MOLECULAR AND CELLULAR BIOLOGY, Sept. 2004, p. 7681–7694
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 17
Single-Chain Estrogen Receptors (ERs) Reveal that the ER?/?
Heterodimer Emulates Functions of the ER? Dimer in
Genomic Estrogen Signaling Pathways
Xiaodong Li, Jing Huang, Ping Yi,† Robert A. Bambara, Russell Hilf,
and Mesut Muyan*
Department of Biochemistry and Biophysics, University of Rochester School of
Medicine, Rochester, New York
Received 31 March 2004/Returned for modification 4 May 2004/Accepted 1 June 2004
The effects of estrogens, particularly 17?-estradiol (E2), are mediated by estrogen receptor ? (ER?) and
ER?. Upon binding to E2, ERs homo- and heterodimerize when coexpressed. The ER dimer then regulates the
transcription of target genes through estrogen responsive element (ERE)-dependent and -independent path-
ways that constitute genomic estrogen signaling. Although ER? and ER? have similar ERE and E2 binding
properties, they display different transregulatory capacities in both ERE-dependent and -independent signal-
ing pathways. It is therefore likely that the heterodimerization provides novel functions to ERs by combining
distinct properties of the contributing partners. The elucidation of the role of the ER heterodimer is critical
for the understanding of physiology and pathophysiology of E2 signaling. However, differentially determining
target gene responses during cosynthesis of ER subtypes is difficult, since dimers formed are a heterogeneous
population of homo- and heterodimers. To circumvent the pivotal dimerization step in ER action and hence
produce a homogeneous ER heterodimer population, we utilized a genetic fusion strategy. We joined the cDNAs
of ER? and/or ER? to produce single-chain ERs to simulate the ER homo- and heterodimers. The fusion ERs
interacted with ERE and E2 in a manner similar to that observed with the ER dimers. The homofusion
receptors mimicked the functions of the parent ER dimers in the ERE-dependent and -independent pathways
in transfected mammalian cells, whereas heterofusion receptors emulated the transregulatory properties of the
ER? dimer. These results suggest that ER? is the functionally dominant partner in the ER?/? heterodimer.
Estrogen hormones, particularly 17?-estradiol (E2), exert
their effects through a complex array of convergent and diver-
gent signaling pathways that mediate genomic and nongenomic
events, resulting in target tissue-specific responses (11, 31).
The E2 information is conveyed by the transcription factors,
estrogen receptor ? (ER?) and ER? (11, 31), which are en-
coded by distinct genes and are expressed in different tissues as
well as in the same tissue at various levels (11, 31).
Upon binding to E2, ER dimerizes and interacts with per-
mutations of a palindromic DNA sequence separated by three
nonspecific nucleotides: 5?-GGTCAnnnTGACC-3?, the con-
sensus estrogen responsive element (ERE) (11, 18, 31). The
E2-ER-ERE complex subsequently recruits coactivators/regu-
lators to promote local chromatin remodeling and to bridge
with general transcription factors for the initiation of transcrip-
tion (11, 31). This pathway is called ERE-dependent ER sig-
naling. The E2-ER complex also regulates gene expression
through functional tethering to a transcription factor bound to
its cognate regulatory element on DNA. This is the DNA-
dependent and ERE-independent signaling pathway (22, 36).
Furthermore, E2 elicits effects through the membrane and
cytoplasmic ERs (24, 39).
ER? and ER? share high amino acid identity (96%) in their
DNA-binding domains (DBDs) (11, 31), which is reflected in
the abilities of ERs to bind to the same spectrum of ERE
sequences with similar affinities (27, 45). The carboxyl-terminal
ligand-binding domains (LBDs) also show considerable ho-
mology (53%) responsible for similar ligand-binding affinities.
Despite comparable biochemical properties, ERs differ in their
transregulation potencies in a ligand, promoter, and cell con-
text-dependent manner (11, 31). Studies have indicated that
the distinct amino-terminal A/B domains, a lesser conserved
region between ERs (30% homology), play critical roles in the
manifestation of transregulatory activity of the receptor sub-
type (5, 12, 28, 44).
In addition to acting as a homodimer, ER? heterodimerizes
with ER? in vitro and in situ when coexpressed (4, 34, 40).
However, the role of the ER?/? heterodimer in E2 signaling
remains largely unknown due to the presence of a heteroge-
neous population of ER dimers. An innovative approach was
introduced to address the functions of ER?/? heterodimer in
the ERE-dependent signaling pathway (40). This approach
changes the DNA-binding specificity of an ER subtype to that
of the glucocorticoid hormone receptor (GR) (10). Coexpres-
sion of a wild-type (WT) ER with a consensus glucocorticoid
(GRE)-binding ER allows the measurement of transcriptional
properties of the ER?/? heterodimer from a hybrid response
element composed of ERE and GRE half-sites without an
interference from the ER homodimers. Using this system, it
was shown that ER?/? heterodimer generates new attributes
to E2 signaling by combining functional properties of both
* Corresponding author. Mailing address: Department of Biochem-
istry and Biophysics, University of Rochester School of Medicine, 601
Elmwood Ave., Rochester, NY 14642. Phone: (585) 275-7751. Fax:
(585) 271-2683. E-mail: firstname.lastname@example.org.
† Present address: Department of Molecular and Cellular Biology,
Baylor College of Medicine, Houston, TX 77030.
contributing partners (40). Although a powerful approach for
structure-function analysis, this system could produce not only
ER?/? heterodimer but also ER? and ER? homodimers. Fur-
thermore, since the approach utilizes a hybrid DNA response
element, analysis of responses from natural EREs would be
precluded. In addition, in the presence of both homo- and
heterodimer ERs, examination of DNA-dependent and ERE-
independent regulation of estrogen-responsive genes by a spe-
cific ER dimer is difficult.
We recently sought an alternative approach to generate a
homogeneous population of homo- or heterodimers of ER? by
circumventing the pivotal dimerization step in receptor action
(30). We engineered a single-chain ?-? protein by genetically
conjugating two ER? cDNAs in tandem. Although a mono-
mer, the homofusion ?-? exhibited biochemical and functional
properties that mimicked those of the ER? dimer. Using this
genetic conjugation approach, we have now generated single-
chain ?-? and ?-? receptors to study the role of the ER?/?
heterodimer in genomic estrogen signaling pathways. The ho-
mofusion ?-? and ?-? simulated the functional properties of
the parent ER? and ER? dimers. The heterofusions ?-? and
?-?, on the other hand, emulated the functions of the ER?
genomic signaling pathways. These results suggest that ER?
dictates the transregulatory properties of the ER?/? het-
MATERIALS AND METHODS
Construction of fusion ER receptors and glutathione S-transferase (GST)
fusion cofactors. The human WT ER? and ER? cDNAs with or without the Flag
epitope were described previously (30, 38, 44). The WT ER? cDNA encodes a
530-amino-acid protein. Construction of fusion ERs was accomplished as de-
scribed previously (30). ERs defective in DNA binding were constructed by
changing two Cys residues in the first zinc finger of the C domain to His residues
at positions 202 and 205 in ER? and at positions 166 and 169 in ER?. Dimer-
ization defective ERs were constructed by changing three Leu residues at posi-
tions 504, 508, and 511 to Glu in ER? and at positions 455, 459, and 462 in ER?.
Mutant fusion receptors were engineered by exchanging mutant ER? and/or
ER? cDNAs in the WT fusion receptor cDNAs.
The generation of GST-cofactor fusion proteins was described previously (30,
38, 44). GST fusion steroid receptor cofactor 1 (SRC-1) and transcription inter-
mediary factor 2 (TIF-2) polypeptides contain nuclear interacting signature mo-
tifs within the region encompassing residues 219 to 399 (SRC-1219-399) and 623
to 986 (TIF-2623-986), respectively. The GST fusion TIF2-Q polypeptide contains
a glutamine-rich region that encompasses residues 1125 to 1325.
Synthesis and detection of fusion ERs in vitro and in situ. The linearized
pBluescript II KS bearing no cDNA or a receptor cDNA was transcribed by using
T3 RNA polymerase and translated by using a rabbit reticulocyte lysate as
directed by the manufacturer (Promega, Madison, Wis.). For quantification of
ERs synthesized in vitro, we used L-[methyl-3H]methionine (72 Ci/mmol; NEN
Life Sciences, Boston, Mass.), followed by fluorography as described previously
(30). Equal aliquots of reaction mixtures (5 ?l/50-?l reaction mixture) were also
subjected to Western blot analysis. Proteins were probed with a polyclonal ER?
(HC-20; Santa Cruz Biotechnology, Santa Cruz, Calif.)- or ER? (PA1-313;
Affinity Bioreagents, Golden, Colo.)-specific antibody directed to the carboxyl
terminus. Proteins were visualized by using the ECL-Plus Western blotting sys-
tem (Amersham Pharmacia). In Western blot analysis with a monoclonal Flag
antibody (M2; Sigma-Aldrich, St. Louis, Mo.), we used a Flag Western detection
kit as directed by the manufacturer (Stratagene, La Jolla, Calif.).
EMSA. Both strands of oligomers were synthesized and purified by the Inte-
grated DNA Technologies (Coralville, Iowa). Regions flanking test sequences
were identical. The oligomers were annealed,32P end labeled, and subjected to
electrophoretic gel mobility shift assay (EMSA) as described previously (30). We
used fivefold greater molar concentrations of ER? to obtain similar amounts of
ERE binding as described below. Equal amounts (10 ?g) of total proteins from
whole-cell extracts were used for EMSA. For the recruitment of cofactors, equal
molar concentrations of in vitro-synthesized receptors were processed as de-
scribed previously (45).
Estimation of relative DNA- and ligand-binding abilities of receptors. The
affinity of ER for various EREs was determined by electrophoretic gel mobility
shift competition assays as described previously (45). Ligand binding was carried
out as described previously (20).
A hydroxyl radical assay was done as previously described (45).
Cell culture and transfections. A receptor cDNA was excised from pBluescript
II KS(?) with XhoI and BamHI and inserted into a mammalian expression
vector (pM2-AH). For transient transfections, Chinese hamster ovary (CHO-
K1), human cervical carcinoma HeLa, and breast adenocarcinoma MDA-MB-
231 (American Type Culture Collection, Rockville, Md.) cells were used. Cells in
48-well tissue culture plates were transfected as described previously (30) by
using 75 ng of expression vector bearing no receptor or a receptor cDNA,
together with 125 ng of a reporter plasmid driving the expression of the firefly
luciferase enzyme cDNA. A reporter plasmid bearing the Renilla luciferase
cDNA (Promega; 2 ng/well) was used to monitor transfection efficiency. Cells
were then treated without or with 10?9M of E2 (Sigma-Aldrich) for 24 h.
Luciferase assays were performed with a dual luciferase assay kit (Promega). The
reporter vectors containing a TATA box promoter without or with one or two
EREs in tandem, the human complementary 3 (C3), and pS2 gene promoters
were described previously (30, 45). Reporter plasmids bearing collagenase (Col)
promoter and the RAR? gene promoter were described recently (15). To assess
the effects of ligands on ER-induced transcriptional responses from Col and
RAR? promoters, we used 125 ng of expression and reporter vectors/well. In
these series of experiments, cells were treated for 40 h in the absence or presence
of various concentrations of E2 or of ICI 182,780 (ICI; Tocris, Inc., Ballwin,
Mo.). The amount of ICI was based on the preliminary studies in which 10?7M
ICI was the optimal concentration to induce a response without eliciting cell
For Western blotting and EMSA, cells were plated onto six-well plates and
transiently transfected with 3 ?g of the expression vector bearing no cDNA or an
ER cDNA/well as described above. Cells were lysed in buffer containing 20 mM
Tris-HCl (pH 7.5), 400 mM KCl, 2 mM dithiothreitol, 1 mM phenylmethylsul-
fonyl fluoride, 20% glycerol, and 1% protease inhibitor cocktail (Sigma-Aldrich)
by a freeze-thaw cycle of three times. Then, 10 ?g of total protein was subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In
cotransfections, a constant amount of an expression vector bearing a receptor
cDNA was cotransfected with increasing concentrations of another expression
vector as described in the text.
For immunocytochemistry, cells were processed as described previously (30).
Heterodimerization of ERs. Previous studies showed that
coexpression of the human “short” ER? of 477-amino-acid
with the human WT ER? leads to the formation of the ER?/?
heterodimer in vitro and in situ (4, 34, 40). We wanted to
address whether the WT ER? (ER?) that contains an addi-
tional 53 amino acids at the amino terminus also heterodimer-
izes with WT ER? (ER?) when cosynthesized. In order to
facilitate the examination of biochemical and functional prop-
erties of heterodimer receptors, we initially examined the abil-
ities of ER homodimers synthesized in vitro and in situ to
interact with a
consensus ERE derived from the vitellogenin A2 gene by
EMSA. ER? and ER? cDNAs contain sequences that encode
a Flag epitope at the amino terminus. [3H]methionine was
used to quantify in vitro-synthesized ERs. Fluorography indi-
cated that ER? migrates at molecular masses of 67 and 46
kDa, the latter being a minor species synthesized at various
levels among experiments and not always observable (Fig. 1,
3H). ER? displayed an electrophoretic migration of 60 kDa.
The detection of proteins by Western blot analysis by using
ER? (HC-20 and HC)- or ER? (PA1-313 and PA1)-specific
antibody or the M2 (Flag) antibody for both receptors con-
firmed the identity of receptors. The absence of detection of
32P-end-labeled DNA fragment bearing the
7682LI ET AL.MOL. CELL. BIOL.
the minor species of ER? migrating at 46 kDa by the Flag
antibody either in vitro or in cell extracts from the transiently
transfected ER-negative CHO cells suggests that this is an
amino-terminally truncated ER? isoform. Two distinct ER?
species were also detected by HC in Western blots of trans-
fected CHO or ER?-positive T47-D cells derived from breast
ductal carcinoma. ER? showed a 60-kDa band by the PA
antibody in cell extracts from transfected CHO or ER?-posi-
tive PA-1 cells of ovarian teratocarcinoma.
Despite similar amounts of receptors as assessed by the Flag
antibody in Western blots (Fig. 1B, WB), the extent of inter-
action of the in vitro-synthesized ER? with an equal concen-
tration of ERE (Fig. 1B, EMSA) was significantly lower than
the in situ-synthesized ER? whether or not a saturating con-
centration (10?7M) of E2 was present (data not shown).
Moreover, the ER? synthesized in vitro showed a diffuse ERE-
binding pattern that gradually formed a distinct complex as a
result of increasing receptor concentration. In contrast, the
ER? synthesized in situ interacted with ERE efficiently. ER?
synthesized in S2 (data not shown) cells or in Sf9 insect cells
FIG. 1. In vitro and in situ synthesis of ER? and ER?. (A) For in vitro synthesis of ERs, 1.5 ?g of linearized pBS-KS bearing no cDNA or a
cDNA for an ER was transcribed by T3 polymerase and translated by rabbit reticulocyte lysate in the presence of 4 ?l of [3H]methionine in a total
reaction volume of 50 ?l. Equal amounts (5 ?l) of reaction mixtures were subjected to SDS–7.5% PAGE, visualized by fluorography (3H) or by
Western blotting with an antibody directed to the carboxyl terminus of ER? (HC), of ER? (PA1) or the amino-terminal Flag epitope (Flag) of
both ERs. For in situ synthesis of ERs, CHO cells were transiently transfected with 3 ?g of expression vector bearing none (V) or a cDNA for
an ER. After 24 h, cells were lysed, and equal amounts (10 ?g) of total cellular proteins were resolved by SDS–7.5% PAGE, followed by Western
blotting with Flag, HC, or PA1 antibody. Endogenously expressed ER? in T47D cells (T47D) and ER? in PA-1 cells (PA-1) were also analyzed
by Western blotting with HC and PA1 antibodies, respectively. NS, nonspecific protein. (B) In vitro (InV, 10 ?l) and in situ (CHO, 10 ?g)
synthesized ER? were analyzed by Western blotting with Flag antibody. The ERE binding of ER? synthesized in vitro (InV) or in CHO cells
(CHO) was assessed by EMSA. Briefly, 1, 2, 4, 7, and 10 ?l of transcription-translation mixture and 1, 2, 4, 7, and 10 ?g of total protein from cell
extract were incubated with 62.5 pmol of32P-labeled ERE-containing DNA fragment for 30 min at 4°C and then subjected to 5% nondenaturing
PAGE. The gel is dried and exposed to a PhosphorImager. Unbound ERE (ERE) and ERE-ER? complexes are indicated. (C) The binding of
ER? (lanes 2, 3, and 4) and ER? (lanes 5, 6, and 7) synthesized in vitro (In Vitro) or in transiently transfected CHO cells (CHO) to the consensus
ERE was assessed by EMSA. In this assay, 10 ?l of in vitro-synthesized ER? and 2 ?l of ER? were used to obtain similar amounts of ERE-bound
receptors. Reactions were incubated in the absence or presence (?) of the Flag (lanes 4 and 7), HC (lane 3), or PA1 (lane 6) antibody. ER-ERE
complexes representing truncated-ER? homodimer (complex 1; the least prominent ER-ERE complex), truncated-ER?–WT-ER? heterodimer
(complex 2), and WT-ER? homodimer (complex 3) are indicated. Lane 1 indicates reactions with the parent expression vector.
VOL. 24, 2004 ER?/? HETERODIMER EMULATES FUNCTIONS OF ER? DIMER7683
(45) also binds efficiently to DNA. Although it is not clear,
differential processing of ER? species in vitro versus in situ
could lead to differences in the extent of homodimerization of
ER? and/or stability of the ER? homodimer. This, in turn,
could be manifested as differences in the amount and pattern
of ER? interaction with DNA.
ER? synthesized in vitro formed three distinct ERE-bound
complexes as detected by EMSA in the absence or presence of
the HC antibody: a major complex and two minor complexes
(Fig. 1C, In Vitro), as reported previously (4, 29). The slowest-
migrating complex (complex 3) likely represents the WT ER?
homodimer. The fastest-migrating band, the least prominent
complex, corresponds to the amino-terminally truncated ER?
homodimer (complex 1). The intermediate ER-ERE complex
appears to be the heterodimer of the WT and the truncated
ER? species (complex 2). This conclusion is suggested because
the Flag antibody specific to the amino-terminal Flag epitope
fails to retard the migration of the complex 1 (lane 4). A
similar ERE-binding pattern was also observed with the ER?
synthesized in situ (Fig. 1C, CHO). The ER? synthesized in
vitro, which we used fivefold more compared to ER? to obtain
similar amount of binding, forms a single ER-ERE complex
that migrates somewhat faster than the complex 2 of the ER?,
whereas the ERE-bound in situ-synthesized ER? migrates
slightly faster than the complex 3 of ER?.
To examine the formation of a functional ER?/? het-
erodimer, we used a constant amount of the ER? expression
vector, together with increasing amounts of ER? expression
vector in transcription-translation reactions or in transfection
into CHO cells. The relative amounts of ER proteins were
assessed in Western blots of equal aliquots of the in vitro
reactions (Fig. 2, WB, In Vitro) or of CHO cell extracts (CHO)
with the Flag antibody. The results indicated that increasing
amounts of ER? were cosynthesized with a constant amount of
ER?. To assess the formation of a functional heterodimer, we
used ER? without the Flag epitope to distinguish ER subtypes
with the use of the Flag antibody. The cosynthesis of ERs in
vitro or in situ led to the formation of a functional ER?/?
heterodimer that showed an electrophoretic migration be-
tween ER?-ERE complex 3 representing the ERE-bound
ER? homodimer and the ER? homodimer in EMSA (Fig. 2,
EMSA). The extent of heterodimer formation was correlated
with a gradual decline in the ER?-ERE complex. It is also
evident that residual ER homodimers were still present.
Thus, the coexpression of WT ER?, as shown previously for
the “short” ER? (4, 34), with ER? leads to the formation the
ER?/? heterodimer, the extent of which is dependent upon the
relative amount of the contributing partners.
Construction of fusion ERs. To study the effects of ER?/?
heterodimer in genomic estrogen signaling, we used a genetic
conjugation approach (30) to circumvent the pivotal dimeriza-
tion step in receptor action. Single-chain ERs were produced
by joining the 3? end of the coding sequence of one ER? or
ER? cDNA to the 5? coding sequence of another to encode
two ER monomers in tandem (Fig. 3A) without or with a Flag
epitope at the amino terminus. Analysis of radiolabeled pro-
teins by fluorography revealed that the homofusion ?-? and
?-? and the heterofusion ?-? and ?-? displayed electro-
FIG. 2. Coexpression of ER? and ER? in vitro and in situ leads to formation of ER?/? heterodimer. For Western blotting of in vitro-
synthesized ERs, 0.3 ?g of pBS-KS vector bearing the Flag ER? cDNA was coexpressed with 0.3, 0.6, 1.2, or 1.8 ?g of vector bearing the Flag-ER?
cDNA that correspond to 1-, 2-, 4-, or 6-fold-greater amounts, respectively, of the expression vector. Equal amounts (5 ?l) of reaction mixtures
were subjected to SDS–7.5% PAGE, followed by Western blotting with the Flag antibody. Migration of ER? and ER? are indicated. The
dimerization of ER? and ER? was assessed by EMSA with equal amounts of transcription-translation mixture (5 ?l). For Western blotting of in
situ-synthesized ERs, 0.5 ?g of the mammalian expression vector bearing the Flag-ER? cDNA was cotransfected with 0.5, 1.0, 2.0, or 3.0 ?g
(corresponding to 1, 2, 4, or 6, respectively) of the expression vector carrying the Flag-ER? cDNA in CHO cells. Equal amounts of cell extracts
(10 ?g of total protein) were subjected to SDS–7.5% PAGE, followed by Western blotting with the Flag antibody. For EMSA, the cosynthesis of
ERs was accomplished as described above, except that the cDNA for ER? does not contain sequences for the Flag epitope to distinguish ER
species by the use of the antibody. Equal amounts of total protein (10 ?g) of CHO cell extracts were subjected to EMSA. ERE-ER complexes were
analyzed by using the HC or Flag antibody. ERE-bound ER? and ER? homodimers and the ER?/? heterodimer are indicated. Unbound ERE
is not shown.
7684LI ET AL.MOL. CELL. BIOL.
phoretic migrations with molecular masses of 120 to 136 kDa
that correspond to two ER molecules (Fig. 3B,3H). The de-
tection of proteins by Western blot analysis with ER? (HC)-,
ER? (PA1)-, or Flag epitope-specific antibody (Flag-InV) con-
firms the identity of receptor species. Since radiolabeled coun-
terparts and the Flag antibody show synthesis at comparable
levels, differences in the extent of detection of ?-? and ?-? by
the receptor-specific antibodies in Western blotting are likely
due to a position-dependent alteration in the epitope recogni-
Similarly, the fusion receptors synthesized in transiently
transfected CHO cells migrated with molecular masses ranging
from 120 to 136 kDa assessed by the Flag antibody, whereas
ER? and ER? migrated with molecular masses of 67 and 60
kDa, respectively (Fig. 3B, Flag-CHO).
Characterization of biochemical properties of the fusion
ERs. (i) DNA binding. We examined by EMSA whether single-
chain receptors synthesized in vitro (Fig. 3C) or in situ (Fig.
3D) interact with the consensus ERE. Equal amounts of the in
vitro-synthesized ER? and fusion receptors retarded the elec-
trophoretic migration of labeled ERE. Due to inefficient ERE
binding of the ER? synthesized in vitro, we used a fivefold
greater concentration of receptor to obtain similar amounts of
ERE-bound receptors. The ERE-bound homo- and heterofu-
sion ERs showed migrations comparable to those of the ER
dimers. The interaction of receptor species with ERE is spe-
cific, because the protein-ERE complex was further retarded
by the Flag antibody. Similarly, equal amounts of cell extracts
of transfected CHO cells synthesizing an ER or a single-chain
receptor interacted efficiently with the consensus ERE in vitro.
Since ERs bind to EREs as dimers, similar migration among
ERE-bound WT ERs and fusion species suggests that the
fusion receptors interact with DNA as monomers. We also
observed that the ERE-bound homo- and heterofusion pro-
teins showed slightly faster electrophoretic migrations than
ER?, possibly reflecting differences in size and in conforma-
tion among ER species. We took advantage of these differ-
ences in the electrophoretic mobility to test the conclusion that
the fusion receptors indeed bind to ERE as monomers and,
implicitly, do not dimerize. We used the ER? expression vec-
tor together with the same (1:1) or a twofold-greater (1:2)
amount of the expression vector bearing the ?-? or the ?-?
(data not shown) cDNA in transcription-translation reactions
with radiolabeled Met (Fig. 4A, upper panel). The ER? cDNA
lacks the amino-terminal Flag epitope to discern ERE-bound
receptor species by the use of Flag antibody. Reactions were
subjected to EMSA (Fig. 4A, lower panel). The Flag antibody
shifted only the ERE-bound ?-? without altering the migration
FIG. 3. Construction and synthesis of fusion ERs. (A) Schematics of ER fusion receptor cDNAs. A PCR-generated NdeI restriction enzyme
site at the 5? or 3? end of an ER was used to genetically fuse two ER cDNAs in tandem. (B) Synthesis of fusion ERs in vitro with the parent vector
bearing no cDNA (V) or a cDNA for an ER was accomplished as described for Fig. 1. Equal amounts of in vitro reaction mixtures (5 ?l) or of
(10 ?g of total protein) of cell extracts from transfected CHO cells (Flag-CHO) were subjected to SDS–7.5% PAGE. The in vitro samples were
visualized by fluorography (3H). The same samples were also probed with the HC, the PA1, or the Flag antibody (Flag-InV). (C) ERE binding
of in vitro or in situ (CHO)-synthesized fusion receptors. Equal amounts (2 ?l) of reaction mixtures, with the exception of the mixture containing
ER? (10 ?l), were incubated with radiolabeled ERE in the absence or presence of the Flag antibody. Reaction mixtures were electrophoresed by
5% nondenaturing PAGE. (D) Equal amounts of CHO cell extracts (10 ?g) were subjected to EMSA. The results from a representative
experiment of two independent experiments are shown.
VOL. 24, 2004 ER?/? HETERODIMER EMULATES FUNCTIONS OF ER? DIMER 7685
of ER?. This result demonstrates that the fusion receptors
bind to ERE as monomers.
Each DBD of ER consists of two zinc finger-like modules
that form a single functional domain. Each module contains a
zinc ion with tetrahedral coordination by four Cys residues (8).
Substitution of two of the zinc-coordinating Cys with His res-
idues of the first zinc finger in the DBD of ER? prevents the
receptor from interacting with DNA (21). If the fusion recep-
tors bind to ERE as monomers, analogous mutations in the
DBD of one ? monomer should prevent the interaction of the
variant fusion ERs with an ERE. Although synthesized at
comparable levels, as assessed by fluorography (Fig. 4B, upper
panel), the mutant homofusion ??-? and heterofusion ??-?
failed, just as the DNA-binding defective ER?? dimer had
failed, to bind to ERE in EMSA in contrast to the WT coun-
terparts (Fig. 4B, lower panel). This result is consistent with
the conclusion that fusion receptors bind to DNA as mono-
The dimerization of ER? is primarily mediated by a surface
located within the LBD of each monomer (21, 23). The ab-
sence of dimerization also suggests that two ER monomers in
a single-chain receptor fold into a compact structure whose
formation may or may not be dependent upon interactions
between LBD dimerization surfaces. To address this point, we
generated dimerization defective ER?. A previous study
showed that changing of three Leu residues to Glu in the helix
11 of the LBD of the murine ER? prevents dimerization (41).
We made analogous mutations by changing three Leu residues
at positions 504, 508, and 511 to Glu. Similarly, we produced
variant homofusion ?-? that bears these same mutations in
either (?dd-? or ?-?dd) or both (?dd-?dd) ER? monomers, and
we produced the heterofusion ?dd-? that has mutations in the
ER? monomer. The synthesis of mutant and WT fusion re-
ceptors was assessed by fluorography (Fig. 4C, upper panel).
EMSA revealed that the dimerization defective ER?dd, as
expected, did not significantly interact with an ERE (Fig. 4C,
lower panel). Although the mutant fusion receptors (?dd-? is
shown in Fig. 4), bound to an ERE, the interaction of these
receptors with the ERE was also compromised (?3-fold) com-
pared to the WT counterparts when the binding was assessed
FIG. 4. (A) The fusion ERs bind to ERE as monomers. The expression plasmids bearing ER? and the same (1:1) or a twofold-greater (1:2)
concentration of the expression vector bearing the heterofusion Flag-?-? were cosynthesized in the presence of [3H]methionine in vitro. Equal
amounts (5 ?l) of reaction mixtures were subjected to SDS–7.5% PAGE or EMSA in the absence or presence (?) of the Flag antibody. (B) Intact
DBDs are required for binding to ERE. Radiolabeled WT or DNA-binding defective (❋) ER?, the homofusion ?-?, or the heterofusion ?-? were
subjected to SDS-PAGE and EMSA in the absence or presence (?) of Flag antibody. (C) Effect of dimerization surfaces in the LBDs on the ability
of fusion receptor to bind to ERE. Equal amounts (5 ?l) of WT or variant, with mutations in dimerization interface (indicated by the “dd”
subscript), ER?, ?-?, or ?-? synthesized in vitro were subjected to SDS-PAGE. Then, 100 pM concentrations of each construct were electro-
phoresed on 5% nondenaturing PAGE (EMSA) in the absence or presence (?) of Flag antibody. (D) Dimerization interfaces in LBDs are
required for an efficient binding of the fusion receptors to ERE. Equal molar concentrations (0, 6, 12, 25, and 50 pM [lanes 1, 2, 3, 4, and 5,
respectively]) of WT and mutant (subscript dd) ?-? or ?-? were subjected to EMSA. For all experimental series, a representative experiment from
at least two independent experiments is shown.
7686 LI ET AL.MOL. CELL. BIOL.
at low concentrations of receptors (Fig. 4D). This indicates
that the interactions between dimerization surfaces of both
ER? monomers of the single-chain species increase the effi-
ciency with which the single-chain receptor interacts with ERE.
Moreover, the ?dd-? heterofusion, or homofusion ?dd-?dd,
as its WT counterpart, did not dimerize with the cosynthesized
ER? (data not shown). Thus, fusion receptors fold to allow
complementary interactions of the dimerization interfaces in
the two LBDs, despite the head-to-tail fusion of two ER mono-
mers in a single-chain configuration.
(ii) DNA-binding specificity. Efficient binding of single-
chain receptors to ERE in a manner similar to the ER dimers
implies that the DNA-binding specificity of ER is also pre-
served in fusion receptors. Therefore, we assessed the interac-
tion of receptor species with DNA fragments that contain
various test sequences (Fig. 5A) by EMSA (Fig. 5B). Previ-
ously, we (45) and others (27) have shown that ER? and ER?
bind to various ERE sequences with similar preference and
affinity. Likewise, WT and fusion receptors bound the consen-
sus ERE and ERE sequences derived from the estrogen-re-
sponsive pS2, complementary 3 (C3), oxytocin (Oxy) genes,
and the thyroid hormone responsive element (TRE). Recep-
tors had very little, if any, interaction with GRE and vitamin D
responsive element (VDRE). There was no observable inter-
action with half-ERE (1/2ERE) or a mutant ERE sequence
that bears three nucleotide substitutions in the consensus
Conservation of the DNA-binding specificity of ER? in sin-
gle-chain receptors also suggests that the fusion receptors uti-
lize both half-sites in an ERE for binding. We tested this
prediction by using the missing nucleoside hydroxyl radical
assay that allows the analysis of DNA-protein interaction at a
single-nucleotide resolution. It is expected that the protein
binding to DNA would be adversely affected if a nucleoside
important for binding were missing (13, 45). A low-intensity or
missing band in the lane containing ERE-bound ERs (B) or,
conversely, a high-intensity band in the lane containing free
ERE (F), identifies a nucleoside important for the formation
of the ER-ERE complex. A high ratio of free to bound ERE is
represented by a long horizontal bar in Fig. 5C; the length of
a bar represents the strength of contacts with ERs. The results
revealed that the fusion receptors, just as for the ER? dimer,
occupied both half-sites of the consensus ERE by contacting
the same nucleosides with similar strength (Fig. 5C).
Moreover, the fusion receptors bound to the consensus, pS2
and Oxy ERE sequences with affinities similar to those of the
FIG. 5. (A) Upper strand of the sequence containing the consensus ERE (in brackets), pS2, and Oxy ERE, GRE, VDRE, and TRE responsive
elements and 1/2 ERE and mERE with the underlined five- and three-nucleotide changes from the ERE, respectively. The central base spacer is
shown in lowercase. (B) Fusion ERs bind to the same spectrum of response elements. Equal molar concentrations of in vitro-synthesized ER?
dimer or a fusion receptor were subjected to EMSA. A representative phosphorimage of two independent experiments is shown. (C) Fusion
receptors bind to ERE in a manner similar to ER?. Critical nucleosides for ER-ERE interaction were identified by missing nucleoside hydroxyl
radical assay. End-labeled DNA fragment containing consensus ERE was randomly cleaved by hydroxyl radical, incubated with 50 ?l of
transcription-translation mixtures, and subjected to 5% nondenaturing PAGE. Radioactive bands containing bound and free DNA were excised,
eluted, and subjected to 15% sequencing gel electrophoresis. The intensities of individual DNA bands from sequencing gels were quantified by
using a PhosphorImager. The ratio of free (F) to bound (B) DNA at each base was plotted as horizontal bars, the length of which approximates
the strength of nucleoside contact with the protein. Uncut (U), Maxam-Gilbert G reaction-cut (G), and randomly cut (C) DNA fragments were
also electrophoresed. A representative phosphorimage from at least two independent experiments is shown.
VOL. 24, 2004ER?/? HETERODIMER EMULATES FUNCTIONS OF ER? DIMER 7687
ER? dimer as assessed by DNA competition assay (http:
//dbb.urmc.rochester.edu/labs/muyan/figure.htm). Likewise, all
receptor species bound to E2 with comparable affinities
(http://dbb.urmc.rochester.edu/labs/muyan/figure.htm). We al-
so found that stoichiometry of E2 binding among ER species
was primarily conserved. For example, as we reported previ-
ously (30), 1 mol of ER? binds 1 mol of E2, whereas 1 mol of
?-? or ?-? binds 1.86 ? 0.11 or 1.77 ? 0.19 (n ? 3) mol of E2,
respectively. Thus, the folding of the fusion ERs allows the
receptors to bind to ERE and E2 efficiently.
(iii) Cofactor interactions. The p160 family of coregulators
interacts with the agonist-nuclear receptor complex through a
LXXLL nuclear receptor interacting motif (NRIM) (14).
Upon E2 binding, the LBD undergoes a conformational
change by which the helix 12 located at the carboxyl terminus
of the LBD is realigned over the ligand-binding pocket (3, 35).
Since the conjugation approach uses a fusion between the
amino terminus of one monomer and the carboxyl terminus of
the other, the critical alignment of helix 12 could be compro-
mised despite the fusion receptors bind to E2 efficiently.
To address this issue, we examined the abilities of the fusion
receptors to interact with cofactors by using EMSA. We (45)
and others (1, 19, 26, 33, 42, 43) showed that the p160 family
of cofactors, including TIF-2 and SRC-1, interact with ER?
and ER? in an agonist-dependent manner. Equal molar con-
centrations of fusion receptors synthesized in vitro and the
ER? dimer were preincubated without or with a saturating
concentration (10?7M) of E2. Samples were then incubated
with an end-labeled ERE. GST alone or the GST fusion
polypeptide of TIF-2623-986containing NRIM was added into
the reaction mixtures at increasing concentrations. Reactions
were subjected to EMSA (Fig. 6A). GST alone at any concen-
tration had no effect on the electrophoretic mobility of the
ERE-ER complexes in the absence or presence of E2 (data not
shown). The electrophoretic mobility of ERE-bound ER? was
quantitatively retarded by TIF-2 in response to E2 (lanes 7 to
10). E2 also enhanced the ability of the fusion receptors to
recruit TIF-2 with affinities about twofold lower than that of
the ER? dimer. SRC-1219-399(SRC-1) interacted with the E2-
bound ER?-ERE complex. The SRC-1 was also recruited by
FIG. 6. (A) Interactions of cofactors (CF) with fusion ERs as assessed by EMSA. Equal molar concentrations of in vitro-synthesized receptors
were preincubated in the absence (?, lanes 1 to 5) or presence (?, lanes 6 to 10) of 10?7M E2 (E2), followed by the addition of the end-labeled
consensus ERE. The reactions were then incubated with 0 (?),1.56, 6.25, 25, or 100 ng (lanes 1 to 5 and 6 to 10, respectively) of GST fusion
TIF-2623-986(TIF-2) or 0, 0.125, 0.25, 0.5, and 1 ?g (lanes 1 to 5 and 6 to 10, respectively) of SRC-1219-399(SRC-1). Reactions were resolved on
5% nondenaturing PAGE. A representative phosphorimage of two independent experiments is shown. Unbound ERE is not shown. (B) Inter-
action of increasing concentrations (0.3, 0.6, and 1 ?g) of TIF21125-1325(TIF2-Q)-GST fusion protein with ERE-bound ERs. A representative
phosphorimage of two independent experiments is presented. Free DNA is not shown.
7688 LI ET AL.MOL. CELL. BIOL.
the fusion receptors, however, with efficiencies ?5-fold lower
than that observed with the ER? dimer. Apparent differences
in the extent of SRC-1 recruitment compared to TIF-2 by the
fusion receptors also suggest differences in the binding affini-
ties of cofactors for fusion receptors.
In addition to NRIM, TIF-2 through a glutamine-rich region
(Q) also interacts efficiently with the amino terminus of ER?,
but not of ER?, independent of E2 (38, 43, 45). To examine
whether this receptor-subtype specific interaction is preserved
in the heterofusion ERs, the interaction of the GST fusion
TIF-2 containing residues 1125 to 1325 (TIF2-Q) was tested in
EMSA with receptors synthesized in vitro (Fig. 6B). The re-
sults revealed that increasing concentrations of the cofactor
gradually retarded the electrophoretic migration of the ERE-
bound ER? and ?-? in the absence or presence (data not
shown) of 10?7M E2. The ?-? bound to the cofactor with
?2-fold-lower affinity than the dimer ER?. The TIF-Q, on the
other hand, did not affect the electrophoretic migration of
ER? and ?-?. TIF2-Q interacted quantitatively with the het-
erofusion receptors, however, with affinities twofold lower than
those observed with the homofusion ?-?.
Overall, these results suggest that cofactor interacting sur-
faces in the fusion receptors are altered.
Biological activities of the fusion ERs. (i) ERE-dependent
ER signaling. We next examined whether the homofusion ?-?
and ?-? simulate the transactivation properties of the parent
ER dimers. Furthermore, we attempted to determine whether
the heterofusions ?-? and ?-? display novel functions by in-
corporating functional properties of both contributing partners
in genomic estrogen signaling pathways. ER? has considerably
less transcription potency than ER? in ERE-dependent signal-
ing pathways independent of the promoter and cell context (11,
31). The expression vector without or with cDNA for an ER
was cotransfected into CHO cells, together with a reporter
plasmid bearing no (TATA), one (1ERE), or two (2ERE)
consensus EREs placed upstream of a simple TATA box
(TATA) promoter that drives the expression of the firefly
luciferase cDNA as reporter. Normalized activity from each
reporter construct was compared to the basal activity from the
reporter bearing no ERE in response to the parent expression
vector (V) in the absence of E2, with the latter value set to one.
Proteins were synthesized at similar amounts (Fig. 3B, Flag-
FIG. 7. (A) Intracellular localization of fusion ERs was examined by immunocytochemistry. The expression vector without (V) or with a cDNA
for Flag ER?, ER?, or fusion ?-? was transiently transfected into CHO cells. The proteins were probed with the Flag antibody and visualized by
using fluorescein-conjugated secondary antibody (FITC). DAPI (4?,6?-diamidino-2-phenylindole) staining indicates the nucleus. There was no
protein detectable in cells transfected with the parent vector. The absence or presence (data not shown) of 10?9M E2 did not affect the
intracellular localization of the ERs. (B) CHO cells were transiently transfected with 75 ng of expression vector bearing a cDNA for an ER,
together with 125 ng of reporter plasmid bearing no (TATA), one (1ERE), or two copies of the consensus ERE (2ERE). The TATA box promoter
drives the expression of the firefly luciferase cDNA. The transfection efficiency was monitored by determining the coexpression of 2 ng of reporter
plasmid bearing the Renilla luciferase cDNA. Cells were treated without (data not shown) or with 10?9M E2 (E2) for 24 h. The data represent
the means ? the standard errors of the mean (SEM) of three independent experiments performed in duplicate. (C) CHO cells transiently
transfected as described above by using a reporter plasmid bearing the C3 or pS2 promoter that drive the expression of the firefly luciferase enzyme
cDNA. Cells were treated in the absence (?E2) or presence (?E2) of 10?9M E2 for 24 h. The results from three independent experiments in
duplicate are represented as the means ? the SEM.
VOL. 24, 2004ER?/? HETERODIMER EMULATES FUNCTIONS OF ER? DIMER 7689
CHO) and localized to the nucleus in the absence (Fig. 7A) or
presence (data not shown) of a physiological concentration
(10?9M) of E2. As we showed previously (44), ER?, but not
ER?, induced transcription synergistically from two ERE-con-
taining reporter constructs in CHO cells (Fig. 7B). Although
?-? increased luciferase activity synergistically in response to
E2, albeit to a lesser extent than that observed with ER?, ?-?
had only an additive effect on luciferase activity. On the other
hand, heterofusion ?-? or ?-? induced synergy at levels com-
parable to that observed with the ?-?. These results show that
the homofusion ?-? and ?-? emulate the activities of the
parent receptors and suggest, as proposed previously (40), that
ER? is the dominant partner in the ER heterodimer when
tested from a simple TATA box promoter construct bearing
only tandem consensus EREs.
The expression of estrogen-responsive genes is the result of
integrated effects of various trans-acting factors that are critical
for gene expression. We examined the biopotencies of the
fusion ERs from constructs bearing the enhancer-promoter
region of estrogen-responsive pS2 and C3 genes that drive the
expression of the luciferase enzyme cDNA. To accomplish this,
we transiently transfected CHO cells with an expression vector
bearing a receptor cDNA (Fig. 7C). Normalized activity from
each reporter was compared to the promoter activity in re-
sponse to the parent expression vector (V) in the absence of
E2, with the latter value set to one. ER? and the homofusion
?-? in response to 10?9M E2 augmented the luciferase activity
from both the pS2 and C3 promoters ?6-fold, whereas ER?
and ?-? increased transcription ?2-fold. The heterofusion
ERs showed biopotencies that were similar to those observed
with ER? and ?-? from the C3 promoter. The biopotencies of
the heterofusions were, on the other hand, intermediate be-
tween the homofusion receptors that simulated the transcrip-
tion activities of the parent ER? and ER? when tested with the
pS2 promoter construct.
In HeLa cells, the heterofusion ERs also induced synergy
with biopotencies that simulate that of ?-?, which showed less
potency than the ER? dimer (Fig. 8). The ER? dimer and ?-?
had little effect on luciferase activity. The magnitude of tran-
scription from the C3 promoter by the heterofusion ERs in
response to 10?9M E2 was similar to that of ?-?, which had
significantly lower potency than the ER? dimer (Fig. 8). Both
ER? and ?-?, on the other hand, had minimal effects on
luciferase activity. The heterofusion receptors showed tran-
scription capacities comparable to those of ?-? and ER? when
tested from the PS2 promoter.
These results suggest that, although the biopotencies of the
fusion receptors are dependent upon the promoter and cell
context, the heterofusions ?-? and ?-? emulate the activity of
?-? rather than the ?-? when tested with estrogen-responsive
gene promoters. Thus, it appears that ER? dictates the mode
of transcription of the ER?/? heterodimer in the ERE-depen-
dent ER signaling pathway.
(ii) Repression of ER? transactivity by ER?. Previous re-
ports indicated that ER? functions as a dominant inhibitor of
ER? transcriptional activity in the ERE-dependent signaling
pathway in transfected mammalian cells (12). Indeed, cotrans-
fections of an expression vector bearing ER? cDNA, together
with increasing amounts of ER? cDNA in transiently trans-
fected HeLa cells, revealed that ER? effectively inhibits the
E2-ER? induced transcription from the C3 or the pS2 gene
promoter (Fig. 9, left panels). At the concentration that in-
duced maximal repression, the inhibition of ER?-induced
transcription by ER? was independent of the E2 amount re-
gardless of the promoter-type (data not shown).
This inhibitory capacity of ER? on ER?-induced transcrip-
tion in coexpression systems could occur at multiple levels.
These include heterodimerization, competition for binding to
DNA, and/or transcriptional silencing through squelching.
Since the fusion receptors do not dimerize, hence generating
homogenous populations of receptor species, we reasoned that
coexpression of the fusion receptors would allow us to address
the underlying mechanism. To accomplish this, we performed
a cotransfection assay using an expression vector bearing the
?-? cDNA with an expression vector carrying the ?-? or het-
erofusion ?-? or ?-? cDNA (Fig. 9, right panels). The results
revealed that the cotransfected ?-? repressed the transregula-
tory activity of ?-?. On the other hand, the heterofusion ?-? or
?-? augmented the luciferase activity induced by ?-?. This
finding also suggests that the repression of the ?-? activity by
?-? is independent of sequestration of factors required for
transactivation under the condition we are testing. Since the
heterofusion ERs simulate the activities of the ER? dimer and
?-?, these results also imply that ER? as a homodimer re-
presses the ER? function in the ERE-dependent signaling
pathway. Indeed, the DNA-binding-defective ?-? (??-??)
FIG. 8. HeLa cells were transiently transfected as described for
Fig. 7. The data represent the means ? the SEM of three experiments
performed in duplicate.
7690LI ET AL.MOL. CELL. BIOL.
bearing two mutant ER? monomers did not affect luciferase
levels induced by ?-?. Thus, an effective repression of ER?-
mediated transcriptional responses by ER? occurs through
competition for ERE binding.
(iii) ERE-independent and DNA-dependent ER signaling.
The ligand-ER complex regulates the expression of the Col
and RAR? genes through the DNA-dependent and ERE-
independent signaling pathway. The functional tethering of the
AF-1 and/or AF-2 domains of both ERs to the Jun/Fos family
of proteins bound to the AP-1 element in the promoter of the
Col gene provides the basis for gene responsiveness to li-
gand-ER signaling (22). Similarly, the functional interaction of
the activation domains of ER?, but not ER?, with the Sp-1
transcription factor bound to the GC box provides responsive-
ness for the RAR? gene expression (36). Using a reporter
plasmid that contains the Col or RAR? promoter as a model
for the DNA-dependent and ERE-independent signaling, we
examined whether or not the heterofusion ERs emulate the
ligand-mediated effects of ER?. The expression vector with
cDNA for an ER was cotransfected into cells with a reporter
plasmid with the Col or RAR? promoter that drives the ex-
pression of the firefly luciferase cDNA. Ligand-mediated re-
sponses from the reporter plasmid by an expression vector
were compared to the activity in the absence of ligand, the
latter value being set to one.
The ligand-mediated responses from reporter vectors were
cell type dependent. Although ligand ER did not alter lucif-
erase activity from either the Col or the RAR? promoter in
transiently transfected CHO cells, the homofusion ERs simu-
lated the transactivation abilities of the parent dimers in re-
sponse to ER ligands in HeLa cells (Fig. 10). ER? and ?-?
induced the transactivation from both the Col and the RAR?
promoters in response to 10?7M ICI (see also Materials and
Methods), whereas ER? or ?-? had no effect on luciferase
FIG. 9. Effects of coexpression of ERs and fusion receptor on transcriptional responses. (Left) HeLa cells were transiently transfected with a
constant amount (in nanograms) of ER? expression vector and increasing amounts of the ER? expression vector. Cells were cotransfected with
a reporter plasmid bearing the C3 or pS2 promoter that drives the expression of the firefly luciferase enzyme cDNA. Cells were treated without
(data not shown for simplicity) or with 10?9M E2 for 24 h. (Right) HeLa cells were transiently transfected with an expression vector bearing a
fusion ER cDNA alone or together with another fusion ER cDNA. Cells were also transfected with the C3 or pS2 reporter plasmid. After
transfection, cells were incubated in the absence (data not shown) or presence of 10?9M E2 for 24 h. V, parent expression vector. The data are
the means ? the SEM of three independent experiments performed in duplicate.
VOL. 24, 2004ER?/? HETERODIMER EMULATES FUNCTIONS OF ER? DIMER7691
activity in the presence of ICI. The heterofusion ERs induced
transcription in response to ICI in a manner similar to the ER?
dimer and ?-?. E2 at any concentration tested, shown at 10?9
M, had little effect on luciferase activity from the Col or the
E2, on the other hand, suppressed reporter enzyme activity
mediated by ER? and ?-? in the ER-negative MDA-MB-231
cell line derived from a breast adenocarcinoma; whereas ICI
had little effect on luciferase activity (Fig. 10, lower panel).
Both the ?-? and the ?-? heterofusions emulated the E2-
mediated effects of ER? and ?-? on transcription from the Col
promoter. The ligand-receptor complexes had no effect on
responses from the RAR? promoter in this cell line (data not
shown). Thus, as observed with the ERE-dependent signaling,
it appears that ER? dictates the mode of transcriptional prop-
erties of the heterofusion proteins in the DNA-dependent and
ERE-independent signaling pathway as well.
In addition to acting as homodimers, the coexpression of
ERs leads to heterodimerization, the extent of which, as we
show here, is dependent upon the relative amount of each
subtype. However, the role of the ER?/? heterodimer in E2
signaling has remained largely unknown. This is because coex-
pression leads to the formation of not only the ER?/? het-
erodimer but also the ER? and ER? homodimers that prevent
examination of the transcriptional ability of the heterodimer
and elucidation of its role in E2 signaling. Using a genetic
conjugation approach, we generated monomeric ?-? and ?-?
receptors to integrate the functions of ER? and ER? into a
single-chain protein in order to study the role of ER?/? het-
erodimer in genomic signaling pathways. The homofusion ?-?
and ?-? simulated the functions of the parent ER? and ER?
dimers, respectively. The heterofusions ?-? and ?-?, on the
other hand, mimicked the function of the ER? dimer. Thus,
ER? defines the transregulatory mode of the ER?/? het-
Single-chain receptors are in a dimer-like configuration. We
show here that single-chain receptors as monomers interacted
with the consensus ERE by utilizing the same nucleosides as
the parent ER dimers. Moreover, single-chain receptors bound
to the same spectrum of responsive elements as the ER dimers
with binding affinities similar to those of ERs. Likewise, the
single-chain receptors bound to E2 with high affinity. These
results suggest that the two ER monomers in fusion proteins
fold to a configuration that juxtaposes two DBDs and LBDs to
interact with EREs and E2 in a manner comparable to the ER
dimers. These results suggest that despite the tail-to-head join-
ing of two receptor monomers in the single-chain receptor,
monomers fold to associate intramolecularly in a manner com-
parable to the ER dimers that dimerize through an intermo-
ER? dictates the functions of the ER?/? heterodimer in
genomic signaling pathways. Conformational changes induced
by the binding of E2 to ER realign helix 12 of the LBD over the
ligand-binding pocket to form a cofactor interacting surface,
together with the contributions from helices 3 and 4/5 (3, 35).
Previous studies indicated that distinct classes of coactivators
recognize distinct but overlapping sites on the agonist occupied
LBD of ERs (7). It was also shown that one molecule of SRC-1
through two LXXLL motifs interacts with both cofactor inter-
acting surfaces of LBDs in an agonist-bound ER? dimer (9,
17), as reported for the PPAR? homodimer (32). The genetic
conjugation fuses the amino terminus of one monomer and the
carboxyl terminus of the other, domains of the protein that are
critical for interactions with cofactors. Although single-chain
ERs bind to EREs and E2 with high affinities, the E2-depen-
dent recruitment of the AF2-dependent cofactors by fusion
ERs was found to be less efficient than that of the ER dimers.
This suggests that the cofactor interacting surfaces are altered.
Remarkably, however, two ER monomers in fusion proteins
apparently form a ligand-dependent cofactor surface sufficient
for interaction with the p160 family of cofactors.
In addition to NRIMs, TIF-2 through a glutamine-rich re-
gion (TIF2-Q) interacts preferentially with the amino-terminal
AF-1 domain of ER? independently from ligands (2, 30, 38, 43,
45). We also observed here that the homofusion ?-?, but not
?-?, recruited TIF2-Q with a lower affinity than that observed
with the ER? dimer. These results indicate that the receptor
subtype-specific interaction of TIF2-Q, although not at the
natural extent, is preserved in single-chain receptors. Similarly,
the heterofusions ?-? and ?-? interacted with the TIF2-Q with
FIG. 10. Transcriptional responses to fusion ERs from the ERE-
independent signaling pathway. HeLa or MDA-MB-231 cells were
transiently transfected with 125 ng of expression vector bearing no
cDNA (V) or a cDNA for an ER, together with 125 ng of reporter
vector that contained the Col or the RAR? promoter driving the
expression of firefly luciferase enzyme cDNA. Cells were treated with-
out (?) or with 10?9M E2 (?E2) or 10?7M ICI (ICI) for 40 h. The
means ? the SEM of four independent experiments are shown.
7692LI ET AL.MOL. CELL. BIOL.
efficiencies twofold lower than that observed with the homo-
fusion ?-?. This indicates that the presence of one ER? mono-
mer is sufficient for the ability of the heterodimer to recruit
ER? has considerably less transcription potency than ER?
in heterologous expression systems that utilize EREs. Studies
indicated that receptor-specific AF-1 defines the transcrip-
tional strength of ER? and ER?. It appears that the ability of
ER? to recruit the p160 family of cofactors through the AF-1
domain (2, 30, 38, 43, 45) and to subsequently integrate the
AF-1 and AF-2 functions (2, 45) is critical for the biopotency
of the receptor. TIF2-Q is also recruited preferentially by the
homofusion ?-? and the heterofusions ?-? and ?-?, in contrast
to ?-?, independently from E2. This implies that the abilities of
these fusion receptors to interact with distinct surfaces of a
cofactor likely underlie their mimicry of ER? functions in the
ERE-dependent signaling pathway. We also observed that the
extent of transcription induction by fusion receptors was de-
pendent upon the promoter and cell context. This could be due
to combinatorial effects of many trans-acting components spe-
cific to each gene and, consequently, differences in the com-
position or concentration of coregulatory proteins, which vary
among E2 target tissues. Since the interaction of fusion recep-
tors with SRC-1 was more severely affected compared to
TIF-2, differences in the binding affinities of cofactors for fu-
sion receptors could also contribute to differences in a pro-
moter and cell type responses to single-chain receptors.
Our observations that the ligand-mediated responses of the
ER dimers are preserved in the corresponding homofusion
receptors from the Col and RAR? promoters representing
ERE-independent genomic signaling are consistent with the
suggestion that single-chain receptors display configurations
that mimic the parent ER dimers. Since heterofusion receptors
emulated the functions of the ligand-ER? complex, ER? ap-
pears to dictate the ligand-mediated functions of ER?/? het-
erodimer in the ERE-independent DNA-dependent pathway
as well. We therefore suggest that the ER?/? heterodimer
mimics the mode of transcription of ER? in the genomic es-
Implications for E2 signaling. Although the consequences
of ER? and ER? cosynthesis in vivo are unknown, a fine
tuning of tissue responses to E2 is likely mediated by the
integrated effects of the ER dimers and the ER?/? het-
erodimer. It is certain that ER? mediates the expression of
ERE-bearing responsive genes in response to E2, albeit less
potently than ER?, when expressed alone. Coexpression of
ER? could attenuate the transregulatory potential of ER? in
the ERE-dependent genomic signaling pathway by het-
erodimerization and could also antagonize the effects of the
ER? dimer as a homodimer. Due to the absence of significant
transcriptional responses to ligand-ER? in experimental ap-
proaches we used, assessing the cross-effects of the ligand-ER
complexes on transcriptional responses from the Col or the
RAR? promoter was difficult. It remains possible that in ad-
dition to the ERE-dependent signaling pathway, the ligand-
ER? complex could also modulate the transcriptional responses
mediated by the ER? dimer from the ERE-independent
genomic signaling pathway. Therefore, an alteration in the
regulatory balance resulting from aberrant synthesis of either
or both ER subtypes could lead to a malignancy in E2 target
tissues wherein both subtypes are synthesized. Studies, for ex-
ample, suggest that ER? and ER? can be coexpressed in
normal breast tissue (6, 16, 25, 37). However, the ratio of ER?
to ER? appears to be altered in samples obtained from breast
cancer patients; while ER? is expressed at higher levels, the
expression of ER? is decreased (25).
The genetic conjugation approach precludes the limitation
of monomer association into biologically active dimers and the
dissociation of dimers into inactive monomer induced by de-
stabilizing mutations. This offers an opportunity to address the
roles of ER dimers as homogeneous populations in the phys-
iology and pathophysiology of estrogen signaling. Moreover,
single-chain ERs could be utilized to further the understanding
of nongenomic effects of ER dimers. Permitting functional
analysis of unique symmetrical or asymmetrical mutations that
simulate variant homo- and heterodimers, genetic conjugation
would also allow us to address the effects of various signaling
pathways that cross talk with ER and fundamentally alter li-
gand-ER functions. The fusion protein approach could be ex-
tended further to other members of multisubunit complexes of
the steroid/thyroid hormone receptor superfamily, and to non-
ligand-dependent transcription factors that act primarily as
We thank Jay Reeder for allowing us to access the fluorescence
microscopy facilities. We are grateful to Cheeptip Benyajati, Jeffrey J.
Hayes, and Mark Sowden for critical reading of the manuscript.
This study was supported by National Institutes of Health grant HD
24459 (R.H., R.A.B., and M.M.) and an American Cancer Society
Institutional Research Grant (M.M.).
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