Positive and negative discrimination of estrogen receptor agonists and antagonists using...

Positive and NegativeDiscrimination of EstrogenReceptor Agonists and AntagonistsUsing Site-Specific DNARecombinase Fusion Proteins

Colin Logie*, Mark Nichols†, Kathy Myles, John W. Funder, andA. Francis Stewart

Gene Expression ProgramEuropean Molecular Biology Laboratory (C.L., M.N., A.F.S.)69117 Heidelberg, Germany

Baker Medical Research Institute (K.M., J.W.F.)Prahran, 3181, Victoria, Australia

Activation of the estrogen receptor (ER) by hor-mone involves at least two steps. First, hormonebinding initially relieves repression, a property im-posed on ER in cis by its ligand-binding domain(EBD). Subsequently, the derepressed ER bindsspecific genomic sites and regulates transcription.In addition to the natural hormone, ER binds abroad range of ligands that evoke a spectrum ofresponses ranging from full ER activation by ago-nists to partial activation and inhibition by partial orcomplete antagonists. How these different ligandsevoke different ER responses remains unclear. Toaddress this issue, we have developed a nontran-scriptional assay for ER ligand responsivenessbased on Flp recombinase/human EBD protein chi-meras. These fusion proteins transduce the tran-sient event of ligand binding into a permanent DNAchange in a human cell line system. A fusion pro-tein including ER D, E, and F domains was acti-vated by all the ER ligands tested, demonstratingthat both agonists and antagonists serve to relieveinitial repression, and that differences betweenthem lie downstream in the activation pathway.Mutant variants of the Flp-ER protein that distin-guish between agonists and antagonists, and amutant EBD that selectively lost the ability to re-spond to 17b-estradiol but not to other ligands,were also identified. Thus, agonists and antago-nists can be functionally distinguished in a non-transcriptional assay. (Molecular Endocrinology12: 1120–1132, 1998)

INTRODUCTION

The nuclear receptor family of transcription factorsincludes members with transcriptional activities al-tered by the binding of small, lipophilic ligands. Li-gands that activate the receptors are termed agonists,and those that bind with high affinity, but do not in-duce full transcriptional activity, are termed antago-nists. Agonist binding induces conformationalchanges in the receptor to alter its interaction withassociated proteins so that it passes from a repressedstate to a transcriptionally active state. Antagonistbinding also induces conformational changes, butsome of the subsequent events required for full tran-scriptional activity do not take place. The proteins thatare involved in mediating the transcriptional activity ofnuclear receptors have been the subject of many re-cent studies, with some found to encode acetylasesand associated factors (1–4). The protein-protein in-teractions that characterize the repressed and antag-onist-bound states are more uncertain.

There are two current explanations for repression ofnuclear receptor activity in the absence of bound ag-onist. The first invokes interactions of receptor with aubiquitous complex of heat shock proteins, termed theHsp90 complex (5–9). This situation is thought to berestricted to vertebrate steroid receptors, a special-ized class of nuclear receptors that homodimerizeupon ligand binding (10–12). The second arises fromrecent work on the mechanism of transcriptional re-pression mediated by unliganded nuclear receptors,such as thyroid and retinoic acid receptors, that het-erodimerize with the retinoid X receptor both in thepresence or absence of bound ligand. In the absenceof bound ligand these heterodimers appear to interactwith transcriptional corepressors, notably N-CoR (nu-clear receptor corepressor) and SMRT (silencing me-

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diator for retinoid and thyroid hormone receptors) (13–15), recently shown to recruit deacetylase complexes(16, 17). Whereas receptors that heterodimerize arenot believed to interact with the Hsp90 complex (18),the relationship of steroid receptors with corepressorsis yet to be fully established (19). Similarly, the effect ofantagonist binding on interactions with the Hsp90complex and corepressors remains unclear (20, 21).

To investigate the relationship between repression,agonists, and antagonists of the human estrogen re-ceptor (ER), we have developed a functional approachthat does not rely on the transcriptional consequencesof ligand binding. The approach exploits our earlierobservation that the ligand-binding domains (LBDs) ofsteroid receptors can regulate the enzyme activity ofsite-specific recombinases (SSRs) when expressed asSSR/LBD fusion proteins (22, 23). In the absence of abound agonist, we showed that the enzyme activity ofan SSR, Flp recombinase, was repressed when ex-pressed as a fusion protein with estrogen, androgen,or glucocorticoid LBDs. Binding of cognate agonistsderepressed Flp recombinase activity, permitting site-specific recombination of recombination reporter sub-strates. By this means, the functional consequence ofligand binding is derepression of an accurately mea-surable enzyme activity, which does not, theoretically,rely on the further protein-protein interactions involvedin steroid receptor transcriptional repression, activa-tion, interference, or cross-talk regulation.

We have thus examined the ability of a selection ofestrogen agonists and antagonists to derepressFlp/ER fusion proteins (Flp/EBDs) in a mammalian cellline. All the estrogens tested derepress Flp recombi-nase activity of Flp/wild-type (wt) and G400V EBDfusion proteins in a titratable manner reflecting thebinding affinities of these two EBDs. This verifies thatthe antagonists tested release the EBD from its initiallyrepressed state. These antagonists must thereforeperturb further downstream events in ER action. Wethen used the Flp/EBD assay to examine the pheno-types of EBD mutations chosen from the literature toselectively impair other EBD functions including ligandspecificity. Thereby Flp/EBD fusion proteins that dis-tinguish between agonists and antagonists by func-tional classification were identified. We also describe anew mutant EBD that responds to all the syntheticligands tested but not the natural hormone, estradiol.

RESULTS

Experimental Design

The experiments described used a derivative of 293human embryonic kidney cells, 293R10, stably modi-fied to contain a single chromosomal copy of a Flprecombination substrate as described (Ref. 22 andFig. 1A). The parent 293 cells do not express endog-enous ER as determined by both Western blotting and[3H]estradiol binding (data not shown). Recombination

can be assessed by expression of b-galactosidase,reflecting deletion of the neomycin resistance geneand concomitant juxtaposition of the b-galactosidasegene to the CMV (cytomegalovirus) promoter, or moreaccurately by Southern blotting. Clone 293R10 waselectroporated with various Flp/EBD constructs andrandom integrants isolated by selection for hygromy-cin resistance (Fig. 1A). Typically, more than 30% ofprimary hygromycin-resistant colonies showed induc-tion of b-galactosidase expression upon ligand induc-tion (Fig. 1B). Isolation, expansion, and characteriza-tion of individual colonies also demonstrated thatmore than 30% showed ligand inducibility (data notshown). For each of the different Flp/EBD constructsused, six independent, hygromycin-resistant, ligand-inducible clones were characterized for ligand induc-tion. Whereas the kinetics of recombination mediatedby Flp/EBDs varied somewhat within each set of six,presumably a reflection of differing expression levelsfrom the different Flp/EBD genomic integration sites,the profile of responsiveness to the different ligandsdid not (data not shown). From each set of six, wechose those clones that showed the most rapid kinet-ics of recombination upon ligand induction for furtheranalysis.

Flp/EBD D/E/F Fusion Proteins Are Activated byER Agonists and Antagonists

Initial time course experiments were performed withFlp/EBD fusion proteins that included the D, E, and Fdomains of the ER, corresponding to human ER aminoacids 251–595. Two EBDs were used, wt (Flp/EBDDEF)and the G400V variant (Flp/EBDG400V), which shows aloss of affinity for ligands at 37 C (24). (Table 1 containsa complete listing of Flp/EBD fusion proteins used inthis study.) At saturating concentrations of ligand,both estradiol and the antagonist ICI 164,384 inducedrecombination mediated by Flp/EBDG400V with ap-proximately linear kinetics for the first 5 h until 50%total recombination was achieved (Fig. 2, A and B). Asdiscussed previously (22), the rate of recombinationbeyond 50% is inherently nonlinear, and therefore weharvested the cells 4 h after ligand administration forSouthern analysis to ensure that percent induced re-combination directly reflected ligand-induced dere-pression. Short time course experiments with bothFlp/EBDG400V and Flp/EBDDEF using saturating con-centrations of six different ligands also showed thatthe 4-h time point was within the linear range for all sixligands (Fig. 2, C and D).

Whereas stable clones expressing Flp/EBDG400V

showed no detectable recombination in the absenceof added ligand (Ref. 22 and Fig. 2A, lane 1), this wasnot the case for stable clones expressing Flp/EBDDEF.In the experiment shown in Fig. 2D, 16% recombina-tion was evident before ligand addition, even thoughthe cells were cultured in phenol red-free, charcoal-stripped FCS medium. We attribute this backgroundrecombination to the residual presence of estrogens

Ligand Specificity of the Estrogen Receptor 1121


and the accumulation of recombined products duringcellular expansion from the initial stable colonies. Al-though the recombined chromosomal product contin-ued to accumulate during further culture of this cellline, the excised, circular product was lost, or diluted,as expected (22).

To evaluate how accurately Flp/EBD D/E/F fusionproteins transduce ligand binding into DNA recom-bination, titration experiments with a selection of ERligands were performed. Figure 3A presents a com-posite figure showing results from five differentSouthern blots. For brevity, the other lanes of theseblots, corresponding to the different titration points,have been omitted, and only the 1 mM titration pointsare shown. Figure 3B plots the full data set andshows that all ligands induce recombination medi-ated by Flp/EBDG400V in a titratable manner thatclosely reflects the known affinities of the G400VEBD for these ligands. Similarly, titrations with Flp/EBDDEF also showed the expected dose responsesaccording to known affinity values for the wt humanreceptor (data not shown). EC50 values for theseFlp/EBDs, taken as the ligand concentration re-quired to induce half-maximal recombination, aresummarized in Table 2.

To establish that recombination mediated by Flp/EBD D/E/Fs faithfully reflects ligand binding, extractsof the Flp/EBD D/E/F stable cell lines were made andassessed for ligand binding in competition experi-ments with [3H]estradiol. Table 2 shows these IC50

data and includes Kd values for the full-length ERtaken from the literature. Ligand binding affinity ofFlp/EBDDEF is very similar to that of full-length ER, andno significant distortions of affinity are introduced ei-ther by omission of the ER A/B and C domains or bythe fusion of Flp recombinase to the ER D/E/Fdomains.

The results presented in Figs. 2 and 3 and Table 2show that all ligands tested induced recombinationregardless of their agonist [estradiol, diethylstilbestrol(DES), hexestrol], antagonist (raloxifene, ICI 164,384,ICI 182,780, 4-hydroxytamoxifen), steroidal (estradiol,ICI 164,384, ICI 182,780), or nonsteroidal (DES, hex-estrol, raloxifene, 4-hydroxytamoxifen) character, in amanner that simply reflects ligand binding. All theseligands thus serve to release ER from its initially re-pressed condition, and differences between these ERagonists and antagonists must lie later in the pathwayof ER activation. This conclusion, in a mammalian cellmodel, extends our previous work based on Flp/EBDexperiments in yeast (23) where the partial exclusion of

Fig. 1. Strategy of Stable Cell Experiments with Flp/EBDsA, The stable cell line system is depicted showing 1) the

unrecombined state, 2) the recombined state before loss ofthe excised circle bearing the neomycin resistance gene, and3) the recombined state after loss of the excised circle. Ineach of the three cell states, the Flp/EBD fusion proteinexpression vector is shown above and consists of a bidirec-tional promoter that expresses the hygromycin resistance(hygro) and the Flp/EBD genes. In the uppermost panel, therecombination substrate is shown below and consists of theconstitutively active SV40 promoter (arrow); the two Flp re-combination targets (FRTs) are indicated by arrowheads sep-arated by the neomycin resistance gene (neo) followed by theb-galactosidase gene (lacZ). Also shown are the two probesused for Southern analysis (1 and 2) and the BamHI restric-tion sites, shown as small open circles, used for the Southernanalyses of Figs. 3, 4, 5, and 7. Before recombination, a5.4-kb BamHI fragment is detected by probe 2. Recombina-

tion excises the fragment and a BamHI between the FRTs siteto yield a 8.2-kb fragment detected by probe 2. The excisedcircle is linearized by BamHI to yield a 1.3-kb fragment. B,Primary hygromycin-resistant colonies transformed with aFlp/EBDG400V expression vector were cultured for 10 days inthe presence or in the absence of 100 nM 17b-estradiol andstained for b-galactosidase activity to monitor induced re-combination.

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certain ligands by the yeast cell surface influenced thedose-response curve.

Although all ligands tested release Flp/EBD D/E/Fsfrom the initially repressed condition, the antagonistsraloxifene, and to a lesser extent, ICI 164,384 and ICI182,780, consistently induced faster rates of recom-bination at early time points than did the agonists or4-hydroxytamoxifen (Figs. 2 and 3). This may reflectthe failure of these antagonists to allow other protein-protein interactions not favoring efficient recombina-tion, such as homodimerization or interactions withtranscriptional cofactors.

Mutations That Impair ER HomodimerizationDo Not Discriminate between Agonists andAntagonists

In the ER activation pathway, release from the initiallyrepressed condition permits ER to dimerize. Sincerelease from initial repression is promoted by all of theagonists and antagonists used here, we askedwhether agonists and antagonists could be distin-guished by their ability to promote homodimerization.Mutations previously described that impair ho-modimerization of mouse ER (25), an interpretationthat has gained recent support from the EBD crystalstructure (26), were introduced into Flp/human EBDD/E/F fusion proteins (Flp/EBDL507R and Flp/EBDR503A/L507R). Figure 4 shows that all six ligandsinduce recombination mediated by these Flp/mutantEBDs, albeit at substantially higher ligand concentra-tions. Ligand binding experiments in extracts (Table 2)showed that recombination efficacy approximately re-flected ligand binding. The reduction of binding affinityshown by these dimerization mutants, together withthe recent EBD crystal structure evidence that thedimerization surface is distinct from the ligand bindingpocket (26), suggests that ER dimerization stabilizesligand binding. Interactions between ligand bindingand dimerization have been described for other nu-clear receptors, notably ecdysone and retinoid X re-ceptors (27, 28) and the vitamin D receptor (29). Aswith both wt and G400V EBDs, raloxifene inducedfaster rates of initial recombination for the L507R andR503A,L507R dimerization mutants than the other li-gands. The persistence of this profile suggests that

these agonists and antagonists cannot be differenti-ated on the basis of selective effects on homodimer-ization. These data also indicate that relief from initialrepression is not reliant on dimerization. We also notethat these dimerization mutations do not result in in-creased Flp/EBD recombinase enzyme activity, indi-cating that EBD homodimerization has little deleteri-ous affect on Flp enzyme activity.

Flp/EBD Fusion Proteins That Distinguishbetween Agonists and Antagonists

The above data show that ligand responsiveness ofFlp/EBD fusion proteins mainly reflects the first step inthe ER activation pathway, relief from initial repres-sion. Those steps after dimerization in the activationpathway, where agonists and antagonists elicit differ-ent responses, are not reported by this assay. Conse-quently we employed known EBD mutations to estab-lish Flp/EBDs that could discriminate betweenagonists and antagonists.

The EBD encompasses at least amino acids 305–548 (26, 30). The Flp/EBD fusions used above in-cluded amino acids 251–595, encompassing notonly the EBD (E domain) but also the D domain(251–304) and the F domain (549–595). As weshowed previously (23), removal of most of the Ddomain (here deletion of amino acids 251–303) tocreate Flp/EBDDD produced a Flp/EBD that was ac-tivatable only by agonists and not antagonists, butshowed less recombinase activity than other Flp/EBD fusions after 4 h of ligand stimulation (Fig. 5, Aand B). In yeast, we also observed the exclusivenature of Flp/EBDDD responsiveness to agonists, aswell as the reduced amount of recombinase activityafter induction (23). Competition experimentsshowed that activation by agonists is abolished in adose-dependent fashion by the three ER antago-nists (Fig. 5C). Therefore, as expected given thepresence of the complete ER E domain, Flp/EBDDD

has not lost the capacity to bind antagonist; rather,it fails to be activated by antagonist binding. Thiswas confirmed by binding experiments (Table 2).Western analysis showed that none of the antago-nists had a negative effect on the steady state levelsof Flp/EBDDD (data not shown).

Table 1. Names and Description of Flp/EBD Fusion Proteins Used

Construct Description

1. Flp/EBDDEF wt human ER aa 251 to 595 fused to the C terminus of Flp by the linkerCVRGS encoded by TGCGTACGCGGATCC.

2. Flp/EBDG400V as 1 except ER G400V.3. Flp/EBDL507R as 1 except ER L507R.4. Flp/EBDR503A/L507R as 1 except ER R503A and L507R.5. Flp/EBDDD as 1 except ER a.a.s 251–303 deleted.6. Flp/EBDG521R as 1 except ER G521R.7. Flp/EBDG521V as 1 except ER G521V.8. Flp/EBDG400V/G521V as 1 except ER G400V and G521V.9. Flp/EBDG400V/G521R as 1 except ER G400V and G521R.



A Flp/EBD Fusion Protein That Is Only Activatedby ER Antagonists

The equivalent of a previously described mouse ERpoint mutation, glycine 521 to arginine (31), was intro-duced into the human EBD D/E/F to create Flp/EBDG521R. Consistent with the mouse ER results (31),Flp/EBDG521R is not activated by estradiol but is acti-

vated by 4-hydroxytamoxifen (Fig. 5, D and E). Inter-estingly, Flp/EBDG521R is also activatable by the othertwo antagonists, ICI 182,780 and raloxifene, but not bytwo nonsteroidal agonists, DES and hexestrol. Thismutation therefore appears to discriminate betweenagonists and antagonists on the basis of their func-tional classification as transcriptional activators. Com-petition experiments between ICI 182,780 and the

Fig. 2. Time Courses of Ligand-Induced Flp/EBD RecombinationA, Southern blots of time courses of recombination mediated by Flp/EBDG400V after induction with 100 nM estradiol or 300 nM

ICI 164,384 as indicated. Probe 2 shown on Fig. 1A was used on NdeI-restricted genomic DNA as previously described (22).Before recombination the NdeI band is 4.9 kb. Recombination reduces this band to 3.6 kb. The asterisk marks an artifactual band,probably the result of relaxed cleavage specificity by NdeI. B, Plot showing PhosphorImager quantification of the Southernanalysis shown in panel A presented as counts in the recombined chromosomal product divided by the sum of recombined andunrecombined counts. C, Plot showing a short time course of recombination mediated by Flp/EBDG400V induced by saturatingconcentrations (1 mM) of five different ligands as indicated. D, Plot showing a short time course of recombination mediated byFlp/EBDDEF induced by saturating concentrations (1 mM) of six different ligands as indicated. Note that the time course startedfrom 16% recombination. The plots of panels B, C, and D were based on PhosphorImager quantification of Southern blots suchas shown in panel A.

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three agonists (Fig. 5F) and ligand binding studies inextracts indicate that the G521R mutant has not onlylost the ability to bind agonists but also considerableaffinity for antagonists. We note, however, that 1 mM

DES does produce a low level of recombination (Fig.5D, lane 3), indicating that at high concentrationssome binding can occur. The presence of an arginineat position 521 therefore appears to be more impor-tant for agonist than antagonist binding.

Selective Loss of Estradiol Activation

The experiments described above show that agonistsand antagonists can be differentiated on a functional,nontranscriptional, basis. Although synthetic estro-

gens can be broadly categorized as either agonists orantagonists, several lines of evidence demonstratethat further subcategories exist (32). We have there-fore begun to identify mutations that would permitfurther subcategorization of ligands based on the Flp/EBD assay. Flp/EBD mutants that systematically com-bined glycine or valine at amino acid 400 with glycine,valine, or arginine at amino acid 521 were generated.These Flp/EBD D/E/Fs were tested in a transient ex-pression assay for qualitative responsiveness to ligand(Fig. 6).

As expected, the wt, G400V, and G521R EBDs (Flp/EBDDEF, Flp/EBDG400V, Flp/EBDG521R) showed ligandinducibility consistent with the data from the stableexpression experiments above. Notably, the wild type

Fig. 3. Ligand Titrations with Cells Expressing Flp/EBDG400V or Flp/EBD251–595 Show that Flp/EBD D/E/F Proteins AccuratelyTransduce Ligand Binding to DNA Recombination

A, Southern blot analyses of ligand titrations of cells expressing Flp/EBDG400V. BamHI-restricted chromosomal recombinationsubstrate and product were visualized with probe 2 (Fig. 1A). Only the 1 mM lanes, taken from five different Southern blots, plusthe no-ligand lane, are shown. B, Plot of ligand-induced recombination using data from all lanes of the five titration experiments.Quantification and plotting were as described in Fig. 2. The symbols used for the different ligands are denoted and are the samefor Figs. 4, 5, and 7. Solid symbols denote agonists; open symbols denote antagonists; F, estradiol; f, DES; Œ, hexestrol; E,raloxifene; h, ICI 182,780; ‚, 4-hydroxy tamoxifen.



fusion protein again showed more background recom-bination than the G400V fusion protein. The G521Vmutation displayed some loss of responsiveness toestradiol; however, when this mutation was combinedwith G400V (G400V/G521V), selective loss of respon-siveness to estradiol was observed. The G400V/G521R double mutant was unresponsive to any li-gand, probably because both mutations alonesubstantially reduce ligand affinity and, when com-bined, reduce it further. In other experiments, thesefusion proteins were examined for differential respon-siveness to raloxifene, 4-hydroxytamoxifen, and ICI182,780. No significant differences were observed(data not shown). None of these EBDs displayed a lossof initial repression, with the possible exception of theG521V mutation.

Cells stably expressing the G400V/G521V protein (Flp/EBDG400V/G521V) were next examined for ligand induc-ibility in dose-response experiments (Fig. 7). Loss ofresponsiveness to estradiol was confirmed, and compe-tition experiments showed that estradiol could not blockinduction by DES (Fig. 7C) or raloxifene (data not shown),demonstrating that the G400V/G521V EBD has lost theability to bind estradiol.

DISCUSSION

Both Agonists and Antagonists Relieve InitialRepression of Flp Activity Imposed by wt andG400V EBDs

In addition to sharing the property of binding hor-mones, steroid receptors display a repressor function

that can impart ligand dependency onto certain otherproteins when expressed as fusion proteins (33). It isbelieved that the protein cis-repressor function of thesteroid receptors is mediated via complex formationwith chaperone molecules, most notably Hsp90 (5–9,18, 34). This dogma has been challenged in the case ofthe progesterone receptor on the basis that it is almostexclusively located in the nucleus even in the absenceof cognate ligands, while Hsp90 is cytoplasmic (35),leaving the exact mechanism of protein cis-repressionopen to question.

To analyze protein cis-repression and ligand spec-ificities of the human ER, we explored the character-istics of the Flp/EBD fusion system (22). This receptorassay does not rely on the transcription activationfunction of the LBD as it measures site-specific re-combinase activity, making it possible to directly ob-serve the consequence of ligand binding on the cis-repressor function of the EBD.

In agreement with data generated in a yeast system(23), all the ligands we tested, including the pure ERantagonist ICI 182,780 (36), the mixed agonist/antago-nists 4-hydroxytamoxifen (37), and raloxifene (38), in-duced Flp recombination in this human cell line-basedsystem. These results imply that all estrogens, regard-less of their agonist/antagonist potential in vivo, induce astructural conformation that releases the EBD from itsinitially repressed condition. Furthermore, our data indi-cate that the second step in the ER activation pathway,dimerization, is also not a point of discrimination be-tween the agonists and antagonists used here. There-fore, the crucial difference between these agonists andantagonists lies later in the pathway of ER activation.

Table 2. Dissociation Constants of Flp/EBDs (determined by Scatchard Analysis), Compared with the Concentration at whichHalf-Maximal Recombination was Induced for the Six Ligands Used Here.

ProteinConcentration (nM)

E2 DES HEX RAL ICI 182 OHT

wtER Kd 0.14 0.71 0.3 0.1 0.89 0.1EBDDEF IC50 0.18 0.06 0.1 0.7 0.8 0.28

EC50 0.1–0.3 ND 0.3 1 1 0.3–1EBDG400V IC50 5 0.7 1.2 13.5 15 5.3

EC50 1–3 ND 0.3–1 10 10–30 10–30EBDDD IC50 1.1 0.31 0.38 2.9 4.3 1.6

EC50 1–3 1–3 1–3 10a 10a 10a

EBDL507R IC50 16 6 3.7 29 62 37EC50 30 30 30 50 100 100

EBDR503A/L507R IC50 14 19.6 4 56 57 95EC50 60 60 50 30 100 100

EBDG521Rb EC50c c c 30–100 300d 30–100

EBDG400V/G521Vb EC50c 30 30 100 300d 30–100

E2, Estradiol; DES, diethylstilbestrol; HEX, hexestrol; RAL, raloxifene; OHT, 4-hydroxytamoxifen. Also shown are the dissociationconstants for full-length ER taken from published data (24, 36, 54, 57–59). Kd, dissociation constant; IC50, inhibitory concentrationof ligand required to compete 50% of [3H]estradiol binding; EC50, effective concentration to achieve 50% recombination after 4 h;ND, not determined.a Concentration required to half maximally compete activation by 3 nM estradiol.b Competition experiments based on [3H]estradiol binding in extracts was not possible due to an absence of estradiol binding.c No activity.d Saturating concentrations may not have been reached at 3 mM.

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In contrast to the near complete reliance on ligandfor Flp/EBDG400V and other mutant EBD forms, wefound that the wt EBD, including the hinge region fromresidues 251–303, does not repress Flp fully in mam-malian cells. Background ER activity has been repeat-edly observed in transcription studies with the wtER inmammalian cells and is likely due to the presence oftrace quantities of estrogens in phenol red-free, char-coal-stripped, mammalian cell culture medium, asused here, combined with the high ligand sensitivity ofthe wtEBD (Refs. 24 and 39 and references therein). Insupport of this explanation, the Flp/EBDDEF proteinused here shows good repression in the absence ofligand induction in yeast (23).

Distinction between Agonists and Antagonists byClass

After dimerization, activated ER affects several tran-scriptional responses through classic and nonclas-sic DNA response elements as well as by cross-talkregulation (for reviews, see Refs. 10 and 12). It isprobable that different agonists and antagonists

elicit different responses via these various mecha-nisms (12, 32, 40). Steroid ligand selectivity is wellillustrated by the spectrum of ligand/ER responses.ER is activated by its cognate agonist, estradiol. Thesynthetic nonsteroid estrogen agonist, DES, alsoelicits agonist responses, although DES derivativeshave been shown to be partial agonists given theirabilities to evoke different subsets of agonist re-sponses (41). 4-Hydroxytamoxifen has been shownto be a partial antagonist based on promoter contextvariability (37) and cross-talk regulation (42). Inter-estingly, the partial antagonist raloxifene shows adifferent profile of agonism/antagonism to that of4-hydroxytamoxifen based on uterine, cholesterol,and bone responses (38, 43, 44). ICI 182,780 isdescribed as a complete ER antagonist because itblocks all ER agonist activities (36) although ICI182,780 agonist activity has been described for ERmutations in the C terminus of the E domain (45, 46).Further evidence for a spectrum of ER responseselicited by different ligands has been presented (32).The fact that ER responses are differentially elicitedby different ligands suggests that binding of ligand

Fig. 4. Mutations that Impair Dimerization Do Not Discriminate between Agonists and AntagonistsA, Southern blot analyses of ligand titrations of cells expressing Flp/EBDL507R. BamHI-restricted chromosomal recombination

substrate and product were visualized with probe 2 (Fig. 1A). Only the 1 mM lanes, taken from six different Southern blots, plusthe no-ligand lane, are shown. B, Plot of ligand-induced recombination using data from all lanes of the six titration experimentsof panel A. Quantification and plotting were as described in Fig. 2. C, Southern blot analyses of ligand titrations of cells expressingFlp/EBDR503A/L507R as described in panel A. D, Plot of ligand-induced recombination using data from all lanes of the six titrationexperiments of panel C. See Fig. 3 for ligand symbols.



by ER results in different conformations each re-flecting the particular ligand bound (12, 26, 32, 47).

Given the complexities involved in ER signaling afterdimerization, the use of transcriptional assays to cat-egorize ER ligands into more refined classes than ag-onist or antagonist will reflect both the activity of theligand and the experimental design of the transcrip-tional assays. Since we have shown (Figs. 2–4) thatFlp/EBD responses are largely independent of thosesteps in the ER activation pathway at which agonistsand antagonists are transcriptionally discriminated, wereasoned that Flp/mutant EBDs that discriminate be-tween agonists and antagonists must do so on thebasis of inherent structural properties of ligand bindingby the EBD. Appropriate mutant EBDs were identifiedand cell lines established that discriminate betweenagonists and antagonists by class. To permit further,nontranscriptional classification of ER ligands, mutantEBDs that permit subcategorization of ligands weresought and a mutant EBD (G400V, G521V), that dis-tinguishes between the natural agonist, estradiol, andsynthetic ligands was identified. This demonstratesthat particular ligand specificity mutations can be dis-tinguished in the Flp/EBD assay. The identification of

other ligand specificity mutations and their use in Flp/EBDs should provide a structural, nontranscriptionalbasis for an even more refined categorization of ERligands.

Antagonism in the Context of Flp/EBD Fusions

Deletion of amino acids 251–303 resulted in a Flp/EBDfusion protein that was activatable by the three ERagonists, estradiol, DES, and hexestrol, but only veryweakly by the three antagonists tested. The steroidnature of the ligands was not a determining factorsince two of the agonists, DES and hexestrol, are notsteroid ring compounds, and one of the antagonists,ICI 182,780, has a steroid structure. By ligand bindingexperiments in cellular extracts and ligand competitionexperiments, we showed that Flp/EBDDD is able tobind agonists and antagonists; however, only agonistsinduce Flp recombination. Lack of recombinase induc-tion by antagonists was not due to selective proteindegradation since Western blot analysis demonstratedthat no ligand had significant effects on expressionlevels of any of the Flp/EBD fusion proteins used here(data not shown). The molecular basis of agonist/an-

Fig. 5. Ligand Titration and Competition Experiments with Cells Expressing Flp/EBD Proteins That Discriminate betweenAgonists and Antagonists

A, Southern analysis of recombination induced by 1 mM of the six indicated ligands in a recombination reporter cell lineexpressing Flp/EBDDD. BamHI-restricted chromosomal recombination substrate and product were visualized with probe 2 (Fig.1A). Only the 1 mM lanes, taken from six different Southern blots, plus the no-ligand lane, are shown. The asterisk denotes anonspecific band. B, Plot of ligand-induced recombination using data from all lanes of the six titration experiments of panel A.Quantification and plotting were as described in Fig. 2. C, Estradiol (3 nM)-induced recombination mediated by Flp/EBDDD iscompeted by coincubation for 4 h with increasing concentrations of raloxifene, hydroxytamoxifen, and ICI 182,780 as shown. D,Southern analysis of recombination induced by 1 mM of the six indicated ligands in a recombination reporter cell line expressingFlp/EBDG521R. BamHI-restricted chromosomal recombination substrate and product were visualised with probe 2 (Fig. 1A). Onlythe 1 mM lanes, taken from six different Southern blots, plus the no-ligand lane, are shown. E, Plot of ligand-induced recombinationusing data from all lanes of the six titration experiments of panel A. Quantification and plotting were as described in Fig. 2. F,Failure of 3 mM estradiol, DES, or hexestrol to compete for Flp/EBDG521R recombination induced by 300 nM ICI 182,780. See Fig.3 for ligand symbols.

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tagonist discrimination by Flp/EBDDD has been ad-dressed elsewhere (47).

Ligand-Selective EBD Mutations

Two Flp/EBD fusions that show ligand binding selec-tivity are documented here. Both involve changes atamino acid 521. The first is the G521R mutation pre-viously described to retain 4-hydroxytamoxifen but notestradiol binding (31), and the second combinesG521V with G400V. Glycine 521 is located in a regionclose to bound ligand in the crystal structures of theliganded human ER, retinoic acid receptor, and thyroidreceptor LBDs (26, 48, 49). Glycine 400 is located atthe start of a b-turn, which when mutated to valine,causes a general destabilization of ligand interactionsat 37 C (24). Both regions have been implicated pre-viously in ER and glucocorticoid receptor ligand bind-

ing, functionally and by chemical cross-linking (31,50–53).

Our observations with the G521R mutation extendprevious work to establish that this mutation discrim-inates between agonists and antagonists by class.This is due to a loss of agonist binding, as demon-strated by binding and competition experiments, sug-gesting that the G521R mutation disrupts a part of theligand binding pocket that is essential to bind ERagonists, but not antagonists, with high affinity.

The G400V/G521V EBD appears to be unable tobind estradiol since high levels of estradiol did notcompete for Flp/EBDG400V/G521V-mediated recombi-nation elicited by subsaturating concentrations ofDES. Flp/EBDG400V/G521V, however, responds to all ofthe other ER ligands we tested, regardless of theiragonist or antagonist activity in terms of the full-lengthER. Thus a glycine at position 521 appears critical toaccommodate estradiol in the ligand binding pocket.However, ICI 182,780 is identical to estradiol exceptfor the addition of the long side chain at the seventhcarbon of the steroid ring structure. ICI 182,780 is notexcluded from the EBDG400V/G521V, implying that it,like raloxifene, binds via an extra set of residues in theEBD (26).

The use of SSR/LBD fusions allows direct studies ofligand/LBD combinations that are transcriptionally in-active. This permits analysis of the cis-repression ac-tivity of LBDs and can provide a new way to classifysteroid receptor ligands as agonists or antagonists.Further work is required to exploit the ligand discrim-inatory potential of Flp/EBD fusions to further distin-guish different subclasses of antagonists (pure vs.tamoxifen-like vs. raloxifene-like antagonists) andagonists [steroid vs. nonsteroid and their enantiomers(54, 55)]. Another potentially fruitful avenue of researchmay involve the use of SSR/LBDs in the study of thecis-repressor activities of other members of the steroidand nuclear receptor superfamily.

MATERIALS AND METHODS

DNA Constructs

Table 1 is a complete list of Flp/EBD fusion proteins used inthis study. The Flp/EBDG400V construct has been described inpHFE (22). To reintroduce a glycine at position 400 of thecloned human EBD (24), the NcoI-BglII fragment spanningthis mutation was replaced with the homologous NcoI-BglIIfragment from the mouse ER (25). This did not result in anyfurther alterations in the amino acid sequence.

To delete the D domain of the estrogen-binding domain, anintermediate plasmid, p44HE1 (22), was cut with BamHI andEagI and a linker with the sequence ggatccaacagcctggcctt-gtccctgacggccg was inserted, thus deleting amino acids251–303 of the EBD.

All point mutations were generated by the method of Bar-ettino et al. (56). The sequence of the 59-primer was ccac-cgagtcctggacaag and that of the 39-primer was ccagtagtag-gttgaggccgttg. The template for the first round ofamplification cycles using one of the mutagenic primers andthe 39- primer was p44HE2. The second template, lacking

Fig. 6. Transient Expression of Flp/EBD D/E/F Fusion Pro-teins Carrying Mutant Combinations at ER Amino Acids 400and 521

E25B2/B2 cells were lipofected with either wt (G400/G521), G400V, G521V, G521R, G400V/G521V, or G400V/G521R Flp/EBDs and treated with 1 mM ligands, or ethanolvehicle, as indicated. E2, estradiol; HEX, hexestrol; DES,diethylstilbestrol; RAL, raloxifene. The histograms shownumber of lacZ-positive cells counted in 1 cm2 after a 50-hexpression period. Note the different values on the ordinate.



sequence homology for the 39-primer, was pHFE1. The prod-ucts of the second PCR reactions were digested with StuIand Eco47III to generate a 499-bp fragment that was insertedinto StuI-Eco47III linearized pHFE2. The sequences of themutagenic primers encoding the G521R, G521V, mutationswere gg.cac.atg.agC.aac.aaa.AgA.atg.gag (MaeIII) andgg.cac.atg.agt.aac.aaa.gTc.atg.gag (not tagged) respec-tively. Uppercase nucleotides represent point mutations; therestriction site that was mutated to tag the mutation is un-derlined, and the restriction enzyme is written in betweenbrackets after each primer sequence.

Cell Lines and Cell Culture Conditions

Cell culture conditions were as previously described in-cluding charcoal stripping of the FCS (22). The Flp recom-bination reporter cell line 293R10 was generated by elec-troporation of linear pNeobGal plasmid (23) into 293 cellsas previously described. 293R10 cells were transformedwith pHFE plasmids bearing the indicated EBD mutationsto generate recombination reporter cell lines as described(22). All the data points shown on any one graph in thepresent paper were collected in the course of a single

experiment to allow accurate comparisons in recombinaseactivities.

Southern Analysis

Southern analyses were carried out as described (22), exceptthat 0.86% agarose gels were used. The genomic DNA sam-ples from P1.4 cells (22) used for Fig. 2A were digested withNdeI, which generates 4.9- and 3.6-kb fragments from re-combined and unrecombined pNEObGAL loci, respectively.DNAs for all the other Southern blots presented in this studywere digested with BamHI, which gives the following restric-tion fragments: unrecombined, 5.4 kb; recombined, 8.2 kb;circle, 1.3 kb.

Transient Expression

The CV1-derived Flp excision recombination reporter cell lineE25B2/B2 (22) was grown on glass coverslips and trans-fected overnight with 5 mg Flp/EBD expression vectors bymeans of lipofectamine (Boehringer Mannheim, Mannheim,Germany), according to the manufacturer’s instructions. Li-

Fig. 7. A Mutation Selectively Insensitive to EstradiolA, Southern analysis of ligand titrations with cells expressing Flp/EBDG400V/G521V. BamHI-restricted chromosomal recombi-

nation substrate and product were visualized with probe 2 (Fig. 1A). Only the 1 mM lanes, taken from six different Southern blots,plus the no-ligand lane, are shown. B, Plot of ligand-induced recombination using data from all lanes of the six titrationexperiments of panel A. Quantification and plotting were as described in Fig. 2. C, Excess estradiol (1 mM) does not inhibitFlp/EBDG400V/G521V recombination induced by a subsaturating concentration (30 nM) of DES.

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gand exposure lasted 50 h. Histochemical detection of b-ga-lactosidase was performed as before (22).

Acknowledgments

We thank P.-O. Angrand, Frank Buchholz, Sophie Chabanis,Hinrich Gronemeyer, Dino Moras, and Henk Stunnenberg fordiscussions; V. Kumar, P. Chambon, and P. Danielian forplasmids; and A. Wakeling (Zeneca Pharmaceuticals) for pro-viding ICI 164,384 and ICI 182,780.

Received February 3, 1998. Revision received April 27,1998. Accepted April 30, 1998.

Address requests for reprints to: A. Francis Stewart, Eu-ropean Molecular Biology Laboratory, Gene Expression Pro-gram, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

This work was supported in part by EU-BIOMED GrantBMH4–96-0181 (to A.F.S.).

* Present address: University of Massachusetts MedicalCenter, Program in Molecular Medicine, Worcester, Massa-chusetts 01604.

† Present address: University of Pittsburgh Cancer Insti-tute, Pittsburgh, Pennsylvania 15213.

REFERENCES

1. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, GlossB, Lin SC, Heyman RA, Rose DW, Glass CK, RosenfeldMG 1996 A CBP integrator complex mediates transcrip-tional activation and AP-1 inhibition by nuclear receptors.Cell 85:403–414

2. Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Na-katani Y 1996 The transcriptional coactivators p300 andCBP are histone acetyltransferases. Cell 87:953–959

3. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, NagyL, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclearreceptor coactivator ACTR is a novel histone acetyltrans-ferase and forms a multimeric activation complex withP/CAF and CBP/p300. Cell 90:569–580

4. Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J,Mizzen CA, McKenna NJ, Onate SA, Tsai SY, Tsai MJ,O’Malley BW 1997 Steroid receptor coactivator-1 is ahistone acetyltransferase. Nature 389:194–198

5. Bohen SP 1995 Hsp90 mutants disrupt glucocorticoidreceptor ligand binding and destabilize aporeceptorcomplexes. J Biol Chem 270:29433–29438

6. Bohen SP, Yamamoto KR 1993 Isolation of Hsp90 mu-tants by screening for decreased steroid receptor func-tion. Proc Natl Acad Sci USA 90:11424–11428

7. Chang HC, Nathan DF, Lindquist S 1997 In vivo analysisof the Hsp90 co-chaperone Sti1 (p60). Mol Cell Biol17:318–325

8. Nathan DF, Lindquist S 1995 Mutational analysis ofHsp90 function: interactions with a steroid receptor anda protein kinase. Mol Cell Biol 15: 3917–3925

9. Kimura Y, Yahara I, Lindquist S 1995 Role of the proteinchaperone YDJ1 in establishing Hsp90-mediated signaltransduction pathways. Science 268: 1362–1365

10. Beato M, Herrlich P, Schutz G 1995 Steroid hormonereceptors: many actors in search of a plot. Cell83:851–857

11. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P,Schutz G, Umesono K, Blumberg B, Kastner P, Mark M,Chambon P, Evans RM 1995 The nuclear receptor su-perfamily: the second decade. Cell 83:835–839

12. Stewart AF, Logie C, Nichols M 1996 Regulation of nu-clear receptors by agonists and antagonists. Curr Opin

Endocrinol Diabet 3:397–40213. Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B,

Kurokawa R, Ryan A, Kamei Y, Soderstrom M, Glass CK,Rosenfeld MG 1995 Ligand-independent repression bythe thyroid hormone receptor mediated by a nuclearreceptor co-repressor. Nature 377:397–404

14. Chen JD, Evans RM 1995 A transcriptional co-repressorthat interacts with nuclear hormone receptors. Nature377:454–457

15. Soderstrom M, Vo A, Heinzel T, Lavinsky RM, Yang WM,Seto E, Peterson DA, Rosenfeld MG, Glass CK 1997Differential effects of nuclear receptor corepressor (N-CoR) expression levels on retinoic acid receptor-medi-ated repression support the existence of dynamicallyregulated corepressor complexes. Mol Endocrinol11:682–692

16. Heinzel T, Lavinsky RM, Mullen TM, Soderstrom M,Laherty CD, Torchia J, Yang WM, Brard G, Ngo SD,Davie JR, Seto E, Eisenman RN, Rose DW, Glass CK,Rosenfeld MG 1997 A complex containing N-CoR,mSin3 and histone deacetylase mediates transcriptionalrepression. Nature 387:43–48

17. Nagy L, Kao HY, Chakravarti D, Lin RJ, Hassig CA, AyerDE, Schreiber SL, Evans RM 1997 Nuclear receptor re-pression mediated by a complex containing SMRT,mSin3A, and histone deacetylase. Cell 89:373–380

18. Dalman FC, Sturzenbecker LJ, Levin AA, Lucas DA, Per-dew GH, Petkovitch M, Chambon P, Grippo JF, Pratt WB1991 Retinoic acid receptor belongs to a subclass ofnuclear receptors that do not form “docking” complexeswith hsp90. Biochemistry 30:5605–5608

19. Smith CL, Nawaz Z, O’Malley BW 1997 Coactivator andcorepressor regulation of the agonist/antagonist activityof the mixed antiestrogen, 4-hydroxytamoxifen. Mol En-docrinol 11:657–666

20. Jackson TA, Richer JK, Bain DL, Takimoto GS, Tung L,Horwitz KB 1997 The partial agonist activity of antago-nist-occupied steroid receptors is controlled by a novelhinge domain-binding coactivator L7/SPA and the core-pressors N-CoR or SMRT. Mol Endocrinol 11:693–705

21. Segnitz B, Gehring U 1990 Mechanism of action of asteroidal antiglucocorticoid in lymphoid cells. J BiolChem 265:2789–2796

22. Logie C, Stewart AF 1995 Ligand-regulated site-specificrecombination. Proc Natl Acad Sci USA 92:940–944

23. Nichols M, Rientjes JMJ, Logie C, Stewart AF 1997 FLPrecombinase/estrogen receptor fusion proteins requirethe receptor D domain for responsiveness to antago-nists, but not agonists. Mol Endocrinol 11:950–961

24. Tora L, Mullick A, Metzger D, Ponglikitmongkol M, ParkI, Chambon P 1989 The cloned human oestrogen recep-tor contains a mutation which alters its hormone bindingproperties. EMBO J 8:1981–1986

25. Fawell SE, Lees JA, White R, Parker MG 1990 Charac-terization and colocalization of steroid binding anddimerization activities in the mouse estrogen receptor.Cell 60:953–962

26. Brzozowski AM, Pike ACW, Dauter Z, Hubbard EH, BonnT, Engstrom O, Ohman L, Greene GL, Gustafsson J,Carlquist M 1997 Molecular basis of agonism and antag-onism in the estrogen receptor. Nature 389:753–758

27. Thomas HE, Stunnenberg HG, Stewart AF 1993 Het-erodimerization of the Drosophila ecdysone receptorwith retinoid X receptor and ultraspiracle. Nature362:471–475

28. Yao TP, Forman BM, Jiang Z, Cherbas L, Chen JD,McKeown M, Cherbas P, Evans RM 1993 Functionalecdysone receptor is the product of the EcR and ultra-spiracle genes. Nature 366:476–479

29. Cheskis B, Freedman LP 1994 Ligand modulates theconversion of DNA-bound vitamin D3 receptor (VDR)homodimers into VDR-retinoid X receptor heterodimers.Mol Cell Biol 14:3329–3338



30. Wurtz J-M, Bourguet W, Renaud J-P, Vivat V, ChambonP, Moras D, Gronemeyer H 1996 A canonical structurefor the ligand-binding domain of nuclear receptors. Na-ture Struct Biol 3:87–94

31. Danielian PS, White R, Hoare SA, Fawell SE, Parker MG1993 Identification of residues in the estrogen receptorthat confer differential sensitivity to estrogen and hy-droxytamoxifen. Mol Endocrinol 7:232–240

32. McDonnell DP, Clemm DL, Hermann T, Goldman ME,Pike JW 1995 Analysis of estrogen receptor function invitro reveals three distinct classes of antiestrogens. MolEndocrinol 9:659–669

33. Mattioni T, Louvion JF, Picard D 1994 Regulation ofprotein activities by fusion to steroid binding domains.Methods Cell Biol 43:335–352

34. Scherrer LC, Picard D, Massa E, Harmon JM, Simons JrSS, Yamamoto KR, Pratt WB 1993 Evidence that thehormone binding domain of steroid receptors confershormonal control on chimeric proteins by determiningtheir hormone-regulated binding to heat-shock protein90. Biochemistry 32:5381–5386

35. Tuohimaa P, Pekki A, Blauer M, Joensuu T, Vilja P,Ylikomi T 1993 Nuclear progesterone receptor is mainlyheat shock protein 90-free in vivo. Proc Natl Acad SciUSA 90:5848–5852

36. Wakeling AE, Bowler J 1992 ICI 182,780, a new anti-oestrogen with clinical potential. J Steroid Biochem MolBiol 43:173–177

37. Berry M, Metzger D, Chambon P 1990 Role of the twoactivating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activityof the anti-oestrogen 4-hydroxytamoxifen. EMBO J9:2811–2818

38. Black LJ, Sato M, Rowley ER, Magee DE, Bekele A,Williams DC, Cullinan GJ, Bendele R, Kauffman RF,Bensch WR, Frolik CA, Termine JD, Bryant HU 1994Raloxifene (LY139481 HCI) prevents bone loss and re-duces serum cholesterol without causing uterine hyper-trophy in ovariectomized rats. J Clin Invest 93:63–69

39. Mattick S, Glenn K, de Haan G, Shapiro DJ 1997 Analysisof ligand dependence and hormone response elementsynergy in transcription by estrogen receptor. J SteroidBiochem Mol Biol 60:285–294

40. Katzenellenbogen JA, O’Malley BW, KatzenellenbogenBS 1996 Tripartite steroid hormone receptor pharmacol-ogy: interaction with multiple effector sites as a basis forthe cell- and promoter-specific action of these hor-mones. Mol Endocrinol 10:119–131

41. Korach KS, Fox-Davies C, Quarmby VE, Swaisgood MH1985 Diethylstilbestrol metabolites and analogs. Bio-chemical probes for differential stimulation of uterine es-trogen responses. J Biol Chem 260:15420–15426

42. Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamox-ifen activation of the estrogen receptor/AP-1 pathway:potential origin for the cell-specific estrogen-like effectsof antiestrogens. Mol Endocrinol 9:443–456

43. Grese TA, Sluka JP, Bryant HU, Cullinan GJ, GlasebrookAL, Jones CD, Matsumoto K, Palkowitz AD, Sato M,Termine JD, Winter MA, Yang NN, Dodge JA 1997 Mo-lecular determinants of tissue selectivity in estrogen re-ceptor modulators. Proc Natl Acad Sci USA 94:14105–14110

44. Evans G, Bryant HU, Magee D, Sato M, Turner RT 1994The effects of raloxifene on tibia histomorphometry inovariectomized rats. Endocrinology 134:2283–2288

45. Mahfoudi A, Roulet E, Dauvois S, Parker MG, Wahli W1995 Specific mutations in the estrogen receptor changethe properties of antiestrogens to full agonists. Proc Natl

Acad Sci USA 92:4206–421046. Montano MM, Ekena K, Krueger KD, Keller AL, Katzenel-

lenbogen BS 1996 Human estrogen receptor ligand ac-tivity inversion mutants: receptors that interpret anties-trogens as estrogens and estrogens as antiestrogensand discriminate among different antiestrogens. Mol En-docrinol 10:230–242

47. Nichols M, Rientjes JMJ, Stewart AF 1998 Different po-sitioning of the ligand binding domain helix 12 and the Fdomain of the estrogen receptor accounts for functionaldifferences between agonists and antagonists. EMBO J17:765–773

48. Renaud JP, Rochel N, Ruff M, Vivat V, Chambon P,Gronemeyer H, Moras D 1995 Crystal structure of theRAR-gamma ligand-binding domain bound to all-transretinoic acid. Nature 378:681–689

49. Wagner RL, Apriletti JW, McGrath ME, West BL, BaxterJD, Fletterick RJ 1995 A structural role for hormone in thethyroid hormone receptor. Nature 378:690–697

50. Simons Jr SS, Pumphrey JG, Rudikoff S, Eisen HJ 1987Identification of cysteine 656 as the amino acid of hep-atoma tissue culture cell glucocorticoid receptors that iscovalently labeled by dexamethasone 21-mesylate.J Biol Chem 262:9676–9680

51. Harlow KW, Smith DN, Katzenellenbogen JA, Greene GL,Katzenellenbogen BS 1989 Identification of cysteine 530as the covalent attachment site of an affinity-labelingestrogen (ketononestrol aziridine) and antiestrogen (ta-moxifen aziridine) in the human estrogen receptor. J BiolChem 264:17476–17485

52. Ekena K, Weis KE, Katzenellenbogen JA, Katzenellenbo-gen BS 1996 Identification of amino acids in the hormonebinding domain of the human estrogen receptor impor-tant in estrogen binding. J Biol Chem 271:20053–20059

53. Ekena K, Weis KE, Katzenellenbogen JA, Katzenellenbo-gen BS 1997 Different residues of the human estrogenreceptor are involved in the recognition of structurallydiverse estrogens and antiestrogens. J Biol Chem272:5069–5075

54. Chae K, Gibson MK, Korach KS 1991 Estrogen receptorstereochemistry: ligand binding orientation and influenceon biological activity. Mol Pharmacol 40:806–811

55. Bocchinfuso WP, Korach KS 1997 Estrogen receptorresidues required for stereospecific ligand recognitionand activation. Mol Endocrinol 11:587–594

56. Barettino D, Feigenbutz M, Valcarcel R, Stunnenberg HG1994 Improved method for PCR-mediated site-directedmutagenesis. Nucleic Acids Res 22:541–542

57. Katzenellenbogen JA, Hsiung HM, Carlson KE, McGuireWL, Kraay RJ, Katzenellenbogen BS 1975 Iodohexes-trols. II. Characterization of the binding and estrogenicactivity of iodinated hexestrol derivatives, in vitro and invivo. Biochemistry 14:1742–1750

58. Jones CD, Jevnikar MG, Pike AJ, Peters MK, Black LJ,Thompson AR, Falcone JF, Clemens JA 1984 Antiestro-gens. 2. Structure-activity studies in a series of 3-aroyl-2-arylbenzo(b)thiophene derivatives leading to(6-hydroxy-2-(4-hydroxyphenyl)benzo(b)thien-3-yl) (4-(2-(1-piperidinyl)ethoxy)-phenyl)methanone hydrochloride(LY156758), a remarkably effective estrogen antagonistwith only minimal intrinsic estrogenicity. J Med Chem27:1057–1066

59. Robertson DW, Katzenellenbogen JA, Long DJ, RorkeEA, Katzenellenbogen BS 1982 Tamoxifen antiestro-gens. A comparison of the activity, pharmacokinetics,and metabolic activation of the cis and trans isomers oftamoxifen. J Steroid Biochem 16:1–13

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