Functional Analysis of a Novel Estrogen Receptor-ß Isoform

Bettina Hanstein1, Hong Liu2, Molly C. Yancisin and Myles Brown

Department of Adult Oncology Dana-Farber Cancer Institute Boston, Massachusetts 02115


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A new level of complexity has recently been added to estrogen signaling with the identification of a second estrogen receptor, ERß. By screening a rat prostate cDNA library, we detected ERß as well as a novel isoform that we termed ERß2. ERß2 contains an in-frame inserted exon of 54 nucleotides that results in the predicted insertion of 18 amino acids within the ERß hormone-binding domain. We also have evidence for the expression of both ERß1 and ERß2 in human cell lines. Competition ligand binding analysis of bacterially expressed fusion proteins revealed an 8-fold lower affinity of ERß2 for 17ß-estradiol (E2) [dissociation constant (Kd ~ 8 nM)] as compared with ERß1 (Kd ~ 1 nM). In vitro transcribed and translated ERß1 and ERß2 bind specifically to a consensus estrogen responsive element in a gel mobility shift assay. Furthermore, we show heterodimerization of ERß1 and ERß2 with each other as well as with ER{alpha}. In affinity interaction assays for proteins that associate specifically with the hormone-binding domain of these receptors, we demonstrate that the steroid receptor coactivator SRC-1 interacts in an estrogen-dependent manner with ER{alpha} and ERß1, but not with ERß2. In cotransfection experiments with expression plasmids for ER{alpha}, ERß1, and ERß2 and an estrogen-responsive element-containing luciferase reporter, the dose response of ERß1 to E2 was similar to that of ER{alpha} although the maximal stimulation was approximately 50%. In contrast, ERß2 required 100- to 1000-fold greater E2 concentrations for maximal activation. Thus, ERß2 adds yet another facet to the possible cellular responses to estrogen.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The estrogen receptor (ER) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that include receptors for steroid hormones, thyroid hormone, vitamin D, retinoic acid, and eicosanoids (1, 2, 3, 4, 5, 6). After diffusion into the cell, estradiol binds to the ER, leading to ER dimerization followed by binding to a conserved estrogen responsive element (ERE) in the regulatory region of target genes. Amino acid sequence comparison of the ER with other nuclear receptors has shown that the receptor is composed of conserved functional regions. The N-terminal transactivating region (AF1) is able to activate transcription in a hormone-independent manner, and this region has been shown to be a target of the mitogen-activating protein kinase-regulatory pathway (7). The DNA-binding domain enables the receptor to bind to its specific DNA target, the ERE. The consensus ERE consists of a palindrome of the half-site sequence 5'-GGTCA-3' separated by 3 bp. The AF2 domain is overlapped by the hormone-binding domain (HBD) and activates transcription in response to estrogen or synthetic estrogen agonists (8).

The mechanism of transactivation by nuclear receptors has recently achieved further complexity by the discovery of an increasing number of coregulators. This group of coregulators can be subdivided into coactivators, corepressors, and integrators. The coactivators were initially biochemically identified as ERAP160 and 140 and RIP160, 140, and 80 (9, 10) by their ability to specifically interact with the HBD of the receptor in a ligand-dependent manner. Specifically for the ER, this interaction was promoted by E2 but antiestrogens such as 4-OH-tamoxifen were able to effectively block this interaction. Yeast two-hybrid screening led to the molecular cloning of the steroid receptor coactivator (SRC) 1, which when cotransfected with nuclear receptors, including ER, was capable of augmenting ligand-dependent transactivation (11). Subsequent cloning and sequence comparison of transcriptional intermediary factor (TIF)2 and glucocorticoid receptor interacting protein (GRIP)1 revealed GRIP1 to be the mouse homolog of human TIF2. More recently, p300/CBP cointegrator protein (p/CIP) [also receptor-associated coactivator (RAC)3 (12) and activator for thyroid hormone and retinoid receptors (ACTR) (13)] were shown to be new members of this family (14). Interestingly, this ER coactivator was also identified as amplified in breast cancer (A1B) (15). In addition, the phospho-CREB-binding protein CBP and the related p300 have been demonstrated to be ER-associated proteins and involved in ligand-dependent transactivation (16, 17). In contrast to the coactivators mentioned above, these proteins are targets of signals mediated by a variety of distinct pathways. Moreover, by interaction with components of the basal transcription machinery, these proteins are thought of as integrators of signals from these diverse pathways. This increasing number of coregulatory factors has added immensely to our understanding of how steroids such as E2 are able to alter the expression of specific genes at the molecular level.

Recently, a new member of the nuclear receptor family with high homology to ER was cloned from rat, mouse, and human and was termed ERß (18, 19, 20). The homology to the rat ER protein (now ER{alpha}) was shown to be 95% in the DNA-binding domain and 55% in the HBD (18). In situ hybridization studies in rat revealed a prominent expression of this novel receptor in the epithelial cells of the secretory alveoli of the prostate and the granulosa cells of the primary, secondary, and mature follicles of the ovary. ERß was found to bind E2 with high affinity and in transient transfection experiments ERß was capable of activating transcription of a reporter gene in an estrogen-dependent manner.

Recently, a partial clone for an alternative splice variant of ERß2 has been described (21). Here we report the complete cloning and functional analysis of this novel rat ERß isoform.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of ERß2, an Alternative Splice Variant of ERß
To obtain clones of ERß we screened a {lambda}gt11 rat prostate cDNA library with two oligonucleotide probes derived from the published ERß sequence, corresponding to the nucleotides 418–477 and 1248–1307. Primary screening of ~900,000 phage revealed four positive plaques that were confirmed in a secondary screening and isolated in a tertiary screening. Inserts of all independently isolated plaques were then subcloned and subjected to nucleotide sequencing. The full-length cDNA clone diverged from the previously published rat cDNA at two positions (496 T to A; 729 C to G). These nucleotide changes result in amino acid changes that are conserved in the published sequence of human ERß. Therefore, it is likely that these result from polymorphisms in the ER. More interestingly, three of four independent clones revealed an insertion of 54 nucleotides at position 1374 of the previously published rat cDNA (Fig. 1Go). This results in an in-frame insertion of 18 amino acids in the predicted HBD of this receptor. We therefore termed this alternative splice variant ERß2, ERß1 being the originally published sequence. The amino acid sequence of this insert exhibits no homology to known proteins or peptide motifs when computer database searches were performed. Recently, the sequence of ERß2 was also reported as an ERß splice variant (21).



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Figure 1. Structure of ERß1 and ERß2

The upper panel shows the functional domains of the previously published rat ERß deduced from its homology to ER{alpha}. Numbers indicate amino acids as described (18 ). The lower panel shows the amino acid sequence encoded by the 54-nucleotide insert found in ERß2.

 
Expression of ERß1 and ERß2 in Different Human Cancer Cell Lines
Since screening of the prostate cDNA library revealed that the majority of clones (3/4) encode ERß2, we examined the expression pattern of this nuclear receptor splice variant in a variety of human cancer cell lines derived from breast, uterus, ovary, and prostate tissue. We isolated RNA from these cells, subjected it to RT using oligo dT primers, and performed PCR reactions with primers derived from the human ERß sequence flanking the insertion site of the 18 amino acids (aa) in the ERß sequence. ER{alpha}-specific primers and primers from the ß2-microglobulin gene were used to control for the quality of the cDNA. As shown in Fig. 2Go, as expected, all cDNAs revealed a product for the ubiquitously expressed ß2-microglobulin (lower panel). The ER{alpha} transcript was detectable in breast cancer cell lines previously described as ER{alpha}-positive such as MCF7, T47D, and BT-20 as well as in the endometrial cancer cell line ECC1 and in the ovarian cancer cell line OVCAR-3. When the PCR reaction was performed with ERß-specific primers, a band corresponding to the expected size for the previously published ERß sequence was detectable in the breast cancer cell lines BT-20, MDA-MB231, and T47D and in primary normal human mammary epithelial cells (HMECs). Moreover, a transcript for ERß1 was detectable in the ovarian cancer cell lines OVCAR-3 and UPN36T. Among endometrium cancer cell lines tested, only the ER{alpha}-negative Ishikawa cell line showed an ERß1 transcript. The human prostate cancer cell lines, PC-3 and DU145, were also positive for ERß1 expression. To confirm that the bands indeed correspond to ERß, we transferred the PCR products onto nitrocellulose and subjected it to Southern blot analysis with a radiolabeled nucleotide probe derived from the previously published ERß sequence. As shown in Fig. 2Go, the band of ~340 bp was indeed reactive with this probe. Interestingly, using PCR primers flanking the insertion in the rat cDNA also revealed a band with the expected size for ERß2 in the human ovarian cancer cell line OVCAR-3 and the osteosarcoma cell line U2OS. To confirm the origin of this PCR product as ERß2, we probed duplicate blots with an oligonucleotide probe corresponding to the unique 54 nucleotides of the ERß2 sequence as shown in Fig. 2Go, top panel. These data indicate that although ERß1 is present in a variety of different cells, the ERß2 transcript is restricted to a minority of these cell lines, suggesting a specific mechanism regulating expression of these splice variants.



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Figure 2. Expression of ER{alpha}, ERß1, and ERß2 in Various Human Cancer Cell Lines

RNA extracted from various human cancer cell lines was subjected to RT using oligo dT primers. Cell lines used were the human ovarian cancer cell lines SW626 (lane 1), OVCAR-3 (lane 2), CAOV-3 (lane 3), UPN36T (lane 4), and the human breast cancer cell lines BT-20 (lane 5), MDAMB231 (lane 6), T47D (lane 7), and MCF7 (lane 8), normal HMEC (lane 9), two human endometrium cancer cell lines ECC1 (lane 10) and Ishikawa (lane 11), the human prostate cancer cell lines PC-3 (lane 12), Du145 (lane 13), and LnCAP (lane 14), the green monkey kidney cell line CV-1 (lane 15), and the human osteosarcoma cell line U2OS (lane 16). Integrity of the cDNAs was verified by PCR using primers specific to human ß2-microglobulin (ß2MG) cDNA (lower panel). Expression of ER{alpha} was analyzed using primers specific to the human ER{alpha} cDNA (second panel from bottom). To analyze expression of ERß1 and ERß2, PCR was performed with primers flanking the alternative splice site. Southern blot analysis on these PCR products was performed using either the ERß2-specific insert as a probe (top panel) or a probe to the common ERß region (second panel from the top).

 
ERß2 Binds Estradiol with Lower Affinity Than ERß1
Since insertion of the 18 aa in ERß2 occurs in the predicted hormone-binding domain of this receptor, we first examined the binding affinity of estradiol for ERß1 and ERß2. Therefore, [3H]E2 was used to conduct competition ligand-binding studies of ERß1 and ERß2. Using bacterial expressed glutathione S-transferase (GST) fusion proteins containing the HBDs of both receptors, dissociation constants (Kd) for E2 were 1 nM for ERß1 but 8-fold higher (Kd = 8 nM) for ERß2 (Fig. 3Go). These data indicate that the 18-aa insertion in the HBD of ERß2 lowers its affinity for E2.



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Figure 3. Estradiol Binds to ERß1 and ERß2

E2 binding to GST fusion proteins of either ERß1 (open squares) or ERß2 (closed squares) HBD was assayed as described in Materials and Methods. Binding is expressed as the percentage of bound radiolabeled E2 at a given competitor concentration compared with binding of labeled E2 in the absence of competitor.

 
ERß2 Binds to an ERE
It has been previously shown that ERß1 is capable of binding to a consensus ERE with high affinity. To test whether the 54-nucleotide insertion of ERß2 had an influence on DNA binding, we conducted electrophoretic mobility shift assays (EMSAs). Receptors were expressed in vitro to comparable levels (data not shown). As demonstrated in Fig. 4Go, both ERß1 and ERß2 were able to bind specifically to the ERE probe in a hormone-dependent manner, under the stringent conditions used (22). Specificity of binding was confirmed by the fact that unlabeled ERE could compete for binding of all receptors to the labeled probe (lanes 5), whereas competition with a mutated ERE (lanes 6), or an unrelated AP-1 sequence (lanes 7), did not have an influence on binding.



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Figure 4. ER{alpha}, ERß1, and ERß2 Bind in an Estradiol-Dependent Manner to an ERE

To analyze DNA binding of the different ERs, EMSA was performed with in vitro translated ER{alpha} (left panel), ERß1 (middle panel), and ERß2 (right panel). Protein/DNA incubation was performed in the absence of hormone (lane 1) or increasing concentrations of E2 (1 nM, 10 nM, 100 nM) (lanes 2–4). Specificity of binding was confirmed by competition using an unlabeled ERE (lanes 5), a mutated ERE (lanes 6), or the unrelated AP-1 site (lanes 7) in the presence of 100 nM E2.

 
When EMSA was performed with increasing E2 concentrations (lanes 2–4), it was interesting to note that maximal binding of ER{alpha} (to the ERE) occurred at the lowest concentration of E2 used (1 nM) (lane 2), whereas both ERß1 and ERß2 showed a dose response with maximal binding at 100 nM. Despite the apparent differences in hormone binding affinity for ERß1 and ERß2, both receptors bound with similar affinity to DNA.

Heterodimerization Occurs between ER{alpha} and Both ERß Isoforms
Since it has been demonstrated that ER{alpha} and ERß1 are capable of forming heterodimers upon ligand binding, we set forth to investigate whether the newly identified splice variant of ERß also forms heterodimers with ER{alpha}. Therefore, we performed EMSA on a labeled ERE probe using in vitro translated full-length receptors in the presence of 100 nM E2 (Fig. 5Go). When ER{alpha} (lanes 1 and 2), ERß1 (lanes 3 and 4), or ERß2 (lanes 5 and 6) were incubated with the labeled ERE probe, antibodies specific for ER{alpha} or ERß were able to supershift the homodimeric ER/DNA complexes (lanes 2, 4, and 6). When in vitro translated ER{alpha} and ERß1 (lanes 7–10) or ER{alpha} and ERß2 (lanes 11–14) were coincubated with the labeled probe, each antibody was able to shift the protein/DNA complex to a different size than the respective homodimeric receptor (lanes 8, 9 and 12, 13). When both antibodies were coincubated either with ER{alpha}/ERß1 or with ER{alpha}/ERß2, a supershifted band was detected, indicating that indeed ER{alpha} forms heterodimers on DNA with ERß1 and ERß2 (lanes 10 and 14). Heterodimers could be detected at E2 concentrations as low as 1 nM (data not shown).



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Figure 5. Heterodimerization of Different ERs

Radiolabled ERE probe was incubated with in vitro translated ER{alpha} (lanes 1 and 2), ERß1 (lanes 3 and 4), or ERß2 (lanes 5 and 6) either in the absence (lanes 1, 3, and 5) or in the presence (lanes 2, 4, and 6) of specific antibodies. Similarly, cotranslated ER{alpha}/ERß1 was incubated with the ERE probe in the absence (lane 7) or in the presence of an ER{alpha}-specific antibody (lane 8), an ERß-specific antibody (lane 9), or both (lane 10). The same experiment was performed using cotranslated ER{alpha} and ERß2 (lanes 11–14).

 
Interaction of SRC1 with ER{alpha} and ERß1, but Not ERß2
The role of putative coactivators in the hormone-dependent transcriptional regulation by ERß1 and ERß2 was addressed by affinity purification of ERß1- and ERß2-associated proteins. The HBD of these receptors was fused to GST and used to purify proteins from the metabolically labeled human breast cancer cell line MCF-7 (Fig. 6Go), the human breast cancer cell line MDA-MB231, and the human osteosarcoma cell line U2OS (data not shown). In the case of ER{alpha} (lanes 1–3), previously described proteins bound GST-HBD-ER{alpha} in the presence of E2 (lane 2), but not in its absence (lane 1) nor in the presence of the ER antagonist tamoxifen (lane 3). Using the GST-HBD-ERß1 fusion protein as an affinity matrix (lanes 4–6), only the ERAP160/SRC1 family of proteins could be detected (lane 5). Interestingly, when performing this experiment with the same amount of GST-HBD-ERß2, we were unable to detect associated proteins that specifically interact with ERß2 (lane 8). As ERß2 ligand-binding analysis had detected an 8-fold lower affinity for E2, we conducted the same experiment using increasing amounts of estradiol (1 µM and 10 µM). Again, using GST-HBD-ERß2 as an affinity matrix, we were unable to detect proteins interacting with the receptor in an estradiol-dependent manner (data not shown). To exclude the possibility that other domains of ERß2 were required for coactivator interaction, we performed GST pull-down experiments using full-length ERß1 and ERß2 as GST fusion proteins and obtained similar results (data not shown). To test whether the proteins in the 160-kDa range, detected in this assay to bind to ER{alpha} and ERß1, include the cloned steroid receptor coactivator SRC1, we performed Western blot analysis with an anti-SRC1-specific antibody on proteins associating with the ligand-binding domain of these receptors (Fig. 7AGo). For this purpose we used whole-cell extracts from MCF-7 cells. Cell lysates were incubated with the GST-HBD affinity matrices of the different receptors in the absence or presence of increasing amounts of E2 (1 µM and 10 µM) or 4-OH-Tamoxifen (1 µM). Specifically bound proteins were resolved by SDS-PAGE and transferred onto nitrocel-lulose. Immunoblotting with a monoclonal antibody directed against human SRC1 revealed significant SRC1 binding only for GST-HBD-ER{alpha} and GST-HBD-ERß1 in the presence of E2 but not for GST-HBD-ERß2 (Fig. 7AGo). Negative control samples consisting of glutathione-sepharose or GST immobilized on glutathione sepharose did not show binding (data not shown). To confirm these results, we tested the ability of recombinant SRC1 to interact with ERß1 or ERß2. Again, in vitro synthesized [35S] methionine-labeled SRC1 associated specifically in an E2 dependent manner only with GST-HBD-ER{alpha} (lane 2) and GST-HBD-ERß1 (lane 5) but not GST-HBD-ERß2 (lane 8) (Fig. 7BGo).



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Figure 6. The HBD of ERß1, but Not ERß2, Interacts with a Protein of 160 kDa in Vitro

Whole cell extracts of MCF-7 cells were metabolically labeled with [35S]methionine and were bound to the GST-HBD fusion of ER{alpha} (lanes 1–3), ERß1 (lanes 4–6), and ERß2 (lanes 7–9). The fusion proteins were previously immobilized on glutathione-Sepharose in the absence (lanes 1, 4, and 7) or the presence (lanes 2, 5, and 8) of 1 mM17ß-estradiol (E) or 4-OH-tamoxifen (T) (lanes 3, 6, and 9). Proteins were eluted in SDS/sample buffer and were resolved on 7.5% SDS/PAGE.

 


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Figure 7. SRC1 Associates with GST-HBD-ER{alpha} and GST-HBD-ERß1, but Not with GST-HBD-ERß2

A, Whole-cell extracts from the human breast cancer cell line MCF-7 were incubated with GST-HBD-ER{alpha} (top panel), GST-HBD-ERß1 (middle panel), and GST-HBD-ERß2 (bottom panel) immobilized on glutathione-Sepharose in the absence (lane 1) or presence of 1 µM E2 (lane 2), 10 µM E2 (lane 3), or 1 µM 4-OH-tamoxifen (T) (lane 4). Specifically associated proteins were recovered by boiling in SDS/sample buffer, resolved on 7.5% SDS/PAGE, and transferred to nitrocellulose. Filters were cut and probed with an SRC1 monoclonal antibody, and specifically bound primary antibody was detected with peroxidase-coupled secondary antibody and chemiluminescence. B, Recombinant 35S-labeled SRC1 produced by in vitro transcription/translation in a rabbit reticulocyte lysate was bound to GST-HBD-ER{alpha} (lanes 1–3), GST-HBD-ERß1 (lanes 4–6), and GST-HBD-ERß2 (7 8 9 ) im-mobilized on glutathione-linked Sepharose in the absence (lanes 1, 4, and 7) or presence of 1 µM estradiol (lanes 2, 5, and 8) and 1 µM tamoxifen (lanes 3, 6, and 9). The translation product (15 ml) was incubated with the different affinity matrices that had been blocked with 3% BSA under the indicated conditions at 4 C for 30 min. After extensive washing, bound proteins were eluted in 20 mM reduced glutathione-containing elution buffer (120 mM NaCl/100 mM Tris-HCl, pH 8.0) and separated by 7.5% SDS-PAGE. Gels were treated with a fluorophore, dried, and visualized by autoradiography.

 
Transcriptional Activity of ERß2 on an ERE
To investigate whether an ERE can mediate transcriptional activity of ERß2, we used a luciferase reporter gene containing two copies of an ERE and the viral thymidine kinase promoter, driving the expression of the luciferase reporter gene for transactivation studies. We performed cotransfection experiments in which U2OS cells were transfected with either an expression plasmid for ER{alpha}, ERß1, or ERß2 and the estrogen-responsive reporter gene construct. E2 maximally stimulates ER{alpha}-mediated transactivation through an ERE of approximately 6-fold at a doses as low as 0.1 nM (Fig. 8Go). ERß1 was less potent in stimulating transcription through an ERE with only 50% of the maximal level seen with ER{alpha}, although transcriptional activation occurs with a similar dose response. In contrast, when the same experiment was performed with an expression plasmid for ERß2, E2 failed to stimulate transactivation through the ERE at 0.1 nM or 1 nM E2 (Fig. 8Go). Increasing doses of E2 to 100 nM were able to stimulate ERß2-mediated activation to a level comparable ERß1 (Fig. 8Go). These data indicate that ERß2 requires 100- to 1000-fold higher concentrations of E2 to stimulate transcription to the same extent as ERß1.



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Figure 8. Transcriptional Activation of ER{alpha}, ERß1, or ERß2

U2OS cells were transiently cotransfected with the ERE reporter plasmid and expression plasmids for either ER{alpha}, ERß1, or ERß2. Cells were left untreated or treated with 0.1 nM, 1 nM, 10 nM, and 100 nM estradiol. Data were normalized as the ratio of raw light units to ß-gal-units and expressed as the fold of induction relative to untreated controls. The data presented are the mean of three independent experiments. Error bars represent SEM.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estradiol mediates its diverse biological effects by binding to the ER, thereby allowing the receptor to bind to and activate transcription through estrogen-responsive elements in the promoter region of target genes. The phenotype of ER{alpha} knockout mice pointed to the potential existence of an alternative mediator for E2 action (23). Recently, a new member of the nuclear receptor family with very high homology to ER{alpha} was cloned and termed ERß (18). The identification of ERß has added a new level of complexity to E2 signaling.

In this report we describe the cloning of an alternative splice variant of ERß, ERß2, which contains an 18-aa insert in the predicted hormone binding domain of this nuclear receptor, adding yet another level of complexity to E2 signaling. We have demonstrated that this isoform is expressed in normal rat prostate as well as various human cancer cell lines. The fact that in some cell lines one isoform appears to be more prominent than the other, and that this relative ratio varies from tissue to tissue examined, suggests a specific mechanism regulating expression of one or the other splice variant.

Interestingly, we demonstrate that both receptors coexist in certain cells and can heterodimerize on the ERE. Further complexity is achieved, since not only do ERß1 and ERß2 heterodimerize, but each can also heterodimerize with ER{alpha}. Since we could not demonstrate that ERß2 binds the coactivator SRC1 one could speculate that heterodimerization of ER{alpha} or ERß1 with ERß2 regulates the recruitment of this coactivator to the transcriptional complex. Since previous studies have demonstrated that other transcriptional cointegrators, namely p300 and the phospho-CREB binding protein (CBP) appear to be rate limiting for active transcription (16), this reduction of SRC1 recruitment might reduce transcriptional activity through the ERE.

Another recent study has shown that while both ER{alpha} and ERß1 activate E2-mediated transcription through an ERE, they exert opposite effects through an AP-1 site (24). While E2 stimulates ER{alpha}-mediated transcription through an AP-1 site, E2 inhibits ERß1-mediated transcription through the same response element. It still has to be demonstrated which effect ERß2 mediates through an AP-1 site, and which effect the different heterodimers of these receptors mediate through this response element in the presence of different ligands.

Interestingly, alignment of the ER{alpha} sequence with ERß, as compared with the predicted structure of nuclear receptors, indicates that the 18-aa insert in ERß2 lies in helix 6 of this receptor. This relatively nonconserved region among different nuclear receptors follows immediately after the {alpha}-turn within the ligand binding domain of the receptor (25). The addition of 18 aa in this region might distort the correct conformation of this receptor for high-affinity E2 binding as supported by our E2 binding data. High physiological E2 concentrations achieved especially in the ovary during pregnancy or the periovulatory phase might be sufficient to activate ERß2. Another interesting possibility is that this insertion creates a new conformational change required for high-affinity binding of a yet unidentified ligand other than E2.

With respect to the basic mechanisms by which nuclear receptors initiate transcription of their target genes, much of recent research in the field has focused on so-called coactivators of these proteins, which bind the nuclear receptors in a ligand-dependent manner to augment AF-2-mediated transactivation. The coactivators have been identified by the in vitro interaction of the ligand-binding domain of ER{alpha} fused to GST as an affinity matrix for proteins interacting in an E2 dependent manner (9, 10). Using the same approach we could demonstrate the interaction of the coactivator of nuclear receptors, SRC1 with both ER{alpha} and ERß1. Interestingly, GST fusion proteins of both the ligand-binding domain and the full-length ERß2 failed to interact with SRC1 in a ligand-dependent manner, despite the fact that both fusion proteins were able to bind both E2 and an ERE. It is striking that this results in a shift in the dose response of ERß2 to E2 but not the maximal level of activation. This suggests the possibility that under certain conditions ERß2 might act to dampen cellular responses to estrogen.

We and others (29) have identified an alternative splice variant of ERß termed ERß2. This protein exhibits interesting properties as a mediator of estrogen action and provides new complexity to the spectrum of potential cellular responses to estrogen.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Library Screening and Plasmids
We screened a {lambda}gt11 rat prostate cDNA library (CLONTECH, Palo Alto, CA), according to the manufacturers guidelines with two radiolabeled oligonucleotides corresponding to nucleotides 418–477 and 1248–1307 of the previously published rat ERß sequence. Labeling of the probe was performed with {gamma}-32P-ATP (6000 mCi/mmol; New England Nuclear, Boston, MA) in the presence of T4 polynucleotide kinase according to standard procedures (26). Positive plaques from the primary screening were isolated by secondary and tertiary screening, and phage DNA was obtained by boiling plaques in 100 ml H2O. Plaque DNA was then amplified by PCR using ERß-specific primers corresponding to nucleotides 402–419 and 1866–1885 of the ERß sequence. One plaque contained the full-length ERß cDNA. The resulting PCR fragment was blunt ended using T4 DNA polymerase and was subcloned into the EcoRV site of pcDNA3.0 (Invitrogen, San Diego, CA), resulting in pERßI and was completely sequenced from both strands by automated sequence analysis (ABI 3000, Molecular Biology Core Facility, Dana Farber Cancer Institute). DNA from the remaining plaques yielded PCR products when a 5'-primer corresponding to nucleotides 922–941 of the ERß sequence was used for amplification, indicating that these were partial cDNA clones. Amplification of the predicted HBD from these different clones was performed with primers

5'-CGTGGATCCGAGCAGGTACACTGCCTG-3'

5'-GATGAATTCTCACTGAGACTGTAGGTTC-3' and the resulting fragments were BamHI/EcoRI digested and subcloned into the corresponding sites of pGEX2TK (27) resulting in pGEX ERß1HBD and pGEX ERß2HBD. These plasmids were also subjected to complete nucleotide sequence analysis, revealing the alternative splice variant in three of these clones. To obtain the full-length ERß2 cDNA we liberated the ERß1 cDNA from pERßI by EcoRI/NotI digest and subcloned the 1.4-kb insert into the corresponding sites of pBluescript SK(-), resulting in pBS ERß1. This subclone was confirmed by partial sequence analysis. The 3'-sequence of ERß2 was then liberated from the pGEX2TK ERß2 plasmid by EcoRI digest, blunt ending, and SmaI digest and subcloned into NotI-digested, blunt ended, and then SmaI-digested pBSERß1, resulting in pBSERß2. Correct orientation of the 3'-end was confirmed by restriction analysis and confirmed by partial sequence analysis, also revealing the presence of the 54-nucleotide insert. To generate eukaryotic expression plasmids for ERß1 and ERß2, the corresponding cDNAs were liberated from pBSERß1 and ERß2 by SacII digest, blunt ending followed by XhoI digest, and subcloned into NotI-digested, blunt ended, and then XhoI-digested pcDNA 3.1(-) vector (Invitrogen), resulting in pcDNA ERß1 and pcDNA ERß2. To obtain full-length GST fusion proteins of ERß1 and ERß2 the corresponding cDNAs were PCR amplified with primers

5'-GAAGATCTATGACATTCTACAGTCCTGC-3'

5'-GATGAATTCTCACTGAGACTGTAGGTTC-3' using pBSERß1 and ERß2 as templates, BglII/EcoRI digested, and subcloned into BamHI/EcoRI-digested pGEX2TK plasmid, resulting in pGEX ERß1fl and pGEX ERß2fl. Both plasmids were verified by complete nucleotide sequence analysis.

Cells and Cell Culture
Cell lines MCF-7, BT-20, MDAMB231, T47D, ECC1, Ishikawa, PC-3, Du145, LnCAP, CV1, and U2OS were obtained from American Type Culture Collection (Manassas, VA). The normal HMECs were purchased from Clonetics (San Diego, CA). The human ovarian cancer cell lines Sw626, OVCAR-3, CAOV-3, and UPN36T were a gift from Dr. S. Cannistra. Cells were maintained in DMEM containing 10% FBS (vol/vol) (Sigma Chemical Co., St. Louis, MO) at 37 C and 5% CO2/95% air.

RT-PCR and Southern Blot
RNA extraction was performed using the Ultraspec RNA isolation system (Biotecx, Houston, TX) according to manufacturer’s guidelines. RT was performed using oligo dT primers. Using the GIBCO BRL RT Kit (GIBCO BRL, Grand Island, NY). Two micrograms of cDNA were used as a template for PCR reactions using two microglobulin-specific primers (CLONTECH, primers specific to human ER (5'-GGGAGCTGGTTCACATGATC-3' and 5'-GTCCAGGACTCGGTGGAT-ATG-3') or primers specific to human ER ((5'-GCCTCCATGATGATGTCCCTG-3' and 5'-GATCATGGCCTTGACACAGAG-3'). PCR cycling was performed using a touch down program: 1) 95 C, 1 min; 2) 95 C, 30 sec; 60 C, 30 sec (-0.5 C/cycle); 72 C, 2 min; 3) 72 C, 2 min, 4 C, for ever, 35 cycles, 60 C to 40 C. Resulting PCR products were subjected to electrophoresis in 2% agarose gels. In case of ER, products were transferred to nitrocellulose and probed with end-labeled oligonucleotides either common to ERß1 and ERß2.

(5'-TCCTCAGAAGACCCTCACTGGCCACGTTGCGCAG-ATGAAGAGTGCTGCCCCAAGG-3') or specific for ERß2 (5'-GCCAAGAAGATTCCCGGCTTTGTGGAGCTCAGCCTG-TTCGACCAAGTGCGGCTCTTGGAG-3'). Both were washed three times in 1x saline sodium citrate, 0.1% SDS at room temperature and for 30 min in buffer containing 0.1x saline sodium citrate, 0.1% SDS at 50 C.

Ligand Competition Analysis
For ligand competition studies GST HBD fusion proteins of ERß1 and ERß2 were diluted in HED buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol) and incubated with 1 nM [3H]17ß-estradiol and various concentrations of unlabeled diethylstilbestrol. The bound and unbound estrogens were separated using dextran-coated charcoal (28). The amount of bound [3H]17ß-estradiol is presented as a percent of total bound in the absence of diethylstilbestrol.

Gel Mobility Shift Assay
Recombinant ER{alpha}, ERß1, and ERß2 cDNAs were transcribed and translated in vitro in TNT-T3 coupled rabbit reticulocyte lysates (Promega, Madison, WI) from the T3 Promoter following the manufacturer’s guidelines. Typically 4 ml of programmed lysate (or 4 ml of a 1:50 dilution of the ERß1 GST-fusion protein for ERß1 and ERß2 heterodimers) were used in each binding reaction. The binding reactions were carried out in binding buffer A100 (20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 1 mM dithiothreitol), 0.5 mg of poly deoxyinosinic-deoxycytidylic acid, 20 mg BSA, 4 ml H2O, 7.5 mM MgCl2 (final concentration), and 4 ng of probe that was labeled by end-filling with Klenow in the presence of [32P]{alpha}-dGTP. Preincubations containing ligand, antibody, and/or cold competitor as indicated were performed at room temperature for 20 min. After the incubation step the probe was added and binding was conducted for 15 min at room temperature. The entire reaction of 17 ml was loaded onto a 4% gel, and electrophoresis was carried out at 110 V for 2 h at room temperature. Gels were dried and exposed for 2–5 h at -80 C. The following antibodies were used: AER 314 (Neomarkers, Fremont, CA), mouse polyclonal serum for ERß1 and ERß2. We used the following oligonucleotides and their compliments as probes and competitors:

ERE, 5'-GATCTCTTTGATCAGGTCACTGTGACCTGACT-TTG-3';

mtERE, 5'-GATCTCTTTGATCAGGACACAGTGTCCTGA-CTTTG-3';

AP1, 5'-GAATGGTGACTCATATTTGAACAAGCCTGCAA-TGCCCAGCAGA-3'.

Metabolic Labeling and Protein-Protein Interaction Assay
Before the metabolic labeling, MCF-7 cells were preincubated with methionine-free DMEM for 10–20 min. Confluent 150-mm diameter dishes were labeled with 1 mCi (1 Ci = 37 GBq) [35S]methionine (New England Nuclear, Boston, MA) for 4 h in methionine-free DMEM. After labeling cells were washed extensively with ice-cold PBS and lysed in 1 ml of buffer A (150 mM NaCl, 50 mM Tris, pH 7.4, 5 mM EDTA, 0.5% Nonidet P-40). After 30 min of rotation at 4 C, cell extracts were clarified by centrifugation at 12,000 rpm, and the supernatant was collected in a fresh tube. Lysates containing 2.5 x 107 cpm were then incubated with a GST fusion protein containing the HBDs of either ER{alpha}, ERß1, or ERß2 (GST-HBD ER{alpha}, ERß1, or ERß2) immobilized on 50 ml of glutathione-Sepharose beads in the presence or absence of the appropriate ligand in buffer B (150 mM NaCl, 50 mM Tris, pH 7.4, 5 mM EDTA) as previously described (9). After washing the beads three times in 1 ml of buffer B and once in 1 ml of buffer A, proteins were eluted in SDS/sample buffer and resolved on 7.5% SDS/PAGE. Gels were fixed in 35% methanol/10% glacial acetic acid, fluorographed in Enhance solution (New England Nuclear), and dried before exposure to film.

Western Blotting
Protein-protein interaction assays were performed as described above using unlabeled cell extracts from MCF-7 cells. Proteins were resolved directly in SDS/polyacrylamide gels after boiling in SDS sample buffer. Immunodetection was performed after blocking the membranes overnight at 4 C in 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.05% Tween 20, and 5% powdered milk by incubating membranes with an anti-SRC1 antibody for 2 h at room temperature. Monoclonal antibody raised against GST-SRC1 was used for immunoblot analysis in a dilution of 1:100. Specifically bound primary antibody was detected with peroxidase-coupled secondary antibody and chemiluminescence.

In Vitro Transcription and Translation
Recombinant SRC1 cDNA in pBluesript was transcribed and translated in TNT-T3 coupled reticulocyte lysates (Promega, Madison, WI) in the presence of [35S]methionine from the T3 promoter following the manufacturer’s guidelines.

Transient Transfection and Luciferase Assay
For transient transfections U2OS cells were seeded in 24-well plates in phenol red-free DMEM supplemented with 10% charcoal dextran-treated FBS. Cells at a density of 40,000/well were transfected with 100 ng of reporter plasmid, 10 ng of receptor expression vector, 10 ng ßActinßGal plasmid and 680 or 690 ng of salmon sperm DNA to a total of 800 ng using the calcium phosphate/DNA precipitation method. After 16 h, cells were washed once with PBS and were left either untreated or treated with 0.1 nM, 1 nM, 10 nM, or 100 nM E2 for 16 h. For luciferase assays, cells were lysed in potassium phosphate containing 1% Triton X-100. Light emission was detected using a luminometer after addition of luciferin. ß-Gal activity was detected using the Galacto-Star (Tropix, Bedford, MA).


    ACKNOWLEDGMENTS
 
We thank S. Cannistra for the gift of the UPN36T cell line, J. DiRenzo and J. DeCaprio for SRC1 antibodies, and J. Brüning for helpful discussions and critical reading of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Myles Brown, M.D., Department of Adult Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115. E-mail: myles-brown{at}dfci.harvard.edu

This work was supported by funds from a Deutsche Forschungsgemeinschaft Fellowship (to B.H.) and by National Cancer Institute Grant CA-57374 (to M.B.).

1 Present address: Frauenklinik, Heinrich Heine Universität, Postfach 10 10 07, D-40001 Düsseldorf, Germany. Back

2 Present address: Robert Lurie Cancer Center, Northwestern University School of Medicine, 745 North Fairbanks, Chicago, Illinois 60611. Back

Received for publication January 29, 1998. Revision received August 24, 1998. Accepted for publication October 1, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Carson-Jurica MA, Schrader WT, O’Malley BW 1990 Steroid receptor family: structure and functions. Endocr Rev 11:201–220[Abstract]
  2. Parker MG 1992 Growth regulation by nuclear hormone receptors. In: Franks LM (ed) Cancer Surveys, 240 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 1–240
  3. Parker MG 1991 Nuclear Hormone Receptors. Academic Press Inc., San Diego, CA, pp 1–404
  4. Beato M, Herrlich P, Schutz G 1995 Steroid hormone receptors: many actors in search of a plot. [Review]. Cell 83:851–857[Medline]
  5. Forman BM, Goode E, Chen J, Oro AE, Bradley DJ, Perlmann T, Noonan DJ, Burka LT, McMorris T, Lamph WW, et al 1995 Identification of a nuclear receptor that is activated by farnesol metabolites. Cell 81:687–693[Medline]
  6. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. [Review]. Cell 83:835–839[Medline]
  7. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494[Abstract]
  8. Durand B, Saunders M, Gaudon C, Roy B, Losson R, Chambon P 1994 Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity. EMBO J 13:5370–5382[Abstract]
  9. Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C, Brown M 1994 Estrogen receptor-associated proteins: possible mediators of hormone-induced transcription. Science 264:1455–1458[Medline]
  10. Cavaillès 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]
  11. Oñate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  12. Li H, Gomes PJ, Chen JD 1997 RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF2. Proc Natl Acad Sci USA 94:8479–8484[Abstract/Free Full Text]
  13. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569–580[Medline]
  14. Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK, Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function [see comments]. Nature 387:677–84[CrossRef][Medline]
  15. Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS 1997 AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277:965–968[Abstract/Free Full Text]
  16. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–14[Medline]
  17. Hanstein B, Eckner R, DiRenzo J, Halachmi S, Liu H, Searcy B, Kurokawa R, Brown M 1996 p300 is a component of an estrogen receptor coactivator complex. Proc Natl Acad Sci USA 93:11540–11545[Abstract/Free Full Text]
  18. 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]
  19. Mosselman S, Polman J, Dijkema R 1996 ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  20. Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, Giguere V 1997 Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor ß. Mol Endocrinol 11:353–365[Abstract/Free Full Text]
  21. Chu S, Fuller PJ 1997 Identification of a splice variant of the rat estrogen receptor beta gene. Mol Cell Endocrinol 132:195–199[CrossRef][Medline]
  22. Brown M, Sharp PA 1990 Human estrogen receptor forms multiple protein-DNA complexes. J Biol Chem 265:11238–11243[Abstract/Free Full Text]
  23. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O 1993 Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 90:11162–11166[Abstract]
  24. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J, Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen receptors ER{alpha} and ERß at AP1 sites [see comments]. Science 277:1508–1510[Abstract/Free Full Text]
  25. Wurtz JM, Bourguet W, Renaud JP, Vivat V, Chambon P, Moras D, Gronemeyer H 1996 A canonical structure for the ligand-binding domain of nuclear receptors. Nat Struct Biol 3:206[Medline]
  26. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K 1997 Current Protocols in Molecular Biology. Greene Publishing Associates, New York
  27. Kaelin WGJ, Pallas DC, DeCaprio JA, Kaye FJ, Livingston DM 1991 Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell 64:521–532[Medline]
  28. Zajchowski DA, Sager R, Webster L 1993 Estrogen inhibits the growth of estrogen receptor-negative, but not estrogen receptor-positive, human mammary epithelial cells expressing a recombinant estrogen receptor. Cancer Res 53:5004–5011[Abstract]
  29. Petersen DN, Tkalcevic, GT, Koza-Taylor PH, Turi TG, Brown TA 1998 Identification of estrogen receptor beta2, a functional variant of estrogen receptor beta expressed in normal rat tissues. Endocrinology 139:1082–1092[Abstract/Free Full Text]