Mutation of Leu-536 in Human Estrogen Receptor-{alpha} Alters the Coupling between Ligand Binding, Transcription Activation, and Receptor Conformation*

Changqing Zhao {ddagger}, Akiko Koide §, Judith Abrams ¶, Sarah Deighton-Collins {ddagger}, Angela Martinez {ddagger}, Janice A. Schwartz {ddagger} ¶, Shohei Koide § and Debra F. Skafar {ddagger} ¶ ||

From the {ddagger}Department of Physiology, Wayne State University School of Medicine, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan 48201 and the §Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637

Received for publication, April 11, 2003 , and in revised form, May 6, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The estrogen receptor (ER), of which there are two forms, ER{alpha} and ER{beta}, is a ligand-modulated transcription factor important in both normal biology and as a target for agents to prevent and treat breast cancer. Crystallographic studies of the ER{alpha} ligand-binding domain suggest that Leu-536 may be involved in hydrophobic interactions at the start of a helix, "helix 12," that is crucial in the agonist-stimulated activity of ER{alpha}, as well as in the ability of antagonists to block the activity of ER{alpha}. We found that certain mutations of Leu-536 increased the ligand-independent activity of ER{alpha} although greatly reducing or eliminating the agonist activity of 17{beta}-estradiol (E2) and 4-hydroxytamoxifen (4OHT), on an estrogen response element-driven and an AP-1-driven reporter. The mutations impaired the interaction of the ER ligand-binding domain with the SRC1 receptor-interacting domain in a mammalian two-hybrid system. When tested in the yeast two-hybrid system, mutation of Leu-536 increased the basal reactivity of ER{alpha} to probes that recognize the agonist-bound conformation but did not significantly alter its reactivity to these probes in the presence of E2. Most interestingly, mutation of Leu-536 reduced the interaction of the 4OHT-bound ER{alpha} and increased the reactivity of the raloxifene- or ICI 182,780-bound ER{alpha}, with probes that recognize the 4OHT-bound ER{alpha} conformation in a yeast two-hybrid system. These results show that Leu-536 is critical in coupling the binding of ligand to the modulation of the conformation and activity of ER{alpha}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The human estrogen receptor-{alpha} (hER{alpha})1 is an allosteric protein whose activity is modulated by the binding of ligands, including estradiol (E2), the cognate ligand, and 4-hydroxytamoxifen (4OHT), the active metabolite of a drug used in the treatment and prevention of breast cancer (13). Like other members of the nuclear receptor superfamily, hER{alpha} is a multidomain protein having an N-terminal domain, a centrally located DNA-binding domain (DBD), and a ligand-binding domain (LBD) that is near the C terminus of the protein (1, 4). The DNA-binding domain mediates the binding of ER to estrogen response elements (EREs) of estrogen-regulated genes (4). Transcriptional enhancement results from the synergistic action of two separate activation functions, AF-1 and AF-2, that are located within the N-terminal domain and the LBD, respectively (59). Whereas the action of AF1 can be independent of hormone, AF2 requires hormone for its activity (6). The binding of agonists produces a conformation change in the receptor that facilitates the interaction of ER{alpha} with coactivator proteins (2, 3, 1012). A surface of the LBD, formed in part by a critical helix, "helix 12," binds to LXXLL sequences that are present in multiple copies within coactivator proteins (1315). AF-1 binds a distinct surface that lies within the C terminus of the p160 group of coactivators (12). The coactivator proteins, in turn, recruit histone acetyltransferases to the complex (2). The ER{alpha} can also modulate transcription at AP-1 sites through protein-protein interactions (16, 17).

Drugs that antagonize estrogen action, such as tamoxifen, raloxifene, and ICI 182,780, are used to treat and prevent breast cancer (18, 19). Because tamoxifen and raloxifene show tissue-specific agonist, as well as antagonist, effects, they are referred to as selective estrogen receptor modulators, or SERMs. The agonist activity of SERMs is due at least in part to AF-1 (20, 21). To develop compounds with desirable profiles of agonist and antagonist activity, particularly for use as breast cancer prevention agents that need to be taken for extended periods of time, it is necessary to fully understand the mechanisms through which these compounds exert their tissue-specific agonist and antagonist activity.

Crystallographic studies of the 4OHT- and raloxifene-bound LBD show that the extended side chains of these ligands displace helix 12, which then occupies the coactivator binding groove and blocks the binding of coactivators to AF2 (1315). The orientation of helix 12 is, therefore, a critical determinant of the activity of the ligand-bound hER{alpha}. Interestingly, the position at which helix 12 initiates is different in the agonist- and antagonist-bound structures; helix 12 starts at Asp-538 in the agonist-bound structure and at Leu-536 in the antagonist-bound structures (1315).

Particular motifs termed "capping motifs" have frequently been found to stabilize the start of {alpha} helices in model peptides and in proteins. These motifs, which are characterized by the dihedral angles of the peptide backbone as well as by the pattern of hydrophilic and hydrophobic residues at the start of the helix, use hydrogen-bonding and hydrophobic interactions to stabilize the start of {alpha} helices (reviewed in Ref. 22). Leu-536 has previously been suggested to be part of a capping motif at the start of helix 12 in the agonist-bound ER{alpha}; it could participate in a hydrophobic interaction with the hydrophobic residues Leu-540 and/or Leu-541 within the helix (23). Examination of the crystallographic structures shows that, in the diethylstilbestrol-bound ER{alpha} LBD (Protein Data Bank code 3ERD [PDB] , the side chain of Leu-536 is oriented toward the ligand and the core of the LBD in one molecule, possibly interacting with the side chains of Leu-540 and Leu-541, but it projects away into the solvent in the other. On the other hand, no side chain of Leu-536 is visible in the estradiol-bound ER{alpha} LBD (PDB code 1ERE [PDB] (13)). In the 4-OHT-bound complex (PDB code 3ERT [PDB] (14)), Leu-536 points toward the core of the LBD. In the complex with raloxifene (PDB code 1ERR [PDB] (13)), the side chain of Leu-536 is in direct contact with the ligand and so is predicted to play a role in sensing the nature of the bound ligand (Fig. 1) (13, 14). We therefore wanted to determine the role of hydrophobic interactions involving Leu-536 in the E2-bound and the tamoxifen-bound states by examining the effects of mutating Leu-536 on the activity and conformation of ER{alpha}.



View larger version (54K):
[in this window]
[in a new window]
 
FIG. 1.
Leu-536 in the DES-bound (left) and 4OHT-bound (right) hER{alpha} The left-hand panel shows an energy-minimized model of the wt hER{alpha} LBD bound with the agonist DES (27). The right-hand panel shows the crystallographic structure of the wt hER{alpha} LBD bound with the SERM 4-OHT (PDB 3ERT [PDB] (14)). The peptide backbone is shown as a ribbon in gray, with the exception that the residues in helix 12 (left, 537–548; right, 537–551) are in turquoise. The ligand (left, DES; right, 4-OHT) is shown as a Connelly surface in chartreuse. The van der Waals surface of Leu-536 is shown in blue; the van der Waals surface of Leu-541 is shown in purple. The Connelly surface of a crystallographic water molecule is shown on the left in orange.

 


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Dulbecco's modified Eagle's/Ham's F-12 medium without phenol red was obtained from Invitrogen; dextran-coated charcoal-treated fetal calf serum was purchased from HyClone. Estradiol and 4-hydroxytamoxifen were purchased from Sigma. ICI 182,780 was purchased from Tocris, and raloxifene was obtained from Eli Lilly. Luciferase activity was measured using the Dual Luciferase Assay kit (Promega). SuperFect and plasmid preparation kits were purchased from Qiagen. Enhanced green fluorescent protein (EGFP) vector and EGFP-specific Color Antibody were purchased from Clontech. ER{alpha}-specific Ab-1, which recognizes an epitope in the AB domains of the receptor, was purchased from NeoMarkers. The ECLTM (Amersham Biosciences) detection system was used to visualize bands in Western immunoblotting. [3H]Estradiol, 40–60 Ci/mmol, was purchased from PerkinElmer Life Sciences.

Construction of Vectors—The expression vector containing the estrogen receptor {alpha} cDNA, HEGO in pSG5, was the generous gift of Drs. Pierre Chambon and Hinrich Gronemeyer. Mutants at Leu-536 were constructed by site-directed mutagenesis using the Gene Editor kit (Promega). The mutations converted leucine 536 to alanine (L536A), glutamic acid (L536E), glycine (L536G), isoleucine (L536I), lysine (L536K), or asparagine (L536N).

The pAP1-luc reporter vector was constructed by cloning a DNA fragment corresponding to the AP-1 consensus sequence from the promoter region of human collagenase 1 (-73 to -52) into the HindIII and XhoI restriction sites of the multicloning region of pLuc-MCS vector, which has a simple TATA box between the multicloning site and the firefly luciferase coding region (Stratagene). The single strands of the AP1 site (sense: AGCTTATGAGTCAGACACCTCTGGCTTC and antisense: tcgagaagccagaggtgtctgactcata) were synthesized by Operon and were annealed into double strands for cloning.

The p2ERE-luc reporter vector was constructed by cloning a DNA fragment containing two estrogen response elements corresponding to the consensus vitellogenin ERE into the HindIII and XhoI restriction sites in the vector pLuc-MCS. The single strands (sense: AGCTTCTAGAGGATCCAGGTCACAGTGACCTGGGCCCGGATCCGGGCCCAGGTCACAGTGACCTGGCCC and antisense: tcgagggccaggtcactgtgacctgggcccggatccgggcccaggtcactgtgacctggatcctctaga) were synthesized by Operon and were annealed prior to cloning. The nature of each mutant ER and the constructed reporter vectors was confirmed by DNA sequencing. The internal control vector pRL-SV40 was purchased from Promega.

The expression vectors for mammalian two hybrid assay, which will produce the Gal4-ER{alpha} LBD and VP16-SRC1 fusion proteins, were generated by cloning ER{alpha} LBD amino acids 264–595 into pBind-CMV and SRC1 amino acids 190–400 into pAct-CMV (Promega). The Gal4-ER LBD fusion protein contains a yeast Gal4 DNA-binding domain, which will bind to the Gal4 binding sites of the pG5luc reporter vector (Promega). The VP16-SRC1 fusion protein possesses the VP16 activation domain. In addition, the pBind-CMV vector expresses Renilla luciferase for normalizing transfection efficiency. For detecting the binding of estradiol to the wt and mutated ER{alpha}, the cDNA encoding Ser-282 to Val-595 of the receptor was inserted into the multicloning site of pET-42b(+) (Novagen).

Construction of Yeast Two-hybrid Vectors—Yeast strains EGY48, MAT{alpha} his3 trp1 ura3 leu2::6LexAop-LEU2, and RFY206, MATa his3{Delta}200 leu2–3 lys2{Delta}201 trp1{Delta}::hisG ura3–52, have been described (24, 25) and were purchased from Origene. The plasmids for B42-monobody, B42-SRC-1 fusions, and pEGER{alpha}297–595 have been described (26). Variants of pEGER{alpha}297–595 with Leu-536 mutations were constructed by subcloning the NcoI-BamHI fragment (BamHI digestion was followed by Klenow treatment) of pSG-hER-Leu-536 mutants into the NcoI-XhoI segment (XhoI digestion followed by Klenow treatment) of pEGER{alpha}297–595.

Cell Transfection and Luciferase Assays—HeLa cells were maintained in Dulbecco's modified Eagle's medium/F-12 with 1% penicillin/streptomycin and 10% dextran-coated charcoal-treated fetal calf serum without phenol red. Cells (3 x 105/well) were seeded in six-well dishes and 20 h later cotransfected by 0.8 µg of ER{alpha} expression vector (wild-type or mutant), 50 ng of pRL-SV40 with either 2 µg of p2ERE-luc or pAP1-luc reporter plasmid using SuperFect as carrier. After 4 h, the transfection medium was removed and replaced with culture medium containing ethanol (vehicle control), 17-{beta} estradiol (0.1, 1, 10, or 100 nM), or 4-hydroxytamoxifen (0.1, 1, 10, or 100 nM and 1 µM). After 48 h, the cells were harvested and the luciferase activity was measured with the Dual Luciferase Assay kit on a Turner 20/20 luminometer. The activity of firefly luciferase reporter was normalized to the activity of Renilla luciferase expressed from the internal control vector pRL-SV40, and the results are expressed as relative luciferase units (RLUs).

Mammalian Two-hybrid Assay—For determination of the interactions between SRC1 and the wt or mutated ER{alpha}, HeLa cells were transfected with 1 µg of pBind-ER{alpha} (wt or mutant), 1.2 µg of pAct-SRC1, and 1.5 µg of pG5luc using SuperFect. The empty vectors, either 1.0 µg of pBind-CMV or 1.2 µg pAct-CMV, were also transfected into parallel cultures of HeLa cells as negative controls. After 3 h, the transfection medium was removed, and cells were washed three times with phosphate-buffered saline and culture medium containing ethanol (vehicle control) or 17{beta} estradiol (100 nM) and then maintained for an additional 24 h. The cells were then harvested, and luciferase activity was measured with the Dual Luciferase Assay kit on a Turner 20/20 luminometer. The activity of firefly luciferase was normalized to the activity of the Renilla luciferase.

Western Blotting—HeLa cells were transfected with vectors expressing the wt or mutated ER{alpha} and the EGFP vector, from which enhanced green fluorescent protein was translated and used as internal control. The whole cell extracts were prepared, and SDS-gel electrophoresis was carried out. The proteins were transferred onto a polyvinylidene difluoride membrane. The membrane was immunoblotted with ER{alpha}-specific Ab-1, and with EGFP-specific Color Antibody. Bands were visualized using the ECLTM system.

Binding of Estradiol to the wt and Mutant ER{alpha}BL21(DE3)pLysS (Novagen) was used as host for expressing a GST-tagged ER{alpha}(S282-V595) fusion protein. After transformation with pET-42b(+)-hER{alpha}, a single colony was cultured in 4 ml of LB broth with glucose (1%), chloramphenicol (35 µg/ml), and kanamycin (35 µg/ml) until the A600 was between 0.6 and 1. Two milliliters of the culture was transferred to 25 ml of the same medium and cultured to a density of 0.6 of A600 at 37 °C with shaking. 20 ml of the culture was induced by isopropyl-1-thio-{beta}-D-galactopyranoside at 0.4 mM for 4 h at room temperature (about 25 °C), and the cells were collected by centrifugation. Cell extracts were prepared following procedures similar to those described in Ref. 27, with the exceptions that cells were lysed using a French press, and cell debris was pelleted by centrifugation at 27,000 x g for 30 min. Aliquots (200 µl) of the cell extracts containing wild-type or mutant ER{alpha}s were incubated with 50 nM [3H]estradiol for 1 h at 0 °C. The nonspecific binding was determined in a parallel set of incubations containing a 200-fold molar excess of unlabeled estradiol. Free and bound steroids were separated by dextran/charcoal assay. The concentration of protein in the cell extracts was measured using the Bradford protein assay.

Yeast Two-hybrid Assay—Yeast was grown in YPD media or YC dropout media following instructions from Origene and Invitrogen. Quantitative assays were performed using the RFY206 strain containing all plasmids as described previously for the interactions of SRC-1, monobodies (small binding proteins) E3#6, E2#23, and the hER{alpha} mutants (26). To measure interactions of monobodies OHT#1 and OHT#33 with the Leu-536 mutants, EGY48 harboring the monobody plasmid and RFY206 harboring the hER{alpha}-EF plasmid and pSH18–34 were mated, and then {beta}-galactosidase assays were performed on the diploid cells. Assays using haploid and diploid cells, respectively, yielded essentially the same results (not shown).

Data Analysis—To analyze the activity of the wt and mutated ER on the ERE-driven and AP-1-driven reporters and in the mammalian two-hybrid assay (Figs. 2, 3, 4, 5, 6), the data were first subjected to a log transformation. Random effects-generalized linear models were used to assess the statistical significance of differences in response; observations from experiments run on the same day were assumed to be correlated. The first model fitted included indicators for mutants, for ligand concentrations, and for their interaction. Ligand concentrations were included in the model as indicator variables to avoid constraining the shape of the relationship between concentration and response. If the simultaneous test for interaction terms was not significant, a reduced model was fitted in which the interaction terms were omitted. Finally, an even simpler model was fitted in which ligand was parameterized as presence or absence, collapsed over different concentrations. If there was significant interaction in the full model, separate models were fitted for each mutant in which ligands were compared. In the case of significant interaction, separate models for each ligand were also fitted, and the mutants were compared. Holm's stepdown procedure was used to control type I errors when making multiple tests among coefficients. Model goodness of fit was assessed graphically.



View larger version (39K):
[in this window]
[in a new window]
 
FIG. 2.
Response of the wt and mutant hER{alpha} to E2 on an ERE-driven promoter. Transcription activation (top) was measured using a transient cotransfection assay with wt or mutant ER{alpha} (HEGO or mutated ER in pSG5), an ERE-driven reporter (p2ERE-luciferase), and a Renilla luciferase transfection control (pRL-SV40) in HeLa cells as described under "Experimental Procedures." The activity of ER is measured by the relative luciferase activity, RLU, which is the ratio of firefly luciferase activity to Renilla luciferase activity. The RLU was measured in the absence of hormone (vehicle control) and in response to increasing concentrations of E2 (10-10, 10-9, 10-8, and 10-7 M E2). The values are the mean ± S.E. of three to four independent experiments, each carried out in triplicate. The level of expression of transfected wt and mutated ERs was assessed by Western immunoblotting as described under "Experimental Procedures" and is shown below. The band corresponding to the hER{alpha} in a single blot, representative of at least three independent transfections, is shown (left to right, wt, L536A, L536E, L536G, L536I, L536K, and L536N).

 


View larger version (23K):
[in this window]
[in a new window]
 
FIG. 3.
Response of the wt and mutant hER{alpha} to 4OHT and ICI 182,780 on an ERE-driven promoter. The activity of ER in response to 4-OHT and ICI 182,780 was measured using transiently transfected HeLa cells as described under "Experimental Procedures" and the legend to Fig. 2, with the exception that single concentrations of the indicated ligands were used. The ligand concentrations used were 4-OHT, 10-7 M, and ICI 182,780, 10-6 M. The values are the mean ± S.E. of three independent experiments, each carried out in triplicate.

 


View larger version (32K):
[in this window]
[in a new window]
 
FIG. 4.
Response of the wt and mutant hER{alpha} to 4OHT on an AP-1-driven promoter. The activity of ER was measured using transiently transfected HeLa cells as described under "Experimental Procedures" and the legend to Fig. 2, with the exception that an AP-1-driven reporter and the ligand 4OHT were used. Concentrations of 10-10,10-9,10-8,10-7, and 10-6 M 4OHT were used. The values are the mean ± S.E. of three to four independent experiments, each carried out in triplicate.

 


View larger version (19K):
[in this window]
[in a new window]
 
FIG. 5.
Response of the wt and mutant hER{alpha} to E2 and ICI 182,780 on an AP-1-driven promoter. The activity of ER in response to E2 and ICI 182,780 was measured using transiently transfected HeLa cells as described under "Experimental Procedures" and the legend to Fig. 2, with the exception that an AP-1-driven reporter and single concentrations of the indicated ligands were used. The ligand concentrations used were E2, 10-8 M, and ICI 182,780, 10-6 M. The values are the mean ± S.E. of three independent experiments, each carried out in triplicate.

 


View larger version (24K):
[in this window]
[in a new window]
 
FIG. 6.
Interactions of hER{alpha} mutants with endogenous coactivators and the SRC-1 RID in a mammalian two-hybrid system. The activity of ER was measured as described under "Experimental Procedures" using transiently transfected HeLa cells in the absence and presence of 10-7 M E2. The values are the mean ± S.E. of three independent experiments, each carried out in triplicate.

 


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We constructed mutants in which leucine 536 was replaced with a series of amino acids to determine the role of this residue in the activity of ER{alpha}. Leu-536 was replaced with residues having a negatively charged (glutamic acid, L536E), a positively charged (lysine, L536K), a polar (asparagine, L536N), a small (alanine, L536A, and glycine, L536G), and the isomeric (isoleucine, L536I) side chain. We then tested the activity of the mutants in a transient cotransfection assay in HeLa cells. Cells were transiently cotransfected with a wt ER{alpha} or a mutant expression vector, and a reporter plasmid containing either two consensus EREs or an AP1 consensus sequence upstream of a simple TATA box linked with the firefly luciferase coding sequence, and a transfection control vector that constitutively expresses the Renilla luciferase (pRL-SV40). Using this system, we examined the transcriptional response of the wt and mutated ERs to the endogenous agonist E2, the SERM 4-hydroxytamoxifen, and the antagonist ICI 182,780.

Activity of the Leu-536 Mutants at an ERE-driven Promoter—Mutation of Leu-536 alters the response of ER{alpha} to E2 (p = 0.03). The wt ER{alpha} and the L536I mutant exhibited 18- and 6-fold higher transcriptional activity in the presence of E2 than their basal activity, respectively (Fig. 2). Remarkably, all other mutants have lost the E2 dependence of their transcription activity (Fig. 2). Furthermore, the activity of the L536A, L536E, L536I, and L536K mutants in the presence of E2 is less than the activity of the E2-stimulated wt ER (p = 0.05 for L536E; all others p < 0.001). No consistent differences in the level of expression of the wt and mutant proteins were detected by Western immunoblotting (Fig. 2). We tested whether the loss of response to E2 was due to an inability of receptors having mutations at Leu-536 to bind hormone. We expressed GST-tagged ER{alpha}(S282-V595) having the wt or mutated ER sequence in E. coli and measured the binding of [3H]estradiol (50 nM) in cell extracts. All GST-ER(S282-V595) fusion proteins bound estradiol in vitro, at levels ranging from 10.6 ± 2.5 pmol of [3H]E2 bound/mg of protein for the non-mutated ER sequence, up to 76 ± 20 pmol of [3H]E2 bound/mg of protein for the L536G mutation (n = 3). Therefore, the lack of effect of E2 on transcription activation was not due to a loss of hormonebinding activity. Moreover, ERs having mutations at Leu-536 exhibit estradiol-dependent activity in yeast and mammalian two-hybrid assays, which provides additional support for their ability to bind E2 (see below). These results show that the presence of a large hydrophobic residue, either leucine or isoleucine, at position 536 of the hER{alpha} is critical for the ability of E2 to stimulate activity on an ERE-driven promoter.

In addition to the effect of mutations at Leu-536 on hormonestimulated transcription, the constitutive activity of the L536N mutant is significantly greater than that of the wt ER (p = 0.04) (Fig. 2). Although the median constitutive activity of the L536A, L536E, L536G, and L536K mutants is greater than that of the wt protein, the differences are not statistically significant because of the variability in these experiments (Fig. 2). Overall, our results are consistent with the previous report that substitution of Leu-536 with proline led to a receptor having increased constitutive activity (28). They are also consistent with the idea that position 536 of the hER{alpha} is involved in maintaining the receptor in an inactive state in the absence of ligand.

Response of the Mutated ERs to 4-OHT and ICI 182,780 on an ERE-driven Promoter—To determine whether antagonists of hER{alpha} modulate the activity of the mutant ERs, we tested the effects of 4OHT and ICI 182,780 on the transcriptional activity of the wt and mutant hER{alpha}s on an ERE-driven promoter using the same transient cotransfection assay (Fig. 3). As reported by others (16, 17), we found that 4OHT could cause a significant increase in transcriptional activity of the wt ER{alpha} (p<0.001), but this increase is much weaker than that stimulated by estradiol (compare Fig. 2 with Fig. 3). By contrast, 4-OHT reduced the transcriptional activity of the L536G, L536K, and L536N mutants by one-half to two-thirds relative to their basal activity (p = 0.04 or less) and had no effect on the activity of the L536A, L536E, and L536I mutants (Fig. 3). Interestingly, although the presence of ICI 182,780 alone had, as expected, no effect on the activity of the wt ER{alpha} (Fig. 3), it reduced the activity of the L536A, L536E, L536G, L536K, and L536N mutants (p = 0.003 or less); the effect on the L536I mutant was not statistically significant (Fig. 3). Thus, mutations at Leu-536 either eliminate or "invert" the activity of 4-OHT on an ERE-driven reporter and cause the strong antagonist ICI 182,780 to reduce the basal activity of the protein. The ability of mutations at Leu-536 to convert 4-OHT from a weak agonist to an "inverse" agonist is reminiscent of the effects of the L540Q mutation on the activity of ER{alpha} (29).

Activity of the Leu-536 Mutants on an AP1-driven Promoter—We next investigated the effect of these same mutations on the activity of ER{alpha} on an AP-1-driven promoter (16, 17, 30). The constitutive activity of the L536A, L536G, L536K, and L536N mutants was greater than the constitutive activity of the wt hER{alpha} on the AP1-responsive reporter (p = 0.03 or less) (Fig. 4). It is especially striking that the L536K and L536N mutants exhibit constitutive activities equal to (L536K) or even greater than (L536N, p < 0.001) the 4-OHT-stimulated activity of the wild-type ER{alpha}.

When compared with the activity of the wt ER in the presence of 4-OHT, the activity of two mutants was greater than the wt protein (L536K and L536N; p < 0.001); the activity of the remaining mutants (L536A, L536E, L536G, and L536I) was indistinguishable from the wt. Because of the increased constitutive activity of the mutated receptors, although 4-OHT increased the activity of the mutated receptors overall (p = 0.007), the degree of stimulation was substantially reduced, from ~6.5-fold in the wild-type ER{alpha} to ~3-fold for the L536E and L536I mutants, and even less for the others (Fig. 4). Comparing Figs. 2 and 4 also shows that the effects of mutations at Leu-536 depend on the promoter: the L536K and L536N mutants display similar levels of activity on the AP-1-driven reporter (p = 0.08, difference not significant), whereas the L536N mutant exerts greater activity on an ERE-driven reporter than does the L536K mutant (p = 0.05). Overall, these data show that Leu-536 in ER{alpha} is a critical position for an AP1-regulated promoter.

Response of the Leu-536 Mutants to E2 and ICI 182,780 on an AP-1-driven Promoter—Because E2 is also an agonist for ER{alpha} at AP-1 sites, we investigated the responses of the wt and mutant hER{alpha}s to E2 on the AP-1-driven reporter. We also wanted to determine whether ICI 182,780 exerts antagonist effects on ER{alpha} acting through AP-1 sites. E2 increased transcription activation by the wild-type ER{alpha} (p = 0.004), whereas as expected ICI 182,780 by itself had no effect (Fig. 5). E2 exerted a marginally significant stimulatory effect on the L536E mutant (p = 0.05). By contrast, E2 reduced the activity of the L536A, L536G, and L536N mutants (p = 0.003 or less) (Fig. 5). E2 exerted no significant effect on the activity of the L536I and L536K mutants (Fig. 5). ICI 182,780 reduced the activity of the L536A, L536G, L536K, and L536N mutants (p = 0.002 or less); it had no effect on the wt ER, the L536E, or the L536I mutant (Fig. 5). These results show that mutations at Leu-536 greatly reduce, and even eliminate or invert, the agonist activity of E2 at an AP-1-driven promoter.

Interaction of the Leu-536 Mutants with SRC1 in a Mammalian Two-hybrid Assay—A critical step in the activation of transcription by ER is the binding of coactivator proteins, such as SRC-1, by the receptor (11, 12). We wanted to determine whether the observed alterations in ER{alpha} function, which are a consequence of mutations of Leu-536, result from alterations in its interaction with coactivators such as SRC-1. We used a mammalian two-hybrid assay system to probe the interaction of the wt and mutated ER{alpha} LBDs with endogenous coactivators as well as their interaction with the nuclear receptor interacting domain of SRC-1.

We constructed vectors to express Gal4DBD-ER{alpha} (amino acids 264–595) and VP16-SRC1 (amino acids 190–400) fusion proteins. The Gal4DBD targets the DEF domains of ER{alpha}, which include the LBD, to the Gal4 promoter on the firefly luciferase reporter vector; this vector also constitutively expresses the Renilla luciferase, which serves as a transfection control. The VP16-SRC1 (amino acids 190–400) fusion protein contains the receptor interacting domain (RID) of SRC-1, including three LXXLL boxes. When the two fusion proteins associate with each other through the ligand-dependent interaction between the hER{alpha} LBD and the SRC1 RID, they recruit the basal transcriptional machinery to the Gal4 promoter, resulting in the production of firefly luciferase. The luciferase activity, therefore, reflects the interaction between the hER{alpha} LBD and the SRC1 RID, or, because the interaction between the LBD and the coactivator is estrogen-dependent, the ability of estrogens to alter the conformation of the hER{alpha} LBD to one that can associate with the SRC1 RID. When the assay is carried out in the absence of the SRC-1 fusion vector, upon estrogen binding the Gal4-ER{alpha} fusion protein will associate with the endogenous coactivators and again stimulate transcription; this provides an indication of the ability of the mutated ER{alpha} to interact with endogenous, full-length coactivators. In both cases, the activity of the firefly luciferase was normalized to the activity of the Renilla luciferase and is expressed in relative luciferase units (RLUs).

In marked contrast to the inability of estradiol to stimulate the activity of the mutated receptors in the transient cotransfection assays, the addition of estradiol (100 nM) increased the activity of the wt and mutated receptors in both the absence and presence of SRC-1 (p < 0.01, Fig. 6). These results show that the wt and mutated Gal4-hER LBDs could interact with endogenous coactivators in the HeLa cells to activate reporter gene transcription in an estradiol-stimulated manner. However, although the presence of SRC-1 increased estradiol-stimulated transcription driven by the wt receptor (p < 0.001), it had no effect on the activity driven by the mutated receptors (p = 0.06 for L536G; p > 0.40 for L536A, L536E, L536I, L536K, and L536N). The 2-fold stimulation of transcription driven by the unmutated Gal4-hER LBD by the SRC-1 RID is similar to the stimulation of transcription driven by the full-length wt hER{alpha} when full-length SRC-1 is overexpressed in HeLa cells (data not shown).

Overall, these findings demonstrate that estradiol binding to the wt and mutated hER{alpha} LBDs increases the association between the receptor and endogenous coactivators and that the mutations introduced at Leu-536 greatly impair the association of the hER{alpha} LBD with the SRC-1 RID in the mammalian two-hybrid system. Again, the ability of estradiol to stimulate the interaction between the mutated hER{alpha} LBDs and endogenous coactivators contrasts with the inability of E2 to stimulate transcription on an ERE-driven reporter (Fig. 2).

Probing the Conformational Changes of the Leu-536 Mutants in Yeast Cells Using Monobodies Recognizing Specific Ligand-bound Structures of the hER{alpha} LBD—Although the previous results demonstrate that mutations at Leu-536 have profound effects on the activity of ER{alpha} and its response to ligands on two different promoters, they provide no direct evidence regarding the effect(s) of these mutations on the conformation of the receptor. To address this question, we used a number of small binding proteins, "monobodies," that can probe ligand-induced conformational changes of the hER{alpha}-LBD (26). Two of the monobodies react with the agonist-bound, wt, ER{alpha} LBD; two react with the 4-OHT-bound, wt, ER{alpha} LBD (26). As an additional probe for the conformation of ER{alpha}, we used a fragment of SRC-1 that contains three NR-boxes (residues 190–400) and binds specifically to the agonist-bound form of hER{alpha} LBD (11, 12). Note that this is the same SRC-1 fragment used in the mammalian two-hybrid experiments. We examined the interactions of these conformation-specific probes with the ligand-binding domain of the wt or mutated ER{alpha}s in the absence and presence of selected ligands using a semi-quantitative {beta}-galactosidase assay in the yeast two-hybrid system.

We first tested the interaction between the mutated ER{alpha}s and probes that specifically recognize the agonist-bound conformation of the wild-type ER{alpha}, the SRC-1 fragment, monobody E2#23, and monobody E2#6. As reported previously (26), these reagents interacted with the wild-type receptor in the presence of E2 but not in the presence of 1 µM concentrations of ICI 182,780, raloxifene, 4-OHT, and progesterone or in the absence of a ligand (Fig. 7, A–C, top panels). Like the wt ER{alpha}, the interactions of the mutants with the SRC-1 fragment and monobody E3#6 were increased in the presence of E2 (Fig. 7, A and B, lower panels). By contrast, marked interactions between the mutated receptors (except for L536I), and either the SRC-1 fragment or monobody E3#6 were detected in the absence of ligand and in the presence of ICI 182,780, raloxifene, and progesterone but not in the presence of 4OHT. Thus, mutating Leu-536 increased the constitutive interaction with probes that recognize the agonist-bound conformation of ER{alpha}, and the increased basal reactivity was blocked by 4OHT but not raloxifene or ICI 182,780. The enhanced SRC-1 interaction of these mutants in the absence of ligand is consistent with the increased constitutive activity of certain mutants on the ERE-driven and AP-1-driven reporters (Figs. 2 and 4). However, the interaction of the mutated ERs with the SRC-1 RID in the presence of 1 µM E2 observed in this system contrasts with the inability of the mutated ERs to interact with SRC-1 in the presence of 100 nM E2 in the mammalian two-hybrid system. Differences in the activity of a mutated ER in yeast and mammalian cells have previously been reported; for example, the S554fs mutant is a potent mediator of E2-stimulated transactivation in yeast, yet its activity in CHO cells is markedly impaired (31).



View larger version (34K):
[in this window]
[in a new window]
 
FIG. 7.
Interactions of hER{alpha} mutants with hER/agonist complex-specific probes as measured using a yeast two-hybrid system. Experiments were carried out as described under "Experimental Procedures." {beta}-Galactosidase activity was measured in the presence of 1 µM of the indicated ligand, estradiol (E2), ICI 182,780 (ICI), 4-hydroxytamoxifen (OHT), raloxifene (RAL), progesterone (PROG), or with vehicle control (EtOH). Column A shows results using residues 190–400 of SRC-1, column B those with monobody E3#6, and C those with monobody E2#23. The data are from triplicate measurements. Different panels represent experiments using different yeast cells, and thus the absolute activity cannot be normalized (or quantitatively compared) across panels.

 

Monobody E3#23 is also specific for the hER{alpha}·LBD·agonist complex. As was observed for SRC-1 fragment and monobody E3#6, the interaction between this reagent and the wt and mutant LBDs is increased in the presence of E2 (Fig. 7C). However, the interaction of the mutant hER{alpha}s with this monobody (Fig. 7C) did not show the increased reactivity in the absence of ligand and in the presence of other ligands observed with the SRC-1 fragment and monobody E3#6 (Fig. 7, A and B). These results indicate that the interactions between hER and a probe depend on the nature of a probe, its affinity for the complex, and the particular site of the interaction. The observation that all the mutants interacted with these probes more strongly in the presence of E2 than in its absence is also consistent with the results of the mammalian two-hybrid assay, and provides additional evidence that these hER{alpha} mutants retain the ability to bind E2. However, unlike the mammalian interaction assay, the yeast interaction assay did not show any impairment of SRC1-LBD interaction by the mutants.

Taken together, the results provide strong evidence that the mutant hER{alpha} LBDs can assume the canonical "agonist" conformation in the presence of 1 µM E2. The increased reactivity of the mutants lacking a large, hydrophobic side chain at Leu-536 with agonist conformation-specific probes in the absence of ligand suggests that removing that side chain facilitates adoption of the agonist-bound conformation by the hormone-free ER{alpha}.

We also found drastic changes in the interaction profile when we probed the mutants with monobodies that recognize the 4OHT-bound conformation of ER{alpha}, OHT#1 and OHT#33 (Fig. 8). Monobody OHT#1 is highly specific to the wild-type hER·OHT complex (Fig. 8A, top panel). Unlike the results with the agonist complex-specific reagents above, no increase in reactivity of the mutants with monobody OHT#1 was observed in the absence of ligand. Rather, the mutants, except for L536I, interacted more strongly with OHT#1 in the presence of raloxifene than in the presence of 4OHT. These results suggest that mutating Leu-536 altered the conformation of the 4OHT-bound ER{alpha}, leading to a reduced interaction with OHT#1; at the same time, mutating Leu-536 altered the conformation of the raloxifene-bound ER{alpha} to more closely resemble that of the 4OHT-bound, wild-type ER{alpha}. Thus, the greatest effect of the mutations at Leu-536 on the SERM-bound ER{alpha} is on the actual conformation of the receptor, rather than a reduction of a barrier between different conformations.



View larger version (28K):
[in this window]
[in a new window]
 
FIG. 8.
Interactions of hER{alpha} mutants with hER·OHT complex-specific probes as measured using a yeast two-hybrid system. Experiments were carried out as described under "Experimental Procedures." {beta}-Galactosidase activity was measured in the presence of 1 µM of the indicated ligand, estradiol (E2), ICI 182,780 (ICI), 4-hydroxytamoxifen (OHT), raloxifene (RAL), progesterone (PROG), or with vehicle control (EtOH). Column A shows results using monobody OHT#1 and B those with monobody OHT#33. Data are from triplicate measurements. Different panels represent experiments using different yeast cells, and thus the absolute activity cannot be normalized (or quantitatively compared) across panels.

 

When probed with monobody OHT#33, the mutations in general increased the reactivity of the receptor to the probe in the presence of either OHT or ICI 182,780 (Fig. 8B). No increase in reactivity was observed in the absence of ligand. However, unlike the results with OHT#1 (Fig. 8A), no significant interaction was found between OHT#33 and the mutants in the presence of raloxifene. As was observed with the agonistspecific monobodies above, these results indicate that the interactions between hER{alpha} and a probe depend on the nature of a probe, its affinity for the complex, or the particular site to which it binds. We have not yet identified the binding sites of these monobodies on hER.

Collectively, the results using the yeast two-hybrid conformation probes indicate that these mutations do not significantly alter the conformation of ER{alpha} when bound to E2 and that they do increase the ability of the ligand-free receptor to adopt the agonist-bound conformation. In addition, these results show that mutations at Leu-536 alter the conformation of hER{alpha} when complexed with 4OHT, raloxifene, and ICI 182,780.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have investigated the role of Leu-536 in the conformation and activity of hER{alpha}. Our results show that a residue having a large hydrophobic side chain, either leucine or isoleucine, at position 536 in the receptor is critical for the ability of ligand to stimulate transcription on an ERE-driven or an AP-1-driven promoter. They are also consistent with the idea that Leu-536 is involved in maintaining the receptor in an inactive state in the absence of ligand. Furthermore, our results showed that not only were E2 and 4OHT unable or limited in their ability to stimulate gene expression through the mutant hERs (Figs. 2 and 4), these ligands actually decreased the activity of certain hER mutants (Figs. 3 and 5). Most importantly, our results show that Leu-536 is a critical determinant of the conformation of the SERM-bound receptor (Fig. 7). Collectively, our results provide strong evidence that mutations at Leu-536 alter the coupling between the binding of ligand, the conformational changes of the receptor, and its ensuing biological activity. To use an automotive analogy, Leu-536 serves as the "tie rod" of hER{alpha}.

The L536N mutant exhibits the highest constitutive activity of all the studied proteins on both AP1- and ERE-driven reporters; indeed, its constitutive activity on the AP-1 reporter is greater than that of the 4OHT-stimulated activity of the wt ER{alpha}. Most of the mutants, however, exhibit differences in activity on the two promoters (Figs. 2 and 4). It is especially striking that the L536K mutant, like the L536N mutant, exhibits constitutive activity greater than or equal to the 4-OHT-stimulated activity of the wild-type ER{alpha} on the AP-1-driven reporter, yet its activity on the ERE-driven reporter remains less than the activity of the L536N. This mutation could therefore be a useful tool for the dissection of ERE-driven and AP-1-driven pathways.

The mutant ER LBDs are impaired in their interaction with SRC1 in the mammalian two-hybrid system but not the yeast two-hybrid system, and they exhibit greater reactivity with agonist conformation-specific reagents in the absence of ligand in the yeast system than in the mammalian system. Differences in the transcriptional activity of mutated ERs in yeast and mammalian cells have previously been reported (31). The mammalian two-hybrid system has a smaller dynamic range (~2-fold) than the yeast two-hybrid system because of the strong activity in the absence of SRC-1. Also, in the mammalian two-hybrid system, SRC-1 is in direct competition with endogenous nuclear receptor coactivators. Thus, the reduced interaction of the mutant receptors with the SRC-1 RID may be caused by a reduction of the affinity of the receptor for the SRC-1 RID relative to endogenous coactivators. In contrast, the yeast two-hybrid system does not have endogenous coactivators, and, in the absence of competing coactivators, it may not be as sensitive to small changes in the affinity of the receptor to SRC-1.

The observed difference in the results between the two systems could also suggest that either 1) the agonist-bound conformation of ER is easier for the mutants to adopt in the yeast environment than in the mammalian cell environment or 2) lower affinity interactions between proteins are more easily detected in the yeast system than the mammalian system. This in turn could result from the differences in other proteins within the two cellular environments, differences in the concentration of E2 used, 100 nM in the mammalian system versus 1 µM in the yeast system, or simply the lower temperature used in the yeast system than in the mammalian system, 30 °C versus 37 °C. The results obtained in both systems, however, are fully consistent with the idea that mutating Leu-536 alters the coupling between the binding of ligand and the conformation of the receptor.

The full-length, mutated, ER{alpha} lost sensitivity to ligand stimulation in a transient cotransfection assay; by contrast, the mutated LBD, when analyzed in either the mammalian or the yeast two-hybrid systems, can be stimulated by ligands. We suggest the difference between the two types of experiments is due at least in part to the presence or absence of AF1 of ER{alpha}, as well as the presence or absence of other receptor-interacting regions besides the three NR boxes of SRC1. AF1 and AF2 of the hER{alpha} functionally interact to drive transcription on ERE- and AP1-driven reporters (12). An alteration in AF2 would change the coordination between AF1 and AF2 of the hER{alpha} in the interaction with cellular coregulators and thereby alter transactivation of the target genes. We suggest that the increased constitutive activity of AF2 in the mutants suffices to interact with AF1 and, thereby, drive transcription at relatively high levels in the absence of ligand. The involvement of AF2 is supported by the ability of 4OHT to reduce the activity of several mutants on an ERE, as well as to block the interaction of the mutants with the agonist conformation-specific probes in the yeast two-hybrid system.

Mutations at Leu-536 caused tamoxifen to repress the activity of ER{alpha} on an ERE, as well as causing E2 to repress the activity of the hER{alpha} on an AP-1-driven reporter (Figs. 3 and 5). We also observed that mutations at Leu-536 greatly reduced binding to SRC1 by ER{alpha} in the mammalian two-hybrid assay (Fig. 6). The loss or inversion of activity of 4OHT and E2 on specific promoters is reminiscent of the effects of deleting the C-terminal 58 amino acids of ER{alpha}, including helix 12 (32). This deletion did not eliminate the ability of ER{alpha} to bind ligand and DNA but caused the hER{alpha} to repress ERE-dependent transaction in a E2- and tamoxifen-dependent manner, to constitutively interact with silencing mediator for retinoic acid and thyroid hormone receptors (SMRT), and to eliminate the binding to SRC1 (32). Similarly, the L540Q mutant of ER{alpha} binds E2 yet responds to ligands in an inverse manner and does not interact with SRC1, but does react with a corepressor (29). The similarities in the phenotypes of the mutants are consistent with a structural model predicting a hydrophobic interaction between Leu-536, Leu-540, and/or Leu-541, so that mutation of these residues would impair formation of the active ER conformation. However, this is contradicted by the striking increase in the constitutive reactivity of the mutated receptors with either SRC1 or the agonist-conformation-specific monobodies in the yeast two-hybrid system, which shows that formation of the active ER{alpha} conformation in the absence of ligand is increased by the mutations at Leu-536. Thus, additional studies are necessary to specifically determine the effects of the mutations on the inactive state of ER{alpha}, the active state of ER{alpha}, and the transition between the inactive and active states in the absence and presence of E2.

Additional support for the idea that residues near the start of helix 12 are important in the function of ER{alpha} comes from recent studies of Asp-538, another residue located near the start of helix 12 in the structure of the agonist-bound ER{alpha}. This residue, although not part of the coactivator interface formed by helices 12, 3, and 5, has been shown to affect the function, stability, and interaction of ER{alpha} with coactivators and corepressors (33). Asp-538, which is necessary for the agonist activity of 4OHT, has been defined as part of a transcription activation region termed "AF2b" (33). It has also been suggested that Asp-538 interacts with AF1 (33). Certain mutations of another neighboring residue, Tyr-537, alter the affinity and kinetics of the interaction between estradiol and the hER{alpha} (27, 34). It will be interesting to determine the effects of mutations at Leu-536 on the affinity and kinetics of hormone binding to ER{alpha}.

Finally, the effects of mutations at Leu-536 on the conformation of the SERM-bound ER{alpha} are strikingly revealed in the experiments with the monobody probes that recognize the OHT-bound wt ER{alpha}. When we studied the interaction between monobodies OHT#1 and OHT#33 with the LBD having mutations at Leu-536, the results demonstrated that the structures of the mutated, ligand-bound LBDs had changed substantially. These experiments strongly support the importance of Leu-536 in maintaining particular conformations of the SERM-bound hER{alpha}; in other words, Leu-536 is "reading" the extended side chain of raloxifene, 4-hydroxytamoxifen, and ICI 182,780.

Overall, our findings provide strong support for the idea that Leu-536 is critical in coupling ligand binding with the conformational changes of ER{alpha} not only in the agonist-bound ER{alpha} but in the SERM-bound ER{alpha} as well. Additional studies of this interesting region of the protein are ongoing.


    FOOTNOTES
 
* This work was supported by Grant 1RO1-DK56934-01A1, the Office of Research on Women's Health, and Grant DAMD 17-00-1-0498 (to D. F. S.) and by Grants R29-GM55042, R01-DK62316, and P30-CA14599 (to S. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201. Tel.: 313-577-1550; Fax: 313-577-5494; E-mail: dskafar{at}med.wayne.edu.

1 The abbreviations used are: hER{alpha}, human estrogen receptor-{alpha}; E2, 17{beta}-estradiol; 4OHT, 4-hydroxytamoxifen; DBD, DNA-binding domain; LBD, ligand-binding domain; ERE, estrogen response element; SERMs, selective estrogen receptor modulators; EGFP, enhanced green fluorescent protein; CMV, cytomegalovirus; RLU, relative luciferase unit(s); wt, wild type; GST, glutathione S-transferase; aa, amino acid(s); RID, receptor interacting domain; DES, diethylstilbestrol. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Evans, R. M. (1988) Science 240, 889-895[Medline] [Order article via Infotrieve]
  2. Nilsson, S., and Gustafsson, J.-A. (2000) Breast Cancer Res. 2, 360-366[CrossRef][Medline] [Order article via Infotrieve]
  3. Hall, J. M., Couse, J. F., and Korach, K. S. (2001) J. Biol. Chem. 276, 36869-36872[Free Full Text]
  4. Kumar, V., Green, S., Stack, G., Berry, M., Jin, J. R., and Chambon, P. (1987) Cell 51, 941-951[Medline] [Order article via Infotrieve]
  5. Berry, M., Metzger, D., and Chambon, P. (1990) EMBO J. 9, 2811-2818[Abstract]
  6. Tora, L., White, J., Brou, C., Tassett, D., Webster, N., Scheer, E., and Chambon, P. (1989) Cell 59, 477-487[Medline] [Order article via Infotrieve]
  7. Bocquel, M. T., Kumar, V., Chambon, P., and Gronemeyer, H. (1989) Nucleic Acids Res. 17, 2581-2595[Abstract]
  8. Tzukerman, M. T., Esty, A., Santiso-Mere, D., Danielian, P., Parker, M. G., Stein, R. B., Pike, J. W., and McDonnell, D. P. (1994) Mol. Endocrinol. 8, 21-30[Abstract]
  9. McInerney, E. M., and Katzenellenbogen, B. S. (1996) J. Biol. Chem. 271, 24172-24178[Abstract/Free Full Text]
  10. Webb, P., Nguyen, P., Shinsako, J., Anderson, C., Feng, W., Nguyen, M. P., Chen, D., Huang, S.-M., Subramanian, S., McInerney, E., Katzenellenbogen, B. S., Stallcup, M. R., and Kushner, P. J. (1998) Mol. Endocrinol. 12, 1605-1618[Abstract/Free Full Text]
  11. Onate, S. A., Tsai, S. Y., Tsai, M. J., and O'Malley, B. W. (1995) Science 270, 1354-1357[Abstract]
  12. Onate, S. A., Boonyaratanakornkit, V., Spencer, T. E., Tsai, S. Y., Tsai, M. J., Edwards, D. P., and O'Malley, B. W. (1998) J. Biol. Chem. 273, 12101-12108[Abstract/Free Full Text]
  13. Brzozowski, A. M., Pike, A. C. W., Dauter, Z., Hubbard, R. E., Bonn, T., Engstrom, O., Ohman, L., Greene, G. L., Gustafsson, J.-A., and Carlquist, M. (1997) Nature 390, 753-758
  14. Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927-937[Medline] [Order article via Infotrieve]
  15. Gangloff, M., Ruff, M., Eiler, S., Duclaud, S., Wurtz, J. M., and Moras, D. (2001) J. Biol. Chem. 276, 15059-15065[Abstract/Free Full Text]
  16. Webb, P., Nguyen, P., Valentine, C., Lopez, G. N., Kwok, G. R., McInerney, E., Katzenellenbogen, B. S., Enmark, E., Gustafsson, J. A., Nilsson, S., and Kushner, P. J. (1999) Mol. Endocrinol. 13, 1672-1685[Abstract/Free Full Text]
  17. Webb, P., Lopez, G. N., Uht, R. M., and Kushner, P. J. (1995) Mol. Endocrinol. 9, 443-456[Abstract]
  18. The Consensus Conference on Treatment of Estrogen Deficiency Symptoms in Women Surviving Breast Cancer (1998) Obstet. Gynecol. Surv. 53, (suppl.) S1-S83; Consensus statement, S2-S10[Medline] [Order article via Infotrieve]
  19. Gradishar, W. J. (2003) Curr. Treat. Options Oncol. 4, 141-150[Medline] [Order article via Infotrieve]
  20. Shang, Y., and Brown, M. (2002) Science 295, 2465-2468[Abstract/Free Full Text]
  21. Smith, C. L., Nawaz, Z., and O'Malley, B. W. (1997) Mol. Endocrinol. 11, 657-666[Abstract/Free Full Text]
  22. Aurora, R., and Rose, G. D. (1998) Prot. Sci. 7, 21-38[Abstract/Free Full Text]
  23. Skafar, D. F. (2000) Cell Biochem. Biophys. 33, 53-62[CrossRef][Medline] [Order article via Infotrieve]
  24. Finley, R. L., Jr., and Brent, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12980-12984[Abstract/Free Full Text]
  25. Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. (1993) Cell 75, 791-803[Medline] [Order article via Infotrieve]
  26. Koide, A., Abbatiello, S., Rothgery, L., and Koide, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 1253-1258[Abstract/Free Full Text]
  27. Zhong, L., and Skafar, D. F. (2002) Biochemistry 41, 4209-4217[CrossRef][Medline] [Order article via Infotrieve]
  28. Eng, F. C., Lee, H. S., Ferrara, J., Willson, R. M., and White, J. H. (1997) Mol. Cell. Biol. 17, 4644-4653[Abstract]
  29. Montano, M. M., Ekena, K., Krueger, K. D., Keller, A. L., Katzenellenbogen, B. S. (1996) Mol. Endocrinol. 10, 230-242[Abstract]
  30. Paech, K., Webb, P., Kuiper, G. G. J. M., Nilsson, S., Gustafsson, J. A., Kushner, P. J., and Scanlan, T. S. (1997) Science 277, 1508-1510[Abstract/Free Full Text]
  31. Wrenn, C. K, and Katzenellenbogen, B. S. (1993) J. Biol. Chem. 268, 24089-24098[Abstract/Free Full Text]
  32. Jung, D. J., Lee, S. K., and Lee, J. W. (2001) J. Biol. Chem. 276, 37280-37283[Abstract/Free Full Text]
  33. Pearce, S. T., Liu, H., and Jordan, V. C. (2003) J. Biol. Chem. 278, 7630-7638[Abstract/Free Full Text]
  34. Yudt, M. R., Vorojeikina, D., Zhong, L., Skafar, D. F., Sasson, S., Gasiewicz, T. A., and Notides, A. C. (1999) Biochemistry 38, 14146-14157[CrossRef][Medline] [Order article via Infotrieve]