From the Diabetes Center and the
§ Department of Medicine, University of California,
San Francisco, California 94143
Received for publication, August 20, 2002, and in revised form, December 11, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Selective estrogen receptor modulators (SERMs)
show differential effects upon ER Estrogen signaling is mediated by two estrogen receptors (ER Selective estrogen receptor modulators (SERMs) are used in treatment
and prevention of estrogen-dependent breast cancer (11). The SERMs inhibit ER action by blocking AF-2 activity, and the mechanism of this effect is now understood at the atomic level (2).
AF-2 is composed of surface-exposed residues from LBD helices (H) 3, 5, and 12, which form a hydrophobic cleft that binds short nuclear
receptor (NR) boxes (consensus
Leu-X-X-Leu-Leu or LXXLL) found
in each p160 (12, 13). Tamoxifen and raloxifene both protrude through
the LBD surface and displace H12, which rotates 110 ° and utilizes a
sequence that resembles the NR box (LLEML) to bind the upper part of
the cleft (14-17). The antagonist ICI 182,780 (ICI) also possesses a
longer extension that protrudes through the LBD surface and displaces
H12, but H12 is not resolved in this structure (18).
While SERMs act as antagonists in breast, they exhibit estrogen-like
effects in other tissues. Tamoxifen shows beneficial agonist effects,
including prevention of osteoporosis, and harmful agonist effects,
including emergence of tamoxifen-stimulated breast cancers and
increased rates of uterine cancer (19). Raloxifene and GW5638 show
agonist effects in bone and other tissues, but lack harmful uterotropic
effects (20). ICI shows agonist effects in bone (21), but generally
acts as a pure antiestrogen (22). Understanding why SERMs exhibit
differential estrogen-like activities will help in identification of
new SERMs with favorable profiles of activity.
Some SERM estrogen-like effects stem from AF-1 (2, 23). ER Other estrogen-like effects of SERMs stem from ER action at genes with
alternate response elements (5-7). ER Why do SERMs show differential effects on ER While SERMs differ in their ability to promote N-CoR recruitment at one
type of gene, it is not clear why. SERM-ER Reagents--
Cells were grown in Dulbecco's modified
Eagle's/F-12 Ham's 1:1 mix without phenol red (Sigma) containing 10%
iron-supplemented calf serum (Sigma) and pen-strep. Estradiol,
diethylstilbestrol (DES), tamoxifen, and trichostatin A were from
Sigma. Raloxifene was a gift from Karo Bio AB, Huddinge, Sweden. ICI
was a gift from Astra/Zeneca, Macclesfield, UK.
Plasmids--
The following plasmids were previously described.
Reporters were ERE-TK-Luc, TK-Luc, Coll73-Luc, and actin
ER Transfections--
Cells were transfected by electroporation
(47). Transfections contained 2 µg of reporters and, where indicated,
1 µg of ER Bacterial Expression and Protein Binding Assays--
GST fusions
were expressed in Escherichia coli BL21 and purified as
before (26). Briefly, cultures were grown to
A600 1.5 at room temperatures
(~22 °C), and protein production was initiated by addition of
isopropyl-1-thio-
Interactions between [35S]methionine-labeled ER Immunoprecipitations--
10-cm dishes of transfected HeLa cells
were treated with ligands for 24 h, chilled on ice, washed with
ice-cold PBS, incubated in lysis buffer (0.2% Triton X-100, 150 mM NaCl, 50 mM Tris pH 7.8, 1 mM
EDTA, 1/1000 dilution Novagen protease inhibitor mixture) for 10 min,
scraped, and centrifuged at 4 °C in an Eppendorf bench top
centrifuge at maximum speed for 15 min. Protein concentration in cell
extract was determined by standard methods. The concentration of each
sample was then normalized by addition of the appropriate volume of
cold lysis buffer to ~5 mg of total protein in 1 ml of total volume.
Each sample was precleared at 4 °C for 2 h with 25 µl of
protein G plus agarose beads (Santa Cruz antibodies, Santa Cruz, CA)
that were prewashed twice with lysis buffer. The beads were then
pelleted by brief centrifugation, and the supernatant was mixed with 25 µl of fresh prewashed protein G plus agarose beads and 5 µg of GAL4
antibody RK5C1 (Santa Cruz antibodies). The mix was shaken overnight in
the cold room. The following day, the beads were pelleted, washed three
times in lysis buffer, dried, and resuspended in SDS-PAGE loading buffer.
Western Blots--
For analysis of immunoprecipitation, protein
from a standard volume of beads was used. For whole cell extracts,
protein concentration was determined in cellular extract by standard
methods, and 20-30 µg of proteins were separated by SDS-PAGE.
Proteins were transferred to a prewetted Immuno-Blot polyvinylidene
difluoride membrane (Bio-Rad, Hercules CA) overnight at 90 mA/30 V
using a standard transfer apparatus. Following transfer, the membrane
was incubated at room temperature in 5% non-fat milk in PBS-T (1×
PBS, 0.1% Tween 20) for 1 h and washed twice in PBS-T for 10 min.
The primary ER A HDAC Inhibitor Eliminates Differential SERM AF-1 Inhibition at an
ERE-responsive Promoter in MCF-7 Cells--
The nuclear receptor
corepressor complex inhibits transcription by recruiting histone
deacetylases, or HDACs (4). To begin this study, we determined whether
inhibition of HDACs would affect SERM inhibition of ER
Fig. 1A shows that, as often
noted, an ERE-responsive reporter (ERE-TK-LUC) exhibited weak
constitutive activity in MCF-7 breast tumor cells and that this
activity was enhanced by estrogens and reduced by SERMs, especially by
ICI. A similar reporter (TK-LUC), which lacked an ERE, was not affected
by ligand. Addition of HDAC inhibitor (trichostatin A, TSA) enhanced
basal activity of both reporters. However, TSA also specifically
blocked the ability of SERMs to inhibit ERE-dependent
transcription, with the result that fairly equivalent levels of
ERE-dependent transcription were obtained in the absence of
hormone and in the presence of each ligand. Similar results were also
obtained in T47D breast cancer cells and GHFT1-5 pituitary cells (not
shown).
To highlight TSA effects upon ER SERMs Show Differential Ability to Sequester Corepressors from
Progesterone Receptor--
Next, we determined whether SERMs would
differentially influence ER
In agreement with previous results, ER Differential Ability of SERMs to Promote ER
As expected (31), Western blotting of cell extracts prior to
immunoprecipitation confirmed that ICI and raloxifene reduced steady
state ER
To further investigate the ligand preference of ER A Mutation (ER
It is known that the nuclear receptor corepressor-binding site lies in
the LBD hydrophobic cleft and that corepressor binding is enhanced by
truncation of H12 (see for example Ref. 48). We first confirmed that
truncation of ER
The effect of the H12 truncation on SERM ligand preference was also
apparent in the context of the isolated ER
Because the 537X mutation allowed N-CoR binding with all SERMs in
vitro, we examined the phenotype of ER An ER
We then tested ER
We then examined whether the L379R mutation would affect the behavior
of the 537X mutation, which, as shown above, increases N-CoR binding
and reduces AF-1 activity. Fig. 8C shows that, in contrast
to the behavior of ER
Finally, ER Mutations That Affect N-CoR Binding Have Parallel Effects on ER
To test these ideas, we examined the effect of the L379R mutation upon
ER
We then examined the effect of the 537X mutation, which increases and
equalizes N-CoR binding in the presence of SERMs. Like ER An Exception to Parallels between N-CoR Binding and AF-1 Activity,
the C3 Complement--
ER
Unlike its activity at EREs and the AP-1 site, however, the
L379R mutation did not preserve any tamoxifen response at C3 in MDA-MB-453 cells (shown here) and other cell types (including HeLa and
HepG2; data not shown). This finding, though not expected, is in line
with previous observations that indicate that ER Differential Corepressor recruitment
underlies differential SERM effects at genes with alternate response
elements in uterine cells (10). However, the structural basis for these
differential SERM effects on corepressor recruitment is unclear, and it
is controversial whether ER In this study, we showed that ICI and raloxifene promote HDAC
repression at an ERE-responsive reporter in MCF-7 cells and ER While SERMs promote N-CoR binding in vitro, the
interactions are weak compared with other nuclear receptors. Between
1-3% of input ER How can we reconcile the paradox that SERMs promote
interactions with N-CoR in vivo, yet ER The diverse effects of SERMs on ER activation function 1 (AF-1).
Tamoxifen allows strong ER
AF-1 activity, whereas raloxifene allows
less and ICI 182,780 (ICI) allows none. Here, we show that blockade of
corepressor histone de-acetylase (HDAC) activity reverses the differential inhibitory effect of SERMs upon AF-1 activity in MCF-7
cells. This suggests that differential SERM repression of AF-1 involves
HDAC-dependent corepressors. Consistent with this, ICI and
raloxifene are more potent than tamoxifen in promoting ER
-dependent sequestration of progesterone
receptor-associated corepressors. Moreover, ICI and raloxifene are more
efficient than tamoxifen in promoting ER
binding to the corepressor
N-CoR in vivo and in vitro. An ER
mutation
(537X) that increases N-CoR binding in the presence of all SERMs blocks
AF-1 activity. An ER
mutation (L379R) that decreases N-CoR binding
increases AF-1 activity in the presence of ICI and raloxifene and
reverses the effect of the 537X mutation. The 537X and L379R mutations
also alter the ligand preference of ER
action at AP-1 sites and C3 complement, an action that also involves AF-1. Together, our results suggest that differential SERM effects on corepressor binding can
explain differences in SERM effects on ER
activity. We propose a
model for differential effects of SERMs on N-CoR binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and ER
),1 which are
conditional transcription factors (1-3). In the best understood
pathway of ER action, the ERs bind specific estrogen response elements
(EREs) in the promoter of estrogen-regulated genes and activate
transcription by recruiting a large coactivator complex composed of
p160 coactivators such as GRIP1 and SRC-1 and the histone
acetyltransferases p300/CREB-binding protein and pCAF (4). Like
other nuclear receptors, the ERs are comprised of an N-terminal
domain (NTD), a central DNA-binding domain (DBD), and a C-terminal
ligand-binding domain (LBD). The DBD mediates ERE recognition, and the
NTD and LBD contain distinct activation functions (AF-1 and AF-2,
respectively) that mediate coactivator recruitment. The ERs also
modulate expression of genes with alternate estrogen response elements,
such as AP-1 sites and SP-1 sites (5-7). ER
acts at AP-1 sites via
protein-protein interactions and recruits p160s in a process that
requires ER
AF-1 and AF-2 and their cognate binding sites within the
p160s (6, 8-10). Thus, estrogen action at classical EREs and alternate
response elements involve similar coactivators.
and ER
AF-1 usually exhibit weak activity at genes with classical EREs, and
consequently, SERMs exhibit little agonist activity at this type of
gene. However, ER
AF-1 is strong in some cells (23-25), in the
presence of high levels of AF-1 coactivators (26), in conditions of MAP
and JNK kinase activation (27-29), and at certain promoters (23, 25,
30). In each of these cases, tamoxifen-liganded ER
exhibits activity
that is equal to isolated AF-1, but raloxifene, GW5638-, and
GW7604-liganded ER
show less activity (20, 31), and ICI-liganded
ER
shows no activity at all (23). Thus, SERMs show differential
effects on AF-1 activity at classical EREs.
enhances AP-1 activity in the
presence of tamoxifen but shows weaker activity in the presence of
raloxifene or other SERMs (5, 6, 32, 33). Moreover, these tamoxifen
effects require the ER
NTD (6). Thus, SERMs differentially regulate
some aspect of ER
AF-1 activity at AP-1 sites, just as they do at
classical EREs. SERMs also enhance AP-1 activity via a second mechanism
that is independent of ER activation functions and up-regulated in
ER
truncations that lack AF-1 and in ER
, which naturally lacks
AF-1 (6, 32, 34). We have suggested that these effects may involve
sequestration of corepressors that inhibit AP-1 activity (35), possibly
of the N-CoR/SMRT family (4). ICI and raloxifene strongly enhance ER
action in this AF-independent pathway, whereas tamoxifen does not.
Thus, SERMs differentially regulate two mechanisms of ER action at
AP-1 sites.
activity? One possible
mechanism involves effects upon ER
turnover. Tamoxifen increases
ER
steady state levels, raloxifene and GW7604 reduce ER
levels,
and ICI reduces ER
levels by more than 90% (31, 36). These effects
correlate well with effects on ER
AF-1 activity, but in
vivo competition assays reveal that the amount of ER
that occupies the ERE is relatively unaffected by SERMs (31, 37, 38).
Moreover, ICI and raloxifene act as ER
agonists at genes with AP-1
sites (6). Thus, raloxifene and ICI inhibition of ER
activity is not
simply a consequence of elimination of functional ERs from the cell.
Another possible mechanism involves differences in corepressor
recruitment. SERMs promote ER
interactions with the corepressors
N-CoR and SMRT (39-42) and N-CoR recruitment to estrogen-regulated
promoters in vivo (10, 43). Several lines of evidence
indicate that corepressors inhibit the activity of the SERM-ER
complexes (reviewed in Ref. 4). Moreover, tamoxifen is less efficient
than raloxifene at recruiting N-CoR to estrogen-regulated genes with
alternate response elements in Ishikawa uterine cells (10), and this
effect may explain why tamoxifen behaves as an agonist in this context
when raloxifene does not (10, 26, 32).
complexes interact
relatively strongly with the corepressor complex in vivo (10, 40, 43, 44) but, at best, only weakly bind to bacterially expressed N-CoR and SMRT in vitro (42, 44). It also remains unclear whether SERMs directly influence ER
interactions with N-CoR
itself, or whether these effects involve N-CoR-associated proteins.
Finally, it is unclear whether differences in corepressor recruitment
explain differences in SERM agonist activity in contexts besides ER
action at genes with alternate response elements in uterine cells.
Here, we examine SERM effects upon ER
interactions with N-CoR and
ask whether these effects underlie differences in SERM agonist activity
in a variety of contexts in vivo.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase (5). TAT3-luc was a gift from K. Yamamoto
(University of California, San Francisco) and C3 Complement-Luc was a
gift from D. McDonnell (Duke University, North Carolina). Mammalian
expression/in vitro transcription-translation vectors were
ER
, ER
Hinge (also known as HE241G), DBD-LBD, AB-DBD, AB, LBD,
LBD537X, G400V, K362A, V376R, E542K, PR, GRIP1, and p300 (12, 26).
Bacterial expression vectors were GST-fused to the N-CoR C terminus
(amino acids 1944-2453) and GRIP1 NR box region (563-1121) (12,
45).
537X and DBD-LBD-537X were prepared by exchanging a restriction
fragment from a 537X mutant LBD into full-length ER
and DBD-LBD
(46). ER
L379R, ER
T371R, and DBD-LBD L379R were prepared by PCR
point mutagenesis (Stratagene). GST-SMRT (amino acids 987-1491) was
made by PCR amplification of SMRT cDNA (gift of Dr. R. Evans, University of California, San Diego) using oligonucleotides with EcoRI and SalI sites. The resulting fragment was
digested and cloned into pGEX-5X-1.
expression vectors and 5 µg of coactivator expression
vectors or control vectors. Luciferase and
-galactosidase activities were measured using luciferase (Promega, Madison, WI) and Galacto-Light assay systems (Tropix, Bedford, MA).
-D-galactopyranoside to 1 mM. After 4 h, bacterial pellets were obtained,
resuspended in 20 mM HEPES, pH 7.9/80 mM, KCl/6
mM, MgCl2/1 mM, dithiothreitol/1 mM, ATP/0.2 mM, phenylmethylsulfonyl fluoride,
and protease inhibitors and sonicated. Debris was pelleted by
centrifugation in an ss34 rotor for 1 h at 12,000 rpm. The
supernatant was incubated with glutathione-Sepharose 4B beads (Amersham
Biosciences) and washed as previously described. Protein
preparations were stored at
20 °C in 20% glycerol.
and
bacterially expressed GST fusions were assessed (5). Labeled ERs were produced using coupled in vitro transcription-translation
(TNT kit, Promega). Briefly, assays were carried out in a volume of 150 µl that contained 137.5 µl of ice-cold protein binding buffer (PBB)
along with 10 µl of GST-bead slurry corresponding to 3 µg of fusion
protein, 1 µl of in vitro translated protein, and 1.5 µl
of ligand or vehicle. PBB was generally freshly prepared in 24-ml
aliquots composed of 20 ml of A-150 (20 mM HEPES, 150 mM KCl, 10 mM MgCl2, and 1%
glycerol), and 2 ml each of phosphate-buffered saline supplemented,
respectively, with 1% Triton X-100 and 1% Nonidet P-40.
Phenylmethylsulfonyl fluoride, dithiothreitol, bovine serum albumin,
and protease inhibitor mixture (Novagen) were added to 0.1 mM, 1 mM, 2 µg/ml, and 1/1000 dilution,
respectively. The mix was incubated for 2 h in the cold room with
gentle agitation, the beads were pelleted by spinning briefly on a
bench top Eppendorf centrifuge and washed four times with PBB
containing no bovine serum albumin, and the pellet was dried under
vacuum for 20 min. Labeled ER was subjected to SDS-PAGE and
autoradiography. The amount of bound protein in GST pulldown assays was
quantified by scanning light exposures of gels into a Hewlett-Packard
Scanjet ADF. Images were converted to PDF format and quantified using NIH image as previously described (8).
antibodies that were used in this study were HC-20
(Santa Cruz antibodies) directed against the ER
C terminus, or
number 1600024 (Geneka, Montreal, Canada) directed against the ER
NTD. The primary N-CoR antibody was N-19 (Santa Cruz antibodies).
Primary antibody was diluted 1:2000 in PBS-T and incubated with the
membrane for 1 h, followed by PBS-T washes, 1 × 15 min and
then 2 × 5 min. The membrane was incubated for 45 min with
horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (Santa
Cruz antibodies) diluted 1:2000 in PBS-T, followed by PBS-T washes
(1 × 15 min and 4 × 5 min). After the last wash, the
membrane was developed with a standard ECL kit (Amersham Biosciences),
covered with Saran wrap, and exposed to x-ray film. Westerns were
quantified as described for GST pulldown assays.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
action. We
chose tamoxifen, raloxifene, and ICI as representative SERMs that
permit, partially inhibit, and completely inhibit AF-1 activity, respectively.
View larger version (28K):
[in a new window]
Fig. 1.
TSA increases ERE-dependent
transcription in the presence of ICI and raloxifene. A,
the panel shows results from MCF-7 cell transfections containing
ERE-TK-LUC reporter or parental TK-LUC. Cells were treated with
ligand ± TSA. B, TSA induction in the presence of each
ligand. To calculate these values, luciferase activity in the presence
of TSA were divided by luciferase activity in the absence of TSA.
C, Western blots performed on extracts of MCF-7 cells
treated with the indicated ER ligands for 24 h or ER ligands and
TSA.
ligand preference, we calculated
fold TSA induction in the presence of each ligand (Fig. 1B).
6-fold TSA induction was obtained in the absence of ligand, 22-fold
induction was obtained with ICI, which does not allow AF-1 activity,
12-fold with raloxifene, which allows intermediate AF-1 activity, and
7.5-fold with tamoxifen, which allows the most AF-1 activity. These
differential TSA effects were not seen at the core TK promoter, and TSA
did not alter the overall profile of SERM effects on ER
steady state
levels (Fig. 1C). Thus, TSA enhances
ERE-dependent transcription more potently in the presence of SERMs. This suggests that SERMs increase TSA-sensitive
HDAC-dependent inhibition at the ERE-responsive promoter
but not at the parental TK core promoter. Moreover, the fact that TSA
induction is larger in the presence of ICI and raloxifene relative to
tamoxifen shows that the magnitude of this HDAC-dependent
effect varies with individual SERMs.
interactions with the corepressor
complex in vivo. To perform this experiment, we investigated
whether SERMs would enhance the ability of ER
to potentiate the
activity of DNA-bound antiprogestin-PR complexes using the system
developed by Bagchi and coworkers (41). This assay measures the ability
of ER
to sequester unspecified N-CoR containing corepressor
complexes and therefore gives an indication of overall ER
interactions with shared corepressor complexes rather than a
direct indication of ER
interactions with particular corepressor
complex components.
potentiated the activity of a
PR-RU486 complex bound to a progestin-responsive reporter gene
(TAT3-LUC) in the presence of tamoxifen but not estradiol (Fig. 2). However, ER
also potentiated
PR activity more potently in the presence of raloxifene and most
strongly with ICI. We conclude that ICI and raloxifene are more
efficient at promoting sequestration of corepressors from the PR-RU486
complex in vivo.
View larger version (16K):
[in a new window]
Fig. 2.
SERMs show differential abilities to promote
titration of corepressors. Components of the experiment are shown
in the schematic at left. The panel shows luciferase
activity that is obtained from the PR-RU486 complex bound to a
progestin-responsive reporter gene (TAT3-LUC) in the absence or
presence of free ER ± SERMs or estradiol.
Interactions with
N-CoR--
N-CoR consists of separable N-terminal repression domains
that interact with HDACs and other components of the corepressor complex and a C-terminal domain that contains the nuclear
receptor-interacting region (4). SERMs promote ER
interactions with
N-CoR in vivo (10, 39, 40, 42, 43). We next determined
whether SERMs would promote ER
interactions with the N-CoR
C-terminal nuclear receptor interacting domain in vivo.
Transiently expressed ER
coprecipitated with the N-CoR C terminus in
the absence of ligand and in presence of tamoxifen and raloxifene
(IP-gal+W ER
, Fig. 3A). A lower, but detectable,
level of ER
coprecipitated in the presence of ICI. This pattern is
similar to that observed between ER
and full-length N-CoR in other
studies ((40, 42) and also our data not shown). Thus, ER
binds to
the N-CoR C terminus in vivo.
View larger version (17K):
[in a new window]
Fig. 3.
SERMs differentially promote
ER interactions with N-CoR in
vivo. A, HeLa cells were transfected with 10 µg of ER
expression vector or 10 µg of empty vector (mock) along
with 10 µg of expression vector for a GAL-N-CoR fusion protein (amino
acids 1944-2453) and treated with ligand for 24 h. The
upper panel shows anti-ER
Westerns blot of transfected
HeLa cell extracts after immunoprecipitation with anti-GAL4 antibody.
The lower panel shows a Western blot of ER
in cell
extracts prior to immunoprecipitation. B, the side panel
shows average results of three separate experiments. Data are expressed
as the ratio of the relative intensity of the ER
band in the IP
(immunoprecipitate, corrected for background and multiplied by a
factor of ten) over the intensity of the ER
band in Western blot
prior to immunoprecipitation (corrected for background).
levels and that tamoxifen increased steady state ER
levels (W ER
). Comparison of the intensity of bands
corresponding to ER
in both blots (Fig. 3B) indicates
that tamoxifen generally increased the amount of ER
in the
N-CoR-enriched fraction by about 2-fold (although there was significant
variation between samples), whereas raloxifene increased the amount of
ER
in the N-CoR-enriched fraction about 3-fold. More surprisingly,
ICI increased the amount of ER
in the N-CoR-enriched fraction by at
least 7-fold relative to the low amount of ICI-liganded ER
in
transfected cells.
interactions with
N-CoR, we examined ER
interactions with bacterially expressed N-CoR
C terminus in vitro. SERMs, but not estradiol, enhanced
ER
interactions with the N-CoR C terminus (Fig.
4A). A similar (albeit weaker)
binding profile was obtained with the SMRT C terminus. Control binding
assays confirmed that ER
interactions with the NR box region of the
coactivator GRIP1 were reduced by SERMs and promoted by estradiol.
Quantification of a series of similar pulldown assays
(n = 15, Fig. 4B) indicated that individual SERMs exhibited differences in their ability to promote N-CoR binding
in vitro. ICI gave the best binding (about 3.5% of input); raloxifene gave intermediate levels (2.2% of input); and tamoxifen gave the least binding (1% of input). Thus, SERMs differ in their ability to promote ER
interactions with N-CoR, and the ligand preference of these effects correlates qualitatively with SERM effects
on repression of AF-1 activity (see the introduction), HDAC inhibition
of an ERE-responsive promoter in MCF-7 cells (Fig. 1B), and
sequestration of corepressors from the PR-RU486 complex (Fig. 2).
View larger version (24K):
[in a new window]
Fig. 4.
SERMs differentially promote
ER interactions with N-CoR in
vivo. A, labeled ER
was bound to GST
fusions (shown in schematic at top) in the presence of vehicle or
ligand, separated by SDS-PAGE, and processed for autoradiography. The
panels show binding to N-CoR C terminus (amino acids 1944-2453), SMRT
C terminus (amino acids 987-1491), and GRIP1 NR box region (amino
acids 563-1121), which contain nuclear receptor-interacting regions
(black stripes). 10% input and ER
retained by GST are
shown as controls. B, the side panel shows
average results from 15 separate experiments involving GST-N-CoR. Data
are expressed as percentage of input ER
retained on GST-corepressor
beads after correction for background binding to GST control
beads.
537X) That Increases N-CoR Binding also Inhibits
AF-1 Activity--
While SERMs show intriguing effects on ER
interactions with N-CoR, it is also clear that these interactions are
relatively weak (see "Discussion"). To further investigate the
significance of these interactions, we sought ER
mutations that
would alter the pattern of ER
binding to N-CoR and then investigated
the properties of these ER
mutants in transfection assays in
vivo.
H12 would enhance N-CoR binding (Fig.
5A). An ER
mutant that
lacked H12 (ER
537X) showed enhanced N-CoR binding in the absence of
ligand and in the presence of tamoxifen and estradiol (see Fig.
5B for quantification). ER
537X did not show appreciably
increased N-CoR binding in the presence of ICI, suggesting that H12
does not influence ER
binding to N-CoR in the presence of ICI.
Surprisingly, ER
537X did exhibit increased binding in the presence
of raloxifene (8% input). This suggests that raloxifene exerts
unspecified effects on core-LBD structure that promote N-CoR
binding.
View larger version (36K):
[in a new window]
Fig. 5.
Helix 12 modulates N-CoR binding with
SERMs. A, labeled ER truncations or mutants (shown
in schematic at left) were bound to GST-N-CoR in the absence
or presence of ligands as in Fig. 1A. B, the
panel shows percentage of input ER
and ER
537X retained on
GST-N-CoR beads. Data are the average of five separate
determinations.
LBD (LBD-537X). Nevertheless, the isolated ER
LBD failed to bind N-CoR suggesting that other regions of ER
contribute to N-CoR binding. The ER
DBD-LBD region did bind N-CoR with a ligand preference that resembled full-length ER
, but an ER
deletion mutant that lacked the hinge domain (ER
hinge) and the isolated AB-DBD and AB regions did not bind
N-CoR. Thus, ER
interactions with N-CoR require the hydrophobic cleft and the DBD-hinge.
537X in vivo.
As expected (24), wild type ER
enhanced transcription of an
ERE-responsive reporter (ERE-TK-LUC) in the absence of ligand in
MDA-MB-453 breast cells, and estradiol gave further stimulation (Fig.
6A). The SERMs tamoxifen and
raloxifene allowed some residual activity that stems from AF-1 (24),
but ICI allowed none. ER
G400V, which exhibits reduced constitutive
activity (49, 50), showed similar activity to ER
in the presence of
each ligand. In parallel, ER
537X showed three differences from wild
type ER
. First, ER
537X showed reduced estrogen response. This is
consistent with the requirement for H12 in p160 binding (15). Second,
ER
537X showed reduced constitutive activity. This phenotype is
common in ER
LBD mutants (24). Third, and most importantly,
ER
537X failed to elicit a tamoxifen response. This lack of response
persisted even in the presence of a 10-fold excess of ER
537X
expression vector (not shown). Fig. 6B shows that ER
537X
was expressed at comparable levels to wild type ER
in the presence
of SERMs. Thus, the lack of tamoxifen response in the presence of
ER
537X is not a consequence of a large reduction in ER
levels.
Moreover, our analysis of the amount of ER
and ER
537X
transfection vectors that are required to elicit responses from the
ERE-responsive reporter indicate that ER
537X has not become
superactive at low levels of transfected receptor and is not squelching
its own activity under these conditions (data not shown). We conclude
that an ER
mutation (537X) that enhances corepressor binding also
eliminates AF-1 activity.
View larger version (33K):
[in a new window]
Fig. 6.
ER 537X shows no AF-1
activity with any tested SERM. A, luciferase activity
in extracts of MDA-MB-453 cells transfected with ERE-LUC, ER
expression vectors, and
-galactosidase internal control. Data are
corrected for
-galactosidase activity. B, Western blot of
MDA-MB-453 extracts transfected with ER
or ER
537X expression
vectors. Antibody was SC-2004 directed against the ER
NTD.
Transfection efficiency was monitored with a
-galactosidase internal
control.
Mutation That Reduces N-CoR Binding Allows Equal AF-1
Activity with SERMs--
The nuclear receptor corepressor-binding
surface consists of residues that overlap the hydrophobic cleft that
comprises part of the AF-2 surface (48, 51-54). In particular, a Leu
residue at the base of H5 is important for corepressor binding (51). We
therefore next determined whether mutation of the equivalent ER
residue (Leu-379) would influence SERM effects on corepressor binding.
Fig. 7 shows that N-CoR binding was
reduced in all conditions in the presence of ER
L379R but that these
effects were especially prominent in the presence of ICI and raloxifene
(about 0.5% of ICI-liganded ER
L379R bound N-CoR relative to 4% of
ICI-liganded wild type ER
; about 0.2% of raloxifene-liganded
ER
L379R bound to N-CoR relative to more than 2% of wild type
ER
). ER
L379R also reduced GRIP1 binding in the presence of
estradiol (Ref. 15 and not shown). Mutations in nearby residues (K362A,
T371R, V376R, and E542K) did not show large effect effects on the
overall ligand preference of N-CoR binding, even though some (K362A,
V376R, and E542K) did reduce GRIP1 binding (Ref. 12 and not shown). Thus, Leu-379 is important for SERM-dependent ER
interactions with N-CoR in vitro, and these interactions are
deficient in the ER
L379R mutant, but not in other mutations in the
AF-2 surface.
View larger version (30K):
[in a new window]
Fig. 7.
An ER mutation in
the hydrophobic cleft, L379R, reduces N-CoR binding. A,
labeled ER
or mutants were bound to GST-N-CoR. Representative gels
are shown. B, the panel represents determinations of average
mutant binding to GST-N-CoR. Averages of three determinations is
shown.
L379R in vivo. Like other ER
AF-2
mutants, ER
L379R showed reduced estrogen response and constitutive
activity (Fig. 8A). More
importantly, ER
L379R showed increased raloxifene and ICI response.
Other ER
AF-2 mutants (K362A, V376R, and E542K) showed reduced
estrogen response and constitutive activity or comparable estrogen
response and constitutive activity to wild type ER
(T371R), but all
retained the same SERM ligand preference as wild type ER
. Fig.
8B shows that ER
L379R showed a modest increase in steady
state levels in the presence of ICI and decreased expression levels in
the presence of raloxifene and tamoxifen. However, modest alterations
in ER
levels were also observed with other mutants (especially
E542K), and these alterations did not translate into alterations in
SERM activity in vivo. Thus, ER
L379R activity is not
simply related to ER
steady state levels. Truncation of the
ER
L379R NTD (DBD-LBDL379R) abolished all
ER
L379R-dependent SERM agonist effects, confirming that
they require AF-1 (not shown). Thus, the L379R mutation increases AF-1
activity at an ERE in the presence of ICI and raloxifene.
View larger version (42K):
[in a new window]
Fig. 8.
The ER L379R mutation
allows increased AF-1 activity in the presence of ICI and
raloxifene. A, an MDA-MB-453 transfection in which
ER
activity was compared with ER
L379R and a variety of ER
AF-2
mutants. B, Western blot of MDA-MB-453 cell extracts
transfected with ER
expression vectors and treated with SERMs or
estradiol. C, ER
L379R reverses the phenotype of
ER
537X. Average of four MDA-MB-453 transfections with ER
vectors
indicated. Data are presented as fold induction. Inset shows
Western blot of extracts of COS cells transfected with empty vector or
ER
mutants.
537X, an ER
L379R/537X double mutant did enhance ERE-dependent transcription in the presence of
SERMs, even though it was poorly expressed (see inset).
Thus, the L379R mutation reverses the phenotype of ER
537X.
AF-1 activity is usually low in HeLa cells (23), but can
be enhanced by the AF-1 target coactivator proteins, GRIP1 and p300 (6,
26). To determine whether alteration of N-CoR binding would affect the
AF-1 activity that is mediated by this coactivator complex, we examined
effects of overexpression of AF-1 target coactivators upon ER
L379R.
As expected (26), ER
and ER
G400V enhanced transcription with
tamoxifen but not with raloxifene or ICI in HeLa cells and did so only
when GRIP1 and p300 were supplied (Fig.
9, compare left and
right panels). In the same conditions, ER
L379R enhanced
transcription with tamoxifen (albeit not quite as strongly as wild type
ER
) but also enhanced transcription with raloxifene and ICI. By
contrast, ER
K362A enhanced transcription only with tamoxifen. Again,
ER
L379R levels were slightly increased in the presence of ICI, but
similar alterations in ER
levels were also obtained with ER
K362A,
which did not show altered SERM ligand preference. Thus, the L379R
mutation allows increased ICI and raloxifene agonist activity in
conditions in which AF-1 activity is mediated by the p160/p300 complex.
Together, our results indicate that the L379R mutation permits AF-1
activity in the presence of a range of SERMs. We conclude that
the amount of ER
binding to N-CoR (whether influenced by SERMs or
mutations) correlates with the amount of ER
AF-1 activity at simple
EREs.
View larger version (29K):
[in a new window]
Fig. 9.
ER L379R enhances
AF-1 activity through the p160-p300 complex. The panel shows a
HeLa transfection with empty vectors (left panel) or
expression vectors for GRIP1 and p300 (right panel). Note
different scales.
Action at AP-1 Sites--
ER
enhances AP-1 activity in the presence
of estradiol and the partial agonist tamoxifen (5, 32), and these
effects require ER
activation functions and p160s (6, 10). However, ER
also enhances AP-1 activity via a second AF-independent mechanism that is up-regulated in ER
deletions that lack AF-1 and strongly potentiated by ICI and raloxifene (6, 32, 34) and may involve corepressors (6, 35). Thus, a mutation that reduces N-CoR binding
should potentiate ER
AF-1 activity at AP-1 sites but should also
inhibit the activity of ER
truncations that are committed to the
AF-independent pathway. Moreover, mutations that enhance N-CoR binding
may show increased ability to potentiate ER
activity in the
AF-independent pathway.
action at AP-1 sites. Fig.
10A confirms that ER
enhanced the activity of an AP-1-responsive promoter (Coll73-LUC) in
the absence of ligand and in the presence of tamoxifen, estradiol, and
DES in HeLa cells and that ER
G400V showed a similar profile. The
same data show that ER
L379R exhibited equivalent activity at the
AP-1-responsive reporter in the absence of ligand and in the presence
of each SERM. The L379R mutation also enhanced estradiol and DES
response at the AP-1 site. This is consistent with the suggestion that
the corepressor complex restricts estrogen-liganded ER
activity in
some contexts (44). Fig. 10B confirms that truncation of the
ER
L379R NTD (DBD-LBDL379R) completely abolished all SERM effects.
Thus, increased SERM activities that are observed with ER
L379R
require AF-1 just as they do at EREs. The same data confirm that the
DBD-LBD enhances AP-1 activity in the presence of ICI and raloxifene
but not tamoxifen or estradiol (6, 24) and that the L379R mutation
abolishes these responses. Thus, the ICI and raloxifene response
through the AF-independent pathway requires the N-CoR binding
surface.
View larger version (27K):
[in a new window]
Fig. 10.
Mutations in ER
that affect N-CoR binding affect SERM activity at an
AP-1-responsive reporter. A, HeLa transfection in which
ER
activity was compared with ER
L379R and ER
537X at an
AP-1-responsive reporter (Coll73-Luc). B, as in A
except that the DBD-LBD truncation, missing AF-1, was used.
L379R,
ER
537X showed equivalent activity at the AP-1-responsive reporter in
the absence of ligand and in the presence of the three SERMs (Fig.
10A). However, unlike ER
L379R, truncation of the NTD did
not abolish these SERM effects (Fig. 10B). Instead, ICI and raloxifene responses were preserved and tamoxifen and estradiol responses were increased. Thus, the 537X mutation equalizes the activity of ligands in the AF-independent pathway. We conclude that
mutations that affect N-CoR binding alter the ligand preference of
ER
action at AP-1 sites through two mechanisms
shows potent AF-1 activity at the C3
complement promoter (C3) (25, 30). Fig.
11 confirms that ER
and ER
G400V
enhanced the C3 activity in the presence of estradiol and tamoxifen but not ICI or raloxifene in MDA-MB-453 cells. ER
537X failed to enhance C3 in the presence of any ligand, just as it did at a simple ERE. Moreover, ER
L379R showed enhanced ICI and raloxifene responses relative to wild type ER
, just as it did at a simple ERE and the
AP-1 site, and enhanced estradiol and DES response, just as did at the
AP-1 site. ICI and raloxifene effects were dependent upon the presence
of the NTD (not shown). Thus, a mutation (L379R) that reduces N-CoR
binding enhances AF-1 activity at C3 in the presence of ICI and
raloxifene.
View larger version (31K):
[in a new window]
Fig. 11.
The L379R mutation allows some AF-1 activity
in the presence of ICI and raloxifene at the complement C3 promoter but
decreases tamoxifen activation. Transfections were performed as in
Fig. 5A except that the C3 complement promoter was
used.
tamoxifen activation of C3 involves an unspecified contribution from the LBD (25,
30). It is possible that this contribution is deficient in the L379R
mutant. We conclude that N-CoR inhibits ICI and raloxifene activity at
C3 but that low levels of N-CoR binding in the presence of tamoxifen do
not fully explain why tamoxifen is a potent activator in this context.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
shows meaningful direct interactions with N-CoR. It is also unclear whether similar mechanisms underlie differences in SERM activity in other contexts.
-dependent sequestration of PR-associated corepressor
complexes, and that they do so more potently than tamoxifen. We also
found that SERMs promote ER
interactions with the nuclear receptor interaction domain of N-CoR (C terminus) in immunoprecipitations, and
estimates of ER
levels in the N-CoR-enriched fraction
versus the cell extract suggested that the ICI and
raloxifene are better than tamoxifen in promoting this interaction.
Likewise, ICI and raloxifene are more potent than tamoxifen in
promoting ER
association with the N-CoR C terminus in GST pulldown
assays in vitro. Thus, SERMs show differential effects on
ER
interactions with N-CoR and do so with a ligand preference that
parallels their effects on suppression of AF-1.
was retained on the GST-N-CoR beads, whereas
20-40% of input TR was retained on the GST-N-CoR beads in parallel
(data not shown, see Ref. 45). Moreover, peptides that correspond to
isolated N-CoR nuclear receptor-interacting domains 1 and 3 (45) failed
to compete for ER
/N-CoR interactions (not shown). We can infer that
the affinity of ER
for these isolated peptides is very low (greater
than 30 µM compared with 1 µM affinity of LXXLL peptides for ER
AF-2, discussed in Ref. 55).
Finally, we were unable to detect ER
LBD interactions with N-CoR in
mammalian two-hybrid assays (not shown). To evaluate the significance
of interactions between ER
and N-CoR, we therefore identified ER
mutations that influence N-CoR binding in vitro and examined
their function in vivo. We found that a mutation (537X) that
increases and equalizes N-CoR binding in the presence of SERMs
eliminates AF-1 activity at simple EREs and the C3 promoter and
equalizes SERM activation of AP-1 sites via the AF-independent
mechanism. A mutation (L379R) that reduces and equalizes N-CoR binding
with SERMs allows equivalent AF-1 activity at simple EREs and AP-1 sites in the presence of SERMs and increases ICI and raloxifene response at C3. The same mutant also reverses the effect of the 537X
mutation and eliminates ER
action through the AF-independent pathway
at AP-1 sites. Thus, mutations that affect N-CoR binding have parallel
effects on ER
activity. We therefore propose that differential
effects on direct corepressor interactions underlie many instances of
differential SERM activities in vivo. This idea is in line
with observations that SERMs enhance ER
interactions with peptides
corresponding to corepressor nuclear receptor-interacting domains
in vivo (43, 44) and that mutations that influence ER
interactions with N-CoR or N-CoR-like peptides alter the ligand preference of ER
action in vivo (42, 44).
only binds weakly
to N-CoR in vitro? We suggest that ER
interactions with
the N-CoR C terminus correspond to the ligand-dependent
component of ER
/corepressor interactions, but that ER
also
contacts other surfaces of the corepressor complex that augment
corepressor recruitment. We also recognize that our binding assays may
not faithfully recreate conditions that are required for maximal
ER
/N-CoR interactions (perhaps ER
or N-CoR need specific
modifications or cofactor interactions) and that our studies do not
address the roles of N-CoR versus SMRT versus
other repressors and certainly do not exclude the possibility that
there are multiple repressors of ER
action. It will be interesting
to ask whether ER
uses a similar surface to recruit other
repressors, such as REA and HET/SAFb (56, 57).
interactions with
N-CoR contribute to an emerging picture in which nuclear receptor
antagonists exert diverse effects upon corepressor binding. The PPAR
antagonist GW6471 promotes corepressor binding (58), whereas the TR
antagonist NH-3 promotes corepressor release (59). PR antagonists show differential effects on corepressor binding (60). ER
residues that
mediate corepressor binding (Leu-379 and others (42, 44, 48)) lie
within the hydrophobic cleft. Estrogens permit docking of H12 against
the lower part of the cleft and should prevent corepressor binding
(Fig. 12, i). By contrast,
SERMs displace H12. H12 can fold over the cleft as in published ER-SERM
crystal structures (Fig. 12, ii) (14, 15, 17, 18). The
results described here suggest that this conformation occludes the
H3-H5 region that mediates corepressor binding. SERMs must therefore
allow H12 to dock in another position that exposes the H3-H5 region and
promotes N-CoR binding (Fig. 12, iii). Perhaps this
configuration resembles the PPAR-SMRT structure (58) or the ER
-ICI
structure (18). We therefore suggest that overall levels on N-CoR
binding depend on equilibrium between H12 positions (ii and iii);
tamoxifen would favor ii, ICI would favor iii,
and raloxifene would permit both. Factors that might influence this
equilibrium include SERM effects upon the length of the H11-H12 loop,
which affect the ease of H12 docking in the cleft (14, 15), contacts
between the SERM extension and either ER
or N-CoR, or unspecified
effects of SERMs on core-LBD structure (see Fig. 5).
View larger version (19K):
[in a new window]
Fig. 12.
Model to explain differential SERM effects
upon N-CoR binding. The ER coactivator/corepressor-binding
surface is shown in schematic, with H3 shown in white, H5 in
dark gray, and H12 in pale gray. N-CoR binding
residues are shown as circles. Three possible liganded
conformations of the ER
-LBD are presented, and their effects on
N-CoR binding are discussed in the text. I, estradiol
promotes formation of the AF-2 hydrophobic cleft (composed of H3, H5,
and H12) and blocks N-CoR binding. ii, tamoxifen or
raloxifene protrudes through the LBD surface, thereby displacing H12,
which folds into the cleft and blocks N-CoR binding. iii,
SERMs (especially raloxifene and ICI) also displace H12 but here, H12
occupies an unusual position that exposes the cleft and allows N-CoR to
bind.
To what extent does differential corepressor binding account for SERM
activity in vivo? The L379R mutation eliminates tamoxifen response at C3 (Fig. 11) and reduces tamoxifen response at EREs and
AP-1 sites in HeLa cells (Figs. 9, 10). Thus, some tamoxifen responses
require a contribution from the LBD that is unrelated to corepressor
binding. Moreover, while SERM activity is not absolutely related to
ER levels over the short time course of our experiments (Figs.
1C, 6B, 8B, and 9B), it is
likely that SERM-dependent alterations in ER
levels play
a more important role over longer times. Nevertheless, differential
SERM effects on corepressor binding contribute to differential SERM
activity in a variety of contexts. It will be especially important to
understand corepressor recruitment to genes with alternate response
elements, which are important for ER effects on proliferation (5, 10,
61, 62).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Stefan Nilsson (Karobio AB, Huddinge, Sweden) for Raloxifene and Alan Wakeling (Astra/Zeneca, Macclesfield, UK) for ICI 182,780.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK51083 and CA80210 (to P. J. K.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Medicine, 2200 Post St., Rm. C442, University of California, San Francisco, CA 94115-1640. Tel.: 415-476-6790; Fax: 415-885-7724; E-mail: kushner@itsa.ucsf.edu.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M208501200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ER, estrogen receptor; ERE, estrogen response element; H, helices; NR, nuclear receptor; PBB, protein binding buffer; ICI, ICI 182,780; SERM, selective estrogen receptor modulator(s); DES, diethylstilbestrol; AP, activator protein; SRC, steroid receptor coactivator; GRIP1, glucocorticoid receptor interacting protein 1; N-CoR, nuclear receptor corepressor; SMRT, silencing mediator of retinoid and thyroid responsive transcription; HDAC, histone deacetylase; AF, activation function; PR, progesterone receptor; TR, thyroid receptor; PPAR, peroxisome proliferator-activated receptor; NTD, N-terminal domain; DBD, DNA binding domain; LBD, ligand binding domain; GST, glutathione S-transferase; PBS, phosphate-buffered saline; REA, repressor of estrogen receptor activity; HET-SAFB, hsp27-ERE-TATA-binding protein/scaffold attachment factor B; MAP, mitogen-activated protein; JNK, c-Jun NH2-terminal kinase; TSA, trichostatin A; C3, C3 complement promoter.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Parker, M. G. (1998) Biochem. Soc. Symp. 63, 45-50[Medline] [Order article via Infotrieve] |
2. | Kushner, P. J., Agard, D., Feng, W. J., Lopez, G., Schiau, A., Uht, R., Webb, P., and Greene, G. (2000) Novartis Found. Symp. 230, 20-40[Medline] [Order article via Infotrieve] |
3. | Katzenellenbogen, B. S., and Katzenellenbogen, J. A. (2000) Breast Cancer Res. 2, 335-344[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Glass, C. K.,
and Rosenfeld, M. G.
(2000)
Genes Dev.
14,
121-141 |
5. | Webb, P., Lopez, G. N., Uht, R. M., and Kushner, P. J. (1995) Mol. Endocrinol. 9, 443-456[Abstract] |
6. |
Webb, P.,
Nguyen, P.,
Valentine, C.,
Lopez, G. N.,
Kwok, G. R.,
McInerney, E.,
Katzenellenbogen, B. S.,
Enmark, E.,
Gustafsson, J. A.,
Nilsson, S.,
and Kushner, P. J.
(1999)
Mol. Endocrinol.
13,
1672-1685 |
7. | Safe, S. (2001) Vitam. Horm. 62, 231-252[Medline] [Order article via Infotrieve] |
8. |
Liu, M. M.,
Albanese, C.,
Anderson, C. M.,
Hilty, K.,
Webb, P.,
Uht, R. M.,
Price, R. H., Jr.,
Pestell, R. G.,
and Kushner, P. J.
(2002)
J. Biol. Chem.
277,
24353-24360 |
9. |
Planas-Silva, M. D.,
Shang, Y.,
Donaher, J. L.,
Brown, M.,
and Weinberg, R. A.
(2001)
Cancer Res.
61,
3858-3862 |
10. |
Shang, Y.,
and Brown, M.
(2002)
Science
295,
2465-2468 |
11. |
Jordan, V. C.,
Gapstur, S.,
and Morrow, M.
(2001)
J. Nat. Cancer Inst.
93,
1449-1457 |
12. |
Feng, W.,
Ribeiro, R. C.,
Wagner, R. L.,
Nguyen, H.,
Apriletti, J. W.,
Fletterick, R. J.,
Baxter, J. D.,
Kushner, P. J.,
and West, B. L.
(1998)
Science
280,
1747-1749 |
13. |
Mak, H. Y.,
Hoare, S.,
Henttu, P. M.,
and Parker, M. G.
(1999)
Mol. Cell. Biol.
19,
3895-3903 |
14. | Brzozowski, A. M., Pike, A. C., Dauter, Z., Hubbard, R. E., Bonn, T., Engstrom, O., Ohman, L., Greene, G. L., Gustafsson, J. A., and Carlquist, M. (1997) Nature 389, 753-758[CrossRef][Medline] [Order article via Infotrieve] |
15. | Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P. J., Agard, D. A., and Greene, G. L. (1998) Cell 95, 927-937[Medline] [Order article via Infotrieve] |
16. |
Pike, A. C.,
Brzozowski, A. M.,
Hubbard, R. E.,
Bonn, T.,
Thorsell, A. G.,
Engstrom, O.,
Ljunggren, J.,
Gustafsson, J. A.,
and Carlquist, M.
(1999)
EMBO J.
18,
4608-4618 |
17. | Shiau, A. K., Barstad, D., Radek, J. T., Meyers, M. J., Nettles, K. W., Katzenellenbogen, B. S., Katzenellenbogen, J. A., Agard, D. A., and Greene, G. L. (2002) Nat. Struct. Biol. 9, 359-364[Medline] [Order article via Infotrieve] |
18. | Pike, A. C., Brzozowski, A. M., Walton, J., Hubbard, R. E., Thorsell, A. G., Li, Y. L., Gustafsson, J. A., and Carlquist, M. (2001) Structure 9, 145-153[Medline] [Order article via Infotrieve] |
19. | Jordan, V. C. (2002) Cancer Cell 1, 215-217[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Willson, T. M.,
Norris, J. D.,
Wagner, B. L.,
Asplin, I.,
Baer, P.,
Brown, H. R.,
Jones, S. A.,
Henke, B.,
Sauls, H.,
Wolfe, S.,
Morris, D. C.,
and McDonnell, D. P.
(1997)
Endocrinology
138,
3901-3911 |
21. |
Sibonga, J. D.,
Dobnig, H.,
Harden, R. M.,
and Turner, R. T.
(1998)
Endocrinology
139,
3736-3742 |
22. |
Wakeling, A. E.
(2000)
Endocr. Relat. Cancer
7,
17-28 |
23. | Berry, M., Metzger, D., and Chambon, P. (1990) EMBO J. 9, 2811-2818[Abstract] |
24. |
Webb, P.,
Nguyen, P.,
Valentine, C.,
Weatherman, R. V.,
Scanlan, T. S.,
and Kushner, P. J.
(2000)
J. Biol. Chem.
275,
37552-37558 |
25. |
Norris, J. D.,
Paige, L. A.,
Christensen, D. J.,
Chang, C. Y.,
Huacani, M. R.,
Fan, D.,
Hamilton, P. T.,
Fowlkes, D. M.,
and McDonnell, D. P.
(1999)
Science
285,
744-746 |
26. |
Webb, P.,
Nguyen, P.,
Shinsako, J.,
Anderson, C.,
Feng, W.,
Nguyen, M. P.,
Chen, D.,
Huang, S. M.,
Subramanian, S.,
McKinerney, E.,
Katzenellenbogen, B. S.,
Stallcup, M. R.,
and Kushner, P. J.
(1998)
Mol. Endocrinol.
12,
1605-1618 |
27. | Kato, S., Endoh, H., Masuhiro, Y., Kitamoto, T., Uchiyama, S., Sasaki, H., Masushige, S., Gotoh, Y., Nishida, E., Kawashima, H., et al.. (1995) Science 270, 1491-1494[Abstract] |
28. |
Font de Mora, J.,
and Brown, M.
(2000)
Mol. Cell. Biol.
20,
5041-5047 |
29. |
Feng, W.,
Webb, P.,
Nguyen, P.,
Liu, X., Li, J.,
Karin, M.,
and Kushner, P. J.
(2001)
Mol. Endocrinol.
15,
32-45 |
30. | Fan, J. D., Wagner, B. L., and McDonnell, D. P. (1996) Mol. Endocrinol. 10, 1605-1616[Abstract] |
31. |
Wijayaratne, A. L.,
Nagel, S. C.,
Paige, L. A.,
Christensen, D. J.,
Norris, J. D.,
Fowlkes, D. M.,
and McDonnell, D. P.
(1999)
Endocrinology
140,
5828-5840 |
32. |
Paech, K.,
Webb, P.,
Kuiper, G. G.,
Nilsson, S.,
Gustafsson, J.,
Kushner, P. J.,
and Scanlan, T. S.
(1997)
Science
277,
1508-1510 |
33. |
Weatherman, R. V.,
and Scanlan, T. S.
(2001)
J. Biol. Chem.
276,
3827-3832 |
34. |
Jakacka, M.,
Ito, M.,
Weiss, J.,
Chien, P. Y.,
Gehm, B. D.,
and Jameson, J. L.
(2001)
J. Biol. Chem.
276,
13615-13621 |
35. | Kushner, P. J., Agard, D. A., Greene, G. L., Scanlan, T. S., Shiau, A. K., Uht, R. M., and Webb, P. (2000) J. Steroid. Biochem. Mol. Biol. 74, 311-317[CrossRef][Medline] [Order article via Infotrieve] |
36. | Dauvois, S., Danielian, P. S., White, R., and Parker, M. G. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4037-4041[Abstract] |
37. | Reese, J. C., and Katzenellenbogen, B. S. (1992) Mol. Cell. Biol. 12, 4531-4538[Abstract] |
38. | Metzger, D., Berry, M., Ali, S., and Chambon, P. (1995) Mol. Endocrinol. 9, 579-591[Abstract] |
39. |
Jackson, T. A.,
Richer, J. K.,
Bain, D. L.,
Takimoto, G. S.,
Tung, L.,
and Horwitz, K. B.
(1997)
Mol. Endocrinol.
11,
693-705 |
40. |
Lavinsky, R. M.,
Jepsen, K.,
Heinzel, T.,
Torchia, J.,
Mullen, T. M.,
Schiff, R.,
Del-Rio, A. L.,
Ricote, M.,
Ngo, S.,
Gemsch, J.,
Hilsenbeck, S. G.,
Osborne, C. K.,
Glass, C. K.,
Rosenfeld, M. G.,
and Rose, D. W.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2920-2925 |
41. |
Zhang, X.,
Jeyakumar, M.,
Petukhov, S.,
and Bagchi, M. K.
(1998)
Mol. Endocrinol.
12,
513-524 |
42. |
Yamamoto, Y.,
Wada, O.,
Suzawa, M.,
Yogiashi, Y.,
Yano, T.,
Kato, S.,
and Yanagisawa, J.
(2001)
J. Biol. Chem.
276,
42684-42691 |
43. | Shang, Y., Hu, X., DiRenzo, J., Lazar, M. A., and Brown, M. (2000) Cell 103, 843-852[Medline] [Order article via Infotrieve] |
44. |
Huang, H. J.,
Norris, J. D.,
and McDonnell, D. P.
(2002)
Mol. Endocrinol.
16,
1778-1792 |
45. |
Webb, P.,
Anderson, C. M.,
Valentine, C.,
Nguyen, P.,
Marimuthu, A.,
West, B. L.,
Baxter, J. D.,
and Kushner, P. J.
(2000)
Mol. Endocrinol
14,
1976-1985 |
46. |
Lopez, G. N.,
Webb, P.,
Shinsako, J. H.,
Baxter, J. D.,
Greene, G. L.,
and Kushner, P. J.
(1999)
Mol. Endocrinol.
13,
897-909 |
47. | Webb, P., Lopez, G. N., Greene, G. L., Baxter, J. D., and Kushner, P. J. (1992) Mol. Endocrinol. 6, 157-167[Abstract] |
48. |
Marimuthu, A.,
Feng, W.,
Tagami, T.,
Nguyen, H.,
Jameson, J. L.,
Fletterick, R. J.,
Baxter, J. D.,
and West, B. L.
(2002)
Mol. Endocrinol.
16,
271-286 |
49. | Tora, L., Mullick, A., Metzger, D., Ponglikitmongkol, M., Park, I., and Chambon, P. (1989) EMBO J. 8, 1981-1986[Abstract] |
50. |
Aumais, J. P.,
Lee, H. S.,
Lin, R.,
and White, J. H.
(1997)
J. Biol. Chem.
272,
12229-12235 |
51. |
Burke, L. J.,
Downes, M.,
Laudet, V.,
and Muscat, G. E.
(1998)
Mol. Endocrinol.
12,
248-262 |
52. | Hu, X., and Lazar, M. A. (1999) Nature 402, 93-96[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Nagy, L.,
Kao, H. Y.,
Love, J. D., Li, C.,
Banayo, E.,
Gooch, J. T.,
Krishna, V.,
Chatterjee, K.,
Evans, R. M.,
and Schwabe, J. W.
(1999)
Genes Dev.
13,
3209-3216 |
54. |
Perissi, V.,
Staszewski, L. M.,
McInerney, E. M.,
Kurokawa, R.,
Krones, A.,
Rose, D. W.,
Lambert, M. H.,
Milburn, M. V.,
Glass, C. K.,
and Rosenfeld, M. G.
(1999)
Genes Dev.
13,
3198-3208 |
55. |
Darimont, B. D.,
Wagner, R. L.,
Apriletti, J. W.,
Stallcup, M. R.,
Kushner, P. J.,
Baxter, J. D.,
Fletterick, R. J.,
and Yamamoto, K. R.
(1998)
Genes Dev.
12,
3343-3356 |
56. |
Montano, M. M.,
Ekena, K.,
Delage-Mourroux, R.,
Chang, W.,
Martini, P.,
and Katzenellenbogen, B. S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6947-6952 |
57. |
Oesterreich, S.,
Zhang, Q.,
Hopp, T.,
Fuqua, S. A.,
Michaelis, M.,
Zhao, H. H.,
Davie, J. R.,
Osborne, C. K.,
and Lee, A. V.
(2000)
Mol. Endocrinol.
14,
369-381 |
58. | Xu, H. E., Stanley, T. B., Montana, V. G., Lambert, M. H., Shearer, B. G., Cobb, J. E., McKee, D. D., Galardi, C. M., Plunket, K. D., Nolte, R. T., Parks, D. J., Moore, J. T., Kliewer, S. A., Willson, T. M., and Stimmel, J. B. (2002) Nature 415, 813-817[CrossRef][Medline] [Order article via Infotrieve] |
59. | Nguyen, N. H., Apriletti, J. W., Cunha Lima, S. T., Webb, P., Baxter, J. D., and Scanlan, T. S. (2002) J. Med. Chem. 45, 3310-3320[CrossRef][Medline] [Order article via Infotrieve] |
60. |
Wagner, B. L.,
Norris, J. D.,
Knotts, T. A.,
Weigel, N. L.,
and McDonnell, D. P.
(1998)
Mol. Cell. Biol.
18,
1369-1378 |
61. |
Philips, A.,
Chalbos, D.,
and Rochefort, H.
(1993)
J. Biol. Chem.
268,
14103-14108 |
62. |
Philips, A.,
Teyssier, C.,
Galtier, F.,
Rivier-Covas, C.,
Rey, J. M.,
Rochefort, H.,
and Chalbos, D.
(1998)
Mol. Endocrinol.
12,
973-985 |