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INTRODUCTION |
Estrogen has a wide range of physiologic activities, including the
control of development, reproduction, and metabolism as well as effects
on cell growth and differentiation. Most, if not all, actions of
estrogen occur through its receptors,
ER
1 and ER
. The
functional domains of the ER are relatively well defined. These domains
include the N-terminal domain (A/B regions), DNA binding domain (C),
hinge (D), ligand binding domain (E), and the C-terminal domain (F). A
ligand-dependent activation function 2 in the C-terminal
region of the ligand binding domain (LBD) and a ligand-independent
activation function 1 in the N-terminal domain have also been
characterized (1, 2).
In the traditional model of ER action, the receptor binds as homodimers
(3) or heterodimers (4-7) to estrogen response elements (EREs) in the
promoters of many, though not all, estrogen-responsive genes. Similar
to other nuclear receptors, the ER recruits an array of transcriptional
cofactors (coactivators and corepressors) that bind to the receptor and
also interact with other transcription factors, including components of
the general transcription factor apparatus. Some of the cofactors also
possess chromatin-remodeling activities or recruit additional proteins
to the complex to mediate transcription (reviewed in Ref. 8).
It is now recognized that the type of ligand bound to the ER influences
its interaction with cofactors. The crystal structures of the ER LBD
when bound to an agonist (estradiol) or an antagonist (raloxifene) have
been solved. Comparison of these structures suggests a molecular basis
for the differential ligand-dependent cofactor binding (9).
The binding of 17
-estradiol induces a major shift in the position of
helix 12, one of several helices that form the coactivator interaction
surface. Substitution of raloxifene or 4-hydroxytamoxifen (10) for
estradiol changes the orientation of helix 12 in a manner that
partially obscures the residues involved in the coactivator
interaction. Antagonist-bound ER binds to corepressors in
vitro (11, 12), but these interactions are not as strong as those
seen with certain other nuclear receptors such as the thyroid hormone
or retinoic acid receptors. The region of cofactor binding has been
localized to a hydrophobic surface of the LBD.
Not all genes that are regulated by the ER contain an ERE. The
mechanism for estrogen action through this "nonclassical" pathway (or pathways) is not clear. However, several lines of evidence suggest
that the ER interacts with other transcription factors bound to their
response elements (e.g. NF-
B, SP1, electrophile response
element, AP1) in these target genes. Repression exerted through the
NF-
B site has been examined in the context of the human interleukin
6 promoter (13, 14). In this case, repression is dependent on two
transcription factors, NF-
B and CAAT enhancer-binding protein
. A direct interaction of NF-
B and ER has been
demonstrated and requires the DBD and the D region of ER (13). This
direct protein binding contributes to interleukin 6 promoter repression by estrogen (14).
The ER has also been shown to affect gene expression from promoters
containing an AP1 site. In some cases, such as the collagenase (15-17), human insulin growth factor 1 (18), or chicken ovalbumin (19)
promoters, estrogen activates expression. Of interest, in the context
of the collagenase promoter ER antagonists also stimulate expression
(15, 17). Other genes containing an AP1 site in their promoters are
negatively regulated by estrogen, including the ovine
follicle-stimulating hormone
(20) and human choline
acetyltransferase gene (21).
In this report, we further examine the mechanism by which the ER acts
through the nonclassical pathway, using the AP1 response element as a
model. We demonstrate, using selective DBD mutations, that DNA binding
by the ER is not necessary for its activity through this nonclassical pathway.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The mouse ER
expression vector was provided by
Malcolm Parker (Imperial Cancer Research Fund, London, United Kingdom)
and subcloned into pcDNA3.1(
) (Invitrogen). Point mutations were introduced using overlapping polymerase chain reactions, and the sequence of the mutated cDNA was confirmed by DNA sequencing. The
Gal4-Jun expression vector contains the Gal4 DNA binding domain fused
to a fragment of human c-Jun lacking its DNA binding domain (amino acid
332 to the C terminus) in pSG424 (22). The Gal4 DNA binding domain in
pSG424 was used as a control. The reporter plasmid
ERE2-tk109-luc has been described previously (23). The AP1-luc reporter contains seven AP1 sites linked to a basal promoter (Stratagene); the 73col-luc reporter contains a fragment of collagenase promoter (
73 to +63) containing one AP1 site (24). The
UAS-E1b-TATA-luc reporter contains five copies of the upstream
activating sequence (UAS) upstream of E1b-TATA in the pA3-luc
vector (25).
Cell Culture--
TSA-201 cells, derived from estrogen
receptor negative human embryonic kidney 293 cells (26), were cultured
in Dulbecco's modified Eagle's medium supplemented with 5%
fetal bovine serum. MCF-7 cells (estrogen receptor positive,
subclone WS8, derived from human breast adenocarcinoma), provided by V. Craig Jordan (Northwestern University Medical School, Chicago, IL),
were cultured in minimum Eagle's medium supplemented with
nonessential amino acids, 10 mM Hepes, and 5% calf serum.
Four days before transfection, cells were harvested using phenol
red-free trypsin-EDTA and cultured in estrogen-depleted media
(prepared without phenol red and supplemented with sera extracted three
times with dextran-coated charcoal).
Transfections and Luciferase Assays--
Cells were transferred
to 12- or 24-well plates in estrogen-depleted medium 1 day prior to
transfection. TSA-201 cells were transfected with calcium phosphate as
previously described (27), and MCF-7 cells were transfected with
liposomes as previously described (23). ERE, AP1, and 73col reporter
plasmids (500 ng/well) were transfected together with 10 ng/well
receptor expression vector or empty vector used as a control. Mammalian
cell two-hybrid experiments used the UAS-E1b-TATA-luc reporter (500 ng/well) to detect Gal4-Jun (50 ng/well) activity and its interaction
with various ER mutants (1 ng/well). 17
-Estradiol was purchased from Sigma and ICI 182,780 was provided by Alan Wakeling (Zeneca
Pharmaceuticals). Estradiol (1 nM) and ICI 182,780 (100 nM) were added to treatment media as stock solutions
in absolute ethanol. Ethanol was added to control media in the
same final solvent concentration (typically 0.1%). Luciferase activity
was determined 24 or 48 h after transfection by using an AutoLumat
LB953 luminometer (EG&G) and expressed as relative light units. The
mean and standard errors of triplicate or quadruplicate samples are
shown for representative experiments. All transfection experiments were
repeated three or more times with similar results.
Electrophoretic Mobility Shift Assays--
The ERE probe and the
conditions for electrophoretic mobility shift assays were previously
described (28). Protein samples were prepared by in vitro
translation (TNT, T7 coupled in vitro translation kit,
Promega), preincubated with a binding buffer containing 10 mM Hepes, pH 7.9, 50 mM KCl, 5% glycerol, 50 ng/µl herring testes DNA, and 1 nM 17
-estradiol at
4 °C for 30 min, and then incubated with labeled ERE probe at
4 °C for 30 min in a total volume of 20 µl. The samples were
loaded and subjected to electrophoresis through 4% nondenaturing
polyacrylamide gels, and radioactivity was visualized by autoradiography.
Western Blots--
TSA-201 cells were transfected with the
indicated expression vectors and cultured in medium supplemented
with regular (unextracted) serum. Nuclear extracts were prepared as
described elsewhere (29). The extracts were fractionated using 10%
SDS-polyacrylamide gel electrophoresis gels and transferred onto
HybondTM-P transfer membranes (Amersham Pharmacia Biotech).
Immunodetection was performed using mouse monoclonal ER antibody D-12
(Santa Cruz Biotechnology) and anti-mouse, horseradish
peroxidase-conjugated IgG (Promega). Proteins were visualized
using an ECL+Plus kit (Amersham Pharmacia Biotech) according to the
manufacturer's instructions.
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RESULTS |
ER Agonists Repress and Antagonists Stimulate Transcription
Mediated by the Nonclassical AP1 Pathway--
The response to ER
agonists and antagonists was examined using classical and nonclassical
pathway reporter constructs. For the classical pathway,
ERE2-tk109-luc, which contains two copies of the
vitellogenin gene ERE upstream of the 109-base pair fragment of
thymidine kinase promoter, was used. For the nonclassical pathway, two
different AP1 luciferase reporters were used: 73col-luc, which contains
a fragment of the collagenase promoter (
73 to +63) and includes a
single AP1 site (24), and AP1-luc, which contains seven AP1 sites
upstream of a basal promoter (Stratagene). Transfections were performed
in ER-negative TSA-201 cells.
In the absence of transfected ER, hormone treatments did not alter the
activity of any of these reporter constructs, confirming the absence of
endogenous ER or other estrogen-responsive pathways (data not shown).
When ER was cotransfected with the ERE reporter, estradiol activated
and the antiestrogen ICI 182,780 repressed transcription (Fig.
1, TSA, ERE
reporter). The responses to agonists and antagonists were
reversed when the AP1-luc reporter was used. Estradiol suppressed
transcription, and the antiestrogen ICI 182,780 stimulated promoter
activity (Fig. 1, TSA, AP1 reporter). These effects were specific for the presence of AP1 sites; no response was
observed using a reporter lacking the AP1 sites but retaining the
remainder of the promoter and the vector backbone (data not shown). A
reporter containing a fragment of the native collagenase promoter (
73
to +63), which contains a single AP1 site, displayed a response pattern
similar to the artificial AP1-luc reporter (Fig. 1, TSA, 73col
reporter), and all subsequent experiments were performed with
AP1-luc. Consistent with the effects of estradiol and ICI 182,780, diethylstilbestrol repressed whereas raloxifene, tamoxifen, and
4-hydroxytamoxifen stimulated AP1-luc transcription (data not
shown).

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Fig. 1.
Estradiol and ICI 182,780 have opposite
effects on the classical (ERE reporter) and the nonclassical pathways
(AP1 reporters). Transient transfection of TSA cells
(TSA column) with mER expression vector and
reporters (ERE, ERE2-tk109-luc; AP1,
AP1-luc; 73col, 73col-luc) and transient transfection of
MCF-7 cells (MCF-7 column) with the indicated
reporters are shown. RLU, relative light unit.
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Reporter constructs were also transfected into ER-positive MCF-7 cells
in the absence of the ER expression vector. Responses mirrored those
observed in TSA cells. Estrogen activated the ERE reporter and
repressed the AP1-luc and 73col-luc reporters, whereas ICI 182,780 repressed the ERE reporter and activated the AP1-luc and 73col-luc
reporters (Fig. 1, MCF-7). The ligand effects on the
nonclassical pathway were enhanced when exogenous ER was transfected into MCF-7 cells along with the AP1-luc reporter (data not shown).
Mutations That Abolish ER Binding to the ERE Do Not Disrupt
Activity through the Nonclassical AP1 Pathway--
To determine
whether the nonclassical pathway requires ER binding to DNA, four
mutations were introduced into the ER DBD. The mutant receptors were
examined for DNA binding and functional activity using the ERE and AP1
reporters. These mutations were hypothesized to preclude ER action
through the classical pathway but to retain the ability to interact
with other transcription factors, thereby potentially mediating actions
through the nonclassical pathway. The structure of the zinc fingers and
the location of introduced mutations are depicted in Fig.
2A. The first three mutants
are all within the "P-box" of the first zinc finger, a region known
to mediate interaction with DNA (30). The E207G/G208S mutant (a double
mutant) has been demonstrated previously to disrupt binding to the ERE
(31). The E207A/G208A mutant was created with the intent of better
preserving the protein structure by substituting with alanine residues.
In the third mutant, Lys-210 was changed to Ala, disrupting the direct
interaction of the positively charged lysine with DNA (32). The fourth
mutant, A277T, is in the "D-box" of the second zinc
finger, a region that has been implicated in homodimerization and
thereby DNA binding (32).

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Fig. 2.
Mutations introduced into the DBD of
mER abolish or diminish ER binding to
DNA. A, schematic illustration of the mER zinc
fingers in the DBD. Amino acids that constitute P-box and D-box are in
bold letters. Residues Glu-207 and Lys-210 within the P-box make direct
contacts with the DNA base pairs. Residues Pro-226, Ala-227, and
Gln-230 within the D-box directly participate in the formation of a
dimer. Mutated amino acids are circled. B,
electrophoretic mobility shift assay using the ERE vitellogenin probe
and WT or mutant mouse ER proteins.
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The DNA binding properties of these mutants were tested in gel mobility
shift assays (Fig. 2B). E207G/G208S had no detectable binding and E207A/G208A exhibited minimal residual binding (0.8%) relative to the wild type (WT) ER. The mutation in the dimerization domain (A227T) retained a small amount of ERE binding (2.4% WT). The
K210A mutation was substantially less effective than the other mutants
at disrupting DNA binding (8.1% WT).
The ability of these mutants to act through the classical and
nonclassical pathways was examined in transient transfection assays
(Fig. 3, A and B).
Three of the mutants (E207G/G208S, E207A/G208A, K210A) showed little or
no estrogen responsiveness through the ERE-mediated classical pathway
(Fig. 3A). The A227T D-box mutant retained significant
estrogen responsiveness through the ERE reporter. The loss of activity
with the E207G/G208S and E207A/G208A mutants was confirmed using
artificial reporters containing EREs from other estrogen responsive
genes such as human pS2 and human oxytocin (data not shown). Western
blot analysis of nuclear extracts demonstrated equal expression of the
WT and mutant proteins (Fig. 3C).

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Fig. 3.
Mutations that eliminate ERE binding preserve
activity through the nonclassical AP1 pathway. A,
transient transfection of TSA cells using the
ERE2-tk109-luc reporter and the indicated WT and mutant
mouse ER constructs. B, transient transfection of TSA
cells using the AP1-luc reporter. C, Western blot analysis
of nuclear extracts of TSA cells transiently transfected with the
indicated constructs of mouse ER , using anti-ER
antiboby. RLU, relative light unit.
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In the AP1 reporter assay (Fig. 3B), the E207G/G208S,
E207A/G208A, and A227T mutants each retained the nonclassical pattern of response. The E207A/G208A mutant was the most active, with responses
similar to those of the WT ER. Thus, E207A/G208A exhibits selective
loss of ERE binding and transcriptional control by the classical
pathway but retains full regulation by the nonclassical pathway. The
K210A mutant lost the ability to act through the nonclassical pathway.
Functional Interaction Between ER and the AP1 Protein Jun--
A
mammalian two-hybrid assay was used to test the hypothesis that ER
interacts with AP1 proteins in the nonclassical pathway. WT or mutant
ER was cotransfected with a Gal4-Jun fusion protein, using
UAS-E1b-TATA-luc as a reporter (Fig.
4B). In control experiments, the Gal4 DBD alone did not activate the UAS-E1b-TATA-luc reporter, either in the absence or presence of ER. Gal4-Jun increased basal expression but did not confer responsiveness to estradiol or ICI 182,780. When WT ER was coexpressed, estradiol repressed
Gal4-Jun-mediated transcription, whereas ICI 182,780 activated
transcription, a pattern that mimics the activity seen in the AP1
reporter assays.

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Fig. 4.
Functional interaction between Jun and
ER. A, schematic illustration of the experimental
design. B, transient transfection of TSA cells using the
UAS-E1b-TATA-luc reporter, Gal4 alone, or the Gal4-Jun fusion protein
with full-length mER or empty vector pcDNA3.1. C,
transient transfection of TSA cells using UAS-E1b-TATA-luc, Gal4-Jun
fusion, and the indicated mouse ER constructs. RLU,
relative light unit.
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The activities of the DBD mutants were also tested in this assay.
E207A/G208A, a mutant that preserved the nonclassical response pattern
for AP1 (Fig. 3B), strongly activated Gal4-Jun-mediated transcription in the presence of ICI 182,780 (Fig. 4C). The
E207G/G208S and A227T mutants, which exhibited less robust activity in
the AP1 reporter assay (Fig. 3B), activated Gal4-Jun to a
lesser extent than E207A/G208A or WT ER (Fig. 4C). K210A
lost all activity in both the AP1 reporter assay and in the interaction
assay. Thus, the transcriptional properties of the ER DBD mutants are
similar in the Gal4-Jun and AP1 reporter assays.
ER Mutations That Abolish Interactions with Coactivators
Disrupt Signaling through the Classical and Nonclassical
Pathways--
The LBD of ER plays an important role in cofactor
interactions, which are central to ER activity through the ERE. Two
different point mutations (I362R and K366D) were introduced into ER
helix 3, which together with helices 4, 5, and 12 forms a hydrophobic cavity that is involved in cofactor interactions. The specific substitutions were chosen based on their high degree of conservation when compared with related nuclear receptors (Fig.
5A) and because similar
mutations have been reported to disrupt interactions with specific
coactivators, including steroid receptor coactivator 1 (SRC-1)
(33, 34). Using a mammalian two-hybrid system, these mutant ERs were
confirmed to lose most (I362R) or all (K366D) of their interaction with
Gal4-SRC-1 and Gal4-GRIP-1 in the presence of estradiol (data
not shown).

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Fig. 5.
Evidence for transcriptional cofactor
interaction in the classical and nonclassical pathways.
A, alignment of the sequence of helix 3 among mER and
other members of the nuclear receptor family. Mutated residues are
shown in bold. B, transient transfection of TSA
cells using the ERE2-tk109-luc reporter and the indicated
mER constructs. C, transient transfection of TSA cells
using the AP1-luc reporter and the indicated mER constructs.
D, transient transfection of TSA cells with UAS-E1b-TATA-luc
reporter, Gal4-Jun, and expression vectors containing WT mER or its
mutants I362R or K366D. RLU, relative light unit; hER ,
human ER ; hRXR , human retinoid X receptor
; rTR , rat thyroid hormone receptor ;
hPR, human progesterone receptor.
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The K366D mutation largely eliminated the effects of estradiol and ICI
182,780 (Fig. 5, B and C). With the ERE reporter,
basal activity was greatly reduced, as were estradiol activation and ICI suppression. With the AP1 reporter, estradiol suppression was
eliminated and ICI stimulation was markedly decreased. The effect of
the I362R mutation was more dependent on the ligand (estradiol
versus ICI). Estradiol stimulation of the ERE reporter and
suppression of the AP1 reporter was retained. ICI 182,780 stimulated
the ERE reporter and elicited a small decrease with the AP1 reporter.
These results indicate that ER mutations that alter binding to
transcriptional cofactors impair ER action through both the classical
and nonclassical pathways.
The effects of the helix 3 mutants in the mammalian two-hybrid assays
with Gal4-Jun (Fig. 5D) were similar to those seen with the
AP1 reporter, consistent with the idea that ER actions on the AP1
reporter are mediated through interactions with Jun.
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DISCUSSION |
In the classical pathway, ER action is mediated by direct receptor
binding to EREs. Agonist binding induces an ER conformation that favors
interactions with coactivators and general transcription factors,
resulting in increased transcription. When bound to an antagonist, the
ER does not interact with coactivators and, in turn, does not activate
transcription. In contrast, the mechanism by which ER regulates
nonclassical pathways is less well established. We hypothesized that
this mechanism involves ER interactions with other proteins, rather
than direct binding to DNA. AP1-regulated genes were used as a model
system for the nonclassical pathway. With these reporter genes,
agonists repress and antagonists activate transcription in the presence
of ER. We identified a DBD mutant of ER (E207A/G208A) that lost
transactivation through the classical pathway but retained regulation
of the nonclassical pathway. These data provide new insights into
mechanisms by which nonclassical ER signaling occurs. First, ligands
traditionally considered estrogen receptor agonists or antagonists have
an opposite effect on the nonclassical AP1 pathway, where agonists
repress and antagonists activate transcription. Such a reversal of
activity suggests that a novel mechanism mediates ER activity through
the nonclassical AP1 pathway. Second, it is possible to abolish the
ability of ER to bind to an ERE and still preserve activity through the
nonclassical pathway. However, as discussed below, the ER DBD likely
interacts with other proteins, such as Jun, and this interaction
requires a structurally intact DBD. Third, the interaction between ER
and cofactors has functional significance both in the classical and the
nonclassical pathways.
In the nonclassical pathway, we found that estradiol represses and ICI
182,780 activates transcription via the AP1 reporter, a pattern that is
the opposite of the ligands' effects on the ERE reporter. Though the
activation of transcription by an antagonist is consistent with other
reports (15-17), the repressive effect of estradiol on the collagenase
promoter has not been observed previously, perhaps because of
differences in experimental conditions. However, estrogen-mediated
suppression of gene expression is a common physiologic phenomenon and
includes genes such as interleukin 6 (13, 14), tumor necrosis
factor
(35), follicle-stimulating hormone
(20), choline
acetyltransferase (21), quinone reductase (36), and lipoprotein lipase
(37). In many cases, negative regulation involves the AP1 site (20, 21,
37).
ER action through the nonclassical pathway does not involve ER binding
to DNA and appears to be mediated by protein-protein interactions. For
example, the P-box mutation E207A/G208A in the first zinc finger
eliminates DNA binding and ERE activation but preserves activity
through the nonclassical pathway. A possible candidate for the
protein-protein interaction in the nonclassical pathway is Jun, a
member of the AP1 protein family. This idea is supported by evidence
for ER interaction with Gal4-Jun in mammalian two-hybrid assays and by
the observation that ER mutants have similar effects when tested in
Gal4-Jun interaction assays or in AP1 reporter assays. The ICI-induced
interaction between Jun and ER detected in a two-hybrid assay may not
be direct, as other cellular proteins (e.g. coactivators)
may participate in the Gal4-Jun-ER interaction. Attempts to supershift
AP1-bound Jun with ER in electrophoretic mobility shift assays or to
coimmunoprecipitate a Jun-ER complex, did not detect direct
interactions (data not shown). It is possible that these interactions
are not strong enough to withstand the experimental conditions.
Alternatively, cofactors such as steroid receptor coactivator 1 or other proteins may bridge or stabilize the Jun-ER complex.
Previous studies have demonstrated a direct interaction between ER and
c-Jun using glutathione S-transferase pull-down
assays (16, 38). It is notable, however, that in the mammalian
two-hybrid assays, the ER-Jun interaction was induced by ICI 182,780 but not by estradiol, suggesting that conformational changes may
influence the protein interactions. The mechanism of estradiol-induced
repression remains unknown, but may be influenced by the promoter
context of the estrogen-dependent regulatory sites. For
example, activation of interleukin 6 promoter requires the synergistic
activity of two transcription factors, CAAT enhancer-binding protein
and NF-
B. In this case, estradiol-induced repression appears to
involve interactions with each of these factors (13, 14). Another possible repression mechanism could involve estrogen-mediated inhibition of the Jun N-terminal kinase pathway (39). For example, estrogen-dependent repression of the receptor activator of
NF-
B ligand appears to involve down-regulation of c-Jun expression and a decrease in Jun phosphorylation by the c-Jun N-terminal kinase
(40).
As with the AP1 reporter, WT ER and the DBD mutants activate Gal4-Jun
dependent transcription in the presence of ICI 182,780. We found that a
construct containing only the D, E, and F domains of ER does not
affect transcription from the AP1 reporter despite hormonal treatment,
whereas the construct lacking the A/B domain but containing the DBD
alters transcription in the same manner as WT ER (data not shown).
These findings are consistent with several published reports (16, 18,
38) and raise the possibility that ER interactions with Jun involve a
region within the DBD. It is also notable that the K210A mutation
eliminates activity through the nonclassical pathway, as well as
interaction with Jun, even though this mutant retains partial activity
through the ERE. Because neither ER zinc finger is embedded within the protein structure, the zinc fingers may participate in protein-protein interactions.
It is well established that coactivators are involved in the classical
pathway. Using selective ER mutants (I362R and K366D) that alter
interactions with coactivators, we found that these mutants not only
affect the classical pathway but also impair ER action through the
nonclassical AP1 pathway. The hydrophobic pocket where these mutations
were introduced interacts with both coactivators and corepressors. For
example, the mutation of residues analogous to Ile-362 and Lys-366 in
thyroid hormone receptor
diminishes triiodothyronine
activation but also impairs basal repression and the interaction with
corepressors nuclear receptor corepressor (NCoR) and silencing mediator
for retinoic acid and thyroid hormone receptors (SMRT) (41). It
is therefore possible that the I362R and K366D mutations affect
interactions with corepressors although we did not detect any
interactions between ER and corepressors in two-hybrid assays (data not
shown), perhaps because these interactions are weaker than the
ER-coactivator interactions.
One can speculate that the reversal of the ligands' effects on the
nonclassical pathway is because of the reversal of cofactors bound to
liganded ER. It is somewhat counterintuitive that the antagonist ICI
182,780 might cause an interaction between the ER and coactivators and
that estradiol would induce an interaction between the ER and
corepressors. However, it is possible that when the ER is involved in a
protein-protein interaction instead of binding to an ERE, the
hydrophobic cofactor binding pocket may assume a different shape,
shifting the interactions of associated proteins. As mentioned above,
the same region of the LBD recognizes both coactivators and
corepressors (41), increasing the likelihood that subtle changes in the
tertiary protein structure could change a subset of proteins recognized
by the ER.
The I362R mutant provides an additional argument in support of the
hypothesis that subtle changes in protein structure can have a profound
effect on the recognition of coactivators. This mutation does not
completely eliminate the interaction with coactivators and therefore
still stimulates transcription through the classical pathway in the
presence of estradiol. However, this mutant also activates
transcription in the presence of an antagonist. In this respect, the
I362R mutation resembles some mutations of helix 12 that "switch"
an antagonist to an agonist. Examples include the mutant L540Q of human
ER
(42) and the double mutants L543A/L544A and M547A/L548A of
mouse estrogen receptor
(mER
) (43), which are activated by ICI
164,384 and 4-hydroxytamoxifen.
We conclude that for the AP1 response element, ER interacts with other
proteins instead of, or in addition to, DNA to exert its
transcriptional effects. The nonclassical pathway retains the
requirement for cofactors. However, the patterns of cofactor binding
change, as demonstrated by the reversal of estradiol/ICI activities
through the AP1 reporter. These data confirm the sensitivity of ER
activity to subtle changes in its structure, whether caused by
artificial mutations or by an altered set of associated proteins.