Estrogen Receptor Domains E and F: Role in Dimerization and Interaction with Coactivator RIP-140

Gregory A. Peters1 and Sohaib A. Khan

Department of Cell Biology, Neurobiology, and Anatomy University of Cincinnati College of Medicine Cincinnati, Ohio 45267


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have used the yeast two-hybrid system to localize the ligand-dependent dimerization domain of the estrogen receptor-{alpha} (ER) to region E in vivo. In this system, the cDNAs corresponding to the A–D, E, E/F, A–E ({Delta}F), and full-length (wtER) domains of the human ER were each cloned into the yeast two-hybrid vectors GAL4 DB and GAL4 TA and expressed in different combinations in yeast harboring a GAL1-lacZ reporter. The reporter was used as a relative measure of the interaction between the ER domains, through reconstitution of GAL4 activity. We found that the interaction of E or E/F domains of the ER with full-length ER is estradiol dependent and estrogen responsive element independent, as measured by the reconstitution of GAL4 activity from GAL4-E domain-containing fusion protein interactions. In the presence of F domain, this activity is reduced 10-fold. The results suggest that sequences in the F domain are inhibitory to the dimerization signal that is present in the E region. We propose that the full-length ER contains intrinsic dimerization restraints contributed by regions outside domain E that are released upon binding hormone agonist. In addition, we have demonstrated that coactivator RIP140 is able to interact with the ER in vivo at the E domain of the receptor in the presence of estrogen. Yeast two-hybrid analysis shows that RIP140 does not homodimerize in the presence or absence of estrogens. We present evidence showing that the ER has the inherent ability to interact with RIP140 in the presence of antiestrogens, but sequences inherent in the ER itself that are present outside of the E domain compromise this ability.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor-{alpha} (ER), a member of the steroid/nuclear receptor superfamily, is a ligand-inducible transcription factor that mediates the actions of estrogens in target cells. Estrogen action on target cells involves a distinct pathway where estradiol freely diffuses across the cell membrane and binds the ER. The ligand-bound ER homodimerizes, binds specific upstream DNA sequences called estrogen-responsive elements (EREs), and activates transcription of its target genes by as yet unknown mechanisms.

All of the steroid receptors possess a modular structure, with discrete regions of the protein (domains) responsible for transcriptional activation, DNA binding, nuclear localization, ligand binding, and dimerization (1, 2). The estrogen receptor can be divided into six functionally independent domains denoted from N- to C-terminal by the letters A to F. Dimerization properties of the estrogen receptor have been primarily localized to the E region (1, 3), a complex domain that integrates several functions including hormone binding and ligand-dependent transcriptional activation (AF-2) (4). The ER also possesses a ligand- independent transcriptional activation function in region A/B that is promoter and cell type dependent (AF-1) (5) and a possible third activation function near the N-terminal end of domain E (AF-2a) (6, 7). The DNA-binding domain (DBD or region C) also possesses a weak dimerization property that stabilizes binding to an isolated ERE in vitro (8) and has been suggested to restrain steroid receptor transcriptional synergy through the DBD-dimer interface (9). A role of the F domain has not been established in dimerization of the receptor, but it has been proposed that F has a specific modulatory function that affects the agonist/antagonist effectiveness of antiestrogens and the transcriptional activity of the liganded ER in cells (10).

Recently, a new concept of steroid hormone action has developed with the discovery of several novel coactivators that increase the ability of the receptors to activate transcription (reviewed in Refs. 11, 12). It is unknown whether the coactivators have a role in the dimerization of the steroid receptors, but it has been suggested that these cofactors may act as a bridging apparatus between the receptor and the transcriptional machinery. Among the coactivators of ER, RIP-140 associates in vitro and in vivo with the ER carboxyl terminus in the presence of estrogen, but not in the presence of antiestrogen (13, 14).

Several in vitro studies have demonstrated that the formation of the ER homodimer after ER-ERE binding is not dependent on estrogen (1, 15, 16, 17, 18). Others suggest roles for ligand in the interaction (1) and that salt conditions, ionic strength, and temperature influence binding (19, 20). In the absence of an ERE, we recently showed that dimerization of full-length ER is ligand dependent in vivo using the yeast two-hybrid system (21).

In the current study, the yeast two-hybrid system was used to localize the ligand-dependent dimerization domain of the receptor and to determine the domains that heterodimerize with the full-length receptor. Yeast has been used extensively to study the estrogen receptor and several other members of the nuclear receptor family of genes (22, 23, 24, 25), taking advantage of the absence of these factors to perform experiments without any cellular background. Here, we show that the E domain can interact strongly with the full-length receptor in the presence of ligand and that the F domain can decrease this interaction. We also confirm that the human ER (hER) forms a protein-protein interaction with RIP-140 and that the interaction is dependent on the presence of ligand. In addition, the presence of ER domain F somewhat disrupts interaction of the full-length receptor with RIP-140 and is a likely factor in the inability of antiestrogen-bound ER to interact with RIP-140.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Neither GAL4 DB nor GAL4 TA Fusions to Estrogen Receptor Domains Alone Activate the Transcription of GAL1 Promoter Driving LacZ
The cDNAs corresponding to the A–D, E, E/F, and A–E ({Delta}F) domains (Fig. 1AGo) of the human estrogen receptor were each cloned into the yeast two-hybrid vectors GAL4 DB (pPC62) and GAL4 TA (pPC86) and expressed in yeast harboring a GAL1-lacZ reporter (26). These particular vectors were chosen to ensure low expression levels to avoid artificial scenarios that often complicate in vitro studies and others that involve protein overexpression. In all of these studies, Western blotting was performed to confirm correct and equivalent expression of GAL4 ER fusion protein constructs (Fig. 1BGo). The lacZ reporter was used as a relative measure of the interaction between the ER domains, through reconstitution of GAL4 activity. Since the estrogen receptor contains three independent transcriptional activation functions (AF-1, AF-2, and AF-2a), it was initially unknown whether GAL4 fusions to A–D and {Delta}F (containing AF-1), or E, E/F, and {Delta}F (containing AF-2 and AF-2a) themselves could activate transcription of the GAL1-lacZ reporter gene in this system. Therefore, each fusion construct was transformed into yeast alone and ß-galactosidase activity was measured by 5-bromo-4-chloro-3-indoyl ß-D-galactoside (X-gal) reaction. GAL4 DB-A-D and GAL4 TA-A-D alone, which contain AF-1 as well as a DBD, showed no activation of the GAL1-lacZ reporter in the presence or absence of 17ß-estradiol (Table 1Go, controls). The AF-2 and AF-2a-containing constructs, represented by domains E, E/F, and {Delta}F, showed no activation of the reporter when expressed as fusions with GAL4 DB or GAL4 TA (Table 1Go, controls), indicating these domains cannot activate the reporter gene by themselves. This finding may be explained by the fact that the AF-1 and AF-2 regions of hER have been found to mediate transactivation by nonacidic amino acids (4), whereas GAL4 transactivation occurs through acidic residues. To address the possibility that the GAL4 DB-wild-type (wt)ER fusion alone is able to activate GAL1-lacZ transcription through the particular AFs present in the estrogen receptor, we reasoned that coexpression of unfused coactivator RIP140 with GAL4 DB-wtER should increase transcriptional activity of the reporter contributed by ER and amplified by RIP140. Our reasoning was based upon the fact that, depending on the estrogen-responsive promoter and expression level, RIP140 enhances ER transactivation between 1.5- to 4-fold (27) or 30- to 100-fold in yeast (our unpublished observations). However, in the presence or absence of estrogen, coexpression of GAL4 DB-wtER with RIP140 did not amplify ß-galactosidase activity through GAL1-lacZ, suggesting that our reporter in the yeast two-hybrid system is measuring relative protein-protein interaction and not transcriptional activity contributed by ER (data not shown). It is therefore reasonable to conclude that AF-1, AF-2, or AF-2a of the ER do not activate GAL1-lacZ transcription by themselves when these regions are fused to GAL4 DB or GAL4 TA.



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Figure 1. Estrogen Receptor-{alpha} Domains Expressed as GAL-4 Protein

A, The human estrogen receptor and its functional domains corresponding to the cDNAs cloned into yeast two-hybrid vectors GAL4 DB and GAL4 TA. The portion of hER{alpha} expressed in PCY2 yeast is shown relative to the full-length hER ( = wtER, wild-type ER; AF-1, AF-2, AF-2a, transactivation function 1, 2, 2a, respectively). B, Western blot analysis of GAL4-ER domain constructs expressed in PCY2 yeast. GAL4 DB and GAL4 TA fusion proteins containing ER domain E were detected using anti-hER antibody H222 using Western-blot analysis. Commercially available hER (Panvera) was loaded as positive controls (lanes 1 and 13). Equal amounts of total protein were analyzed from the yeast carrying no vector(s) (lane 2), GAL4 TA-E/F (lane 3), GAL4 DB-E/F (lane 4), GAL4 TA-E (lane 5), GAL4 DB-E (lane 6), GAL4 TA-{Delta}F (lane 7), GAL4 DB- {Delta}F (lane 8), GAL4 TA-wtER (lane 9), and GAL4 DB-wtER (lane 10). GAL4 DB and GAL4 TA fusion proteins containing ER domains A/B were detected using anti-hER antibody ERC314 (Santa Cruz). Equivalent protein samples were analyzed from the yeast carrying GAL4 TA-A–D (lane 11) and GAL4 DB-A–D (lane 12). Empty yeast carrying no plasmid was used as a negative control (lanes 2 and 14).

 

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Table 1. Plasmid Fusion Vectors Cotransformed in Yeast (PCY2) and the Percentage of ß-Galactosidase-Positive Colonies after Treatment on the Filter for X-gal Reaction

 
The Interaction of GAL4DB Fusions with GAL4 TA Fusions as a Measure of Dimerization of Estrogen Receptor Domains
The estrogen receptor domain constructs (Fig. 1AGo) were coexpressed as fusions with GAL4 DB and GAL4 TA in PCY2 yeast treated without and with 17ß-estradiol. The ß-galactosidase activity was determined as a relative measure of homodimerization of the individual domains of the ER. Throughout all of our experiments, in yeast treated with vehicle alone, none of the ER domains tested using two-hybrid analysis showed any activation of ß-galactosidase activity. However, upon treating PCY2 yeast with 17ß-estradiol, full-length ER (wtER) as well as ER missing the F domain ({Delta}F) both reconstituted ß-galactosidase activity in individual homodimerization assays (Table 1Go and Fig. 2AGo). Estrogen receptor missing the F domain had almost twice the ability as full-length receptor in reconstituting reporter gene activity. GAL4 fusions containing the A–D, E, or E/F domains could not individually homodimerize in the presence or absence of estrogen (Fig. 2AGo). This finding is in contrast to in vitro evidence showing that the hormone-binding domain (region E/F) homodimerizes in the absence of hormone (28). It is possible that the in vitro data may be due to an artificial scenario due to the greatly overexpressed estrogen receptor necessary for in vitro studies and may be complicated by minute concentrations of estrogen present in the media. In vivo, however, our results clearly show that estrogen induces the formation of a full-length estrogen receptor homodimer and demonstrate that an ER dimer also forms when the receptor is missing domain F (Fig. 2AGo). Furthermore, the {Delta}F dimer formation results in twice the ß-galactosidase activity as the wild-type full-length receptor.



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Figure 2. Estrogen Induction of ß-Galactosidase Activity in Yeast Expressing GAL4 DB and GAL4 TA Domain Protein Fusions as a Relative Measure of Dimerization

A, Homodimerization of ER domains; B, wild-type ER interaction with ER domains. The yeast liquid culture was treated with 0.2% ethanol or 1 µM 17ß-estradiol for 18–22 h as indicated, and ß-galactosidase activity was determined by ONPG reaction. The figure is representative of three independent experiments. Each concentration was run in triplicate, and the values shown are means ± SEM.

 
The Interaction of GAL4DB-wtER with ER Domains: Effect of ER Domain Deletions
The various domains of the ER fused to GAL4 TA were coexpressed with the full-length ER (wtER) fused to GAL4 DB, and ß-galactosidase activity was measured as a indicator of heterodimerization. When fused to GAL4 TA, the A–D region of the ER showed no interaction with full-length ER in the presence or absence of hormone (Table 1Go and Fig. 2BGo). These results are consistent with evidence that the dimerization signal in region C is influenced or stabilized by the presence of an ERE (8, 9) not present in PCY2 yeast, or that this dimerization signal is masked by accessory factors. Individual expression of GAL4 TA fusions to E, E/F, or {Delta}F domains showed estrogen-dependent interaction with GAL4 DB-wtER (Table 1Go and Fig. 2BGo), indicating the dimerization signal in region E is functional in yeast and activated by estrogen. When the relative ß-galactosidase activities of the constructs containing this signal are compared, in the presence of estrogen, E domain interaction with wtER influences an 8-fold increase in activity compared with levels of the full-length ER homodimer (Fig. 2BGo). When F domain is added back to E domain (indicated by GAL4 TA-E/F), the activity drops to similar GAL4-wtER homodimer levels. Estrogen receptor missing domain F ({Delta}F) reconstituted ß-galactosidase activity at more than twice the level of the full-length ER homodimer. The interactions of GAL4 DB-wt ER with GAL4 TA-wtER, GAL4 TA-E, or with GAL4 TA-E/F are all estradiol dose dependent, with 1 nM concentrations of estradiol sufficient to elicit a response (Fig. 3Go). Similar estradiol dose dependency was observed between GAL4 DB-wtER and GAL4TA-{Delta}F, with the maximum ß-galactosidase activity at an intermediate level (~25 U, data not shown). The data suggest that the presence of F domain is inhibitory to the ligand-dependent dimerization signal in the ligand-binding domain.



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Figure 3. Estrogen Dose Response Induction of ß-Galactosidase Activity in Yeast Carrying GAL4 DB-wtER and GAL4 TA ER Domain Fusions

The yeast liquid culture was treated with various concentrations of 17ß-estradiol (10-3 to 104 nM) for 18–22 h as described. The ß-galactosidase activity was determined by ONPG reaction. The figure is representative of three independent experiments. Each concentration was run in triplicate, and the points shown are means ± SEM.

 
RIP-140 Interactions with the Estrogen Receptor and with ER Domains
To determine whether RIP-140 can make a protein-protein interaction with the estrogen receptor in yeast, full-length ER (wtER) and RIP-140 were coexpressed as fusion proteins with GAL4 DB and GAL4 TA, respectively, and GAL1-lacZ reporter activity was measured. When expressed as a fusion protein with GAL4 DB in the absence of hormone, RIP-140 interaction with the full-length ER fused with GAL4 TA could not be detected. However, upon addition of 17ß-estradiol, ß-galactosidase activity was induced (Fig. 4AGo), indicating estrogen is required for the protein-protein interaction of RIP140 with ER.



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Figure 4. ß-Galactosidase Activity in Yeast Expressing GAL4 DB-RIP140 and GAL4 TA Fusions to ER Domains or RIP140

A, Estrogen induction of ß-galactosidase activity in yeast expressing GAL4 DB-RIP140 and GAL4 TA ER domain fusions.The yeast liquid culture was treated with 0.2% ethanol or 1 µM 17ß-estradiol for 18–22 h as indicated, and ß-galactosidase activity was determined by ONPG reaction. The figure is representative of three independent experiments. Each concentration was run in triplicate, and the values shown are means ± SEM. B, Estrogen dose response induction of ß-galactosidase activity in yeast carrying GAL4 DB-RIP140 and GAL4 TA fusions with E or E/F domains. The yeast liquid culture was treated with various concentrations of 17ß-estradiol (10-3 to 104 nM) for 18–22 h as described. The ß-galactosidase activity was determined by ONPG reaction. The figure is representative of three independent experiments. Each concentration was run in triplicate, and the points shown are means ± SEM.

 
To determine the estrogen receptor domain responsible for the interaction of hER with RIP140 in vivo, we expressed the various ER domains fused to GAL4 TA with GAL4 DB-RIP140. In yeast treated with vehicle alone, none of the ER domains tested using two-hybrid analysis showed any activation of ß-galactosidase activity. In the presence of estradiol, reconstitution of ß-galactosidase activity was observed by hER fusion proteins containing region E, the hormone-binding domain (Fig. 4AGo), similar to mouse ER hormone-binding domain/RIP 140 interaction using mammalian two-hybrid assays (13). Strikingly, truncation of carboxy- and amino-terminal ends of the ER to express the hormone-binding domain fusion, GAL4 TA-E, resulted in extremely high ß-galactosidase activity (Fig. 4AGo). Comparing relative interactions of RIP-140 with the full-length ER shows that truncating ER to the E domain increased ß-galactosidase activity 20-fold. Addition of the ER domain F significantly decreased the interaction with RIP140 relative to E domain alone (compare GAL4 TA-E with GAL4 TA-E/F, Fig. 4AGo), although the activity is still 14 times higher relative to RIP140 interaction with the full-length receptor. The interaction of RIP140 with E or E/F domain is estrogen dose dependent, again showing the presence of the F domain decreases the relative amount of interaction of RIP-140 with E domain (Fig. 4BGo).

To determine whether RIP140 can form a homodimer, RIP140 fused to GAL4DB and GAL4 TA was coexpressed in PCY2 yeast, and GAL1-lacZ reporter gene activation was measured. In the presence or absence of estradiol, RIP140 was unable to form a homodimer in vivo (Fig. 4AGo).

In our assay, RIP-140 also associates with E domain in the presence of the antiestrogens tamoxifen and ICI 182,780, and with E/F in the presence of ICI only (Fig. 5Go). When the E domain fusion of the ER is coexpressed with the RIP140 fusion in the presence of antiestrogens, the ß-galactosidase activity is significantly higher than in yeast treated with vehicle alone. Treatment of yeast with ICI 182,780 evokes a 10-fold increase in reporter activity relative to yeast treated with tamoxifen. However, relative to yeast treated with estradiol, tamoxifen and ICI 182,780 decreased ß-galactosidase activity 100-fold and 10-fold, respectively.



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Figure 5. Estrogen and Antiestrogen Induction of ß-Galactosidase Activity in Yeast Expressing GAL4 DB-RIP140 and GAL4 TA Fusions with E Domain or E/F Domains

The yeast liquid culture was treated with 0.2% ethanol or 1 µM 17ß-estradiol, tamoxifen, or ICI 182,780 for 18–22 h as indicated, and ß-galactosidase activity was determined by ONPG reaction. The figure is representative of three independent experiments. Each concentration was run in triplicate, and the values shown are means ± SEM. EtOH, Ethanol; E2-17ß, 17ß-estradiol; Tam, tamoxifen; ICI, ICI 182, 780.

 
Addition of the ER domain F decreased the interaction with RIP140 relative to E domain alone in the presence of estrogen and the ICI compound and abolished activity in the presence of tamoxifen (Fig. 5Go). In the presence of physiological concentrations of antiestrogens, no interaction of RIP-140 with the full-length receptor could be detected in our system. These data suggest an inherent ability of ER at the E domain to interact with coactivator RIP140 in the presence of antiestrogens and estrogens that is somehow repressed by ER sequences outside the E domain.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have used the yeast two-hybrid system to localize the ligand-dependent dimerization domain of the estrogen receptor to region E in vivo. In this system, the interaction of E or E/F domains of the ER with full-length estrogen receptor is estradiol dependent and ERE independent, as measured by the reconstitution of GAL4 activity from GAL4-E domain-containing fusion protein interactions. The results suggest that sequences in the F domain are inhibitory to the dimerization signal that is present in the E region. In addition, we demonstrate that coactivator RIP140 is able to interact with the estrogen receptor in vivo in yeast at the E domain of the receptor in the presence of estrogen. We present evidence showing that the estrogen receptor has the inherent ability to interact with RIP140 in the presence of antiestrogens, but sequences inherent in the estrogen receptor itself that are present outside of the E domain compromise this ability.

In using this system to measure protein-protein interactions, the levels of ß-galactosidase measured are relative levels of interaction and do not represent the dissociation constant (Kd) of the interaction of the two proteins in question (29). When the individual GAL4 fusions with the domains of ER are transformed alone into yeast or cotransformed with empty GAL4 vectors, no ß-galactosidase transcription is measured (Table 1Go). This indicates that the ER transcriptional activation functions (AF-1, AF-2, and AF-2a) do not activate GAL1-lacZ transcription alone or that the assay is not sensitive enough to detect the activation. However, it may be that, by adding several functional AFs to the region of the promoter, the reporter gene will be activated. In this case for example, to address the possibility that the GAL4 DB-wtER fusion alone is able to activate GAL1-lacZ transcription through the particular AFs present in the estrogen receptor, we coexpressed unfused coactivator RIP140 with GAL4 DB-wtER, reasoning that we would see an increase in transcriptional activity of the reporter contributed by ER and amplified by RIP140. This reasoning was based on evidence demonstrating that RIP140 enhances ER transcriptional activity in yeast between 4- to 100-fold, depending on promoter context (Ref. 27 and our unpublished observations). However, coexpression of GAL4 DB-wtER with RIP140 did not amplify ß-galactosidase activity through GAL1-lacZ, suggesting that our reporter in the yeast two-hybrid system is measuring relative protein-protein interaction and not transcriptional activity contributed by ER. It is important to note that ER AF-1 and AF-2 activity depend on cellular and promoter context (5), and in our promoter context, RIP140 was unable to amplify any background activity contributed by wtER on GAL1-lacZ. However, as in most in vivo approaches, we cannot exclude the possibility of other endogenous proteins contributing to the interactions. In any case, in yeast two-hybrid analysis, protein-protein interaction is required to bring GAL4 DB and GAL4 TA together to reconstitute the full activity that switches on the reporter.

It is apparent that by truncating the full-length estrogen receptor to only the E region, the dimerization or interaction potential of the receptor is accentuated. With the addition of F domain, we observed that the ability of E to dimerize with ER repressed to levels relative to the full-length ER homodimer. Possibly sequences in the E domain essential to dimerization are masked by intramolecular folding or secondary structure contributed by the F domain. If this is the case, crystal structure data of the ligand-binding domain (30) and DBD (8) of the ER must be reexamined in the context of the full receptor. Crystal data of the E domain dimer show that the structure is essentially {alpha}-helical, with a major repositioning of helix 12 (at the carboxy terminus of E domain) as the receptor binds hormone. We propose that the binding of hormone agonist to the receptor changes the conformation in the ligand-binding domain so that the intrinsic dimerization restraint is released. Lui et al. (9) have suggested that the estrogen receptor contains a DBD dimerization restraint that once released, allows AF-1 and AF-2 synergistic activity. This release of the restraint could be hypothesized to be facilitated by the binding of a coactivator such as RIP-140, which would be expected to alter ER conformation upon binding. In this respect, RIP140 differs in the fact that it contains two distinct sites having a LXXLL motif that facilitates interaction with the estrogen receptor (14, 31), while coactivators mSUG1 and TIF1 contain single sites of interaction with nuclear receptors. This additional site of interaction could be required to release the dimerization restraint inherent in the full-length estrogen receptor.

Alternatively, sequences within the ER may interact with factors such as the heat shock proteins or other chaperones that direct the proper folding of the full-length receptor or play a role in the dimerization of the receptor. It is possible that putative ER corepressor proteins may also mask the dimerization signal in E, either directly or by altering the conformation of the receptor. The extreme C terminus of the progesterone receptor (PR) has been recently shown to contain a transcriptional repressor domain that functions through a putative corepressor (32).

In this report, we have shown GAL4 DB-wtER does not interact with GAL4 TA-A–D in the presence or absence of estrogen, suggesting the DBD of ER (domain C) does not contribute to the dimerization of the receptor in the absence of an ERE. In heterodimerization experiments, the dimerization signal was localized to the E domain, which was repressed when F domain was added back. It is possible that F domain protein residues fold back onto residues in domain E that are essential to dimerization, similar to the mechanism by which synthetic peptides have been engineered as antiestrogens to disrupt ER dimerization by interfering with phosphorylation site Tyr537 (33). Previous studies have shown that domain F is not required for transcriptional response to estrogen (2, 3), and that this region does not affect the turnover rate of the ER in target cells (34). A role of domain F, however, has been proposed in modulating the affects of agonist/antagonist effectiveness of antiestrogens and the transcriptional activity of the liganded ER in cells (10). In certain cells and promoter contexts, antiestrogens, which stimulate transcription of ERE reporter constructs with the full-length ER, were unable to stimulate transcription with {Delta}F ER. In this regard, our experiments show that {Delta}F exhibits a 4-fold decrease in the ability to interact with coactivator RIP140 compared with full-length ER in the presence of agonistic hormone. It appears that one mechanism that antiestrogens use in modifying gene transcription is related to inducible changes in conformation of the carboxy-terminal tail of the receptor, as has been the case with the influence of RU486 on PR (35).

The effects of RIP140 and other coactivators and corepressors on modulating the conformational structure of nuclear receptors deserve further attention. In one model, coactivators could act as factors that remodel nuclear receptor protein structure, exposing dimerization signals and directing an optimal ER complex conformation to the transcriptional machinery. The ability of each coactivator to optimize receptor conformation could be directly related to the ability of the coactivator to enhance transcription, and the stochiometric amount present in each cell may play a significant role in transcriptional regulation of the tertiary ER complex. In this regard, RIP140 has been proposed to indirectly regulate AF-2 transcriptional activity by competing with SRC-1 for receptor binding (36).

With the discovery of several truncated ER variants in normal tissue (37, 38, 39) or tumors (40, 41, 42, 43, 44), the effects of removing dimerization restraints of the ER would give cells the ability to form ER dimers even if coactivators were absent from the cell. The results of these experiments may also have implications on the development of potential dominant negative ERs to impair dimerization in the case of ER-positive breast cancer. Comparison of the carboxy-terminal ends of ER{alpha} and the recently described ERß (45, 46) show that hERß F domain is missing 16 amino acids at the extreme C terminus relative to ER{alpha} (Fig. 6Go). The remaining part of ERß is not well conserved with ER{alpha}, showing homology with only 5 of 27 amino acid residues. Since the domain structures of the other human receptor proteins for gonadal and adrenal hormones (PR-A, PR-B, glucocorticoid receptor, mineralocorticoid receptor, and androgen receptor) show the absence of F domain (47), it is possible that sequences in F domain may play a unique role specific for ER{alpha} and/or ERß.



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Figure 6. Alignment and Comparison of the F Domains of hER{alpha} and hERß

Homologous amino acid sequences are indicated by a line between residues.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Yeast Strains and Methods
The Saccharomyces cervisiae yeast strain PCY2 (MATa {Delta}gal4 {Delta}gal 80 URA3::GAL1-lacZ lys2–810amber his 3- {Delta} 200 trp1- {Delta}63leu2ade2–101ochre) was used for all assays (26). Yeast strains were grown in yeast extract/peptone/dextrose (YEPD) or supplemented synthetic dextrose medium (-leu,-trp). Transformation of yeast was carried out using dimethylsulfoxide/polyethylene glycol/lithium acetate method with plasmid DNA (48).

cDNA and Constructs
Construction of GAL4-wtER fusion vectors was described (21). Yeast two-hybrid vectors pPC62 (GAL4DB) and pPC86 (GAL4TA) were used to allow expression of the various domains at a low level (26). To subclone E domain into GAL4 DB (pPC62) and GAL4 TA (pPC86) (26), E domain cDNA was amplified with PCR using oligonucleotides 5'-ACGCACGTCGACGAAGAAGAACAGCC-3' and 5'-GGGGGTTGAACTAGTGGGCGCATGTA-3' containing SalI and SpeI sites, respectively. After restriction enzyme digestion, the PCR product was directionally cloned into GAL4 DB at the SalI/SpeI sites to create GAL4DB-E. GAL4DB-E was digested with SalI/SpeI to release domain E; the cDNA was placed into GAL4 TA to create GAL4TA-E.

Previously, we subcloned the full-length hER cDNA digested with SalI into pBluescript II SK+ at the SalI site such that its transcription is dependent on T7 polymerase (T7-hER) (21). T7-hER domains E and F were amplified using T3 primer and the same SalI-containing oligonucleotide used for E domain cloning above. The PCR product was digested with SalI and NotI and placed into the DB fusion vector to create GAL4DB-E/F. GAL4DB-E/F was digested with SalI/NotI to release domains E–F; the cDNA was placed into GAL4TA to create GAL4TA-E/F.

Similarly, PCR was used to amplify the cDNA encoding A–D from pCMV (a gift from B. Katzenellenbogen), using primers 5'-AATCGTCGACAATGACCATGACCCTCC-3' (SalI) and 5'-GGACTAGTTAAGAGCGTTTGATCATGAG-3' (SpeI). The PCR product was digested with SalI/SpeI and placed into both GAL4 vectors to create GAL4DB-A–D and GAL4TA-A–D.

Human {Delta}F domain cDNA was amplified using the SalI-containing primer (used for A–D cloning) and the SpeI-containing primer (used for E-domain cloning), and each PCR product was directly cloned into GAL4 DB and GAL4 TA at the SalI/SpeI site. Each GAL4 DB and GAL4 TA fusion cDNA construct was sequenced to confirm correct reading frame before transforming yeast.

RIP-140 cDNA in pEF-BOS [kindly provided by Dr. Malcolm Parker (13)] was amplified by PCR with primers 5'-GCGTCGACGCTTCTATTGAACATGACTCAT-3' (SalI) and 5'-GGACTAGTCCAAAACTGGATGGCAGGT-3' (SpeI). The PCR-amplified fragment was cloned into pBluescript II SK+ at SalI/SpeI restriction sites. The RIP-140 coding region was released from pBluescript II SK+ by SalI/SpeI and cloned into GAL4DB and GAL4TA vectors. Each GAL4 DB and GAL4 TA fusion cDNA construct was sequenced to confirm correct reading frame before transforming yeast.

Ligand Treatment and ß-Galactosidase Activity Assay
ß-Galactosidase activity in PCY2, which was the product of LacZ driven by the GAL1, was used to indicate reconstitution of GAL4 transactivation activity via the interaction of the two fusion proteins. Transformed yeast were selected and cultured in synthetic medium. Yeast were grown in 1% ethanol and then transferred to 2% glucose medium containing ligand. After treating with ligand (18–22 h), yeast cells were resuspended in Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4) containing 0.03% SDS. The reaction was started with the addition of 0.2 ml of 4 mg/ml o-nitrophenol-ß-D-galactoside (ONPG) at 30 C and stopped by adding 0.5 ml of 1 M Na2CO3. ß-Galactosidase activity was determined by measuring the values at A420 and A550 using the following equation: U = 1000 x[(A420) - (1.75 x A550)]/[t x v x A600] [(t = time of reaction (min); v = volume of yeast culture used in reaction mixture (ml)].

For X-gal reaction, paper filter lifts of colonies were transferred to selection medium containing 1 µM 17ß-estradiol or 0.1% ethanol for 6 h, submerged in liquid nitrogen, and transferred to X-gal. The number of blue and/or white colonies were counted.

Immunoblotting
Yeast were collected by low-speed centrifugation, resuspended in 0.25 M NaOH and 1% ß-mercaptoethanol, and placed on ice for 10 min. Then, 0.16 ml of trichloroacetic acid (50%) was added to 1 ml of suspension on ice, and yeast were pelleted. After washing with acetone, the pellet was dried and resuspended in SDS-PAGE sample buffer. Equal amounts of total protein were analyzed on 9% SDS-PAGE and transferred to polyvinylidene difluoride membrane. After the transfer, blots were stained with 0.5% Ponceau S Red to monitor transfer efficiencies and subsequently probed with either ERC314 (Santa Cruz Biotechnology, Santa Cruz, CA) or H222 (Abbott Diagnostics, North Chicago, IL) antibody.


    ACKNOWLEDGMENTS
 
We thank Dr. Benita Katzenellenbogen for providing full-length ER cDNA, Dr. Malcolm Parker for supplying the RIP 140 cDNA, and Drs. Pierre Chevray and Daniel Nathans for the two-hybrid vectors pPC62, pPC86, and yeast strain PCY2.


    FOOTNOTES
 
Address requests for reprints to: Sohaib A. Khan, Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, PO Box 670521, Cincinnati, Ohio 45267-0521. E-mail: Sohaib.Khan{at}uc.edu

This work was supported by National Aeronautics and Space Administration U95–002 Predoctoral Fellowship (to G.P.) and NIH Grant CA-72039 and American Cancer Society Grant CN-77110 (to S.K).

1 Present address: Department of Molecular Biology, NC-20, The Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195. Back

Received for publication August 6, 1998. Revision received October 23, 1998. Accepted for publication November 9, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Kumar V, Chambon P 1988 The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer. Cell 55:145–156[Medline]
  2. Kumar V, Green S, Staub A, Chambon P 1986 Localization of the oestradiol-binding and putative DNA binding domains of the human oestrogen receptor. EMBO J 5:2231–2236[Abstract]
  3. Kumar V, Green S, Stack G, Berry M, Jin J-R, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[Medline]
  4. Tora L, White J, Brou C, Tasset D, Webster N, Scheer E, Chambon P 1989 The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell 59:477–487[Medline]
  5. Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8:21–30[Abstract]
  6. Norris JD, Fan D, Kemer SA, McDonnell DP 1997 Identification of a third autonomous activation domain within the human estrogen receptor. Mol Endocrinol 11:747–754[Abstract/Free Full Text]
  7. Pierrat B, Heery DM, Chambon P, Losson R 1994 A highly conserved region in the hormone-binding domain of the human estrogen receptor functions as an efficient transactivation domain in yeast. Gene 143:193–200[CrossRef][Medline]
  8. Schwabe JWR, Chapman L, Finch JT, Rhodes D 1993 The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell 75:567–578[Medline]
  9. Liu W, Wang J, Yu G, Pearce D 1996 Steroid receptor transcriptional synergy is potentiated by disruption of the DNA-binding domain dimer interface. Mol Endocrinol 10:1399–1406[Abstract]
  10. Montano MM, Muller V, Trobaugh A, Katzenellenbogen BS 1995 The carboxy-terminal F domain of the human estrogen receptor: role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists. Mol Endocrinol 9:814–825[Abstract]
  11. Horowitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:1167–1177[Abstract]
  12. Glass CK, Rose DW, Rosenfeld MG 1997 Nuclear receptor coactivators. Curr Opin Cell Biol 9:222–232[CrossRef][Medline]
  13. Cavailles V, Dauvois S, L’Horset F, Lopez G, Hoare S, Kushner PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor. EMBO J 14:3741–3751[Abstract]
  14. L’Horset F, Dauvois S, Heery DM, Cavailles V, Parker MG 1996 RIP-140 interacts with multiple nuclear receptors by means of two distinct sites. Mol Cell Biol 16:6029–6036[Abstract]
  15. Fawell SE, Lees JA, White R, Parker MG 1990a Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor. Cell 60:953–962
  16. Nardulli AM, Greene GL, Shapiro DJ 1993 Human estrogen bound to an estrogen response element bends DNA. Mol Endocrinol 7:331–340[Abstract]
  17. Curtis SW, Korach KS 1990 Uterine estrogen receptor interaction with estrogen-responsive DNA sequences in vitro: effects of ligand binding on receptor-DNA complexes. Mol Endocrinol 4:276–286[Abstract]
  18. Murdoch FE, Meier DA, Furlow JD, Grunwald K, Gorski J 1990 Estrogen receptor binding to a DNA response element in vitro is not dependent upon estradiol. Biochemistry 29:8377–8385[Medline]
  19. Brown M, Sharp P 1990 Human estrogen receptor forms multiple protein-DNA complexes. J Biol Chem 265:11238[Abstract/Free Full Text]
  20. Beekman JM, Allan GF, Tsai SY, Tsai M-J, O’Malley BW 1993 Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain. Mol Endocrinol 7:1266–1274[Abstract]
  21. Wang H, Peters GA, Zeng X, Tang M, Ip W, Khan S 1995 Yeast two-hybrid system demonstrates that estrogen receptor dimerization is ligand-dependent in vivo. J Biol Chem 270:23322–23329[Abstract/Free Full Text]
  22. Metzger D, White JH, Chambon P 1988 The human oestrogen receptor functions in yeast. Nature 334:31–36[CrossRef][Medline]
  23. Wittcliff JL, Wenz LL, Dong J, Nawaz Z, Butt TR 1990 Expression and characterization of an active human estrogen receptor as a ubiquitin fusion protein from Escherichia coli. J Biol Chem 265:22016–22022[Abstract/Free Full Text]
  24. Pham TA, Hwung YP, Santiso-Mere D, McDonnell DP, O’Malley BW 1992 Ligand-dependent and -independent function of the transactivation regions of the human estrogen receptor in yeast. Mol Endocrinol 6:1043–1050[Abstract]
  25. Lyttle CR, Damian-Matsumura P, Juul H, Butt TR 1992 Human estrogen receptor regulation in a yeast model system and studies on receptor agonists and antagonists. J Steroid Biochem Mol Biol 42:677–684[CrossRef][Medline]
  26. Chevray PM, Nathans D 1992 Protein interaction cloning in yeast: identification of mammalian proteins that react with the leucine zipper of Jun. Proc Natl Acad Sci 89:5789–5793[Abstract]
  27. Joyeux A, Cavailles P, Balaguer P, Nicolas JC 1997 RIP 140 enhances nuclear receptor-dependent transcription in vivo in yeast. Mol Endocrinol 11:193–202[Abstract/Free Full Text]
  28. Salomonsson M, Haggblad J, O’Malley B, Sitbon G 1994 The human estrogen receptor hormone binding domain dimerizes independently of ligand activation. J Steroid Biochem Mol Biol 48:447–452[CrossRef][Medline]
  29. Estojak J, Brent R, Golemis EA 1995 Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol 15:5820–5829[Abstract]
  30. Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson J-A, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–757[CrossRef][Medline]
  31. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  32. Xu J, Nawaz Z, Tsai SY, Tsai MJ, O’Malley BW 1996 The extreme C terminus of the progesterone receptor contains a transcriptional repressor domain that functions through a putative corepressor. Proc Natl Acad Sci USA 29:12195–12199[CrossRef]
  33. Arnold SF, Notides AC 1995 An antiestrogen: a phosphotyrosyl peptide that blocks dimerization of the human estrogen receptor. Proc Natl Acad Sci USA 92:7475–7479[Abstract]
  34. Pakdel F, LeGoff P, Katzenellenbogen BS 1993 An assessment of the role of domain F and pest sequences in estrogen receptor half-life and bioactivity. J Steroid Biochem Mol Biol 46:663–672[CrossRef][Medline]
  35. Vegeto E, Allan GF, Schrader WT, Tsai MJ, McDonnell DP, O’Malley BW 1992 The mechanism of RU486 antagonism is dependent on the conformation of the carboxy-terminal tail of the human progesterone receptor. Cell 69:703–713[Medline]
  36. Treuter E, Albrektsen T, Johansson L, Leers J, Gustafsson J-A 1998 A regulatory role for RIP140 in nuclear receptor activation. Mol Endocrinol 12:864–881[Abstract/Free Full Text]
  37. Friend KE, Ang LW, Shupnik MA 1995 Estrogen regulates the expression of several different estrogen receptor mRNA isoforms in rat pituitary. Proc Natl Acad Sci USA 92:4367–4371[Abstract]
  38. Gotteland M, Desauty G, Delarue JC, Liu L, May E 1995 Human estrogen receptor messenger RNA variants in both normal and tumor breast tissues. Mol Cell Endocrinol 112:1–13[CrossRef][Medline]
  39. Hu C, Hyderm SM, Needleman DS, Baker VV 1996 Expression of estrogen receptor variants in normal and neoplastic human uterus. Mol Cell Endocrinol 118:173–179[CrossRef][Medline]
  40. Murphy LC 1990 Estrogen receptor variants in human breast cancer. Mol Cell Endocrinol 74:C83–C86
  41. Sluyser M 1992 Role of estrogen receptor variants in the development of hormone resistance in breast cancer. Clin Biochem 25:407–414[Medline]
  42. Fuqua SAW, Fitzgerald SD, Chamness GC, Tandon AK, MacDonnell DP, Nawaz Z, O’Malley B, McGuire ML 1991 Variant human breast tumor estrogen receptor with constitutive transcriptional activity. Cancer Res 51:105–109[Abstract]
  43. Fuqua SAW, Allred DC, Auchus RJ 1993 Expression of estrogen receptor variants. J Cell Biochem 17G [Suppl]:194–197
  44. Fuqua SAW, Wiltschke C, Castles C, Wolf D, Allred DC 1995 A role for estrogen receptor variants in endocrine resistance. Endocr Relat Cancer 2:19–25
  45. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  46. 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]
  47. Jensen EV 1996 Steroid hormones, receptors, and antagonists. Ann NY Acad Sci 784:1–17[Abstract]
  48. Hill J, Ian KA, Donald G, Griffiths DE 1991 DMSO-enhanced whole yeast transformation. Nucleic Acids Res 19:5791[Medline]