Delineation of a unique protein-protein interaction site on the surface of the estrogen receptor.
ABSTRACT Recent studies have identified a series of estrogen receptor (ER)-interacting peptides that recognize sites that are distinct from the classic coregulator recruitment (AF2) region. Here, we report the structural and functional characterization of an ERalpha-specific peptide that binds to the liganded receptor in an AF2-independent manner. The 2-A crystal structure of the ER/peptide complex reveals a binding site that is centered on a shallow depression on the beta-hairpin face of the ligand-binding domain. The peptide binds in an unusual extended conformation and makes multiple contacts with the ligand-binding domain. The location and architecture of the binding site provides an insight into the peptide's ER subtype specificity and ligand interaction preferences. In vivo, an engineered coactivator containing the peptide motif is able to strongly enhance the transcriptional activity of liganded ERalpha, particularly in the presence of 4-hydroxytamoxifen. Furthermore, disruption of this binding surface alters ER's response to the coregulator TIF2. Together, these results indicate that this previously unknown interaction site represents a bona fide control surface involved in regulating receptor activity.
- SourceAvailable from: Sonia L Planey[Show abstract] [Hide abstract]
ABSTRACT: Abstract Most of the biological effects of estrogens are mediated via the estrogen receptors (ERs) at the level of gene regulation. Recently, new information regarding the role of ERs in physiology, pathology and the mechanisms through which estrogens bring about these functions has emerged. The physiological effects of estrogen are manifested through two ER isoforms - ERα and ERβ - which display distinct regions of sequence homology. The crystal structures of these receptors bound to their specific ligands (e.g. agonists or antagonists) have revealed much about how ligand binding alters receptor structure/conformation and the interaction with coactivators or corepressors as well as how it determines the cellular response to a ligand. ERs are involved in the variety of physiological and pathological activities and different cells and tissues have shown divergent responses to these two receptor isoforms. The discovery of sub-isoforms of ER alpha and beta has further complicated our understanding of how the interaction between ERs and its ligands contribute to the development of disease. Nevertheless, continuing efforts in the study of ERs have helped us to more clearly define their role in disease and to develop novel, ER-targeted therapeutics.Journal of Receptor and Signal Transduction Research 11/2013; · 1.63 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The estrogen receptors (ERs) are a group of versatile receptors. They regulate an enormity of processes starting in early life and continuing through sexual reproduction, development, and end of life. This review provides a background and structural perspective for the ERs as part of the nuclear receptor superfamily and discusses the ER versatility and promiscuity. The wide repertoire of ER actions is mediated mostly through ligand-activated transcription factors and many DNA response elements in most tissues and organs. Their versatility, however, comes with the drawback of promiscuous interactions with structurally diverse exogenous chemicals with potential for a wide range of adverse health outcomes. Even when interacting with endogenous hormones, ER actions can have adverse effects in disease progression. Finally, how nature controls ER specificity and how the subtle differences in receptor subtypes are exploited in pharmaceutical design to achieve binding specificity and subtype selectivity for desired biological response are discussed. The intent of this review is to complement the large body of literature with emphasis on most recent developments in selective ER ligands.International Journal of Environmental Research and Public Health 09/2014; 11(9):8709-8742. · 1.99 Impact Factor
Delineation of a unique protein–protein interaction
site on the surface of the estrogen receptor
Eric H. Kong*†, Nina Heldring†‡, Jan-Åke Gustafsson‡, Eckardt Treuter‡, Roderick E. Hubbard*, and Ashley C. W. Pike*§
*Structural Biology Laboratory, Chemistry Department, University of York, York YO10 5YW, United Kingdom; and‡Department of Biosciences at Novum,
Karolinska Institutet, S-14157 Huddinge, Sweden
Edited by Pierre Chambon, Institut de Genetique et de Biologie Mole ´culaire et Cellulaire, Strasbourg, France, and approved January 24, 2005 (received for
review September 28, 2004)
Recent studies have identified a series of estrogen receptor (ER)-
interacting peptides that recognize sites that are distinct from the
classic coregulator recruitment (AF2) region. Here, we report the
structural and functional characterization of an ER?-specific pep-
tide that binds to the liganded receptor in an AF2-independent
manner. The 2-Å crystal structure of the ER?peptide complex
reveals a binding site that is centered on a shallow depression on
the ?-hairpin face of the ligand-binding domain. The peptide binds
in an unusual extended conformation and makes multiple contacts
with the ligand-binding domain. The location and architecture of
the binding site provides an insight into the peptide’s ER subtype
specificity and ligand interaction preferences. In vivo, an engi-
neered coactivator containing the peptide motif is able to strongly
enhance the transcriptional activity of liganded ER?, particularly in
the presence of 4-hydroxytamoxifen. Furthermore, disruption of
this binding surface alters ER’s response to the coregulator TIF2.
Together, these results indicate that this previously unknown
interaction site represents a bona fide control surface involved in
regulating receptor activity.
coregulator ? phage display ? structure
iological effects of the steroid hormone 17?-estradiol (E2). Tran-
scriptional activation is facilitated by two distinct activation func-
tions (AF), the constitutively active AF1 located at the N terminus
of the receptor and a ligand-dependent AF2 that resides in the
ER agonists promote association with coactivators, whereas antag-
onists favor recruitment of corepressors.
The LBD serves as the major interaction point between nuclear
receptors (NRs) and coregulatory proteins. Coactivator recruit-
ment is mediated by short ?-helical, leucine-rich LxxLL consensus
motifs (NR-box) that bind to the AF2 region of the LBD (3–5).
stabilizes a receptor conformation in which elements of AF2 form
a hydrophobic groove that can accommodate the LxxLL motifs
found within coactivators (6–8). In contrast, ER antagonists affect
disrupting the LxxLL-binding site and preventing coactivator re-
cruitment (7, 9). The resultant antagonist-induced conformation
favors recruitment of corepressor complexes (10). Although the
details of corepressor binding to ERLBD are not known, antago-
nist-induced displacement of H12 in other NRs promotes core-
pressor recruitment to an interaction surface that overlaps with the
AF2 groove (11–13).
Although this conformational mechanism can be used to ratio-
nalize the differential recruitment of coactivators and corepressors
alone can account for the diverse pharmacology of selective ER
receptor modulators (SERMs). SERMs, such as raloxifene and
exhibit tissue-dependent pharmacology, imitating the action of
he estrogen receptor (ER) functions as a ligand-activated
transcription factor and is responsible for mediating the phys-
estrogens in certain tissues while opposing their action in others
(14). These compounds appear to elicit their effects by inducing
distinct conformational changes in ER that favor interaction with
specific subsets of coactivators (15). The mechanisms of such
coactivator binding are unclear but are likely to involve recognition
surfaces, which are unrelated to AF2, that are revealed in response
to a particular modulator.
The screening of random peptide libraries is a powerful method
Phage display techniques using both focused and random peptide
libraries have been used to investigate the interaction of coregula-
tors with liganded ER (13, 16–19). These studies have isolated a
number of short peptides with characteristic sequence motifs that
specifically recognize different liganded states of ER. Such pep-
shown to interfere with receptor activity both in vitro and in
cell-based assays (18, 19). These observations have led to the
suggestion that such motifs represent biologically relevant interac-
tion modules or, alternatively, that they mimic receptor–cofactor
interactions. Although some of the isolated motifs are directed to
the region around ER’s AF2 binding groove, several possess
with distinct regions of the receptor.
To address whether ERLBD harbors additional protein–protein
specifically to ER?. The 11-residue peptide antagonist, referred to
as ?II, was initially isolated from a random peptide phage display
library by using E2- or OHT-bound ER? and found to interact in
the presence of a broad spectrum of receptor modulators (16). In
this study, we have used a combination of techniques to both
delineate the ?II binding site on ER?LBD and demonstrate that
this surface is involved in ER’s response to certain ligands.
Materials and Methods
Protein Expression and Purification. The H12 truncated mutant
(ER??H12) used for structural analysis was generated by inserting
a stop codon after Val-533 in a pET15b-ER?LBD construct (20).
ER??H12 was expressed in Escherichia coli strain C41 (DE3).
ER??H12 was extracted from inclusion bodies with a buffer (150
mM NaCl?1 mM EDTA?2 mM DTT?10% glycerol?1 mM
PMSF?50 mM Tris, pH 8.0) containing 1% (wt?vol) Zwittergent
3-12. The detergent was removed by dialysis, and the extract was
applied to an E2–Sepharose column. The E2-affinity matrix was
prepared as described (21). Bound ER??H12 was carboxymethy-
lated overnight with 10 mM iodoacetic acid and eluted with 50 ?M
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: ER, estrogen receptor; E2 17?-estradiol; AF, activation function; LBD, li-
gand-binding domain; NR, nuclear receptor; OHT, 4-hydroxytamoxifen; SPR, surface plas-
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.pdb.org (PDB ID code 2BJ4).
†E.H.K. and N.H. contributed equally to this work.
§To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
www.pnas.org?cgi?doi?10.1073?pnas.0407189102 PNAS ?
March 8, 2005 ?
vol. 102 ?
no. 10 ?
OHT in a buffer containing 1 mM DTT, 1 mM EDTA, 250 mM
NaSCN, and 25 mM Tris (pH 8.5). ER??H12 was further purified
ER??H12 were combined with a 1.5-fold molar excess of ?II
peptide and concentrated to 10 mg?ml. Additional peptide was
added to obtain a final peptide?LBD molar ratio of 3:1.
Crystallization and Structure Determination. Hanging drops com-
posed of equal volumes of ER??H12-OHT-?II complex and
reservoir solution [20% (wt?vol) polyethylene glycol (PEG) 2000
monomethyl ether (Mme)?620 mM sodium formate?3% (wt?vol)
solution at 19°C. Crystals were cryoprotected in mother liquor
containing 32.5% (wt?vol) PEG 2000 Mme and vitrified in liquid
nitrogen. Crystals belong to space group P212121and have a single
LBD homodimer per asymmetric unit. Diffraction data, collected
to a resolution of 2 Å on beamline ID29 at the European Synchro-
and scaled by using the HKL suite of programs (22). The structure
was solved by molecular replacement using AMORE (23). Initial
?II peptide. The structure was refined with REFMAC5 (24) using all
available data. All model building was carried out with QUANTA
(Accelrys, San Diego). The final model has an Rcrystof 18.7% and
in Table 1, which is published as supporting information on the
PNAS web site.
Surface Plasmon Resonance (SPR). Measurements were performed
by using a BIAcore X instrument and streptavidin-coated sensor
chips. Experiments were carried out as described (8). Biotinylated
responses (60–160 relative units). Qualitative binding experiments
with liganded ER???LBD and LBD mutants were performed by
flowing each complex (1 ?M dimeric concentration) over the ?II
chip for 2 min at 5 ?l?min?1. A sensor chip comprising an immo-
bilized biotinylated SRC2–2 peptide (EKHKILHRLLQDS) (8)
was used as a control binding surface to assess the structural
LBD mutants, the kinetic analysis and preparation of protein used
as supporting information on the PNAS web site.
Mammalian Two-Hybrid and Reporter Assays. Details of the ER and
TIF2 expression plasmids used are described in Supporting Text.
Mammalian two-hybrid experiments were carried out as described
(19). For the transcriptional assays with the engineered TIF2
coactivator, Cos-7 cells were cotransfected with expression vectors
(pSG5) for wild-type ER? and either TIF2-NR12 or TIF2?II
Experiments were performed in triplicate and contained 30 ng of
ER? construct, 30 ng of ?-gal construct, 150 ng of reporter
construct, and 150 ng of TIF2 expression plasmid. After transfec-
tion, cells were treated with 10 nM E2, 100 nM OHT, or dimeth-
ylsulphoxide for 16 h before analyzing luciferase and ?-gal activity.
For the transcriptional assays comparing wild-type and mutant
expression vectors (pSG5 or Gal4DBD) for wild-type ER?, ER?
G442H, Gal4-tagged ER? DEF?H12, or Gal4-tagged ER?
DEFG442H?H12 and either empty expression vector (pSG5) or
TIF2wt expression vector together with either the estrogen respon-
sive 3? ERE-TATA-luc reporter or the Gal4 responsive reporter
5? UAS-TATA-luc. Each triplicate contained 30 ng of ER?
30 ng of TIF2 expression plasmid. After transfection, cells were
treated with 100 nM OHT or dimethylsulphoxide for 16 h before
analyzing luciferase and ?-gal activity.
?II Peptide Motif Interacts Specifically with Liganded ER?LBD. Pre-
vious reports of ?II binding to ER? showed that the interaction
for crystallization (9), we carried out real-time analysis of the
binding between ERLBD and the ?II interaction motif by using
SPR. A biotinylated ?II peptide immobilized onto a streptavidin-
on the PNAS web site). The ?II peptide interacts with ER?LBD
bound to both agonists (E2, diethylstilbestrol, genistein) and an
to that observed for full-length ER? (16). As previously reported
lacking the C-terminal activation helix H12 (ER??H12) retains its
ability to bind to the ?II peptide (Fig. 5B). Kinetic analysis
affinity and has an apparent dissociation constant (Kd) of 34 ?M
Structure of the ER?LBD-?II Peptide Complex. To get a better
understanding of the nature and location of the ?II-binding site on
ER?, we cocrystallized ER?LBD with an 11-residue peptide
corresponding to the ?II interaction motif (LTSRDFGSWYA).
Initial crystallization trials with various ER?LBD-?II complexes
did not yield crystals suitable for structural analysis. Based on our
observations that ?II binding was independent of H12, we also set
up trials using the truncated ER??H12 mutant. The resultant
complex, liganded with OHT, produced well diffracting crystals.
The overall ER??12 mutant structure is very similar to that seen
previously for ER?LBD in complex with either E2 (9) or OHT (7)
(rms deviation for ?-carbons of 0.51 and 0.69 Å, respectively).
Removal of H12 and the preceding loop has no discernable effect
on the integrity of the LBD fold.
Peptide-Binding Site. The ER??H12-OHT-?II peptide complex
crystallises with a single LBD homodimer in the asymmetric unit.
Each monomer within the homodimer interacts with a peptide
motif (Fig. 6A, which is published as supporting information on the
PNAS web site). Difference Fourier maps revealed well defined
electron density for the peptide, allowing us to model the entire
11-residue ?II sequence (Fig. 6B). The ?II motif binds across the
entrance of the solvent-filled channel that leads to the buried
receptor’s AF2 coactivator recruitment site (Fig. 1B). Residues
from H2, the H2–H3 loop, H6, the S1–S2 ?-hairpin, H8–H9 loop,
H9, and H10 contribute to an extensive binding surface (10 Å by 24
The ?II peptide adopts an extended conformation that closely
matches the surface topology of the LBD (Fig. 2A). Residues
turns 90° and climbs toward H10. This bent conformation is
stabilized by an intramolecular interaction between the backbone
carbonyl of Phe-6 and the indole nitrogen of Trp-9. Gly-7 appears
to fulfil a structural role because of the strained backbone dihedral
angles required at this position.
LBD recognition is mediated by an extensive network of
hydrogen-bonded and nonpolar interactions. No single interaction
appears to dominate, and the observed peptide conformation is
reliant on multiple intra- and intermolecular contacts. Binding of
the ?II motif buries 650 Å2of predominantly hydrophobic surface
area on the LBD. Residues from the peptide make a total of eight
direct hydrogen bonds with the LBD (Fig. 2B). The N terminus of
www.pnas.org?cgi?doi?10.1073?pnas.0407189102 Kong et al.
the peptide is anchored to the loop between H2 and H3. Ser-3
is completely buried and plugs the entrance to the solvent
channel. Contacts between the central portion of the motif and
the LBD are mainly hydrophobic in nature (Fig. 2C). Phe-6 lies
at the periphery of the site and packs against the aliphatic side
body of the LBD and packs against Gly-442 (H9) and Trp-393
(H6). Residues at the N-terminal end of H9 (Gln-441, Gly-442,
Glu-443, and Glu-444) make multiple hydrogen-bonded inter-
actions with C-terminal half of the ?II motif (Fig. 2B). The
C-terminal end of the motif is anchored by two separate inter-
actions. The main chain amides of Tyr-11 and Ala-12 interact
with carboxylate side chain of Glu-443. In addition, the side
chain of Tyr-10 projects over the H8–H9 loop and binds in a
narrow cleft between Gln-441 and Ala-493 (Fig. 2 A–C).
Comparison with all other ER?LBD ligand complexes demon-
strates that the ?II-binding site is structurally invariant. ?his
observation underlies ?II’s ability to bind to any agonist-, AF2
antagonist-, or pure antagonist-bound ER?. The motif’s specificity
for ER? appears to derive from the fact that parts of the site are
poorly conserved in ER? and other NRs (Fig. 2D). In particular,
sequence changes at the N-terminal end of H9 are likely to affect
?II binding. In ER?, Gly-442 and Glu-443 are replaced by a
histidine and lysine, respectively.
Mutagenesis Studies of LBD Binding to ?II. To validate the location
of the interaction site delineated in the crystal structure and to
investigate the ER?-specific nature of the interaction, we made a
series of single site LBD mutants and evaluated ?II-binding by
fact that residues lining the binding site fulfil key roles in main-
taining the structural integrity of the LBD. Consequently, only
residues that anchored the termini of the ?II motif were targeted
alanine (Q441, E443) or with the corresponding ER? residue type
(I326H, G442H, E443K).
Fig. 2E shows the binding response of each mutant (liganded to
and severely impaired the interaction. In contrast, binding of the
mutants to an immobilized LxxLL peptide, derived from the
NRbox2 region of TIF2 (SRC2–2) (8), was similar to that observed
for the wild-type LBD (Fig. 2E Inset). These control binding
experiments suggest that none of the mutations impacted on the
LBD’s ability to bind E2 and adopt an activated conformation
capable of recruiting coactivator. The mutant binding data
indicates that the inability of the ?II motif to interact with ER?
derives primarily from steric clashes with the bulkier residues
found at positions 326 and 442 (279 and 394 in ER?) in this
isoform (Fig. 2D).
Binding of ?II Peptide in Mammalian Cells. Previous studies have
demonstrated ?II binding to ER in a cellular context (18, 25). To
confirm that the ?II binding site observed in the crystal structure
we investigated the interaction by using a well established mam-
malian two-hybrid (M2H) assay (19). As demonstrated in Fig. 3A,
the ?II peptide interacted with full-length ER? in the presence of
both E2 and OHT, but not in the absence of ligand. Furthermore,
whereas this interaction was independent of ER’s AF2 domain
mutation abolished receptor–peptide association (Fig. 3 B and C).
In contrast, an LxxLL peptide (19) bound equally well to both the
wild-type and G442H-mutated full-length receptor in the presence
To elucidate which amino acid residues of the ?II motif are
essential for functional peptide–receptor interactions, we per-
formed limited alanine scanning mutagenesis on the ?II sequence
and tested variants for ER interaction in the M2H system (Fig. 3 D
and E). Replacement of Phe-6, Gly-7, Ser-8, Trp-9, or Tyr-10
completely abolished receptor interaction (Fig. 3E). In contrast,
substitution of Ser-3 had a moderate effect on ?II binding. In the
crystal structure, the O? of Ser-3 is buried and makes a single
hydrogen bond with the carboxylate of Glu-323. Although this
interaction appears to be dispensable for binding, the motif is
otherwise highly sensitive to sequence changes. Such a high degree
of sequence dependence reflects the nature of ?II binding, which
relies on multiple interactions with the LBD.
Enhancement of OHT-Dependent Transcriptional Activity by an Engi-
neered Coactivator. To investigate whether the ?II interaction
attempted to modify the behavior of the p160 coregulator TIF2
(GRIP1, SRC2, NcoA-2). TIF2 enhances ER-mediated transcrip-
tional activity in an AF2- and hormone-dependent manner (26).
TIF2’s receptor-interacting domain contains three NR boxes that
are involved in ligand-dependent NR interaction. Of these, NR box
1 and 2 have been implicated in high-affinity binding to ER (8, 27).
We inactivated NR box3 (TIF2–NR12) and replaced NR box 1 and
defined in the experimental electron density. Secondary structure elements are labeled, and the AF2 region of the LBD (H3–H5) is colored green. (B) Composite
surface representation of the ER?LBD monomer highlighting the location and extent of the known AF2 (green) and proposed ?II (cyan) coregulator sites. The
molecule is viewed in an identical orientation to the right ribbon representation in A.
Overall structure of ER?LBD in complex with the ?II interaction motif. (A) Ribbon representation of the ER??H12-OHT-?II structure. Two perpendicular
Kong et al.
March 8, 2005 ?
vol. 102 ?
no. 10 ?
When Cos-7 cells were cotransfected with expression vector for
full-length ER? along with reporter plasmids, we found that
coexpression of TIF2?II, but not TIF2–NR12, could markedly
increase ER activity in the presence of OHT (Fig. 4B). Identical
behavior was also observed with a truncated ER lacking the AF1
domain (ER?–DEF; data not shown). Similar results were ob-
served in HuH7 and HeLa cells. As expected, when the G442H
mutant receptor was analyzed under similar conditions, the mod-
whereas the observed coactivation effect of wild-type TIF2 was
unaffected (data not shown).
Mutation of ?II Surface Alters ER’s Sensitivity to Coregulators. Fi-
nally, we examined whether mutations of the ?II-binding surface
are able to affect ER?’s transcriptional behavior. The transactiva-
tion characteristics of the G442H mutant receptor were studied by
using either an ERE-luciferase (full-length ER?) or a Gal4-
mutant receptors responded in an identical manner to E2. Surpris-
ingly, the mutant receptor exhibited a substantial increase in
transcriptional activity in response to OHT when TIF2 was coex-
pressed (Fig. 4C). Removal of AF1 resulted in similar behavior for
the wild-type or mutant receptors regardless of whether TIF2 was
coexpressed (data not shown). However, although the wild-type
receptor requires AF2 for reporter activation, the G442H mutant
responds to the addition of TIF2 even in the absence of H12 (Fig.
4D). These data suggest that the ?II-binding surface mediates the
observed OHT response via TIF2 itself or via unknown cellular
coregulators that antagonize TIF2’s effects.
in complex with the phage display-derived ?II motif defines a
site. The ability of the ?II motif to recognize ER? regardless of the
bound ligand derives from the fact that its binding site is located in
a region of the LBD that is not affected by the distinct conforma-
tional effects induced by receptor agonists and antagonists. Previ-
that make key contacts with the peptide are represented in ball-and-stick form with green carbons. (B) Schematic representation of the interactions made by the ?II
peptide. The ?II peptide (purple bonds) and LBD residues (black bonds) are shown. Water molecules are depicted as cyan spheres. For clarity, only intermolecular
program LIGPLOT (33). (C) Surface representation of ?II site. The molecular surface of ER??H12 is shown and colored according to residue type (purple, hydrophobic;
with the ?II motif are represented asterisks and circles respectively. Key sequence differences are boxed. (E) Binding of mutant E2-liganded ER?LBDs. Sensorgrams
obtained from injection of 1 ?M wild-type and mutant ER?LBDs over a surface immobilized with ?II peptide. (Inset) Maximum binding response for each mutant to
control SRC2–2 (LxxLL) peptide and ?II peptide coated sensor chips. Responses shown are after 2-min continuous injection at a flow rate of 5 ?l?min?1.
www.pnas.org?cgi?doi?10.1073?pnas.0407189102Kong et al.
ous studies have shown that the AF2 region of the LBD serves as
13). The ?II site identified here is located on the opposite face of
the LBD (Fig. 1B). Both surfaces are predominantly hydrophobic
in character but differ in topology. The LxxLL binding groove is
compact, encompasses a relatively small surface area (450 Å2), and
is conserved in most NRs. In contrast, the ?II site covers a larger
area (650 Å2) and is specific to ER?. The concave ?II binding
surface incorporates conserved regions of the LBD that flank the
entrance to a channel that leads to the buried hormone binding
and creates a 300-Å3water-filled chamber. It is noted this chamber
corresponds to the so-called ‘‘second-binding site’’ that has been
evaluated as an alternative ligand-binding cavity (28). However, no
of ER?LBD, and its role in ligand-binding remains controversial.
Consequently, it is highly unlikely that the ?II site is involved in
binding small molecule ligands.
Biological Significance of the ?II Sequence. What is the likelihood
relevant recruitment motif? In the original phage display study, the
?II motif was only observed once and, therefore, no consensus
information is available (16). Our mutagenesis studies demonstrate
tion of GAL4-DBD-tagged ?II with VP16-tagged ERs. Full-
length ER? (A), ER??H12 (B), and ER?G442H (C). (D and E)
Amino acid requirements for ?II interaction. (D) Amino acid
sequences of peptides used. (E) Mammalian two-hybrid re-
ceptor binding assay of GAL4-DBD-tagged wild type and mu-
tant peptides to VP16-ER? wild type. Binding of the ?II and
LxxLL peptides were evaluated both in the absence of ligand
activity is shown.
ER?-peptide interaction in mammalian cells. Interac-
tional activity. (A) Schematic representation of NR-box
modified coregulator TIF2 containing the ?II peptide se-
quence. (B) Cell-based reporter assay of transcriptional ac-
tivity of engineered TIF2 variants in the presence of full-
length ER?. (C and D) Reporter activity of wild-type and
mutant ER in the presence of 100 nM OHT. Data are pre-
sented as percent activation, where the activity of full-
length ER? (C) and ER?DEF?H12 (D) are set to 100%.
Enhancement of tamoxifen-dependent transcrip-
Kong et al.
March 8, 2005 ?
vol. 102 ?
no. 10 ?