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s t e r o i d s 7 2 ( 2 0 0 7 ) 949–954

avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate /s tero ids

ptical probes to identify the glucocorticoid receptor ligandsn living cells

uhammad Awaisa, Moritoshi Satob,c, Yoshio Umezawad,∗

Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science, 38 Nishigonaka, Myodaiji, Okazaki,apanGraduate School of Arts and Sciences, The University of Tokyo, JapanPRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama, JapanDepartment of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

r t i c l e i n f o

rticle history:

eceived 19 June 2007

eceived in revised form

August 2007

ccepted 16 August 2007

ublished on line 22 August 2007

eywords:

lucocorticoids

lucocorticoid receptor

oregulators

a b s t r a c t

Glucocorticoids act through glucocorticoid receptor (GR) and are used for the treatment

of several diseases. Ligand-induced recruitment of coregulator protein(s), coactiva-

tor/corepressor, to GR is an initial step in transcriptional activation/inhibition of GR. We

describe herein genetically encoded fluorescent probes for screening of glucocorticoids, nat-

ural and synthetic, in single living cells. The GR ligand binding domain was connected to

the GR interacting peptide sequence from coactivator or corepressor protein via a flexible

linker sequence. This fusion protein was sandwiched between cyan and yellow fluorescent

proteins (CFP and YFP, respectively) to complete the construct of the probe. This con-

struct functions as an optical probe for imaging ligand-induced interaction between the

glucocorticoid receptor and the coregulator protein (GLUCOCOR) in live cells. The interaction

between GR LBD and coregulator peptide within GLUCOCOR brings CFP in close proximity

onformational change

luorescence resonance energy

ransfer

of YFP to induce fluorescence resonance energy transfer from CFP to YFP. The GLUCOCORs

can identify functionally active GR ligands, rapidly and conveniently, in a high-throughput

screen; and are capable of distinguishing GR agonists, antagonists, and selective GR mod-

ulators in intact living cells. Therefore, the present method may play a significant role in

developing new glucocorticoids for clinical use.

corticoids for clinical use [4]. The GR is a ligand-dependent

. Introduction

lucocorticoids are steroid hormones that are essential forumerous physiological processes such as endocrine home-stasis, lipid metabolism, stress responses, and inflammation.lucocorticoids are widely used to treat immune and inflam-atory diseases including asthma, rheumatoid arthritis, and

llergic rhinitis. The biological activities of glucocorticoidsre mediated by binding to glucocorticoid receptor (GR). Theeceptor is expressed in a wide variety of tissues including

∗ Corresponding author. Tel.: +81 42 468 9292; fax: +81 42 468 9292.E-mail address: umezawa@chem.s.u-tokyo.ac.jp (Y. Umezawa).

039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.steroids.2007.08.006

© 2007 Elsevier Inc. All rights reserved.

bones (osteoblasts and osteocytes), liver, brain, T and B cells,and macrophages. Therefore, GR is widely recognized as atherapeutically important target [1,2]. A recently solved crys-tal structure of the GR has provided a better understanding ofthe receptor mechanism of action [3], which has acceleratedthe efforts to identify or discover new and improved gluco-

transcription factor of the nuclear receptors (NRs) superfam-ily [5,6]. Like other NRs, GR is comprised of several separablefunctional domains (A–F). The E region constitutes the lig-

950 s t e r o i d s 7 2 ( 2 0 0 7 ) 949–954

Fig. 1 – Fluorescent probes for the ligand-induced coactivator/corepressor recruitment to the GR LBD in living cells. (a)Principle of the GLUCOCOR, based on intramolecular FRET, to visualize the ligand-dependent interaction between the GRLBD and the transcription intermediary factor 2 (TIF2)/silencing mediator for retinoid and thyroid hormone receptor (SMRT)[8,11,12]. Upon ligand binding, the GR LBD and coactivator/corepressor interact with each other. Consequently, YFP isoriented in close proximity to CFP; this results in an increase in the FRET response. Magnitude of the FRET increase stronglydepends on the relative orientation and distance between the donor (CFP) and acceptor (YFP) fluorophore. (b) Construct ofthe optical probes for expression and imaging in mammalian cells. Shown at top of each bar are restriction sites. TIF2peptide contains 11 residues (742–752 aa). Linker consists of five residues (GGNGG). The GR LBD contains residues from521–777 aa. CFP and YFP are different-colored mutants of green fluorescent protein derived from Aequorea victoria withmammalian codons and the following additional mutations: CFP, F64L/S65T/Y66W/N146I/M153T/V163A/N212K, and YFP,

k seqOR w

S65G/V68L/Q69K/S72A/T203Y. Kz is an abbreviation of Kozamammalian cells. For GLUCOCOR1, TIF2 peptide in GLUCOC

and binding domain (LBD) that contains a ligand-dependentactivation function AF-2 (helix 12) in its carboxy terminalregion. The GR LBD plays important roles in addition to ligandbinding, including receptor dimerization, coregulator binding,transcriptional activation, and repression.

The LBD of all NRs have a common overall three-dimen-sional structure [7]. A ligand binding to the NR induces aconformational change in the NR LBD, which allows theligand-bound NR to interact with coregulator proteins, coacti-vators, and corepressors. The coactivator binding to the NRresults in the activation of gene expression related to theNR functions, however, corepressor binding to the NR sup-presses the gene expression in the cell. Pure agonists recruitcoactivators to NRs, while pure antagonists inhibit the recruit-

ment of coactivators to NRs or recruit corepressor proteinsto NRs [3,8–12]. However, in the case of selective NR modu-lators (SNRMs), the ligands can recruit both coactivators andcorepressors to NRs to stimulate or repress the NR transcrip-

uence, which allows optimal translation initiation inas replaced with SMRT peptide (2340–2350 aa).

tional activity [13–15]. The agonistic or antagonistic characterof a SNRM depends upon the expression levels of coactivatorand corepressor proteins in a particular cell/tissue of the body[14,15]. The expression levels of coactivator and corepressorproteins are known to be very different between tissues. Thedose of SNRMs thus results in the tissue-specific recruitmentof coactivators or corepressors to NR. The molecular basis ofthe agonist, antagonist, and SNRM functions provide us withan idea for a rational method for high-throughput screening ofGR ligands. In the present report, we developed optical probesfor the screening of GR ligands using the GR LBD and the pep-tide sequence from GR-interacting coactivator or corepressorprotein that is expressed in the target tissue.

The principle of the optical probes is shown in Fig. 1a.

The GR LBD is attached with a coregulator peptide, coactiva-tor/corepressor, via a flexible linker sequence. The resultantprotein was inserted between cyan and yellow fluorescentproteins (CFP, donor; and YFP, acceptor fluorophore, respec-

2 0 0

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2

2

FaT(ceaiTklebg

2

Tf(iaKcrst

s t e r o i d s 7 2 (

ively) in such a way that excitation and emission spectra ofFP and YFP are suitable for fluorescence resonance energy

ransfer (FRET) from CFP to YFP [16–18]. This fusion proteinunctions as an optical probe for imaging ligand-induced inter-ction between the glucocorticoid receptor and the coregulatorrotein (GLUCOCOR) in live cells in an intramolecular FRETashion. The addition of an agonist to cultured cells expressedith GLUCOCOR promotes interaction between the GR LBD

nd coactivator within GLUCOCOR; this results in an increasen the FRET from CFP to YFP. By contrast, an antagonist inhibitsR LBD/coactivator interaction within GLUCOCOR. The addi-

ion of an SNRM ligand that has mixed agonist/antagonistharacter promotes the GR LBD interaction with the coac-ivator as well as the interaction between the corepressorithin GLUCOCOR and GLUCOCOR1 (Fig. 1b), respectively, to

ncrease the FRET response. The strategy was used to dis-riminate among GR agonists, antagonists and selective GRodulators (SGRMs). We have demonstrated that mifepris-

one (RU486) is capable of inducing interaction of GR withoactivator and corepressor peptide, respectively therebyunction as a SGRM. Knowledge about the conformationalhange(s) that are induced by a ligand in the GR LBD, whichn turn enables the interaction with coregulator proteinss crucial for the understanding of the physiological actionf the ligand, and would be useful for the development ofR ligands to use as therapeutic agents for an cure of dis-ases.

. Experimental

.1. Materials

etal calf serum (FCS), Hank’s balanced salt solution (HBSS)nd LipofectAMINE 2000 reagents were purchased from Lifeechnologies (Rockville, MD). Minimum essential medium

MEM), trypsin–EDTA, dexamethasone (DEX), cortisol, corti-osterone, RU486, cyproterone acetate (CPA), progesterone,stradiol (E2), testosterone, genistein (Gen), nonylphenol (NP),nd bisphenol A (Bis-A) were purchased from Sigma Chem-cals Co. (St. Louis, MO). All cloning enzymes were fromakara Biomedical (Tokyo, Japan). The human GR� was aind gift from Dr. Shimizu (Institute of Molecular and Cel-

ular Biosciences, The University of Tokyo). A mammalianxpression vector pcDNA3.1 (+) was from Invitrogen Co. (Carl-ad, CA). All other chemicals used were of analytical reagentrade.

.2. Plasmid construction

o prepare the cDNAs for the constructs shown in Fig. 1b,ragment cDNAs of ECFP (1–238 aa), EYFP (1–238 aa), GR LBD521–777 aa), a flexible linker (GGNGG), and transcriptionalntermediary factor 2 (TIF2) peptide (742–752 aa) were gener-ted by standard polymerase chain reaction (PCR) to attach aozak sequence and restriction sites shown in constructs. To

onstruct GLUCOCOR1, the TIF2 peptide in GLUCOCOR waseplaced with the peptide of a corepressor (2340–2350 aa),ilencing mediator for retinoid and thyroid hormone recep-or (SMRT). All PCR fragments were sequenced with an ABI310

7 ) 949–954 951

genetic analyzer. The cDNAs were inserted at HindIII and XhoIsites of mammalian expression vector pcDNA3.1 (+).

2.3. Cell culture and transfection

Porcine epithelial kidney cell line PK-15 cells were cultured inMEM supplemented with 10% FCS, 1.0 mM sodium pyruvateand 0.1 mM nonessential amino acids, respectively, at 37 ◦Cin a humidified atmosphere of 5% CO2. Cells were transfectedwith an expression vector pcDNA3.1 (+) containing GLUCOCORin the presence of lipofectAMIN 2000 reagent in 3.5 cm glass-bottom dishes.

2.4. Imaging of cells

Culture medium was replaced with HBSS for fluorescenceimaging experiments. Within 12–24 h after transfection, thecells expressed with GLUCOCOR/GLUCOCOR1 were imaged atroom temperature on a Carl Zeiss Axiovert 135 microscopewith a cooled charge-coupled device camera MicroMAX (RoperScientific Inc., Tucson, AZ), controlled by MetaFluor (Univer-sal Imaging, West Chester, PA). Set the glass-bottomed dishonto the 40× oil immersion objective (Carl Zeiss, Jena, Ger-many) equipped on the fluorescence microscope. The cellswere observed with a 440 nm excitation filter, 455 nm dichroicmirror, and 535 nm emission filter. By browsing the cells onthe dish, we selected moderately bright cells in which thefluorescence is well distributed in the cytosol. The desiredobservation field that contained cell(s) of interest was selected.The region of interest within the cell was selected to examinetime course of CFP/YFP emission ratio during the followingimage acquisition, and started to acquire images every 10–20 sfor 10–15 min with the 440 nm excitation filter (CFP), 455 nmdichroic mirror, 480 nm emission filter (CFP), and 535 nm emis-sion filter (YFP). During the image acquisition, added stimuliof interest, for example, DEX.

3. Results and discussion

3.1. Interaction between the GR LBD and thecoactivator within GLUCOCOR can be observed in real time

To evaluate response of the GLUCOCOR indicator for theinteraction between GR LBD and coactivator upon gluco-corticoid stimulation, PK15 cells expressed with GLUCOCORwere stimulated with DEX (100 nM, a potent synthetic GRagonist) and this event was observed by following the timecourse of the changes in FRET. The emission ratio of CFP toYFP (CFP/YFP) was observed to decrease steadily and thenlevel off after ∼12 min, but no detectable change in theCFP/YFP emission ratio was noted with HBSS (carrier with-out DEX) under otherwise identical experimental conditions(Fig. 2a ).

According to the crystal structure data of DEX-GR LBD com-plex, DEX is completely buried in the ligand binding pocket

of GR and all the hydrophobic groups of DEX form hydrogenbonds with the GR LBD, which helps the GR LBD to adoptan active conformation. In the active conformational changeof GR LBD, the helix 12 folds back over the ligand binding

952 s t e r o i d s 7 2 ( 2 0

Fig. 2 – FRET was expressed as emission ratio of CFP to YFPsignals. (a) Time course of the FRET responses uponaddition of DEX or HBSS (carrier without ligand) to livePK-15 cells expressed with GLUCOCOR. Each time course isone of the five independent experiments. For eachexperiment, 0.8 �g of the expression vector encodingGLUCOCOR was transfected to cultured cells in 3.5 cmglass-based dishes. A single cell was selected from eachdish to monitor the effect of DEX on GLUCOCOR. (b)

the GR within GLUCOCOR thereby showed an agonistic behav-ior. The present GLUCOCOR assay is neither intended as aread-out of the binding affinity of ligands to the GR nor tran-

Fig. 3 – FRET responses of GLUCOCOR for variousconcentrations of DEX, cortisol, corticosterone, RU486,progesterone, CPA, testosterone, cortisol, E2, Bis-A, NP, andGen. The results are the means ± S.D. of emission ratios of

Pseudocolor images of the CFP/YFP emission ratio ofGLUCOCOR before (t = 0 min) and after addition of DEX.

pocket and generates a hydrophobic groove on the surfaceof the LBD to accommodate a coactivator [3]. A coactivatorinteracts with the agonist-bound LBD by using its leucinerich motif (LXXLL, L = leucine, X = any amino acid) [3,9]. In thepresent study we used TIF2 peptide, 742NALLRYLLDKD752, thatinteracts specifically with the GR LBD. The LRYLL sequenceof the TIF2 motif forms a two-turn � helix that orients thehydrophobic leucine side chains into groove formed in partby the helix 12 and residues from helices 3, 3′, 4, and 5. TheN- and C-terminal ends of the coactivator are clamped bya positively charged lysine residue of helix 3 and a nega-tively charged glutamic acid residue of helix 3. The dockingmode of the TIF2 LXXLL motif is similar to that seen inthe coactivator complexes with other NRs [7,9]. However,GR residues aspartic acid (D590) and arginine (R585) form asecond charge clamp that interacts with residues R and Dof TIF2 motif [7]. Because of this additional charge clamp,the TIF2 motif develops a strong interaction with the GRLBD. This interaction between the GR LBD and TIF2 resultedin a decrease in the distance and/or change in orientationbetween CFP and YFP within GLUCOCOR, thereby increased

the efficiency of FRET from CFP to YFP. This allows realtime imaging of DEX-induced interactions between GR LBDand the coactivator in live cells. Fig. 2b shows pseudocolorimages of cells expressed with GLUCOCOR when stimulated

0 7 ) 949–954

with DEX, and it illustrates a DEX-induced change in theCFP/YFP emission ratio of the GLUCOCOR throughout thecell.

3.2. Response of GLUCOCOR with natural andsynthetic steroids and endocrine disruptors

Next, DEX (a potent synthetic glucocorticoid), cortisol (anendogenous GR ligand), corticosterone, RU486 (a GR and PR-targeted drug), progesterone (an endogenous PR ligand), CPA(a synthetic progestin and anti-prostate cancer), testosterone(an androgen receptor ligand), estradiol (an endogenous estro-gen receptor ligand), and endocrine disrutors, EDs, [19] suchas Bis-A, NP, and Gen were applied to GLUCOCOR at var-ious concentrations from 0.01 to 100 �M to evaluate theirabilities to promote GR LBD/coactivator interaction withinGLUCOCOR. A dose-dependent increase in the FRET responsewas observed with the addition of DEX, cortisol, corticos-terone, RU486 or progesterone to the GLUCOCOR expressingcells (Fig. 3 ). The CPA, testosterone, Bis-A, NP, and Gen didnot display any considerable response. DEX exhibited maxi-mum response followed by cortisol; RU486 and progesteroneshowed significant but equal responses. Corticosterone didnot reach a saturation level at the concentrations tested.Previously, RU486 was considered as an antagonist for the pro-gesterone and glucocorticoid receptor. Recently, it has beenproved that RU486 is a selective modulator for the proges-terone receptor having mixed agonistic/antagonistic behaviordepending upon the cellular concentrations of coregulatorproteins [14]. We have also demonstrated the ability of theRU486 to promote interaction between coactivator and pro-gesterone receptor in living cells [20]. The increase in theFRET response by RU486 in Fig. 3 depicted the ability ofRU486 to promote interaction between the coactivator and

three cells from three different experiments. For eachexperiment, a single cell was imaged from a 3.5 cmglass-based dish to visualize the effect of eachconcentration of the tested ligand.

s t e r o i d s 7 2 ( 2 0 0

Fig. 4 – Emission ratio change for 100 nM DEX in theabsence and presence of various concentrations of CPA toassess the inhibitory effect of CPA on the DEX activity topromote receptor-coactivator interaction within GLUCOCOR.The results are the means ± S.D. of emission ratios fromthree different cells in three experiments. To determine theinhibitory effect of a concentration of CPA, the compoundwas added to three glass-based dishes containing theGLUCOCOR expressing cells. Each dish was incubated for1w

smFeGfGda

3iw

TiihtiaietNhistE

new NR ligands including glucocorticoids that maintain theirefficacy and beneficial actions such as anti-cancer, anti-diabetes, and anti-inflammatory but with reduced side effects[4]. The design of such compounds will unquestionably be

Fig. 5 – Emission ratio change upon addition of each RU486,

0–15 min at room temperature. Cells were imaged and DEXas added to the same dish without washing the inhibitor.

criptional activity of the GR in response to a ligand. Theagnitude of the FRET responses and the differences in the

RET observed by the tested ligands might be because of sev-ral factors, such as, (1) differences in ligand affinity for theR LBD, (2) differences in the ligand’s ability to induce con-

ormational change in the GR LBD, and consequently, in theR LBD’s ability to interact with the coactivator peptide, (3)ifferences in the rates of cellular influx or efflux of the lig-nds.

.3. Antagonist-induced inhibitory effect on thenteraction between the GR LBD and the coactivatorithin GLUCOCOR

he compounds that did not show any considerable responsen Fig. 3 are either inactive for the GR or inhibit GR–coactivatornteraction by acting as antagonists. To confirm this fact, weave evaluated the response of DEX in the presence of CPA,estosterone, E2, Bis-A, NP, and Gen, respectively. The CPAnhibited the DEX-induced GR LBD–coactivator interaction indose-dependent manner as shown in Fig. 4 , thereby behav-

ng as an antagonist for the GR. No considerable inhibitoryffect on the activity of DEX to induce GR–coactivator interac-ion was observed in the presence of testosterone, E2, Bis-A,P, or Gen (data not shown). The EDs Bis-A, NP, and Genave been shown to activate the ER but suppress AR activ-

ty [18,21]. In the case of GR, these EDs neither activate noruppress the GR function to interact with the coactivator;herefore Bis-A, NP and Gen and steroids testosterone and2 can be classified as inactive for the GR. Therefore, by

7 ) 949–954 953

using the GLUCOCOR we can discriminate between an inac-tive (neither agonist nor antagonist) and an antagonist ofGR.

3.4. Ligand-induced interaction between the GR LBDand the corepressor peptide within GLUCOCOR1

Steroids such as RU486, cortisol, corticosterone, DEX, andCPA were applied, at various concentrations (0.1–100 �M) toGLUCOCOR1 (Fig. 1b) to evaluate their ability to promoteGR LBD/corepressor interactions. RU486 displayed maximumFRET response; cortisol, corticosterone, and DEX showed weakresponses compared to RU486; and CPA did not elicit a FRETresponse as shown in Fig. 5 . The results demonstrate thatRU486 is a SGRM that has the ability to induce recruitmentof corepressor as well as coactivator proteins to a GR LBD(Figs. 3 and 5) depending upon the availability/relative con-centration of coactivator and corepressor proteins in a certaincell/tissue of the body to stimulate or block the transcriptionalactivities of the GR. CPA in Fig. 3 showed antagonistic effectby inhibiting coactivator peptide recruitment to the GR LBDwithin GLUCOCOR. The CPA did not also promote corepressorpeptide recruitment to the GR LBD within GLUCOCOR1 (Fig. 5).Because CPA lacks a bulky side chain, which is present in mostof steroid receptor antagonists, the observed functional effectsof CPA may occur by a different mode of receptor antagonism,which is not clear yet. Most probably, CPA antagonizes GR bystabilizing helix 12 in such a conformation, which is neitherfavorable for coactivator nor corepressor peptide binding tothe GR LBD within GLUCOCORs. There is a possibility thatin the presence of CPA, a corepressor interacts with the N-terminal of the receptor to exert its repressive effect on theactivity of the receptor.

A common goal of pharmaceutical industry is to develop

cortisol, corticosterone, DEX, and CPA to cultured cellsexpressed with GLUCOCOR1. The results are themeans ± S.D. of emission ratios from three different cells inthree experiments.

( 2 0

r

954 s t e r o i d s 7 2

aided by a structural knowledge of ligand-induced confor-mational changes in the NR LBDs and an understandingof how the ligand-regulated interactions between NRs andcoregulator proteins contribute to transactivation or transre-pression of genes. Crystal structures, although offering thehigh-resolution view of structures, provide a static view ofthe receptor. Moreover, it is not always possible to crystal-lize a NR LBD/coregulator complex with a number of ligandsto evaluate ligand-induced conformational changes. There-fore, some alternative, high-throughput method is neededto monitor the NR LBD/coregulator interactions in the pres-ence of various natural and synthetic ligands. The live-cellimaging tool provides an important complement to biochem-ical and structural biology studies, extending the analysis ofprotein–protein interactions, protein conformational changes,and behavior of signaling molecules to their natural environ-ment within the intact cells. Although the FRET technologyusing CFP and YFP fluorophores is not novel, the idea to usethis technology in glucocorticoid field and the design of theGLUCOCORs for glucocorticoids screening are innovative. Inthe present study, we have demonstrated the ability of severalligands to promote/inhibit the coregulator recruitment to theGR LBD in the physiological environment of single living cells.We have shown that a SNRM, RU486, induces a conforma-tional change in the GLUCOCOR and GLUCOCOR1 to promotethe coactivator and corepressor recruitment to the GR LBD,respectively, thus indicating both agonist- and antagonist-likebehavior.

In summary, ligand-induced GR/coregulator interactionscan be imaged in real time, rapidly and conveniently, in singleliving cells using GLUCOCORs. The GLUCOCOR optical probesare not intended as a read-out of the binding affinity of aligand/drug, but rather it probes the efficacy of drugs as anagonist, antagonist or SGRM in living cells. The permeabil-ity of a drug into cells and the conformational changes thatare induced in a receptor to regulate interaction between thereceptor and coactivator and/or corepressor proteins all deter-mine efficacy of a drug much more than a simple bindingassay.

Acknowledgements

This work was supported by grants from Japan Science andTechnology Agency (JST), and Japan Society for the Promotionof Science (JSPS).

e f e r e n c e s

[1] Joels M, Vreugdenhil E. Corticosteroids in the brain. Cellular

and molecular actions. Mol Neurobiol 1998;17:87–108.

[2] Buckingham JC. Glucocorticoid: exemplars of multi-tasking.Br J Pharmacol 2006;147:S258–68.

[3] Bledsoe RK, Montana VG, Stanley TB, Delves CJ, Apolito CJ,Mckee DD, et al. Crystal structure of the glucocorticoid

0 7 ) 949–954

receptor ligand binding domain reveals a novel mode ofreceptor dimerization and coactivator recognition. Cell2002;110:93–105.

[4] Rosen J, Miner JN. The search for safer glucocorticoidreceptor ligands. Endocr Rev 2005;26:452–64.

[5] Rechavi MR, Garcia HE, Laudet V. The nuclear receptorsuperfamily. J Cell Sci 2003;116:585–6.

[6] Gronemeyer H, Gustafsson JA, Laudet V. Principles formodulation of the nuclear receptor superfamily. Nat RevDrug Discov 2004;3:950–64.

[7] Greschik H, Moras D. Structure–activity relationship ofnuclear receptor–ligand interactions. Curr Top Med Chem2003;3:1573–99.

[8] Heery DM, Kalkhoven E, Hoare S, Parker MG. A signaturemotif in transcriptional co-activators mediates binding tonuclear receptors. Nature 1997;387:733–6.

[9] Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, AgardDA, et al. The structural basis of estrogenreceptor/coactivator recognition and antagonism of thisinteraction by tamoxifen. Cell 1998;95:927–37.

[10] Bourguet W, Vivat V, Wurtz JM, Chambon P, Gronemeyer H,Moras D. Crystal structure of a heterodimeric complex ofRAR and RXR ligand-binding domains. Mol Cell2000;5:289–98.

[11] Nagy L, Kao HY, Love JD, Li C, Banayo E, Gooch JT, et al.Mechanism of corepressor binding and release from nuclearhormone receptors. Gene Dev 1999;13:3209–16.

[12] Xu HE, Stanley TB, Montana VG, Lambert MH, Shearer BG,Cobb JE, et al. Structural basis for antagonist-mediatedrecruitment of nuclear corepressors by PPAR�. Nature2002;415:813–7.

[13] Wang Q, Blackford Jr JA, Song LN, Huang Y, Cho S, Simons JrSS. Equilibrium interactions of corepressors andcoactivators with agonist and antagonist complexes ofglucocorticoid receptors. Mol Endocrinol 2004;18:1376–95.

[14] Liu Z, Auboeuf D, Wong J, Chen JD, Tsai SY, Tsai MJ, et al.Coactivator/corepressor ratios modulate PR-mediatedtranscription by the selective receptor modulator RU486.Proc Natl Acad Sci USA 2002;99:7040–944.

[15] Smith CL, O’Malley BW. Coregulator function: a key tounderstanding tissue specificity of selective receptormodulator. Endocr Rev 2004;25:45–71.

[16] Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, IkuraM, et al. Fluorescent indicators for Ca2+ based on greenfluorescent proteins and calmodulin. Nature 1997;388:882–7.

[17] Sato M, Hida N, Umezawa Y. Imaging the nanomolar range ofnitric oxide with an amplifier-coupled fluorescent indicatorin living cells. Proc Natl Acad Sci USA 2005;102:14515–20.

[18] Awais M, Sato M, Lee X, Umezawa Y. A fluorescent indicatorto visualize activities of the androgen receptor ligands insingle living cells. Angew Chem Int Ed 2006;45:2707–12.

[19] Campbell CG, Borglin SE, Green FB, Grayson A, Wozei E,Stringfellow WT. Biologically directed environmentalmonitoring, fate, and transport of estrogenic endocrinedisrupting compounds in water: a review. Chemosphere2006;65:1265–80.

[20] Awais M, Sato M, Umezawa Y. Imaging of selective nuclearreceptor modulator-induced conformational change in the

nuclear receptor to allow interaction with coactivator andcorepressor proteins in living cells. ChemBioChem2007;8:737–43.

[21] Sohoni P, Sumpter JP. Several environmental oestrogens arealso anti-androgens. J Endocrinol 1998;158:327–39.