Estrogen Receptor, a Common Interaction Partner for a Subset of Nuclear Receptors
Soo-Kyung Lee,
Hueng-Sik Choi,
Mi-Ryoung Song,
Mi-Ock Lee and
Jae Woon Lee
College of Pharmacy (S-K.L., J.W.L.) Hormone Research Center
(H-S.C., J.W.L.) Chonnam National University Kwangju, 500757
Korea
Department of Microbiology (M-R.S., M-O.L.) College
of Medicine Yonsei University Seoul, 120752 Korea
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ABSTRACT
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Nuclear receptors regulate transcription by
binding to specific DNA response elements as homodimers or
heterodimers. Herein, the yeast and mammalian two-hybrid tests as well
as glutathione-S-transferase pull-down assays
were exploited to demonstrate that estrogen receptor (ER) directly
binds to a subset of nuclear receptors through protein-protein
interactions between ligand-binding domains. These receptors include
hepatocyte nuclear factor 4, thyroid hormone receptor (TR), retinoic
acid receptor (RAR), ERß, and retinoid X receptor (RXR). In yeast
cells, a LexA fusion protein to the human ER ligand-binding domain
(LexA/ER-LBD) was an inert transactivator of a LacZ
reporter gene controlled by upstream LexA-binding sites. However,
LexA/ER-LBD differentially modulated the LacZ reporter gene expression
when coexpressed with native TRs, RARs, or RXRs. Similarly,
cotransfection of these receptors in CV1 cells up- or down-regulated
transactivations by ER. From these results, we propose that ER is a
common interaction partner for a subset of receptors, and these
interactions should mediate novel signaling pathways in
vivo.
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INTRODUCTION
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The nuclear receptor superfamily is a group of transcriptional
regulatory proteins linked by conserved structure and function (1). The
superfamily includes receptors for a variety of small hydrophobic
ligands such as steroids, T3, and retinoids, as well as a
large number of related proteins that do not have known ligands,
referred to as orphan nuclear receptors (2). The receptor proteins are
direct regulators of transcription that function by binding to specific
DNA sequences named hormone response elements (HREs) in promoters of
target genes. Nearly all the superfamily members bind as dimers to
HREs. A dimerization interface has previously been identified within
the DNA-binding domains (DBDs) of retinoid X receptors (RXRs), retinoic
acid receptors (RARs), vitamin D receptor, and thyroid hormone
receptors (TRs) that selectively promotes DNA binding to cognate HREs
(3, 4, 5, 6, 7, 8). An additional dimerization interface that mediates cooperative
binding to DNA, referred to as the I-box, has recently been mapped to a
40-amino acid region within the C-terminal ligand-binding domains
(LBDs) of RAR, TR, chicken ovalbumin upstream promoter
(COUP-TF), RXR, and hepatocyte nuclear factor 4 (HNF4) (9, 10). The two
dimerization domains appear to work in tandem and led to a two-step
hypothesis for binding of dimers to DNA (2, 9). According to this
hypothesis, the LBD dimerization interface initiates the formation of
solution dimers that, in turn, acquire the capacity to bind to a number
of differently organized HREs. However, formation of a second dimer
interface within the DBD restricts receptors to bind to only
cognate HREs (2, 9).
While some nuclear receptors apparently bind HREs only as
homodimers, TRs, vitamin D receptor, RARs, the peroxisome
proliferator-activated receptors (PPARs), and several orphan nuclear
receptors bind their specific response elements with high affinity as
heterodimers with RXRs (11, 12, 13, 14, 15, 16). Based on this high-affinity binding,
such heterodimers have been considered to be the functionally active
forms of these receptors in vivo. However, RXR is not the
only receptor capable of forming heterodimers with other nuclear
receptors. TR (17, 18), COUP-TF (19, 20, 21), PPAR (22), and small
heterodimer partner (SHP) (23) have been reported to form heterodimers
with other receptors. Interestingly, although heterodimerization with
RXR usually results in enhanced transactivation, COUP-TF and SHP seem
to function primarily as transcriptional repressors (23, 24, 25, 26, 27, 28).
Heterodimer formation can generate many different combinations of
receptors, particularly in the light of the fact that multiple genes
and multiple isoforms exist for RAR, RXR, TR, COUP-TF, and PPAR. As the
expression of each isoform depends on cell type and the stage of growth
and development, heterodimerization among all these receptors results
in the formation of an extremely diverse group of receptors. In
principle, the enormous number of heterodimer combinations could cover
a wide range of transcriptional activities, thus generating significant
diversity in gene regulation.
Herein, we show that estrogen receptor (ER) directly binds, through
protein-protein interactions between ligand-binding domains, to HNF4,
TR, RAR, and RXR, adding a complexity to the already complicated list
of the heteromeric interactions. We have also confirmed the results
(29, 30) that newly cloned ER named ERß interacts with ER. In
parallel, COUP-TF (31), estrogen-related receptor 1 (ERR1) (32), and
SHP (D. D. Moore, personal communications) were shown to
interact with ER, while this manuscript was in preparation. We further
show that coexpression of native TR, RAR, or RXR differentially
regulates transactivations by ER in yeast and CV1 cells. These results
suggest that ER is a common interaction partner for a subset of
receptors, and these interactions mediate novel signaling pathways
in vivo.
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RESULTS
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ER Interacts with a Subset of Nuclear Receptors in Yeast
We found that coexpression of a previously described B42/RXR-LBD
hybrid (33) stimulates the LexA/ER-LBD (a LexA fusion protein with the
LBD of ER)-mediated LacZ reporter gene expression in the
yeast two-hybrid tests, suggesting that ER-LBD interacts with RXR-LBD
(Fig. 1A
). This LacZ expression
was further stimulated approximately 1.5-fold in the presence of 1
µM 9-cis-RA. We further found that the
LexA/ER-LBD-mediated LacZ expression was stimulated when
coexpressed with analogous B42 fusions to several receptors, including
RAR, TR
, HNF4, and ERß, suggesting that ER also interacts
with these receptors (Fig. 1A
). The expression was slightly enhanced by
1 µM T3 with the B42/TR
coexpression,
whereas it was significantly stimulated by 1 µM
E2 with the B42/ERß coexpression (
3-fold) (Fig. 1A
).
In addition, we showed that an inert transactivator LexA/ERß
stimulated the LacZ reporter gene expression approximately
6.5-fold when coexpressed with B42/ERß, confirming the recently
reported ERß homodimerization (29, 30) in this yeast system (Fig. 1A
). In contrast, coexpression of B42 alone or B42 fusions to the LBDs
of TRß, glucocorticoid receptor (GR), or the orphan receptor CAR (34)
did not stimulate the LexA/ER-LBD-mediated LacZ expression,
attesting to the specificity of these interactions. Similarly,
expressions of all the B42 fusions alone, with no LexA fusion
coexpressed, were transcriptionally inert with the LacZ
reporter (data not shown). However, the B42/TR-LBD coexpression
stimulated the LexA/ER-LBD-mediated LacZ expression
approximately 2.5-fold in the presence of 1 µM
T3, raising the possibility that TR-LBD may contain a
T3-inducible interaction domain with ER. From these
results, we concluded that ER-LBD may interact with RAR, RXR-LBD,
TR
, TRß-LBD (only in the presence of T3), HNF4-LBD,
and ERßL, in which LBDs are likely to be sufficient. It is noteworthy
that the LacZ expression strength by coexpression of these
ER heterocomplexes was generally comparable to that of the previously
described TR/RXR heterodimer (33) (Fig. 1B
, compare with the results of
LexA/TR-LBD and B42/RXR-LBD). Overall, the similarities in the pattern
of interactions observed with LexA/ER-LBD and LexA/RXR-LBD (33, 35, 36)
strongly suggest that ER, like the RXRs, is a common interaction
partner for members of the receptor superfamily.

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Figure 1. ER Binds to a Subset of Nuclear Receptors in Yeast
EGY48 cells, in which ß-galactosidase expression is dependent on the
presence of a transcriptional activator with a LexA DNA-binding domain,
were transformed with plasmids expressing the indicated LexA and B42
chimeras. These cells were grown in liquid culture containing
galactose, since expression of the B42 chimeras is under the control of
the galactose-inducible GAL1 promoter (33 ). ß-Galactosidase readings
were determined and corrected for cell density and for time of
development (A415/A600) x 1000/min. The result
is the average of at least three different experiments, and the
SDs are less than 5%. A, Open bars indicate
no hormone added. Light-hatched bars indicate the
presence of 1 µM E2. Thick-hatched
bars indicate the presence of 1 µM
9-cis-RA (B42/RAR and B42/RXR-LBD) or T3
(B42/TR and B42/TR-LBD). Black bars indicate the
presence of 1 µM E2 plus 1 µM
T3 (B42/TR and B42/TR-LBD) or 1 µM
E2 plus 1 µM 9-cis-RA (B42/RAR
and B42/RXR-LBD). B, Open bars indicate no hormone
added. Light-hatched bars indicate the presence of 1
µM T3. Thick-hatched bars
indicate the presence of 1 µM 9-cis-RA.
Black bars indicate the presence of 1 µM
E2 plus 1 µM 9-cis-RA.
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To confirm whether various ligand-dependent stimulations of the
LacZ expressions observed reflect genuine ligand-dependent
protein-protein interactions or simply ligand-dependent
transactivations mediated by the AF2 domains (1), we reexamined the
LacZ expressions with two previously defined AF2 mutant
receptors, LexA/TR-LBD459 (33) and B42/RXR-LBD
AF2 (36). As shown in
Fig. 1B
, the 9-cis-RA-dependent induction of the
LacZ expression observed with coexpression of B42/RXR-LBD
and LexA/TR-LBD was lost when B42/RXR-LBD
AF2 was coexpressed
instead, whereas the T3-dependent induction was
specifically lost when LexA/TR-LBD459 was used. When LexA/TR-LBD459 and
B42/RXR-LBD
AF2 were coexpressed, neither T3 nor
9-cis-RA-depedent induction of the LacZ
expression was observed. These results, along with our previous finding
(33) in which the interactions of LexA/TR and B42/RXR were not affected
by mutations into the AF2 domain of TR, clearly demonstrate that
protein-protein interaction of TR and RXR are ligand independent.
Therefore, results of the yeast two-hybrid tests should be taken with
caution that they may not represent true ligand-dependent interactions.
Thus, we further characterized these interactions using two additional
methods (i.e. mammalian two-hybrid tests and
glutathione-S-transferase (GST)-pull downs).
ER Interacts with TR, RAR, and RXR in Mammalian Cells
To examine whether ER binds to these receptors in mammalian cells,
we employed a mammalian two-hybrid system that reproduces the
heteromeric receptor-receptor interactions (37). As shown in Fig. 2
, cotransfection of CV1 cells with
expression vectors for VP16/TR, VP16/RAR, and VP16/RXR significantly
increased the Gal4/ER-LBD-mediated induction of the Gal4-TK-Luc
reporter gene activity (
4-, 7.5-, and 9.5-fold induction,
respectively). Similar results were obtained in the presence of 0.1
µM cognate ligand, suggesting that they interact with
each other in a ligand-independent manner. As expected, VP16 alone
was ineffective. Thus, we concluded that ER is capable of
constitutively binding to TR, RAR, and RXR in mammalian cells. These
results suggest that the various ligand-dependent stimulations of the
LacZ expression in yeast (Fig. 1A
) may have reflected
ligand-dependent transactivations of the AF2 domains, as suggested by
the results shown in Fig. 1B
.

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Figure 2. ER Binds to a Subset of Nuclear Receptors in
Mammalian Cells
CV1 cells were transfected with ß-galactosidase expression vector and
VP16 alone, VP16/TR, VP16/RAR, or VP16/RXR-expression vectors along
with a reporter gene Gal4-TK-Luc (37 ). Open bars
indicate no hormone added, whereas hatched and
black bars indicate addition of 0.1 µM
T3 and 9-cis-RA, respectively. Normalized
luciferase expressions from triplicate samples are presented relative
to the ß-galactosidase expressions, and the SDs are less
than 5%.
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ER Binds to TR, RAR, and RXR in Vitro
To further characterize these interactions in vitro,
GST-fusions to full-length rat TRß, human RAR
, and RXR
were
expressed, purified, and tested for interaction with an in
vitro-translated full-length human ER or luciferase labeled with
[35S]methionine. As expected, luciferase was not able to
interact with any of the GST proteins (data not shown). However, ER
readily interacted with GST/TR, GST/RAR, and GST/RXR either in the
presence or absence of ligand (Fig. 3
, A, B
and C). ER didnt interact with GST alone or GST/GR, as expected. In
contrast, ER bound to GST fusion to the receptor-binding domain of
SRC-1 (GST/SRC-R) (38) in an E2-stimulated manner (Fig. 3D
). The percentage of total [35S]ER bound by the fusion
proteins were approximately 1530% of the inputs (data not shown),
and the E2-dependent fold-increase in the pull-down of
[35S]ER by GST/SRC-R was approximately 4- to 5-fold. From
these results, we concluded that ER constitutively binds to TR, RAR,
and RXR through direct protein-protein interactions. In particular,
these results, along with the mammalian two- hybrid tests (Fig. 2
),
suggest that at least some of the ligand-dependent stimulation of the
LacZ expression observed in the yeast two-hybrid tests (Fig. 1
) should have resulted from ligand-induced transactivations rather
than ligand-induced protein-protein interactions.

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Figure 3. Pull-Down Assays
Luciferase and ER-LBD labeled with [35S]methionine by
in vitro translation were incubated with glutathione
beads containing GST alone, GST fusions to TRß (A), RAR (B),
RXR (C), SRC-R and GR (D). Beads were washed, and specifically bound
material was eluted with reduced glutathione and resolved by SDS-PAGE.
(-) denotes no hormone added; 0.1 µM of each
ligand was added as indicated.
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Function of the ER Heterocomplexes
We have previously shown that LexA/TR-LBD requires coexpression of
native RXR for an efficient T3-dependent transactivation in
yeast (33). Similar to LexA/TR-LBD, LexA/ER-LBD alone was an inert
transactivator, either in the presence or absence of E2, of
the previously described LacZ reporter gene controlled by
upstream LexA-binding sites in yeast (33). To gain insights into the
functional consequences of these interactions in vivo, we
coexpressed holo-proteins of these ER-interacting receptors in the
LexA/ER-LBD-expressing yeast cells. Interestingly, RAR
, RARß,
RXRß, TR
, or TRß dramatically enhanced the basal transcriptional
activities of LexA/ER-LBD (Fig. 4
).
Coexpression of RAR
led to approximately 2-fold induction in the
basal transcriptional activities, which was further enhanced
approximately 2-fold by addition of 1 µM
9-cis-RA. Interestingly, coexpression of RXR
or RXR
induced the LexA/ER-LBD-mediated LacZ expression only in the
presence of 1 µM 9-cis-RA; i.e. the
basal level of expression was not affected by coexpression of these two
receptors (Fig. 4
). These results indicate that coexpression of these
native receptors can differentially regulate transactivations by ER in
yeast, attesting to the possible functional significance of these
interactions in vivo.

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Figure 4. Modulation of LexA/ER-LBD by Coexpressed
Holo-nuclear Receptors in Yeast
EGY48 cells, in which ß-galactosidase expression is dependent on the
presence of a transcriptional activator with a LexA DBD, were
transformed with plasmids expressing LexA/ER-LBD and the indicated
holo-nuclear receptors. Open bars indicate no hormone
added. Light-hatched bars indicate the presence of 1
µM E2. Thick-hatched bars
indicate the presence of 1 µM 9-cis-RA
(RARs and RXRs) or T3 (TRs). Black bars
indicate the presence of 1 µM E2 plus 1
µM T3 (TRs) or 1 µM
E2 plus 1 µM 9-cis-RA (RARs
and RXRs). ß-Galactosidase readings were determined and corrected for
cell density and for time of development
(A415/A600) x 1000/min. The result is the
average of at least three different experiments, and the
SDs are less than 5%.
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To assess the functional consequences of these interactions in
mammalian cells, TR
, RAR
, or RXR
was cotransfected into CV1
cells along with an ER expression vector and a reporter construct
controlled by ERE (ERE-TK-CAT or ERE-TK-Luc). Addition of 0.1
µM E2 stimulated the ER-mediated reporter
gene expression approximately 5- to 7-fold in the absence of
cotransfected receptors. Coexpression of TR
, however, significantly
stimulated both the basal and E2-induced transactivations
by ER (
4- and 2-fold, respectively). Interestingly, a weak additive
effect was observed with the TR
coexpression; i.e.
E2 plus T3 resulted in approximately 1.5-fold
higher activities than E2 alone (Fig. 5A
; see the results with 50 ng TR
cotransfected). Coexpression of RAR
did not affect the basal level
of transactivations by ER. However, it repressed the
E2-induced transactivation almost to the basal level (Fig. 5B
). In contrast, coexpression of RXR
modestly stimulated the
E2-induced transactivations by ER. The basal level of
transactivations by ER, however, was not affected by the RXR
coexpression. Similarly, coexpression of ER inhibited the
9-cis-RA-induced transactivations of a reporter construct
controlled by ß-retinoic acid response element (data not
shown). In addition, cotransfection results varied depending on cell
types and receptor isotypes, suggesting that other factors contribute
to the final phenotype of each ER heterocomplex (M.-O. Lee,
unpublished results). These results, along with the yeast coexpression
results (Fig. 4
), suggest that these ER heterocomplexes can
differentially modulate the ER-mediated transcriptional activites
in vivo.
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DISCUSSION
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It has been demonstrated that control of E2-responsive
gene regulation can be quite complex (reviewed in Ref. 39). Several
lines of evidence indicated that gene regulation by E2 and
ERE involves not only ER but also other transcription factors. For
example, other nuclear receptors such as TR, RXR, COUP-TF, ERR1, and
RAR have been shown to interact with EREs in addition to their cognate
response elements (40, 41, 42). In this report, we have clearly shown that
ER, along with RXR, is a common interaction partner for a subset of
receptors, suggesting that E2-responsive gene regulation
may also involve direct protein-protein interactions with other nuclear
receptors. In particular, it is an interesting possibility that these
different ER heterocomplexes may mediate novel signaling pathways
through their own DNA-binding sites that are distinct from the
conventional EREs. In contrast to the broad positive effects of RXR,
these ER heterointeractions led to differential regulation of the
transactivations by ER (Figs. 4
and 5
). In addition, cotransfection
results dramatically varied depending on cell types (CV1 vs.
MCF7 cells, for instance) and receptor isotypes (M.-O. Lee,
unpublished results). These differential regulations may result from
direct bindings of ER heterodimers to ERE, which either positively or
negatively regulates E2-induced gene expression.
Alternatively, formation of a non ERE-binding heterodimer may simply
inhibit E2-induced gene expression or allow other positive-
or negative-acting proteins to occupy EREs instead. Despite our
extensive efforts, however, we were not able to find any intermediate
heterodimer bindings or inhibitions of ER bindings to ERE by these
ER-interacting receptors in electrophoresis mobility shift assays
(EMSA) (data not shown). Similarly, heterodimers of COUP-TF/ER (31),
ERR1/ER (32), and SHP/ER (D. D. Moore, personal communications)
readily detected in solution were not observed to bind EREs in EMSA. In
parallel, two recent reports showed interaction of COUP-TF with orphan
receptors nurr77 (20) and HNF4 (21) only in solution but not when bound
to DNA. These results are consistent with the possibility that these
ER/receptors may bind EREs as a heterodimer or a higher-order complex
not stable enough to sustain in EMSA. These weak complexes may show
distinct phenotypes by differentially interacting with other proteins
such as recently defined transcription cofactors, which are often
receptor- and cell type-specific (reviewed in Ref. 43). Consistent with
this possibility, xSRC-3, an SRC-1 homolog we recently isolated from
Xenopus, was found to show unexpected specificity in tissue
distribution and receptor interactions (44).
Even though the oligomeric status of the ER heterocomplexes are not
currently known, two lines of evidence attest to the specificity of the
protein-protein interactions between ER and these receptors. First, a
series of chimeric HNF4s we recently constructed to define the HNF4
homodimeric interactions and RXR heterodimeric interactions (10)
differentially interacted with ER (our unpublished results).
Second, we isolated a few mutant RXRs that show specifically impaired
interactions with ER while maintaining wild-type interactions with
other receptors (our unpublished results). Similar to the chimeric
HNF4s, these mutations also reside within the I-box of the RXR, which
was recently found to be essential for homo- and heterodimer formations
(9, 10), suggesting that HNF4 and RXR exploit the conventional
dimerization domains (the I-box) to interact with ER. Recently, the
DNA-binding pattern of the RXR was shown to be regulated by
ligand-dependent modulation of its oligomeric state (dimer
vs. tetramer), and the tetramerization domain was defined to
be distinct from the dimerization domain I-box (45). Thus, further
mutational studies within this newly defined tetramerization domain of
RXR, as well as mapping the regions of ER essential for these
interactions, will be required to clearly decode the oligomeric status
of these ER heterocomplexes.
The LexA-based yeast two-hybrid system presents two possible
complications to study protein-protein interactions. First, the
full-length LexA used in this system contains its own dimerization
domain, which could complicate interpretation of the interaction
results, particularly where dimerization is a prerequisite for
two target proteins to interact with each other. Second, this system
cannot distinguish between genuine ligand-dependent protein-protein
interactions and ligand-dependent transactivations. For instance, the
ligand-induced stimulations of the LacZ expressions when
LexA/TR and B42/RXR-LBD were coexpressed were shown to reflect
ligand-dependent transactivations mediated by the AF2 domains of TR and
RXR (Fig. 1B
). With these complications, other methods such as the GST
pull-downs should be employed to probe protein-protein interactions.
The interactions of ER with TR, RAR, and RXR were
hormone-independent in both mammalian two-hybrid tests and GST
pull-downs, suggesting that the seemingly hormone-dependent
interactions in yeast (Fig. 1
) may have been the hormone-dependent
transactivations of the AF2 domains. The T3-dependent
stimulation of the LacZ expression with TR-LBD and ER-LBD
coexpressed (Fig. 1A
) may have also reflected the
T3-dependent transactivations of the AF2 domain of TR. The
basal interactions between these proteins might have been too low to
detect in the yeast system, which, however, became detectable with an
aid from the activated transactivations of the AF2 domain upon
T3 addition (Fig. 1A
; see the results with LexA/ER-LBD and
B42/TR-LBD).
The physiological implications for these interactions have been
accumulating. For instance, manipulations of T3 and E2in vitro were shown to potentiate or mutually inhibit
effects of gene expression (40, 46, 47). Environmental conditions that
alter levels of circulating T3, such as cold temperature,
were also shown to alter E2-dependent female reproductive
behavior (48, 49, 50, 51). TR
was recently shown to attenuate ER-mediated
transactivations from the vit A2 consensus ERE, which is not
necessarily dependent on DNA binding, as the TR
DNA-binding mutant
was still able to inhibit E2-dependent transactivation (52, 53). However, under similar conditions, we observed a modest
enhancement of the ERE-mediated transactivations by either liganded or
unliganded ER when cotransfected with TR
(Fig. 5
). The reasons for
this discrepancy are not currently known. In addition, retinoids have
been shown to inhibit the growth of hormone-dependent breast cancer
cells both in vivo and in vitro (54, 55, 56).
Nonadditive inhibition has been demonstrated between retinoids and
antiestrogens, suggesting that each agent may produce common
antiestrogenic effects. Recently, synergistic interactions between HNF3
and HNF4 mediating apolipoprotein AI gene expression were shown to be
further potentiated by cotransfected ER (57), in which the physical
association of ER and HNF4, as described in this report, may play a
role. However, the physiological implications for the ER/SHP (D. D.
Moore, personal communications), ER/COUP-TF (31), and
ER/ERR1 (32) interactions are not currently known.
In conclusion, we found that ER directly binds to a subset of nuclear
receptors through protein-protein interactions between ligand-binding
domains, and these ER heterocomplexes led to differential regulation of
transactivations by ER. From these results, we propose that ER is a
common interaction partner for a subset of receptors, and these
different ER heterocomplexes should mediate novel signaling pathways
in vivo.
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MATERIALS AND METHODS
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Hormones, Yeast Cells, and Plasmids
E2, T3, and 9-cis-RA were
obtained from Sigma Chemical Co. (St. Louis, MO). EGY48 cells, the
lexA-ß-galactosidase reporter construct, and the LexA- and
B42-parental vectors were as reported (33, 58). LexA or B42 fusions to
the LBDs of GR, CAR, HNF4, RXR
and TRß as well as a B42 fusion to
full-length RAR
were as previously described (10, 33, 35, 36). The
entire hinge and ligand-binding domains (D, E, and F; amino acids
245595) of the human ER were subcloned into EcoRI and
SalI sites of the LexA-vector by PCR using EcoRI
and XhoI-site bearing primers to construct LexA/ER-LBD. LexA
or B42 fusions to full-length mouse ERß (a gift from Dr. Vincent
Giguere) or human TR
were similarly constructed. Yeast expression
vectors, YEP-RXR
, YEP-RXRß, YEP-RXR
, YEP-RAR
, YEP-RARß,
YEP-RAR
, YEP-TR
, and YEP-TRß were as described (33, 59).
Expressions of all of these holo- or chimeric receptor constructs were
confirmed by Western analyses (33, 58, 59). T7 vector to express
full-length hER
and expression vector for GST-fusions to full-length
TRß, RXR
and RAR
were gifts from Dr. David Moore at Baylor
College of Medicine (36). A PCR fragment encoding GR-LBD (human GR
amino acids 484777) was cloned into EcoRI and
SalI restriction sites of pGEM4T (Pharmacia, Piscataway, NJ)
to construct GST/GR-LBD. Mammalian expression vectors for ER, TR
,
RXR
, and RAR
, the reporter constructs ERE-TK-CAT and ERE-TK-Luc,
and the transfection indicator construct pRSV-ß-gal are as described
(34, 52, 53, 58). Vectors for the mammalian two-hybrid tests were gifts
from Dr. Ron Evans at Salk Institute (37). All the PCR-based constructs
were sequenced against any mutation that might have been erroneously
introduced during amplification.
Yeast ß-Galactosidase Assays
The cotransformation and transactivation assays in yeast as well
as quantitative liquid ß-galactosidase assays were performed with the
following changes as described previously (33). The yeast culture were
initially diluted to an A600 of 0.05, and plated
into 96-well culture dishes with the various concentrations of hormone.
The cultures were then incubated in the dark at 30 C for 16 h. The
A600 was determined, and then cells were lysed and
substrate was added and A415 was read after 1030 min. The
normalized galactosidase values were determined as follows:
(A415/A600) x 1000/min developed. For each
experiment, at least six independently derived colonies expressing
chimeric receptors were tested.
Pull-Down Assays
GST fusion proteins were produced in Escherichia coli
and purified using glutathione-Sepharose affinity chromatography
essentially as described (36). GST proteins were bound to
glutathione-Sepharose 4B beads (Pharmacia) in binding buffer (50
mM KPO4, pH 6.0, 100 mM KCl, 10
mM MgCl2, 10% glycerol, 10 mg/ml E.
coli extract and 0.1% Tween 20). Beads were washed once with
binding buffer and incubated for 60 min at 4 C in the same buffer with
equivalent amounts of various proteins labeled with
[35S]methionine by in vitro translation.
Nonbound proteins were removed by three washes with binding buffer
without E. coli extract, and specifically bound proteins
were eluted with 50 mM reduced glutathione in 0.5
M Tris, pH 8.0. Eluted proteins were resolved by PAGE and
visualized by fluorography.
Cell Culture and Transfections
CV1 cells were grown in 24-well plates with medium supplemented
with 10% charcoal-stripped serum. After 24 h incubation, cells
were transfected with 150 ng of ß-galactosidase expression vector
pRSV-ß-gal and 100 ng of a reporter gene ERE-TK-CAT or ERE-TK-Luc,
along with 10 ng ER- and increasing amounts (50100 ng) of TR
,
RAR
, or RXR
expression vector. Total amount of expression vectors
were kept constant by adding decreasing amounts of the CDM8 expression
vector to transfections containing increasing amounts of the TR
,
RAR
, or RXR
vector. For the mammalian two-hybrid tests, a
reporter gene Gal4-TK-Luc, vectors expressing VP16/-, VP16/TR,
VP16/RAR, or VP16/RXR, and Gal4DBD/ER-LBD expression vector were used
as described (37). After 12 h, cells were washed and refed with
DMEM containing 10% charcoal-stripped FBS. After 12 h, cells were
left unstimulated or stimulated with 0.1 µM ligand. Cells
were harvested 24 h later, and CAT or luciferase activity was
assayed as described (58), and the results were normalized to the
ß-galactosidase expression. Similar results were obtained in more
than two similar experiments.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Wongi Seol, Yoon Kwang Lee, and David D. Moore for
valuable advice, plasmids, and communications of their results before
publication. We are also grateful to Dr. Vincent Giguere for mERß and
Dr. Ron Evans for the mammalian two-hybrid vectors.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jae Woon Lee, Ph.D., College of Pharmacy, Hormone Research Center, Chonnam National University, Kwangju, 500757 Korea. E-mail: jlee{at}chonnam.chonnam.ac.kr
This research was supported by grants from KOSEF
(960401-0801-3) and HRC.
Received for publication January 13, 1998.
Revision received April 6, 1998.
Accepted for publication April 13, 1998.
 |
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