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Endocrinology Vol. 139, No. 3 1082-1092
Copyright © 1998 by The Endocrine Society

ARTICLES

Identification of Estrogen Receptor ß2, A Functional Variant of Estrogen Receptor ß Expressed in Normal Rat Tissues

Donna N. Petersen, George T. Tkalcevic, Petra H. Koza-Taylor, Tom G. Turi and Thomas A. Brown

Department of Molecular Sciences and Osteoporosis Group, Pfizer Central Research, Groton, Connecticut 06340

Address all correspondence and requests for reprints to: Thomas A. Brown, Department of Molecular Sciences and Osteoporosis Group, Pfizer Central Research, Eastern Point Road, Groton, Connecticut 06340. E-mail: thomas_a_brown{at}groton.pfizer.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of estrogen and estrogen agonists can be mediated by estrogen receptor {alpha} (ER{alpha}) and estrogen receptor ß (ERß). We now report the identification and initial characterization of several novel isoforms of rat ERß messenger RNA (mRNA). The most abundant of these mRNA variants we have called ERß2. ERß2 had an in-frame insertion of 54 nucleotides that resulted in the predicted insertion of 18 amino acids within the ligand binding domain. We demonstrated by semiquantitative RT-PCR and RNase protection that ERß2 mRNA was expressed at levels equal to those of the previously published ERß (ERß1) in ovary, prostate, pituitary, and muscle. In tissues of the nervous system, including frontal cortex, hippocampus, and hypothalamus, ERß1 was present in a 2- to 6-fold greater abundance than ERß2. We have also detected variants of both ERß1 and ERß2 mRNAs that contained deletions of 117 bp encompassing the region encoding the second zinc finger of the DNA binding domain. All four mRNA species were efficiently translated into functional protein in a heterologous system. ERß2 bound estradiol with a lower affinity (Kd 5.1 nM) than either ER{alpha} (0.19 nM) or ERß1 (0.14 nM). The binding of ERß2 was selective in that cortisol, testosterone, aldosterone, and progesterone among other agents did not compete for estradiol binding. However, a variety of known estrogenic agents, including physiological estrogens (estrone and estriol), plant and environmental estrogens (genistein, coumestrol, bisphenol A, methoxychlor), and pharmocological agents (tamoxifen, 4-hydroxytamoxifen) did effectively compete for estradiol binding to both ERß1 and ERß2. Interestingly, the binding pharmacology differed among the agents tested. For example, genistein competed effectively for estradiol binding to ERß1 but was >150-fold weaker at competing from ERß2. In contrast, 4-hydroxytamoxifen competed equally well at both receptors. We have also demonstrated by a gel shift assay that both ERß1 and ERß2 bound specifically to DNA containing a consensus estrogen response element. ERß1 and ERß2 could heterodimerize with each other and with ER{alpha}. Both ERß1 and ERß2 activated transcription in response to estradiol, however, ERß2 required a 1000-fold greater estradiol concentration for activity than did ERß1. Cotransfection of ERß2 had no effect on ERß1 activation when used in a equal ratio. A 10-fold excess of ERß2 did raise the half-maximal dose of estradiol required for transcriptional activation, whereas the maximal level of induction did not change. The ERß complementary DNAs deleted within the DNA binding domain could not bind to DNA or activate transcription from this reporter in the cell backgrounds tested. In conclusion, although the physiological significance of these ERß variants warrants further investigation, ERß2 mRNA encodes a specific, functional receptor for estradiol and estrogenic agents. We propose that ERß2 should also be considered in addition to ERß1 and ER{alpha} when describing the effects of estrogen, estrogen agonists/antagonists, or environmental estrogens.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MOLECULAR mechanisms mediating the effects of estrogen and estrogenic compounds have significance in health and disease. Currently, hormone replacement therapy (HRT) is recommended for the treatment of symptoms associated with menopause, including hot flushes and osteoporosis (1, 2). A major added benefit of HRT is a decreased mortality attributable largely to a significant decrease in cardiovascular disease (3, 4). Increasing clinical evidence also indicates that HRT improves cognition and is associated with a decreased incidence of Alzheimer’s disease (5, 6). Unfortunately the use of HRT remains relatively low, partly due to concerns about possible side effects of increased incidence of breast and endometrial cancers (7, 8). As a result, researchers are now attempting to develop second generation selective estrogen agonists for use in postmenopausal women. For example, raloxifene and droloxifene are currently in advanced clinical trials (9). These agents selectively prevent bone loss and lower serum cholesterol, while antagonizing the action of estrogen in reproductive tissues (10, 11). An additional reason to study estrogen action is the concern regarding the adverse effects of environmental estrogens on wildlife and man (12, 13). Both the development of new therapies and the in vitro assessment of environmental estrogens would be aided by additional descriptions of the molecular mechanisms mediating estrogen action.

Estrogen can act through the classical estrogen receptor, now called ER{alpha} (14, 15). ER{alpha}, a member of the steroid/thyroid/retinoid hormone receptor superfamily, binds estrogen with high affinity. Ligand binding induces a conformational change in ER{alpha} and subsequent DNA binding to specific estrogen response elements (EREs) present in target genes. ER{alpha} then activates transcription initiation through protein:protein interactions with accessory factors. Conclusive evidence that ER{alpha} mediates specific in vivo effects of estrogen has been obtained using estrogen receptor knockout animals (ERKO) (16). For example, classic estrogen responses in the uterus such as water imbibition and hyperemia, robust proliferation, and gene induction are absent in the ERKO animals (16, 17). Tamoxifen can also induce these responses in normal mice but is similarly ineffective in ERKO animals (16). ERKO mice also implicate ER{alpha} in less appreciated physiological functions such as male fertility and male sexual behavior (18, 19). On the contrary, other experiments in ERKO mice have suggested that some estrogenic effects are not mediated solely by ER{alpha}. For example, progesterone receptor messenger RNA (mRNA) is induced in the preoptic area of ERKO animals by estrogen (20, 21). Also, estrogenic compounds such as 4-hydroxyestradiol and kepone were shown to induce lactoferrin mRNA in the uterus of ERKO animals (22). These data suggest the existence of an additional receptor capable of mediating estrogenic action. The recent cloning of a new estrogen receptor, called ERß, by Kuiper et al. (23) supports this suggestion, although the physiological role of this new receptor remains undetermined. The human (24) and mouse (25) homologs of ERß have also been reported.

ERß possesses the properties expected of a physiological relevant estrogen receptor (23, 24, 25, 26). It is a member of the steroid hormone superfamily; it binds estrogen with high affinity; it activates transcription in response to estrogen; and it is expressed in estrogen target tissues such as pituitary and uterus. ERß is quite distinct from ER{alpha}. In the conserved DNA binding and ligand binding domains, the amino acid identities are 95% and 55%, respectively (23, 24, 25). The amino terminal AF-1 domain and hinge region exhibit little primary sequence homology. ERß binds estradiol with a dissociation constant (Kd) of 0.4 nM vs. the 0.1 nM reported for ER{alpha} under the same assay conditions (26). In general, other known ligands including estrogen metabolites and partial agonists/antagonists such as tamoxifen do not discriminate between ER{alpha} and ERß (26). The complete tissue distribution of ERß has not yet been described; however, initial reports indicate a distinct but overlapping pattern of expression relative to ER{alpha} (21, 26). ERß is highly expressed in prostate and ovary with lower expression in brain, uterus, and testis (24, 26). As reviewed by Katzenellenbogen and Korach (27), the discovery of ERß raises important questions regarding the mechanisms of action of estrogen. The action of antagonists such as ICI164384 and agonists/antagonists such as droloxifene and raloxifene must now be interpreted with respect to their actions on this new receptor. In addition, it would be prudent to consider action of ERß when measuring the effects of potential environmental estrogens. Our primary focus is to unravel the physiological roles of ER{alpha} and ERß to design the appropriate selectivity to treat osteoporosis, cardiovascular disease, Alzheimer’s, and other disease states while minimizing the undesirable effects.

In the process of cloning rat ERß, we identified several ERß mRNA variants. One version, termed ERß2, encoded a receptor with an 18-amino acid insertion within the ligand binding domain. ERß2 exhibited altered ligand binding and transcriptional activation properties. Given that ERß2 mRNA was expressed in various normal rat tissues at levels equal to those of the previously published version of ERß, we propose that ERß2 should also be considered in addition to ERß1 and ER{alpha} when describing the effects of estrogen, estrogen agonists/antagonists, or environmental estrogens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Oligonucleotides
Primer 1 5'-AACCGCCATGAGTATTCAGC-3' (rERß 5' PCR primer) Primer 2 5'-CTTCCTCCTACGTAGACAACCG-3' (rERß 5' PCR primer) Primer 3 5'-TCCTGGTATGAGGAACACCG-3' (rERß 3' PCR primer) Primer 4 5'-ACCATCTGTGTAGTCTGTCCGC-3' (rERß 3' PCR primer) Primer 5 5'-GCCATGACCATGACCCTTCAC-3' (rER{alpha} 5' PCR primer) Primer 6 5'-GGAGTTCTCAGATGGTGTTGG-3' (rER{alpha} 3'-primer) Primer 7 5'-AAGCTTGCCATGGACTACAAGGACGACGATGACAA-AATGACATTC-TACAGTCCTGC-3' (5' FLAG-rERß PCR primer) Primer 8 5'-AAGCTTGCCATGGACTACAAGGACGACGATGACAA-AATGACCAT-GACCCTTCACACC-3' (5' FLAG-rER{alpha} PCR primer) Primer 9 5'-AAGCTTGCCATGGACTACAAGGACGACGATGACA-AAATGGAGTC-TGCCAAGGAGACTCG-3' (5' FLAG-rER{alpha}{Delta}A/B PCR primer) Primer 10 5'-AAGCTTGCCATGGACTACAAGGACGACGATGACA-AAATGGGACC-AAACGCAAAGAGGGATGCTC-3' (5' FLAGrERß{Delta}A/BPCR primer) Oligonucleotide 1 5'-GATCCAGGTCACAGTGACCTA-3' (ERE top strand TK-lux vector) Oligonucleotide 2 5'-GATC-TAGGTCACTGTGACCTG-3' (ERE bottom strand TK-lux vector) Oligonucleotide 3 5'-GATCTGCCCGTCAGGTCACAGTGACCTGAT-CAAAGTTAAG-3' (ERE Top strand, gel shift) Oligonucleotide 4 5'-GTACCTTAACTTTGATCAGGTCACTGTGACC-TGACTTTAG-3' (ERE bottom strand, gel shift)

Plasmid constructs
ERE-3-TK-lux had 3 EREs in the same orientation and was constructed by tandem cloning of a double-stranded oligonucleotide (oligonucleotides 1 and 2) upstream of a minimal thymidine kinase promoter driving luciferase expression. CMVß was purchased from ClonTech (Palo Alto, CA). All mammalian expression vectors were constructed in pcDNA3 containing the CMV promoter (Invitrogen, San Diego, CA). Rat ER{alpha} was cloned by RT-PCR using primers 5 and 6. Mammalian expression constructs designed to express FLAG epitope tagged receptors were created by PCR from the appropriate plasmid template as follows: FLAGrERß1 and FLAGrERß2 with primers 7/3, FLAGrERß1{Delta} and FLAGrERß2{Delta} primers 10/3, FLAGrER{alpha} primers 8/6; FLAGrER{alpha}{Delta} primers 9/6. All plasmid constructs were confirmed by DNA sequencing of the entire receptor coding region.

Complementary DNA (cDNA) cloning of ERß isoforms
The coding region of ERß was cloned by RT-PCR from rat ovary and pituitary RNA using Expand High Fidelity PCR System according to manufacturer’s instructions (Boehringer-Mannheim, Mannheim, Germany). Oligonucleotide primer pairs (Primers 1/3, 1/4, 2/3, 2/4) were designed from the published sequence (23). PCR products were cloned into pCR2.1 TA Cloning Kit (Invitrogen) and sequenced.

Mammalian cell expression
Receptor proteins were overexpressed in 293T cells. These cells, derived from HEK293 cells, have been engineered to stably express large T antigen and can therefore replicate plasmids containing a SV40 origin of replication to high copy numbers. 293T cells were transfected using lipofectamine as described by the manufacturer (GIBCO-BRL, Bethesda, MD). Cells were harvested in PBS with 0.5 mM EDTA at 48 h post transfection. Cell pellets were washed once with PBS/EDTA. Whole cell lysates were prepared by homogenization in TEG buffer (50 mM Tris pH 7.4, 1.5 mM EDTA, 50 mM NaCl, 10% glycerol, 5 mM DTT, 5 µg/ml aprotinin, 10 µg/ml leupeptin, 0.1 mg/ml Pefabloc) using a dounce homogenizer. Extracts were centrifuged at 100,000 x g for 2 h at 4 C, and supernatants were collected. Total protein concentrations were determined using Bio-Rad reagent (Bio-Rad, Hercules, CA).

Estradiol binding assay
Transfected 293T cell extracts were incubated overnight at 4 C with tritiated estradiol (141 Ci/mmol or 40 Ci/mmol, New England Nuclear, Boston, MA). Bound estradiol was recovered using a modified hydroxylapatite binding assay (28). Briefly, labeled extracts were incubated with hydroxylapatite for 30 min at 4 C. Bound estradiol was recovered by centrifugation at 5000 x g for 5 min. The hydroxylapatite was washed 3 times with 2 vol of 50 mM Tris-HCl pH 7.5/100 mM NaCl/1 mM EDTA/0.5% Triton X-100. The hydroxylapatite was transferred to a scintillation vial with ethanol and the recovered tritium was determined by scintillation counting. Dissociation constants were calculated by curve fitting using a simple Langmuir isotherm model. Linear transformation of the data to a Scatchard plot resulted in approximately the same dissociation constants.

Competition binding assay
The ability of various compounds to inhibit [3H]-estradiol binding was measured by a competition binding assay using dextran-coated charcoal as has been described (29). 293T cell extracts expressing either rERß1 or rERß2 were incubated in the presence of increasing concentrations of competitor and a fixed concentration of [3H]-estradiol (141 Ci/mmol) in 50 mM Tris, pH 7.4, 1.5 mM EDTA, 50 mM NaCl, 10% glycerol, 5 mM DTT, 0.5 mg/ml ß-lactoglobulin in a final volume of 0.2 ml. All competitors were dissolved in dimethylsulfoxide. The final concentration of rERß1 was 50 pM with 0.5 nM [3H]-estradiol and rERß2 was 200 pM with 2 nM [3H]-estradiol. After 16 h at 4 C, dextran-coated charcoal (20 µl) was added. After 15 min at room temperature, the charcoal was removed by centrifugation and the radioactive ligand present in the supernatant was measured by scintillation counting. IC50 values were converted to Ki according to the Cheng-Prusoff equation, Ki = IC50/(1 + [L)/Kd)(29). The values used in these equations were Ki = IC50/(1 + 0.5/0.14) and Ki = IC50/(1 + 2.0/5.1) for ERß1 and ERß2, respectively.

Transcriptional activation assay
Transient transfection assays were performed in 293S cells, a derivative of HEK293 cells that can easily be adapted for suspension culture. One day before transfection, 293S cells were plated at 1 x 106 cells/10cm culture dish. The cells were transfected with 7 µg of ERE-TK-lux plasmid plus 0.5 µg of the appropriate expression construct plasmid (rERß1pcDNA3, rERß2pcDNA3, rERß1{delta}3pcDNA3, or rERß2{delta}3pc-DNA3) for 4 h using lipofectamine reagent according to the manufacturer’s protocol (GIBCO-BRL). As an internal control, 0.5 µg of CMVß (ß-galactosidase expression plasmid) was included in the transfection. Immediately after transfection, the cells were trypsinized and harvested in phenol red-free DMEM supplemented with 10% charcoal-stripped FBS. Transfected cells were replated into 24-well culture dishes. After 1 h, 17ß-estradiol was added to the indicated final concentrations. Cells were lysed after 36 h in 100 µl of lysis buffer (25 mM Tris phosphate pH 7.8/1 mM EDTA/8 mM magnesium chloride/1% Tween-20/15% glycerol). Luciferase activity was measured by combining 20 µl lysate with 100 µl of luciferin reagent (Promega, Madison, WI) in a white-pigmented Dynatech Microlite-1 plate (Dynatech Lab, Inc., Chantilly, VA) Luciferase readings were obtained using a Dynatech ML 2250 microtiter plate luminometer. All luciferase results were normalized to ß-galactosidase activity.

Western blot analysis
Fifty micrograms of each protein extract was fractionated on 10% polyacrylamide SDS gels (Novex, San Diego, CA) under reducing conditions. Proteins were then transferred to nitrocellulose (Novex) and subjected to Western blot analysis. After transfer, nitrocellulose membranes were blocked overnight at 4 C in 5% nonfat dry milk/0.1% Tween-20. After blocking, anti-FLAG-M2 monoclonal antibody (Kodak, New Haven, CT) was added for 1 h in 0.25% nonfat dry milk/0.1% Tween-20 at a concentration of 1 µg/ml. The membranes were then washed 3 x 10 min in TBS/0.1% Tween-20. Goat antirabbit IgG-peroxidase conjugated secondary antibody (Pierce, Rockford, IL) was added at a 1:2000 dilution in 0.25% nonfat dry milk/0.1% Tween-20, and membranes were incubated for 1 h. Membranes were again washed 3 x 10 min in TBS/0.1% Tween-20. Immunoreactive protein was detected on autoradiographic film using an ECL chemiluminescence kit (Amersham, Arlington Heights, IL).

Semiquantitative PCR
Primers 1 and 3 flanking the published coding sequence of rat ERß were used to amplify the entire coding region in the presence of [{alpha}-32P]-dCTP. Aliquots were taken at alternate cycles beginning at cycle 20 and the amplified product was restriction digested with SacI. The digestion products were separated by nondenaturing PAGE, dried, and subjected to autoradiography. Size standards were generated by PCR of 25 ng plasmid DNA containing the coding sequence of rat ERß1, ERß2, ERß1{delta}3, or ERß2{delta}3. Control reactions with actin specific primers (5'-AGAGAGAAGATGACACAGATCATGTTTGAG; 5'-GAAGCATTTG-CGGTGGACAATGGA) were performed for 30 amplification cycles. The actin specific products were separated using agarose gel electrophoresis and stained with ethidium bromide.

RNase protection assay
Total RNA was extracted using guanidinium/cesium chloride gradients and quantified by spectrophometric determination at 260 nm. A 317-bp cDNA fragment beginning within the 54 nucleotide insertion of rERß (nucleotides 992-1309, Fig. 1Go) was subcloned into pBlueScript II SK+ (Stratagene, La Jolla, CA). The plasmid was linearized with XbaI and transcribed with T7 RNA polymerase in the presence of 50 µCi [{alpha}-32P]-CTP using the Maxiscript T7/T3 kit (Ambion, Austin, TX). Total RNA (50 µg) was hybridized with 1 x 105 cpm probe for 16 h at 45 C. Unhybridized probe was removed with RNase as described (30). Samples were then resolved on a 6% PAGE in the presence of urea. Gels were dried and exposed to autoradiographic film. Control 18S probes were generated using the vector pTRI RNA 18S as described by the manufacturer (Ambion).



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  		Figure 1. A, Amino acid sequence comparison of rat ERß isoforms. B, Schematic of ERß isoforms with the positions of the 18 amino acid insertion and 39-amino acid deletion depicted. Nucleotide sequences have been deposited in GenBank nos. AF042058, AF042059, AF042060, and AF042061.

 
Gel shift assay
Whole cell extracts from 293T cells transfected with the indicated receptor were incubated in final volume of 20 µl in 10 mM HEPES, pH 7.9/50 mM KCl/1 mM DTT/1 mM MgCl2/20% glycerol/1 µg poly dI-dC/100 µg BSA for 30 min at room temperature. Where indicated, estradiol was added before the first incubation to a final concentration of 10 nM. 32P-labeled ERE containing double stranded oligonucleotide (oligonucleotides 3 and 4) was added and the binding reaction was allowed to proceed for an additional 30 min at room temperature. Receptor-DNA complexes were resolved by PAGE in 0.25 x TBE with buffer recirculation. Gels were dried and subjected to autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of rat ERß isoforms
Rat ERß cDNA was isolated by RT-PCR from ovary and pituitary RNA (Fig. 1Go). Several clones encoded a predicted protein sequence identical to the published rat ERß with the exception of 2 amino acid changes (23) (GenBank U57439). We report the sequence as glutamine at amino acid 27 and alanine at amino acid 105, rather than the leucine and proline previously reported at these positions. These two changes were detected in multiple, independent RT-PCR reactions. Therefore, we believe this sequence is correct from this rat strain. Supporting this data are the fact that both the human (X99101) and mouse (U81451) ERß were also reported to encode a glutamine and alanine at these two positions (24, 25).

In addition to the previously published version, we also isolated several variants of ERß (Fig. 1Go). It is likely that these mRNAs are products of alternative splicing events. We propose renaming the previously published sequence ERß1, and naming the other versions ERß2, rERß1{delta}3, and rERß2{delta}3. ERß2 had an insertion of 54 nucleotides. This insertion is predicted to encode an in-frame addition of 18 amino acids, leaving the rest of the protein as described previously. These 18 amino acids lie within the conserved ligand binding domain. ERß1{delta}3 contained a deletion of sequences encoding the second zinc finger of the DNA binding domain. This deletion is analogous to the exon 3 deletion of ER{alpha} previously reported in breast cancer cell lines (31, 32). ERß2{delta}3 exhibited both the insertion and deletion.

Expression of ERß spliced variants in normal rat tissues
The relative expression in normal rat tissues of the four ERß variants was determined by semiquantitative RT-PCR (Fig. 2Go). The coding region of rERß was amplified in the presence of [{alpha}32P]-dCTP, and aliquots were removed after the indicated number of cycles. The amplified products were digested with SacI and separated by nondenaturing PAGE. Plasmid DNAs containing each of the four isoforms were also amplified as standards. As expected, ERß1 amplification resulted in fragments of 851, 605, and 185 bp after SacI digestion (185-bp fragment not shown). Amplification of ERß2 also gave the predicted products of 851 and 185 bp, whereas the middle fragment was increased to 659 bp due to the 54 nucleotide insertion. The digestion of ERß1{delta}3 resulted in restriction fragments of 734, 605, and 185 bp, whereas ERß2{delta}3 yielded 734-, 659-, and 185-bp fragments. In tissues with the highest levels of ERß expression, including prostate, ovary, muscle, and pituitary there was little detection of the 734 bp fragment (arising from the 117 bp deletion of ERß1{delta}3 and ERß2{delta}3) at low cycle numbers. This allowed us to compare the ratio of the 659 and 605 bp fragments to determine the relative proportion of rERß1:rERß2. As shown, the ratio of rERß1:rERß2 is approximately 1:1 in prostate, ovary, muscle, and pituitary. This experiment confirms that the 54 nucleotide insertion is expressed within the context of an mRNA capable of encoding ERß protein. Furthermore, the ERß2 transcript accounted for approximately 50% of the ERß mRNA expressed in these tissues. Interestingly, in tissues of the nervous system, including frontal cortex, hippocampus and hypothalamus ERß1 mRNA was 2- to 6-fold more abundant than ERß2 mRNA. Transcripts encompassing the coding region of ERß were also detected in periosteum, marrow and aorta. However, in these tissues greater than 30 amplification cycles were necessary for detection. Our experience has been that the relative ratios of ERß1:ERß2 were not reproducible when amplifying from these sources of low message abundance. This data are presented only to indicate the relative abundance of ERß in the various tissues. Transcripts containing the 117-bp DNA binding domain deletion were also detected although generally at lower abundance than full-length mRNAs. It is important to note that the absence of ERß amplification in breast and other tissues should not be interpreted as a lack of expression. The PCR primers were designed to amplify the entire previously reported coding region to ensure the integrity of the mRNA species containing the insertion. This large PCR amplification product was not optimal for PCR. In fact, using primers to amplify shorter segments we have successfully amplified ERß from all of the tissues shown in Fig. 2Go (data not shown).



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  		Figure 2. Analysis of ERß isoform mRNA expression in normal rat tissues by RT-PCR. A, Aliquots of [{alpha}32P]-dCTP labeled RT-PCR products were restriction digested and separated by nondenaturing PAGE as described in Materials and Methods. The RT-PCR reactions using plasmid standards for rERß1, rERß2, ERß1{delta}3, and ERß{delta}3 are on the left four lanes of each gel. The tissue source of the template RNA is shown on the right. B, Ethidium bromide staining of RT PCR amplified actin fragment after 30 cycles. Lanes 1–16 correspond to molecular weight standards, prostate, ovary, muscle, pituitary, frontal cortex, hippocampus, hypothalamus, uterus, tibial periosteum, tibial bone marrow, aorta, breast, liver, tibial metaphysis, and tibial growth plate, respectively. C, A schematic of the amplified fragments and SacI restriction sites illustrates the predicted pattern of labeled DNA fragments.

 
We also employed RNase protection assays to more precisely quantitate the ratio of ERß1/ERß2 using total RNA from a variety of tissues (Fig. 3Go). The probe was designed such that a protected fragment of 317 bp represented transcripts containing the insertion of ERß2, whereas transcripts with no insertion resulted in a 297-bp protected fragment. The relative abundance of ERß mRNA observed by RNase protection was consistent with the RT-PCR results in Fig. 2Go and published reports (24, 26). Highest levels were observed in prostate and ovary, with lower levels in a number of tissues. The RT-PCR and RNase protection assays were consistent in that ERß2 was expressed at significant levels in normal rat tissues. Prostate and ovary exhibited a 1:1 ratio, whereas in hippocampus and frontal cortex ERß1 mRNA was more abundant than ERß2 mRNA.



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  		Figure 3. Analysis of ERß1 and ERß2 mRNA expression in normal rat tissues by RNase protection. RNase protection assays were performed on isolated RNA as described in Materials and Methods. Ovary and prostate were exposed to autoradiographic film for 1 day. Samples from other tissues were exposed for 10 days. The lower panel depicts RNase protection of an 18S ribosomal RNA fragment as a control for RNA integrity and recovery.

 
Expression of ERß isoforms
We next addressed the question of whether the ERß transcripts could be expressed in mammalian cells and translated into functional proteins. Constructs of ERß isoforms were designed to incorporate a FLAG epitope tag at the amino terminus and these constructs were transfected into 293T cells. Western blotting with anti-FLAG antibodies demonstrated that all four isoforms were expressed at efficiencies roughly equal to that of ER{alpha} (Fig. 4Go). Therefore, ERß1, ERß2, ERß1{delta}3, and ERß2{delta}3 mRNAs were efficiently transcribed and translated. This analysis also implied that the mRNA and protein products were of approximately equal stability in this cell context.



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  		Figure 4. Protein expression of ERß isoforms. Epitope-tagged rER{alpha} and rERß isoforms were expressed by transient transfection in 293T cells as described in Materials and Methods. Expressed proteins were detected by Western blot analysis using anti-FLAG M2 monoclonal antibody.

 
Estradiol binding affinity
Scatchard analysis with tritiated estradiol was performed to determine the binding affinity of rERß isoforms (Fig. 5Go). The Kd of rERß1 for estradiol was 0.14 nM. The deletion of 39 amino acids within the DNA binding domain did not appreciably alter the ligand binding affinity in that ERß1{delta}3 had a Kd of 0.3 nM. In contrast, the addition of 18 amino acids within the ligand binding domain in ERß2 and ERß2{delta}3 reduced the affinity for estradiol approximately 35-fold to Kd values of 5.1 and 6.4 nM, respectively. Rat ER{alpha} protein was also expressed in 293T cells under identical conditions and the binding affinity was determined to be 0.19 nM (data not shown). Therefore, ER{alpha} and ERß1 expressed in mammalian cells had an equivalent binding affinity for estradiol. Previous reports have suggested that ERß1 had a slightly lower affinity for estradiol than ER{alpha} (0.4 nM vs. 0.1 nM) (23, 26). Differences in expression systems or the fact that iodinated ligand was employed in these earlier studies could account for these slightly disparate results.



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  		Figure 5. Estradiol binding by ERß isoforms. ERß isoforms were expressed by transient transfection in 293T cells. Extracts were prepared and ligand binding affinity for tritiated estradiol was determined as described in Materials and Methods. The inset shows linear transformation of the data by Scatchard analysis for illustrative purposes. Affinity constants were calculated by curve fitting of bound vs. free ligand. Receptors used in these experiments were not epitope tagged.

 
Ligand binding specificity
Competition binding experiments were performed to assess the ligand binding specificity of ERß2 (Table 1Go). Estradiol and other physiological estrogens, estriol and estrone, competed for binding to both ERß1 and ERß2. Weak binding competition was observed with DHT and DHEA. No competition was observed with other hormones such as testosterone, progesterone, aldosterone, cortisol, or corticosterone. Pharmacological estrogens, including tamoxifen, 4-hydroxytamoxifen and nafoxidene, also competed effectively for binding to ERß2. The plant derived estrogens (coumestrol and genistein) and environmental estrogens (methoxychlor and bisphenolA) were also able to compete for binding to both receptors. A comparison of the relative binding affinity of ERß2 vs. ERß1 revealed interesting differences among these compounds. Most of these compounds were similar to estradiol in that they competed for ERß1 binding more effectively than for ERß2. Some ligands, such as genistein and coumestrol, had very poor competition at ERß2 relative to that observed with ERß1. On the contrary, 4-hydroxytamoxifen, tamoxifen, and nafoxidene competed effectively at both receptors. In fact, 4-hydroxytamoxifen was equally effective at both receptors.


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  		Table 1. Binding affinity of selected compounds for rERß1 and rERß2

 
DNA binding
The ability of ERß isoforms and ER{alpha} to bind to a consensus ERE was examined by gel shift analysis. ER{alpha}, ERß1, and ERß2 were able to bind specifically to the ERE containing oligonucleotide (Fig. 6Go). Binding could be competed by unlabeled oligonucleotide but not by a control oligonucleotide possessing a point mutation within the consensus ERE (data not shown). The binding of ER{alpha} to this oligonucleotide appeared greater than that observed for the two ERß isoforms, in spite of the fact that equal amounts of receptor proteins were added as judged by both Western blot analysis and Scatchard plots. The lower binding of ERß has been observed previously with the mouse receptor and likely represents a lower affinity of ERß homodimers for this consensus ERE (25). Constructs deleted in the second zinc finger of the DNA binding domain, ERß1{delta}3 and ERß2{delta}3, did not bind to DNA under these conditions. The addition of 10 nM estradiol did not alter the amount of DNA binding under these conditions. Estradiol did alter the mobility of the ER{alpha}:DNA complex but had no discernible effect on the migration of ERß1 or ERß2 DNA complexes.



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  		Figure 6. DNA binding by ERß isoforms and ER{alpha}. Gel shift analysis using an oligonucleotide containing a consensus ERE was employed to determine the ability of 293T cell expressed proteins to specifically bind to DNA. All receptors were FLAG-epitope tagged. The + symbol indicates the addition of 10 nM estradiol.

 
Dimerization of receptors on DNA
To determine the ability of ERß1 and ERß2 isoforms to heterodimerize with each other and with ER{alpha} we performed gel shift analysis (Fig. 7Go). The presence of a shifted band of intermediate mobility between full-length FLAG-ER{alpha} and FLAG-ERß1 was evidence that these proteins form heterodimers on a consensus ERE containing oligonucleotide. Similarly, ERß2 heterodimerized with ER{alpha}. ERß1 and ERß2 migrated with equivalent mobility in this gel system and because of this we could not determine if ERß1:ERß2 heterodimers were formed using the full-length constructs. To overcome this limitation, we constructed mutant versions of ER{alpha}, ERß1, and ERß2 that were deleted in their amino terminal A/B domain. These proteins, depicted in Fig. 7Go as ER{alpha}{Delta}, ERß1{Delta} and ERß2{Delta}, were significantly lower molecular weight and therefore migrated faster during the electrophoresis. As expected ER{alpha}{Delta} heterodimerized with both ERß1 and ERß2. ERß1{Delta} heterodimerized with full length ER{alpha}, ERß1, and ERß2. ERß2{Delta} heterodimerized with full-length ER{alpha}, ERß1, and ERß2. Therefore, ERß1 and ERß2 were able to heterodimerize with each other and ER{alpha}.



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  		Figure 7. Heterodimerization of ERß isoforms and ER{alpha}. Gel shift analysis using an oligonucleotide containing a consensus ERE was employed to determine the ability of 293T cell expressed proteins to heterodimerize on DNA. Mutant receptors lacking the amino terminal A/B domain are depicted as ER{alpha}{Delta}, ERß1{Delta} and ERß2{Delta}. All receptors were FLAG epitope tagged. The + symbol indicates the addition of 10 nM estradiol.

 
Effect of estradiol on heterodimerization of ERß1, ERß2, and ER{alpha}
Quantitative analysis of the gel shift experiments shown in Fig. 7Go revealed a small but reproducible effect of estradiol on heterodimer formation (Fig. 8Go). Estradiol shifted the equilibrium to favor heterodimerization of ER{alpha}/ERß1 and ER{alpha}/ERß2 over the formation of ERß1: ERß1, ERß2:ERß2, or ER{alpha}:ER{alpha} homodimers. This was most clearly seen by comparing the vehicle vs. plus estradiol samples with ERß1/ER{alpha}{Delta} and ER{alpha}/ERß2{Delta} (34 and 27% increase, respectively; Figs. 7Go and 8Go). The effect was measurable, albeit to a lesser degree, using the full-length constructs. No effect of estradiol was observed on the amount DNA bound homodimers when only one receptor type was added. As would be expected, no effect on heterodimer formation was observed with ER{alpha}/ER{alpha}{Delta}. There was also no evidence for an effect of estradiol on heterodimer formation between ERß1 and ERß2. An anomalous result was that the truncated ERß1 construct did not demonstrate an effect of estradiol on heterodimerization with full length ER{alpha}. Perhaps this truncation of this receptor interfered with its ability to respond to estradiol under these assay conditions.



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  		Figure 8. Quantitation of the estradiol effect on heterodimer formation. The gel shift analyses shown in Fig. 7Go were analyzed with a phosphoimager. The radioactivity present in the indicated heterodimer band in the presence of 10 nM estradiol is expressed as percent of the value with vehicle treatment.

 
Activation of transcription
The ability of ERß isoforms to activate transcription in 293S cells was tested by cotransfection with an estrogen responsive luciferase reporter gene (Fig. 9Go). Consistent with earlier reports, ERß1 was able to activate transcription in response to estradiol. ERß1 was comparable to ER{alpha} in its ability to activate transcription (not shown). ERß2 was also able to activate transcription in response to estradiol, but the half-maximal response required approximately 1000-fold greater estradiol concentrations than was observed for ERß1. It was expected that ERß2 would require greater concentrations of estradiol given its reduced affinity as determined by Scatchard analysis. However, the effect on transcriptional activation is considerably larger than the 35-fold lower estradiol affinity would suggest. Receptors deleted within the DNA binding domain, ERß1{delta}3 and ERß2{delta}3, were not able to activate transcription in this system.



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  		Figure 9. Transcriptional activation by ERß isoforms. The ability of ERß isoforms to activate transcription was assessed by a luciferase reporter assay. All data were normalized by measurement of cotransfected ß-galactosidase activity and expressed as relative light units/ß-galactosidase activity (RLU/ß-gal). Closed circles, rERß1; open circles, rERß2; closed squares, rERß1{delta}3; and open squares, rERß2{delta}3. Error bars represent SD (n = 3).

 
The effect of ERß2 on ERß1 transcriptional activation was demonstrated by cotransfection of both receptor isoforms (Fig. 10Go). When transfected in a 1:1 ratio, ERß2 did not alter the half-maximal dose of estradiol necessary for induction. The maximal level of induction was also comparable to ERß1 alone. When transfected in a 10:1 ß2/ß1 ratio the maximal level of induction again remained comparable. However, under these conditions of excess ß2, the concentration of estradiol necessary for induction was intermediate between that observed for ERß1 and ERß2 alone. Therefore, in this in vitro system the relative expression of ERß1 and ERß2 can modulate the effective dose of estradiol required but has no or minimal effects on the maximal response.



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  		Figure 10. Effect of ERß2 on transcriptional activation by ERß1. The ability of ERß isoforms to activate transcription was assessed by a luciferase reporter assay. All data were normalized by measurement of cotransfected ß-galactosidase activity and expressed as relative light units/ß-galactosidase activity (RLU/ß-gal). Closed circles, rERß1 (0.25 µg); open circles, rERß2 (0.25 µg); closed squares, rERß1 plus rERß2 (0.25 µg each); and open triangles, rERß1 plus rERß2 (0.25 µg, and 2.5 µg, respectively). Note 0.5 µg ERß1 was not significantly different from 0.25 µg, not shown. Error bars represent SD (n = 3).

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we have identified several variant mRNAs encoding versions of rat ERß. These transcripts are predicted to encode ERß protein altered by the addition of 18 amino acids within the ligand binding domain (ERß2), a deletion of the second zinc finger of the DNA binding domain (ERß1{delta}3), or a deletion within the DNA binding domain combined with the addition within the ligand binding domain (ERß2{delta}3). We have demonstrated that all four transcripts can be translated into functional proteins. Finally, we have determined that the ERß2 isoform selectively bound estradiol, specifically bound to a consensus ERE, heterodimerized, and activated transcription.

Semiquantitative RT-PCR was used to demonstrate that the ERß2 mRNA was expressed at levels approximately equal to those of ERß1 in prostate, ovary, pituitary, and muscle. Complementary to these studies, we present RNase protection data demonstrating quantitatively that transcripts containing the addition of ERß2 were as abundant as those of ERß1 in prostate and ovary. Clearly, the ERß2 variant did not merely represent a minor, abherently spliced product. Interestingly, both RT-PCR and RNase protection demonstrated that tissues of the nervous system, including frontal cortex and hippocampus expressed lower levels of ERß2 relative to ERß1. We have not determined if the tissue-specific differences in the steady state ratio of ERß1/ERß2 mRNA were the result of different synthesis and/or different degradation rates. The physiological consequences of varying ERß2/ERß1 ratios is also unknown. Given the differences in the response of the two receptor isoforms to estradiol and other estrogenic compounds, it is possible that regulation of receptor isoform expression could govern the tissue effects of specific estrogenic agents. The expression of ERß1{delta}3 and ERß2{delta}3 mRNAs was less than that of ERß1 and ERß2 as determined by RT-PCR.

Ample precedent has been described for alternatively spliced forms of the classical estrogen receptor, ER{alpha}. The majority of this work has investigated the multiple spliced forms that arise in breast cancer cell lines and tumor samples (33, 34). It has been proposed that the alternatively spliced forms of ER{alpha} might be clinically significant in the progression of breast cancer and the resistance to therapy (31, 33, 35). Most of these ER{alpha} variants are thought to result from exon skipping (33, 34). The ERß1{delta}3 and ERß2{delta}3 variants reported here are directly analogous to the exon 3 deletion detected in ER{alpha} by several investigators (31, 32). This variant of ER{alpha} has a deletion that truncates the DNA binding domain. Interestingly, this ER{alpha} variant was reported to possess dominant negative activity (32). The exon 3 deleted ER{alpha} inhibited DNA binding and transactivation of wild-type ER{alpha}. In our experiments, ERß1{delta}3 and ERß2{delta}3 proteins were similarly unable to bind to DNA or activate transcription from a simple estrogen response element. We have not yet been able to demonstrate a dominant negative activity on this simple promoter. Given results reported here that ER{alpha} and ERß can heterodimerize, it should be interesting to further test the ability of the ERß1{delta}3 and ERß2{delta}3 to inhibit both ER{alpha} and ERß activity. Although our data indicated that ERß1{delta}3 and ERß2{delta}3 mRNAs are expressed at lower levels than the full-length ER{alpha} and ERß mRNAs, it will be important to measure the relative protein concentrations present in a variety of tissues and tumor cells. It is possible that in specific cell types or under specific physiological conditions the abundance of these proteins would be increased relative to the full-length versions. Under these conditions, potential dominant negative activity of ERß1{delta}3 or ERß2{delta}3 could affect the tissue response to estrogen or estrogenic agents.

An addition of 23 amino acids in-frame within the ligand binding domain of the ER{alpha} mRNA has been reported in human breast cancer biopsy samples (36). This insertion occurred at the boundary of exons 5 and 6 of ER{alpha}, a position exactly analogous to the insertions within ERß2. The functional properties were not evaluated. Therefore, it is difficult to make comparisons. This ER{alpha} variant was detected in only 3 of 212 breast tumors analyzed. ERß2 is clearly different in that it was expressed at levels equal to that of ERß1 in normal rat tissues. Nonetheless, it would be interesting to compare the functional properties and splicing events of ER{alpha} and ERß.

The 18-amino acid insertion of ERß2 lies within helix 6 of the structural model of nuclear receptors proposed by Wurtz et al. (37). By this model, helix 6 lies immediately following the ß-turn within the ligand binding domain. It is thought that ligand binding induces a conformational change, whereby the ß-turn shifts toward the surface and the {Omega}-loop flips underneath helix 6. Helix 12 is consequently shifted to cap the ligand binding pocket, exposing the AF2 domain to the surface. Residues within helix 6 are not among those highly conserved across the nuclear receptor family and would not be predicted to be in immediate vicinity to bound ligand (37). However, the added residues could distort the ligand binding pocket and change the mouse trap-like transition that is thought to occur upon ligand binding. Our data are consistent with this in that ERß2 had reduced affinity for estradiol and was relatively insensitive to estradiol in the induction of transcription.

The physiological role of ERß2 is unclear. One possibility is that ERß2 provides a cellular mechanism to respond to elevated estradiol concentrations higher than those required to fully activate ER{alpha} and ERß1. Results in Fig. 10Go suggest the possibility that titration of the relative receptor concentrations could allow a tissue to govern the level of estradiol that is required for responsivity. An alternative possibility is that estradiol is not the only relevant ligand for ERß2. Perhaps a steroid metabolite selectively functions through ERß2. It is also possible that the additional 18 amino acids affect the ability of the ERß to interact with accessory transcription factors. These amino acids could serve as a domain for protein:protein interactions, allowing interactions with additional factors and perhaps eliminating interactions with others. By these mechanisms and others, the promoter selectivity of ERß2 could be altered relative to that of ERß1. Just as ongoing efforts are attempting to unravel the role of ERß1, further investigations will be required to incorporate the description of ERß2 into our understanding of estrogen action at target tissues.

The role of ERß2 in the response to pharmacological agents also warrants further investigation. Even before the identification of ERß, a number of models based on the molecular biology of ER{alpha} had been proposed to explain the tissue selective effects of partial estrogen agonists such as tamoxifen and raloxifene. For example, it has been proposed that partial agonists selectively activate ER{alpha} AF1 function but are unable to induce the precise conformational change required for ligand dependent AF2 function (38, 39). In this way, agents such as tamoxifen were proposed to act preferentially in cell types and promoters that are primarily driven by the AF1 function. In addition to direct DNA binding by receptor, alternative mechanisms whereby ER{alpha} is tethered to the target promoter through protein:protein interactions have been proposed (40, 41). For example, the tissue specific effects of raloxifene have been suggested to arise from selective effects at raloxifene response elements in the TGFß3 promoter (42). More recently, researchers have determined that the ratio of coactivators to corepressors modulates the degree of partial agonism observed with various compounds (43). In spite of the elegant experimental evidence that has supported these models, it is possible that different in vivo relevant mechanisms are as yet undiscovered. In this regard, it is not yet known how ERß1 or ERß2 respond to these agents. We have demonstrated that ERß2 possesses altered binding of estradiol. In addition, the pharmacologically important ligand, 4-hydroxytamoxifen, does not discriminate significantly between ERß1 and ERß2. Future experiments will be required to determine if some ligands act preferentially at ERß2. Perhaps some ligands exhibit potent agonism of ERß2 while antagonizing ER{alpha} and ERß1. The effect of different ligands on the ability of ER receptors to homo- and heterodimerize could be of interest given the results reported here that estradiol slightly shifts the equilibrium toward ER{alpha}:ERß1 heterodimers. Abundant ERß2 mRNA expression in rats suggests relevance in this preclinical model for estrogen action. High quality specific antibodies will be required to determine expression of the protein product in vivo. However, the significance in humans will require the demonstration of analogous spliced variants in human tissues.

In conclusion, ERß2 mRNA was expressed in normal rat tissues at levels equal to those of the published ERß. This mRNA can be translated into functional protein with altered pharmacology. The ERß1{delta}3 and ERß2{delta}3 isoforms were expressed at lower levels in normal rat tissues. Further characterization of these receptor isoforms may increase our understanding of the mechanisms of action of medically and environmentally important estrogenic compounds.

Received August 15, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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