receptor-{alpha} splicing variant mediates both positive and negative effects on gene transcription. Mol Endocrinol 14:634–649[Abstract/Free Full Text]
  21. Wang Y, Miksicek RJ 1991 Identification of a dominant negative form of the human estrogen receptor. Mol Endocrinol 5:1707–1715[Abstract]
  22. Ohlsson H, Lykkesfeldt AE, Madsen MW, Briand P 1998 The estrogen receptor variant lacking exon 5 has dominant negative activity in the human breast epithelial cell line HMT-3522S1. Cancer Res 58:4264–4268[Abstract/Free Full Text]
  23. Fuqua SA, Fitzgerald SD, Allred D, Elledge RM, Nawaz Z, McDonnell DP, O’Malley BW, Greene GL, McGuire WL 1992 Inhibition of estrogen receptor action by a naturally occurring variant in human breast tumors. Cancer Res 52:483–486[Abstract/Free Full Text]
  24. Wang H, Zeng X, Khan SA 1999 Estrogen receptor variants ER{delta}5 and ER{delta}7 down-regulate wild-type estrogen receptor activity. Mol Cell Endocrinol 156:159–168[CrossRef][Medline]
  25. Garcia Pedrero JM, Rio B, Martinez-Campa C, Lazo PS, Ramos S 2002 Calmodulin is a selective modulator of estrogen receptors. Mol Endocrinol 16:947–960[Abstract/Free Full Text]
  26. Frangioni JV, Neel BG 1993 Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. Anal Biochem 210:179–187[CrossRef][Medline]
  27. Rato AG, Pedrero JG, Martinez MA, del Rio B, Lazo PS, Ramos S 1999 Melatonin blocks the activation of estrogen receptor for DNA binding. FASEB J 13:857–868[Abstract/Free Full Text]
  28. Cavailles V, Dauvois S, Danielian PS, Parker MG 1994 Interaction of proteins with transcriptionally active estrogen receptors. Proc Natl Acad Sci USA 91:10009–10013[Abstract/Free Full Text]
  29. Ogawa S, Inoue S, Orimo A, Hosoi T, Ouchi Y, Muramatsu M 1998 Cross-inhibition of both estrogen receptor {alpha} and ß pathways by each dominant negative mutant. FEBS Lett 423:129–132[CrossRef][Medline]
  30. Pfeffer U, Fecarotta E, Vidali G 1995 Efficient one-tube RT-PCR amplification of rare transcripts using short sequence specific reverse transcription primers. Biotechniques 18:204–206[Medline]
  31. McDonnell DP, Clemm DL, Hermann T, Golman ME, Pike JW 1995 Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol Endocrinol 9:659–669[Abstract]
  32. Beato M, Sanchez-Pacheco A 1996 Interaction of steroid hormone receptors with the transcription initiation complex. Endocr Rev 17:587–609[CrossRef][Medline]
  33. McInerney EM, Tsai MJ, O’Malley BW, Katzenellenbogen BS 1996 Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator. Proc Natl Acad Sci USA 93:10069–10073[Abstract/Free Full Text]
  34. Joel P, Traish AM, Lannigan DA 1998 Estradiol-induced phosphorylation of serine 118 in the estrogen receptor is independent of p42/p44 mitogen-activated protein kinase. J Biol Chem 273:13317–13323[Abstract/Free Full Text]
  35. Villa E, Camellini L, Dugani A, Zucchi F, Grottola A, Merighi A, Buttafoco P, Losi L, Manenti F 1995 Variant estrogen receptor messenger RNA species detected in human primary hepatocellular carcinoma. Cancer Res 55:498–500[Abstract/Free Full Text]
  36. Hu C, Hyder SM, Needleman DS, Baker VV 1996 Expression of estrogen receptor variants in normal and neoplastic human uterus. Mol Cell Endocrinol 118:173–179[CrossRef][Medline]
  37. Chambraud B, Berry M, Redeuilh G, Chambon P, Baulieu EE 1990 Several regions of human estrogen receptor are involved in the formation of receptor-heat shock protein 90 complexes. J Biol Chem 265:20686–20691[Abstract/Free Full Text]
  38. Meyer ME, Gronemeyer H, Turcotte B, Bocquel MT, Tasset D, Chambon P 1989 Steroid hormone receptor compete for factors that mediate their enhancer function. Cell 57:433–442[CrossRef][Medline]
  39. Dauvois S, Daniellian PS, White R, Parker MG 1992 Antiestrogen ICI 164, 384 reduces cellular estrogen receptor content by increasing its turnover. Proc Natl Acad Sci USA 89:4037–4041[Abstract/Free Full Text]
  40. Park W, Choi JJ, Hwang ES, Lee JH 1996 Identification of a variant estrogen receptor lacking exon 4 and its coexpression with wild-type estrogen receptor in ovarian carcinomas. Clin Cancer Res 2:2029–2035[Abstract]
  41. Desai AJ, Luqmani YA, Walters JE, Coope RC, Dagg B, Gomm JJ, Pace PE, Rees CN, Thirunavukkarasu V, Shousha S, Groome NP, Coombes R, Ali S 1997 Presence of exon 5-deleted oestrogen receptor in human breast cancer: functional analysis and clinical significance. Br J Cancer 75:1173–1184[Medline]
  42. Fasco MJ, Keyomarsi K, Arcaro KF, Gierthy JF 2000 Expression of an estrogen receptor {alpha} variant protein in cell lines and tumors. Mol Cell Endocrinol 166:156–169[Medline]
  43. Leygue E, Huang A, Murphy LC, Watson PH 1996 Prevalence of estrogen receptor variant messenger RNAs in human breast cancer. Cancer Res 56:4324–4327[Abstract/Free Full Text]
  44. Poola I, Williams DM, Koduri S, Ramprakash J, Taylor RE, Hankins WD 1998 Quantitation of estrogen receptor mRNA copy numbers in breast cancer cell lines and tumors. Anal Biochem 258:209–215[CrossRef][Medline]
  45. Ince BA, Zhuang Y, Wrenn CK, Shapiro DJ, Katzenellenbogen BS 1993 Powerful dominant negative mutants of the human estrogen receptor. J Biol Chem 268:14026–14032[Abstract/Free Full Text]
  46. Wood JR, Greene GL, Nardulli AM 1998 Estrogen response elements function as allosteric modulators of estrogen receptor conformation. Mol Cell Biol 18:1927–1934[Abstract/Free Full Text]
  47. Zhuang Y, Katzenellenbogen BS, Shapiro DJ 1995 Estrogen receptor mutants which do not bind 17 ß-estradiol dimerize and bind to the estrogen response element in vivo. Mol Endocrinol 9:457–466[Abstract]
  48. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  49. Erenburg I, Schachter B, Miray Lopez R, Ossowski L 1997 Loss of an estrogen receptor isoform (ER{alpha}{delta}3) in breast cancer and the consequences of its reexpression: interference with estrogen-stimulated properties of malignant transformation. Mol Endocrinol 11:2004–2015[Abstract/Free Full Text]<