Breast Cancer Research and Treatment 72: 61–68, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Report

Functional mutations of estrogen receptor protein: assay for detection

Mark Nichols1 and Kenneth S. McCarty Jr 2

1Department of Pharmacology, 2Medicine and Pathology, University of Pittsburgh Cancer Institute, University ofPittsburgh, Pittsburgh, PA, USA

Key words: antihormones, coactivators, color plate assay, estrogen receptor, FLP recombinase, helix 12, hormones,ligand binding domain, S. cerevisiae

Summary

Antiestrogens block the function of estrogen receptor (ER) by binding and misfolding the AF-2 transcriptionalactivation region in the ligand-binding domain, inhibiting or altering its association with coactivator proteins. Wedescribe a novel assay uniquely configured to identify aberrations in this function that may lead to antiestrogenresistance. The identification of mutations of ER that affect its function is important to current breast cancertherapies. Standard methods to detect these mutations are cumbersome and the number of described mutationsis limited, reflecting this difficulty. Conventional ER analysis in the clinic demonstrates the presence of antigenicdeterminants of the receptor protein or estrogenic ligand binding without reflection on the critical ability of theliganded receptor to interact with transcription cofactors. Here, we describe the use of estrogenic regulation ofa site-specific recombinase activity, measuring deletion of a color marker gene via FLP-ER fusion proteins, todetect functional changes in ER protein folding that affects the site where cofactors interact. The assay provides amethod to readily detect single amino acid changes in ER, some with biologically important consequences. Withoutsuch a functional assay as described, phenotypic changes are likely to remain undetected and under-evaluated. Itis probable that some human tumors have antihormone resistance resulting from ER mutations that either blockantihormone binding or transmit antihormone binding as a positive transcriptional signal via cofactor interaction.An assay to evaluate functional ER will lead to better predictive tests of treatment modalities.

Introduction

The transcription AF-2 region of the estrogen receptor(ER) hormone-binding domain is of key importancefor transcription and is the prime target of antiestro-gen/SERM action [23]. We have developed a uniqueassay in yeast to screen ER from breast tissues. Thisassay is configured to detect mutations that alter thecritical AF-2 domain and thus alter ER activity withhormones and antihormones. Such mutations can bethen selected to study the effect of the mutation onantiestrogen efficacy, coactivator protein interactions,and transcription control with profound implicationsfor tumor classification and treatment selection.

The analysis of ER protein is a routine compo-nent in the evaluation of breast cancer specimens [13],yet few functional mutations of the estrogen receptor

have been described. Current methods of clinical ana-lyses usually provide information on the presence ofa receptor protein antigen but not on the functionalcapacity of the receptor to elicit an estrogenic re-sponse. Despite this limitation, these assays of tumortissues are used to select breast cancer patients forhormone therapy [13]. Such hormone therapy ne-cessarily requires functional receptors. This therapyoften makes use of compounds that block or antag-onize the effects of estrogen in specific tissues, whileretaining the positive estrogenic character in othertissues [29]. Conventional estrogen receptor assayshave proven to be of limited predictive value for re-sponse to these agents. This ultimately results fromthe fact that steroid hormone signaling is a com-plex interaction of ligand, receptor, cofactors, andchromatin.

62 M Nichols et al.

Figure 1. Schematic diagram of the human ERα gene and protein. The conserved domains, A–F, are shown above the gene with the amino acidposition numbers at each border. The C domain (zinc fingers for DNA binding) and E domain (ligand binding domain) are conserved amongnuclear receptors. The exon structure is also shown with the amino acid positions at each intron-exon boundary.

Mutations of ER have been found in breast can-cers [7, 14, 20], however, technical limitations haveprevented extensive analysis of the frequency of suchmutations or their role in breast tumor biology. This isespecially true for mutations involving single aminoacid changes in the receptor protein. Controversyexists regarding the character of the ER in tumorsdeveloping while patients are actively receiving selec-tive estrogen receptor modifiers (SERMs) as chemo-prevention, or recurring in those who received SERMsas adjuvant therapy [30].

Steroid hormone receptors, including ER, are mod-ular transcription factors composed of domains A–F(Figure 1) [15]. Domain E, the ligand binding do-main (LBD), mediates many other key functions in-cluding dimerization, Hsp90 binding and repression,and hormone-activated transcriptional activity (AF-2)[16]. X-ray crystallography of ER LBDs with boundestradiol, raloxifene, diethylstilbestrol or 4- hydroxy-tamoxifen [4, 27], provide valuable, though staticpictures for understanding receptor function.

The consequence of estrogen binding to the ERLBD induces a conformational change in the receptor,with dissociation of the repressing Hsp90 complex,to allow receptor dimerization and binding to specificDNA response elements upstream of regulated genes[3, 11]. Once bound to DNA, two domains of thereceptor protein have transcriptional activation func-tions (AFs): a hormone-independent AF-1 in the A/Bdomain and the hormone-inducible AF-2 within theLBD [19]. The activity of AF-2 is dependent on theparticular ligand bound (hormone vs. antihormone)[3, 4, 19, 27]. Only hormone bound receptors bindp160 coactivator proteins (e.g., GRIP1, SRC1, AIB1,and RAC3) to the AF-2 region, which greatly en-hances transcription at ER responsive genes [1, 4,8–10, 12, 23, 25, 27].

Generally p160 coactivators contain interactiondomains known as nuclear receptor (NR) boxes, hav-ing the sequence LXXLL, which bind to hormonebound receptors, including ER [12]. Recent structuraldata have shown that the NR boxes bind to a cleftformed in the region of helix 12 of ER, as it is foldedover the hormone bound pocket [12, 27]. The X-raystructures of ER show a different final position of helix12 when an antihormone is bound [4, 27]. Bound4-hydroxytamoxifen leads to occlusion of the coactiv-ator binding cleft by the misaligned helix 12 [27].Mutations in the AF-2 helix 12 sequence or antihor-mone binding have been shown to alter the interactionof SRC1 with ER [18, 26].

Results and discussion

Steroid receptor LBDs can confer ligand dependencein Trans

LBDs interact with Hsp90 protein complexes in theabsence of ligand [24]. Ligand binding reconfiguresthe LBD and releases the Hsp90 complex, freeingthe activity to report binding (Figure 2(A)). We haveshown that estrogen regulation has been fused to theactivity of the yeast site- specific recombinase FLP [5,21], an enzyme that leads to precise deletion of DNAbetween preset FLP binding sites (Figure 2(B))[5].FLP-ER fusion proteins are inactive in the absence ofligand, yet respond to hormone or antihormone bind-ing by deleting a reporter gene, resulting in a shorterreporter band in Southern blots, and a yeast colonycolor change [Figure 3]. These fusions, therefore,faithfully reflect the initially repressed, unligandedER, and are similarly activated by ligand binding inyeast.

Functional mutations of estrogen receptor protein 63

Figure 2. (A) The LBD confers ligand dependence on activity ‘X’, by relieving Hsp90 repression. (B) FLP recombinase binds as a homodimerto its 34 bp recognition sequence (triangles). Two such dimers can interact through a tetrameric intermediate to exactly delete the DNA betweenthe two 34 bp targets. Thus, a selectable marker on B-C DNA is lost.

Figure 3. (A) The FLP-ER expression plasmid. A galactose promoter drives expression of the gene for the FLP-ER LBD fusion protein on asingle-copy plasmid in yeast. (B) The FLP recombination deletion assay. Before recombination, the ADH1 promoter expresses the URA3 gene,which lies between FLP recombination targets (FRTs, triangles) as does the SUP11 tRNA gene. After recombination, the URA3/SUP11 regionis excised. Expression of the endogenous ade2− gene relies on SUP11, producing white colonies before, red colonies after recombination.

Regulation of FLP recombinase activity in yeastwas achieved by fusing the human ER ligand-bindingdomain (domains D, E, and F; aa 251–595) to theC-terminus of FLP [21] (see Figures 1, 3). Ligandtitration experiments with a variety of known estrogenhormones and antihormones were performed and ana-lyzed by Southern hybridization to the deletion target.These confirm that response of FLP-ERs to ligands isa simple reflection of ligand binding by the ER LBD[21, 22].

Detection of estrogen binding in yeast plate assays

Estradiol mediated deletion of the single target in yeastresults in the loss of the marker gene, which leadsto red colonies. After plating a lawn of the reporteryeast containing a FLP-ER clone on a single copyplasmid, an estrogenic ligand was added to the plate(Figure 4). It diffuses and derepresses the recombinaseactivity, leading to red color, where the estrogen con-centration is sufficient to bind the estrogen-bindingdomain (EBD) (KM ∼ 1 nM for the wild-type (wt)EBD). We also tested the accuracy of this assay byusing a FLP-ER with the G400V mutation, known toreduce estradiol binding affinity by about 100 × [16].This indeed reduced red circle size as expected, and toapproximately the same size as the wt FLP-ER withonly 1% as much hormone (compare top row right

vs. bottom row center, Figure 4). Therefore, both theamount of ligand plated and the affinity of the receptorfor that ligand define the size of the red circle.

Assay to visually differentiate ER folding byhormones and antihormones

The D domain ‘linker sequence’ has a profound affecton FLP-ER activity when assayed by altered targetsize in Southern blots [21]. We extended this ob-servation directly by making use of the yeast plateassay (Figure 5). Fusion proteins containing the entireD domain (WT251) are activated by all three hor-mones and three antihormones tested, while fusionscontaining 53 fewer amino acids of ER (WT304) areonly activated by the three estrogen hormones, estra-diol, hexestrol, and diethylstilbestrol. Both forms ofthe fusion protein bind ligands with equivalent affini-ties [22]. Antihormones are known to block receptortranscriptional function by misfolding the LBD andinhibiting subsequent coactivator binding [4, 27]. Thesimplest model for reconciling antihormone bindingyet no recombinase activity is that the conformation ofan antihormone bound LBD interferes with the tetra-meric intermediate of FLP recombination (Figure 6).This interference was caused by removal of the Ddomain, moving the misfolded LBD closer to FLP.In contrast, the proper, hormone bound conformation

64 M Nichols et al.

Figure 4. Color plate assay for estrogen ligand activation. A lawn of unrecombined yeast cells carrying a single copy plasmid with a FLP-EBDclone is plated on 10 cm petri plates containing galactose. The top row shows yeast with the wild-type FLP-EBD sequence and the bottom rowshows a FLP-EBD with the G400V mutation, known to lower estradiol (E2) binding affinity [16]. The middle column has 10 nmol of E2 placedat the center, while the right column has 0.1 nmol E2. The left column shows the ethanol only control.

Figure 5. Detection of hormone versus antihormone conformation of the ER LBD. The D domain selectively alters ligand activity as seenby plate assay results for FLP fused to the estrogen binding domain at amino acid 251, which includes the D, E, and F domains of ER, orfused at amino acid 304, which includes only the E and F domains. Without the D domain ‘spacer’, antihormones are not able to activaterecombination. The ligands plated are CW from 11 O’clock: estradiol (E2), 4-hydroxytamoxifen (Z-OHT), raloxifene (RAL), diethylstilbestrol(DES), tamoxifen (TAM), and hexestrol (HEX).

does not result in interference by the LBD (Figures 5,6). Varying the fusion point amino acid of the EBDwas used to test this idea further. In all, FLP fusionsto ERα amino acids 251, 286, 290, 295, 299, 304, and309 were tested for the ability to be activated by agon-ists and antagonists. Interestingly, the fusion forms atamino acids 286, 290, 295, and 299 showed decreas-

ing activation by antihormones in the plate assays, thatis, progressively smaller red circles (not shown). The304 and 309 forms never showed activity with antihor-mones, while hormones worked with all fusion forms.These data are consistent with the model of stericinhibition by the C-terminal part of the antihormonebound EBD that depends on distance from FLP.

Functional mutations of estrogen receptor protein 65

Figure 6. FLP-ER recombination reflects C-terminal ER structure. FLP-ER recombinases without the 53 amino acid D domain (WT304) showactivation by hormones, but not by antihormones, which misfold helix 12. FLP-ER recombinases including the D domain (WT251) showactivation by hormones and antihormones, though less so for antihormones (see Figure 5). The intermediate for recombinase activity involvesa tetramer of proteins (Figure 2(B)), and is sensitive to ER conformation. The correctly folded hormone bound form of the ER domain allowsrecombination with or without the D domain. Likewise, ER mutations affecting helix 12 (AF-2) folding also lead to ligand-specific blocks orpartial blocks to recombination [22].

Color plate assay is sensitive to ER proteinconformation of the helix 12 (AF-2) domain

Isoforms of FLP-ER can differentiate hormone andantihormone positioning of the C-terminal part (helix12, AF-2) of the LBD (Figure 6), and they correlateto hormone and antihormone effects on ER transcrip-tion [4, 27]. Helix 12 is a key structural motif to bindcoactivators for hormone-induced ER transcription [4,8, 9, 23, 27]. We tested this plate reporter system withclones containing mutated ER domains. The diameterand intensity of the circle of red yeast, induced by dif-fusing ligand, directly reflects the relative activationof the recombinase fusion by that ligand. The 6-ligandpattern on plates creates a distinct ‘fingerprint’ of redcircles. Yeast carrying a wild-type or a mutant FLP-ERexpression clone can be easily differentiated (Fig-ure 7), as shown for a selection of ER mutations thatinvert hormone and antihormone induced recombina-tion [22]. Mutations in helix 12 (e.g., L540P) greatlydiminish all circle sizes. Many distinctive patterns ofthe 6-ligand panel occur (Figure 7), primarily withmutations in or around helices 3, and 10–12, com-ponents of the p160 coactivator binding site of ER[27].

AF-2 altered conformations in breast tissues

Having established a novel FLP-ER screening sys-tem to report ER folding, we then applied it to ERfrom breast tumor samples. Analysis was performed

on RNA isolated from frozen breast cancer tissues. ERsequences were recovered from isolated RNA usingRT PCR, to generate DNA encoding the C-terminalhalf of the ER LBD [6]. From this DNA pool, theER LBD was cloned onto the FLP fusion protein inthe yeast expression plasmid (Figure 2). For eachRNA sample, multiple isolates [30–40] were analyzed.Single individual clones were analyzed by performingplate assays with a panel of ligands, and a wild-typeFLP-ER control was included to allow direct com-parison of red circle size, very indicative of ligandbinding and ER conformation in single experiments.We have examined several breast cancer samples andas expected for complex tissue samples, a numberof the isolated FLP-ERs had the normal wild-typepattern and sequence. However, from among thesetumors, several very interesting plate assay pheno-types were also identified (Figure 8). These ERsreflect several types of defects, such as blockingall ligand binding (all white plates), altering confor-mation after binding (altered circle-size pattern), orby premature termination codons that result in con-stitutive ER function, without the AF-2 domain orbinding of the repressive Hsp90 complex (all redplates).

Mutations of the ER LBD can be screened quickly

The X-ray crystal structures of ER ligand bindingdomains with hormones versus antihormones clearly

66 M Nichols et al.

Figure 7. Color plate assay detects various in vitro mutatedFLP-ERs. The hormones and antihormones are plated in the pat-tern shown [E2 (estradiol), Z-OHT (4-hydroxytamoxifen), RAL(raloxifene), DES (diethylstilbestrol), TAM (tamoxifen), and HEX(hexestrol)]. The first plate is the control (WT304), with wild-typeER sequence, and is activated only by hormones [E2, HEX, andDES]. An ‘inversion’ mutant (#24 = L508E) is activated only byantihormones [Z-OHT, RAL, and TAM] [22]. Each of the numberedvariants of FLP-ER have mutations in the ER domain that affectsligand interaction/protein conformation [#6-L466A, #10-L509R,#11-L508H, #21-L508D, and #24-L508E]. Numerous conservativeamino acid substitutions showed no change from the wild-typeFLP-ER pattern.

show the importance of helix 12 position for co-activator binding and transcription activity [4, 27].Mutational disruption of ER LBDs alters ligand ac-tivation of FLP-ER proteins (Figure 7). An in vitrogenerated mutation was found in the center of helix12 that abolishes its α-helical structure, and it hindersrecombination by FLP-ER [22]. These data are con-sistent with a model, where the C-terminal structureof FLP-ER is an excellent reporter of ligand inducedconformation. This is highly significant because ab-normal structures at the helix 12 C-terminus willalso block effective coactivator binding by GRIP1,SRC1, etc., and subsequent transcription [9, 12, 17,23, 28, 31]. Some isolates from the initial cohortof tumors examined were particularly interesting be-cause they changed the relative red circle size/ratiofor estradiol versus 4-hydroxytamoxifen in the yeastplate assay (Figure 8). Hence, we have developeda unique assay of ER conformation in the regionknown to be needed for hormone inducible transcrip- F

igur

e8.

Col

ony

colo

ras

say

toev

alua

telig

and

resp

onsi

vene

ssre

veal

sch

ange

sin

ER

from

hum

anbr

east

canc

ers.

Lig

ands

are

the

sam

eas

Figu

re7

exce

ptth

at2-

met

hoxy

estr

one

(2-M

E)

has

repl

aced

ralo

xife

ne.

The

isol

ates

show

nar

ecl

oned

into

the

form

ofFL

P-E

R,w

here

both

horm

ones

and

antih

orm

ones

activ

ate

reco

mbi

natio

n(W

T25

1).T

hefir

stpl

ate

cont

ains

the

wild

-typ

ecl

one

WT

251,

asco

ntro

l.T

heot

hers

show

reco

vere

dis

olat

esfr

omtis

sues

cont

aini

ngm

utat

edE

Rs.

The

2nd

plat

esh

ows

nore

pres

sion

(all

red)

inth

eab

senc

eof

ligan

ds,

and

the

4th

plat

esh

ows

nolig

and

activ

atio

nw

hile

reta

inin

gH

sp90

bind

ing

repr

essi

on.T

he3r

dpl

ate

dem

onst

rate

san

inte

rest

ing

ER

phen

otyp

e,w

here

antih

orm

one

4-hy

drox

ytam

oxif

en(r

edar

row

)is

apo

tent

activ

ator

whi

leho

rmon

eses

trad

iol

(bla

ckar

row

)an

dhe

xest

rolh

ave

lost

mos

tact

ivity

.

Functional mutations of estrogen receptor protein 67

tion, and the target of its inhibition by agents such astamoxifen.

Not only does an antihormone remove ER frompotential positive stimulation by a hormone molecule,but it may also block an estrogen response element(ERE) at a gene, to limit the expression of thatgene. In the presence of antihormones, coactivatorsin cells are primarily presented with noninteracting,misfolded LBDs. If a mutation or overexpression of acoactivator would allow a significant coactivator/ER-antiestrogen interaction, the antihormone, such as4-hydroxytamoxifen, may result in stimulated ERtranscription.

An assay to allow detection of mutations of ERthat are involved in alteration of estrogen signal-ing is requisite to understand at least a subset ofclinical tamoxifen failures. These genetically markedyeast strains allow rapid screening, using a simplered/white colony color assay, to functionally distin-guish estrogen hormone and antihormone and thefunctional capacity of the ER protein. Use of thisunique assay should facilitate the identification offunctional ER mutations in breast cancer and dys-plasias. The assay provides a basis for the selec-tion for study of breast tumors with mutations thatinfluence coactivator interactions and, therefore, es-trogenic versus antiestrogen effects of SERMs, andother hormonal therapies. Our method to assay forfunctional status of the ER protein will allow morespecific characterization of response potential forbreast cancers beyond that offered by conventionalsystems.

Materials and methods

Strains and chemicals

The S. cerevisiae strain used for these experiments(MAT aaa, leu2-3,112, his3-11,15, ura3-52, trp1-1::(TRP1,URA3,SUP11), ade2-1ochre, and can1-100)was derived from RS453 (R. Serrano, Valencia, Spain)by integrating the target of recombination (Figure 3)at the trp1 locus. Transformation of yeast by thestandard lithium acetate method was performed asdescribed [2]. Transformed yeasts were grown andmaintained with selection for leucine and tryptophanin glucose or galactose supplemented synthetic mediafrom BIO 101, Inc. The hormones and antihormoneswere purchased from Sigma, except 4- hydroxytamox-ifen (Research Biochemicals International), and ICI

182,780 (a gift from Dr A. Wakeling, Zeneca Pharma-ceuticals).

Color plate assay

Yeast cells containing the integrated SUP11 recom-bination substrate (Figure 3) were transformed byplasmids containing the various FLP-ER genes. Cul-tures were grown in glucose with leucine selectionand then plated at high density on synthetic galactoseplates lacking leucine and tryptophan. A 2 µl dropof ethanol containing each ligand was placed on theplate, as shown (Figures 4, 6–8). An adenine markergene in yeast gives a color phenotype (red) if the targetgene has been deleted by the recombinase. The plateswere grown at 30◦C for 4 days to maximize red colorformation.

Isolation of ER from breast tissues

Analysis was performed on RNA isolated from breastcancer tissues that have been snap frozen and storedat −70◦C. Recovery of ER sequences from isolatedRNA was done by RT PCR. RNA was directly iso-lated from snap frozen tissue (100 mg), homogenizedin a guanidinium thiocyanate lysis buffer and eventu-ally precipitated. Reverse transcriptase (Clontech) wasadded to generate first strand DNA for PCR, whichthen made use of primer sets [6] to generate DNAencoding the C-terminal half of the ER LBD. The oli-gonucleotides used surrounded sequences correlatingto amino acids 240–595 of the ER alpha mRNA toallow cloning of the entire LBD and F domain. CorrectRT PCR generated a DNA fragment of 1060 bp for theER LBD. Fragments of other sizes were checked tosee if they result from exon deletions. From this DNApool, the ER LBD was cloned onto the FLP fusionprotein in the yeast expression plasmid (Figure 3(A)).For each RNA sample, multiple isolates [20–40] wereanalyzed.

Acknowledgements

We thank Dr A.P. Monaghan and Dr K.S. McCarty Sr.for comments, Birgit Suppé for technical support, andDr A. Wakeling for providing ICI 182,780. This workwas supported by funds from the USAMRMC BreastCancer Research Program, Fort Detrick, MD, grantDAMD17-99-1-9353 # BC981166 and the Competi-tive Medical Research Fund (CMRF) of the Universityof Pittsburgh Medical Center.

68 M Nichols et al.

References

1. Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM,Guan XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS:AIB-1, a steroid receptor coactivator amplified in breast andovarian cancer. Science 277: 965–968, 1997

2. Ausubel FM, Brent R, Kingston RE, Moore DD, SeidmanJG, Smith JA, Struhl K (eds): Current Protocols in MolecularBiology. Vol 1–3, Wiley, New York, NY, USA, 1995

3. Beekman JM, Allan GF, Tsai SY, Tsai MJ, O’Malley BW:Transcriptional activation by the estrogen receptor requiresa conformational change in the ligand binding domain. MolEndocrinol 7: 1266–1274, 1993

4. Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T,Engstrom O, Ohman L, Greene GL, Gustafsson J-A, CarlquistM: Molecular basis of agonism and antagonism in the oestro-gen receptor. Nature 389: 753–758, 1997

5. Chen JW, Lee J, Jayaram M: DNA cleavage in trans by the ac-tive site tyrosine during Flp recombination: switching proteinpartners before exchanging strands. Cell 69: 647–658, 1992

6. Chevillard S, Mueller A, Levalois C, Laine-Bidron C, Vielh P,Magdelenat H: Reverse transcription-polymerase chain reac-tion (RT-PCR) assays of estrogen and progesterone receptorsin breast cancer. Breast Cancer Res Treat 41: 81–89, 1996

7. Clarke CL, Balleine RL, Auchus RJ, Fuqua SAW: Estrogenand progesterone receptor variants in human breast cancer.Curr Opin Endocrin Diabet 2: 398–403, 1995

8. Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kush-ner PJ, Baxter JD, Fletterick RJ, Yamamoto KR: Structure andspecificity of nuclear receptor-coactivator interactions. GenesDevel 12: 3343–3356, 1998

9. Ding XF, Anderson CM, Ma H, Hong H, Uht RM, KushnerPJ, Stallcup MR: Nuclear receptor-binding sites of coactiva-tors glucocorticoid receptor interacting protein1 (GRIP1) andsteroid receptor coactivator 1 (SRC1): multiple motifs withdifferent binding specificities. Mol Endocrinol 12: 302–313,1998

10. Eng FCS, Barsalou A, Akutsu N, Mercier I, Zechel C, MaderS, White JH: Different classes of coactivators recognize dis-tinct overlapping binding sites on the estrogen receptor ligandbinding domain. J Biol Chem 273: 28371–28377, 1998

11. Fawell SE, Lees JA, White R, Parker MG: Characteriza-tion and colocalization of steroid binding and dimerizationactivities in the mouse estrogen receptor. Cell 60: 953–962,1990

12. Feng W, Ribeiro RCJ, Wagner RL, Nguyen H, Apriletti JW,Fletterick RJ, Baxter JD, Kushner PJ, West BL: Hormone-dependent coactivator binding to a hydrophobic cleft onnuclear receptors. Science 280: 1747–1749, 1998

13. Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kava-nah M, Cronin WM, Vogel V, Robidoux A, Dimitrov N,Atkins J, Daly M, Wieand S, Tan-Chiu E, Ford L, WolmarkN: Tamoxifen for prevention of breast cancer: report of thenational surgical adjuvant breast and bowel project P-1 study.J Natl Cancer Inst 90: 1371–1388, 1998

14. Fuqua SAW, Chamness GC, McGuire WL: Estrogen receptormutations in breast cancer. J Cell Biochem 51: 135–139, 1993

15. Green S, Chambon P: Nuclear receptors enhance our under-standing of transcription regulation. Trends Genet 4: 309–314,1988

16. Gronemeyer H, Laudet V: Transcription factors 3: nuclearreceptors. Prot Prof 2: 1173–1308, 1995

17. Hong H, Kohli K, Garabedian MJ, Stallcup MR: GRIP1, atranscriptional coactivator for the AF-2 transactivation domainof steroid, thyroid, retinoid, and vitamin D receptors. Mol CellBiol 17: 2735–2744, 1997

18. Kalkhoven E, Valentine JE, Heery DM, Parker MG: Isoformsof the steroid receptor co-activator 1 differ in their ability topotentiate transcription by the oestrogen receptor. EMBO J 17:232–243, 1998

19. Lees JA, Fawell SE, Parker MG: Identification of two trans-activation domains in the mouse oestrogen receptor. NuclAcids Res 17: 5477–5488, 1989

20. Murphy LC, Dotzlaw H, Leygue E, Douglas D, Coutts A,Watson PH: Estrogen receptor variants and mutations. J SterBiochem Mol Biol 62: 363–372, 1997

21. Nichols M, Rientjes JMJ, Logie C, Stewart AF: FLP recom-binase/estrogen receptor fusion proteins require the receptorD domain for responsiveness to antagonists, but not agonists.Mol Endocrinol 11: 950–961, 1997

22. Nichols M, Rientjes JMJ, Stewart AF: Different positioningof the ligand binding domain helix 12 and the F domain of theestrogen receptor accounts for functional differences betweenagonists and antagonists. EMBO J 17: 765–773, 1998

23. Norris JD, Fan D, Stallcup MR, McDonnell DP: Enhancementof estrogen receptor transcriptional activity by the coactivatorGRIP-1 highlights the role of activation function 2 in de-termining estrogen receptor pharmacology J Biol Chem 273:6679–6688, 1998

24. Picard D: Regulation of protein function through expression ofchimaeric proteins. Curr Opin Biotechnol 5: 511–515, 1994

25. Robyr D, Wolffe AP, Wahli W: Nuclear hormone receptorcoregulators in action: diversity for shared tasks. Mol Endo-crinol 14: 329–347, 2000

26. Schwartz JA, Brooks SC: Changes in the structure of theligand or substitutions to AF2 residues in the estrogen re-ceptor make independent contributions to coactivator sen-sitivity by SRC-1. J Ster Biochem Mol Biol 67: 223–232,1998

27. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ,Agard DA, Greene GL: The structural basis of estrogenreceptor/coactivator recognition and the antagonism of thisinteraction by tamoxifen. Cell 95: 927–937, 1998

28. Smith CL, Oñate SA, Tsai MJ, O’Malley BW: CREB bindingprotein acts synergistically with steroid coactivator-1 to en-hance steroid receptor dependent transcription. Proc Natl AcadSci USA 93: 8884–8888, 1998

29. Tonetti DA, Jordan VC: Targeted anti-estrogens to treat andprevent diseases in women. Mol Med Today 2: 218–223,1996

30. Tonetti DA, Jordan VC: The role of estrogen receptor muta-tions in tamoxifen-stimulated breast cancer. J Ster BiochemMol Biol 62: 119–128, 1997

31. Xu J, Qui Y, DeMayo FJ, Tsai SY, Tsai MJ, O’Malley BW:Partial hormone resistance in mice with disruption of thesteroid receptor coactivator-1 (SRC-1) gene. Science 279:1922–1925, 1998

Address for offprints and correspondence: Dr Mark Nichols,Department of Pharmacology, University of Pittsburgh Cancer In-stitute, University of Pittsburgh, 200 Lothrop Street, Pittsburgh,PA 15213, USA; Tel.: 412 383 7344; Fax: 412 624 7737; E-mail:mnichols+@pitt.edu