From the Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
Received for publication, January 17, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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The nuclear receptor for retinoic acid
(RAR) forms a heterodimeric complex with the retinoid X receptor (RXR).
This RXR/RAR heterodimer binds to the promoter of retinoic acid target
genes and recruits coactivators and corepressors to regulate gene
expression. Currently, the relative role of each receptor
monomer in regulating coactivator and corepressor recruitment remains
unclear. Here we show that the receptor-associated coactivator 3 (RAC3)
uses two separate LXXLL motifs to bind RAR and RXR. The
mutation of the coactivator-binding pockets of RAR and RXR abolishes
RAC3 binding. Although the coactivator pocket of RXR is essential for the function of the RXR homodimer, it has a minor role for the recruitment of RAC3 and trans-activation by the RXR/RAR heterodimer. Consistently, deletion of the activation helix of RXR enhances binding
of RAC3 to the heterodimer, and mutation of the coactivator pocket of
RXR had little effect on RXR/RAR activity. In contrast, the
coactivator pocket and the activation helix of RAR are absolutely required. We also show that different residues of the RAR coactivator pocket are used differently for interactions with the corepressor silencing mediator for retinoid and thyroid hormone
receptor (SMRT) and coactivator. These results
indicate a differential role for each retinoid receptor to the overall
binding of cofactors and regulation of transcription by the retinoid
receptor heterodimer.
The steroid/nuclear hormone receptor superfamily is a large class
of ligand-dependent transcription factors that plays
critical roles in regulating genes involved in a wide array of
biological processes including development and homeostasis (1). In the absence of a ligand, several receptors are able to repress basal transcription via functional interactions with the corepressors SMRT1 and N-CoR (nuclear
receptor corepressor) (2, 3). Ligand binding triggers the release of
corepressors and subsequent recruitment of coactivators, which enhance
transcription by recruiting chromatin-modifying activities such as
histone acetylation and methylation (4). Coactivators directly
recruited by liganded receptors include members of the steroid receptor
coactivator/p160 family such as SRC-1, transcriptional
intermediary factor 2/glucocorticoid receptor interacting
protein 1, and RAC3/activator of thyroid and retinoic acid
receptors/amplified in breast cancer 1 (5). These coactivators contain highly conserved Further insight into the biochemical basis of these interactions comes
from crystal structures of the receptor ligand-binding domain complexed
with the LXXLL peptide (13-16). These studies suggest a
ligand-dependent formation of a hydrophobic pocket in the
ligand-binding domain consisting of helices 3, 4, 5, and 12. The
leucines of the The receptors for retinoic acid (RAR), thyroid hormone (TR), vitamin D3
(VDR), and peroxisome proliferators (PPAR) form heterodimeric complexes
with the retinoid X receptor (RXR). These RXR heterodimers bind DNA and
regulate gene expression. However, the role of each receptor monomer in
the context of a receptor dimer in regulating coactivator and
corepressor recruitment remains largely unknown. In this study, we have
characterized the mechanism of recruitment of the coactivator RAC3 and
corepressor SMRT to the RXR/RAR heterodimer. We demonstrate that the
multiple LXXLL motifs of RAC3 are differentially required
for interactions with RAR and RXR. The coactivator and corepressor
binding pockets of RAR overlap extensively. However, differences in
contribution from each helix of the pocket are evident. We find that
although the coactivator pocket of RXR is essential to coactivator
binding and transcriptional activation by the RXR homodimer, this
pocket is not sufficient for the RXR/RAR heterodimer. In contrast, the
coactivator pocket of RAR is absolutely required and sufficient for the
function of the RXR/RAR heterodimer. Consistently, deletion of the AF-2
helix from RXR enhances RAC3 binding to the RXR/RAR heterodimer,
whereas deletion of the AF-2 helix from RAR abolishes RAC3 binding and
transcriptional activation by the receptor heterodimer.
GST Pull-down Assay--
GST fusion proteins were expressed in
Escherichia coli BL21 cells and purified with
glutathione-Sepharose beads. Approximately 5 µg of purified GST
fusion protein was incubated with 5 µl of 35S-labeled
protein with moderate shaking at 4 °C overnight in binding buffer (20 mM HEPES, pH 7.7, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 0.05%
Nonidet P-40, 1 mM dithiothreitol, and 1 mg/ml bovine serum albumin). The bound protein was washed three times with binding buffer,
and the beads were collected by centrifugation. The bound protein was eluted in SDS sample buffer, subjected to
SDS-polyacrylamide gel electrophoresis, and detected by autoradiography.
Site-directed Mutagenesis--
Site-directed mutagenesis was
conducted by the QuickChange site-directed mutagenesis system
(Stratagene). All mutations were confirmed by DNA sequencing.
Gel Electrophoresis Mobility Shift Assay--
The sequence of
the direct-repeat (DR) 5 element is
AGCTTAAGAGGTCACCGAAAGGTCACTCGCAT. The
sequence of the DR1 element is
AGCTTAAGAGGTCAAAGGTCACTCGCAT. The
double-stranded oligonucleotide probe was end-labeled with [32P]dCTP by using the standard Klenow fill-in reaction.
The purified probe was incubated with in vitro
transcribed/translated receptors in a binding buffer containing 7.5%
glycerol, 20 mM HEPES, pH 7.5, 2 mM
dithiothreitol, 0.1% Nonidet P-40, 1 µg of poly[d(I·C)], and 100 mM KCl. The DNA-protein complex was formed on ice for 1 h and resolved on a 5% native polyacrylamide gel, which was subsequently dried and subjected to autoradiography.
Partial Proteolysis Assay--
The partial proteolysis assay was
conducted as described previously (21). Briefly, wild-type and mutant
RXR were transcribed/translated in reticulocyte lysate and incubated
with 1 µM 9-cis-retinoic acid
(9-cis-RA) for 1 h on ice. After that, trypsin was
added to a concentration of 10 µg/ml and incubated for 10, 30, and 50 min. An equal concentration of solvent (80% Ethanol plus 20%
Me2SO) was added in control. After digestion, the
reaction was stopped by boiling in SDS sample buffer, subjected to
SDS-polyacrylamide gel electrophoresis, and detected by autoradiography.
Cell Culture and Transient Transfection--
HEK293 cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum at 37 °C in 5% CO2.
Cells were plated for transfection in Dulbecco's modified Eagle's
medium supplemented with 10% resin charcoal-stripped fetal bovine
serum in 12-well plates 1 day prior to transfection. HEK293 cells were
transfected using the standard calcium-phosphate method. Twelve hours
after transfection, the cells were washed with phosphate-buffered saline and re-fed fresh medium containing indicated concentrations of
ligand. After 24 h, the cells were harvested for LXXLL Motif Preferences for RAR
The above experiments revealed the LXXLL motif preferences
for RAR and RXR in solution. To examine this preference in the context
of a transcriptionally relevant heterodimer on DNA, we performed gel
mobility supershift assays with the RXR/RAR heterodimer at a DR5
element and the RXR homodimer at a DR1 element. The RXR/RAR heterodimer
bound the DR5 probe strongly in the presence of atRA (Fig.
1C, lane 1). The addition of GST did not affect
the heterodimer, whereas the wild-type RAC3-ID shifted a part of the
complex to a slower-migrating form (lane 3). The RAC3-ID
with a mutation of LXXLL motif i or iii slightly reduced
this interaction. By contrast, the mutation of motif ii abolished the
binding completely (lane 5), suggesting that motif ii is
most critical to interaction with the RXR/RAR heterodimer on DNA.
Similarly, the RXR homodimer bound specifically to the DR1 probe, which
was supershifted after the addition of wild-type RAC3-ID in a
9-cis-RA-dependent manner (Fig. 1D).
Consistent with the GST pull-down data, the LXXLL motif i or
ii mutation each inhibited RAC3 binding to the RXR homodimer, whereas
the motif iii mutation had minimal effects.
We next analyzed the contribution of each AF-2 helix of RAR and RXR in
recruiting RAC3 to the RXR/RAR heterodimer (Fig. 1E). Strikingly, when wild-type RAR was heterodimerized with the AF-2 helix-deleted mutant RXR443, this complex interacted with RAC3 much
more strongly than did the wild-type heterodimer in the presence of
atRA (Fig. 1, compare C, lane 3 with
E, lane 2; also see Fig. 3D,
lanes 2 and 4). This suggests that the AF-2 helix
of RXR may inhibit the binding of RAC3 to the RXR/RAR heterodimer on
DNA. In contrast, when the AF-2 helix-deleted mutant RAR403 was
heterodimerized with wild-type RXR, the interaction with RAC3 was
completely inhibited (Fig. 1E, lanes 3-4),
suggesting that the AF-2 helix of RAR is absolutely required for
interaction with RAC3. The wild-type and mutant receptors were
expressed at approximately equal levels as determined by
SDS-polyacrylamide gel electrophoresis and autoradiography (Fig.
1F). Also, the mutant receptors can form heterodimers with the partnering receptor and bind DNA efficiently (8), suggesting that
there were about equal amounts of active protein in each preparation.
In addition, the enhancing effect of RXR AF-2 helix deletion on binding
of RAC3 to the RXR/RAR heterodimer was also observed with the
RAR
We then used the above stable complex to re-examine the
LXXLL motif requirement for RAC3 binding to the RXR/RAR
heterodimer (Fig. 1E, lanes 5-8). Consistently,
mutation of motif i reduced RAC3 interaction, whereas mutation of motif
ii abolished the binding completely. In contrast, mutation of motif iii
had no effect. These results strongly suggest that motif ii is most
critical to RAC3 interaction with the RXR/RAR heterodimer, with motif i also contributing but to a lesser extent.
Identification of Critical Coactivator-binding Residues of RAR
Similarly, we analyzed the conserved residues in the coactivator pocket
of RXR Contribution of Each of the RAR and RXR Coactivator Pockets to the
Recruitment of RAC3--
To determine the relative contribution of
each of the two coactivator-binding pockets of the RXR/RAR heterodimer
to the interaction with RAC3, we conducted a gel mobility shift assay
with coactivator pocket-mutated receptors. Consistent with the above
experiment, the wild-type RXR/RAR heterodimer bound to the DR5 element
was significantly shifted by the wild-type RAC3-ID in the presence of
atRA (Fig. 3A,
lane). Interestingly, each of the four coactivator pocket
mutations in RAR
To further investigate the role of the RXR coactivator pocket in the
recruitment of RAC3, the effect of each of the two RXR coactivator
pocket mutations on binding of RAC3 to the RXR homodimer on DNA was
tested (Fig. 3B). As demonstrated above, the wild-type RXR
homodimer strongly bound the DR1 probe, and this complex was shifted by
RAC3-ID in the presence of 9-cis-RA (Fig. 3B,
lanes 1 and 2). The RXR coactivator pocket
mutants retained the ability to form homodimers and bind DNA,
suggesting that these mutations did not disrupt the structure of the
receptor. However, we found that the m1 mutation had a weaker DNA
binding activity, and the m2 mutation seemed to have an enhanced DNA
binding (lanes 3-6). Nonetheless, both RXR mutants showed
no evidence of binding RAC3-ID (lanes 4 and 6),
indicating that the coactivator pocket of RXR is required for the
recruitment of RAC3 to the RXR homodimer on DNA.
From the above experiments, it is evident that the RAR coactivator
pocket is absolutely required for recruiting RAC3 to the RXR/RAR
heterodimer and that the RXR coactivator pocket is required for
recruiting RAC3 to the RXR homodimer. However, it is not clear whether the RXR coactivator pocket contributes to RAC3 binding to the
RXR/RAR heterodimer. Therefore, we compared the ability of RAC3 to bind
the wild-type RXR/RAR heterodimer versus heterodimers in
which the coactivator pocket of RXR or RAR was mutated (Fig. 3C). Intriguingly, when wild-type RAR was dimerized with the
mutant RXR, RAC3-ID was still capable of binding the complex
significantly (lanes 5-8), contrasting to the lack of
binding to the complex containing mutant RAR and wild-type RXR. This
interaction was abolished when the RAR coactivator site was also
mutated (lanes 9 and 10). These data suggest that
the RAR coactivator pocket is the primary binding site to RAC3, whereas
the RXR coactivator pocket is less critical.
To confirm the above observations, we repeated the experiment using
RXR443, which dramatically enhances the interaction of the RXR/RAR
heterodimer with RAC3-ID (Fig. 3D). When RXR443 harboring the m2 mutation was dimerized with RAR, a much more enhanced
interaction with the RAC3-ID was still evident (lanes 5 and
6). In fact, nearly the entire RXR443/RAR complex was again
shifted by RAC3-ID. A similar result was obtained with the RXR443-m1
double mutant (data not shown). These results suggest that the RXR
coactivator pocket plays a minor role in recruiting RAC3 to the RXR/RAR
heterodimer, in contrast to the essential role of the RAR coactivator pocket.
Contribution of Each of the RAR and RXR Coactivator Pockets to
Transcriptional Activation--
To assess the functional significance
of mutating the coactivator pockets of RAR and RXR in vivo,
we performed reporter gene assays to investigate the transcriptional
activity of these coactivator pocket mutants. First, HEK293 cells were
transfected with wild-type or mutant RAR along with a luciferase
reporter containing DR5 response elements (Fig.
4A). In the absence of ligand,
the wild-type and mutant RAR had little effect on reporter expression,
except the wild-type RAR and E412K had a slight repressive activity. In
the presence of atRA, the reporter alone was stimulated about 2-fold,
whereas transfection of wild-type RAR strongly stimulated the reporter
expression about 10-fold. In contrast, all four RAR mutants failed to
enhance reporter expression above the endogenous level. These
observations correlate well with the above in vitro data in
implicating a critical role of the RAR coactivator pocket in mediating
transcriptional activation from responsive promoters.
We next sought to investigate the role of the RXR coactivator pocket in
supporting transcriptional activation by the RXR homodimer or RXR/RAR
heterodimer in vivo. HEK293 cells were transfected with
wild-type or coactivator pocket-mutated RXR and a luciferase reporter
driven by a DR1-containing promoter (Fig. 4B). The wild-type RXR displayed strong 9-cis-RA-dependent
transcriptional activation on the DR1 promoter as expected. In
contrast, the RXR m1 and m2 mutants were significantly impaired in
their abilities to activate reporter gene expression. We noted that the
m2 mutant retained some weak ligand-dependent activity;
however, this may correlate with its residual binding to RAC3 in
vitro (Fig. 2E) and/or enhanced homodimerization and
DNA binding ability (Fig. 3B). Overall, these data suggest
that the coactivator pocket of RXR is critical to transcriptional
activation by the RXR homodimer, correlating to the binding of RAC3
in vitro.
The above data suggest a requirement of the RXR coactivator pocket for
RAC3 binding and transcriptional activation by the RXR homodimer.
However, our data also suggest that the RXR coactivator pocket is not
as important in recruiting RAC3 to the RXR/RAR heterodimer as the RAR
pocket or as the recruitment of RAC3 to the RXR homodimer. To
investigate the functional significance of this differential requirement of the RXR coactivator pocket in different dimer
configurations, we analyzed the effects of the RXR coactivator pocket
mutations on transcriptional activation of the RXR/RAR heterodimer from a DR5-driven promoter (Fig. 4C). Coexpression of wild-type
RXR and RAR enhanced reporter expression above the expression of RAR alone. Strikingly, both RXR coactivator pocket mutants were capable still of sustaining transcriptional activation from the DR5 promoter, in contrast to their severely impaired function at the DR1 promoter. Similarly, coexpression of RXR443 with RAR was also capable of sustaining transcriptional activation by RXR/RAR and coactivation by
RAC3 (Fig. 4D). In fact, RXR443 slightly enhanced
RAC3-mediated transcriptional coactivation (1.5-fold). Although, this
effect is not as dramatic as the enhancement of binding to RAC3
in vitro, we have found that RXR443 also enhances
recruitment of the SMRT corepressor to the RXR/RAR
heterodimer.2 Furthermore, it
has also been shown that RXR443 decreases ligand-dependent dissociation of SMRT (23). Therefore, the ability of RXR443 to support
transcriptional activation in vivo may be compromised by
enhanced corepressor association. These data suggest that the RXR
coactivator pocket is critical to the activity of the RXR homodimer but
is less important to the RXR/RAR heterodimer.
Involvement of the RAR Coactivator Pocket for Corepressor
Interactions--
Several recent studies have determined that SMRT and
N-CoR contain LXXLL-like motifs that are required for
interaction with unliganded TR and RAR (18-20). Therefore, we wished
to determine whether the same residues within the RAR coactivator
pocket that were required for RAC3 binding were also critical to
binding of the corepressor SMRT (Fig.
5A). As expected, GST-SMRT-ID
pulled down significant amounts of wild-type RAR in the absence of
hormone. Interestingly, the V240R, F249R, and L261R mutations each
inhibited the interaction substantially, with F249R and L261R more or
less abolishing the binding; V240R had a more modest effect. In
contrast, the E412K mutation in helix 12 did not alter the SMRT-RAR
interaction, with was opposite of the strong effect on the RAC3-RAR
interaction. These results suggest that the RAR coactivator pocket
overlaps with a proposed corepressor pocket. However, distinct
contributions of individual residues do exist, because V240R had a more
modest effect on SMRT binding relative to RAC3 binding, whereas F249R displayed an opposite effect.
We then analyzed the recruitment of SMRT to the RXR/RAR heterodimer
bound to DNA (Fig. 5B). As expected, the wild-type
heterodimer was significantly shifted to a slower-migrating form by the
SMRT-ID (lane 2). Intriguingly, when RAR harboring the
V240R, F249R, or L261R point mutations were substituted for the
wild-type receptor, recruitment of SMRT was abolished (lanes
3-8). On the other hand, the E412K mutation did not affect SMRT
binding (lane 10), which was consistent with the above GST
pull-down data. Overall, these data suggest that corepressors bind to a
surface of RAR that overlaps with the coactivator-binding site.
Finally, we assessed the functional consequences of the RAR
corepressor-binding mutations on transcriptional repression by the
receptor. We introduced these mutations into the context of RAR403
because RAR403 can strongly repress basal transcription (3), allowing
sensitive assay for repression activity. The E412K mutation was not
included because it is not contained within RAR403. These mutant
receptors were tested for transcriptional repression by transient
transfection in HEK293 cells (Fig. 5C). Co-transfection of
RAR403 with the DR5-driven reporter resulted in significant repression
of basal activity in the absence of hormone, relative to empty vector.
However, the expression of RAR403 V240R, F249R, or L261R each abolished
this repression activity. These results demonstrate that the
corepressor-binding residues are also critical for transcriptional
repression by unliganded RAR.
We have investigated the mechanisms by which the coactivator RAC3
and corepressor SMRT are recruited by retinoid receptors and how these
interactions correlate with transcriptional activities of the
receptors. We find that RAC3 preferentially utilizes the LXXLL motifs i and ii to bind RXR and RAR, with highest
affinity of motif ii with RAR. We identify specific residues within the coactivator-binding pockets of RAR and RXR that are required for coactivator and corepressor bindings. We demonstrate that mutation of
these coactivator pocket residues disrupts recruitment of RAC3 and
transcriptional activities of the receptors. Interestingly, we also
find that the integrity of the RXR coactivator pocket is not sufficient
for the RXR/RAR heterodimer to recruit RAC3 and activate transcription,
whereas this RXR pocket is absolutely required for the function of the
RXR homodimer. Consistently, deletion of the AF-2 helix from RXR
enhances rather than inhibits coactivator binding to the RXR/RAR
heterodimer. Additionally, we demonstrate that several
coactivator-binding residues in RAR are also involved in the binding of
corepressor and regulation of transcriptional repression.
The coactivator RAC3 contains three separate LXXLL motifs
within its receptor-interacting domain. Based on previous studies of
the crystal structure of liganded receptor with LXXLL
peptide (15), it is likely that each receptor binds one
LXXLL motif. Therefore, a receptor dimer may selectively
utilize two LXXLL motifs to recruit a coactivator.
Accordingly, we find that the RAC3 LXXLL motif ii and motif
i, to a lesser degree, are both important for interactions with RAR in
solution or as part of the DNA-bound RXR/RAR heterodimer. In contrast,
motifs i and ii are equally important to interactions with RXR in
solution or when homodimerized and bound to DNA. Therefore, in the
RXR/RAR heterodimer, motif ii may bind to RAR first. The subsequent
interaction with RXR may be mediated by motif i. In the case of the
RXR/VDR heterodimer, motif iii may bind to VDR first, followed by a
secondary interaction between RXR and motif ii (9). Thus, the existence of three LXXLL motifs in RAC3-ID likely provides the
coactivator with flexibility to adapt to different structural
conformations that each receptor dimer assumes.
Recent crystallographic evidence has detailed the formation of a
hydrophobic pocket induced by ligand binding to the receptor that
serves as the docking surface for the LXXLL motif of
coactivators (14-16). This pocket consists of helices 3, 4, 5, and 12 of the ligand-binding domain including a charge clamp formed by a
conserved glutamate from helix 12 and a lysine from helix 3, which
together precisely position the LXXLL motif within the
pocket. Based on the interactions observed in the crystal structures of
estrogen receptor We also have investigated the ability of RXR, the common heterodimeric
partner for nonsteroid receptors, to interact with RAC3 because it is
not known what role RXR plays in recruiting coactivators to the RXR/RAR
heterodimer on DNA. Although heterodimerization with RXR is essential
for DNA binding, RXR has long been considered as a transcriptionally
silent partner for partnering receptors (1, 24, 25). However, the
RXR-specific ligand SR11237, when in combination with the RAR
antagonist BMS453, can induce differentiation of NB4 acute
promyelocytic leukemic cells and transcriptional activation (26). It
was demonstrated further that the RXR-specific ligand LG268 can
synergize with a low dose concentration of RAR-specific ligand TTNPB on
recruiting the coactivator SRC-1 (27), suggesting that RXR may play a
role in coactivator recruitment and transcriptional activation of the
RXR/RAR heterodimer. We find that RXR was able to bind RAC3 in
solution, and mutation of specific coactivator-pocket residues inhibits
this interaction. Intriguingly, however, the RXR pocket contributes
differently to coactivator recruitment to DNA-bound nuclear receptors
depending on the particular dimer examined. In the case of the RXR/RXR
homodimer bound to a DR1 element, the RXR pocket is required to recruit RAC3. This is likely caused by the loss of both pockets in the RXR
homodimer. Consistently, mutation of this pocket drastically reduces
the ability of RXR to activate transcription in vivo from a
DR1-driven reporter. A different pattern is evident upon examining the
RXR/RAR heterodimer. Here, the RXR coactivator pocket is not sufficient
to recruit RAC3 to a DR5 element, whereas the RAR pocket is absolutely
required and seems sufficient under these conditions. In support of
these in vitro data, the RXR mutants have only modest effects on RXR/RAR transcriptional activity at a DR5-driven reporter in vivo, in contrast to the strong effect on the RXR
homodimer. Our data suggest that RAR may act as a primary docking point
for coactivator binding to RXR/RAR heterodimer, whereas RXR may play a
secondary role in the recruitment of coactivator.
The finding that the AF-2 helix of RXR can interfere with RAC3 binding
to a DNA-bound RXR/RAR heterodimer is intriguing and consistent with
our previous studies with RXR/VDR (9). This inhibition may be the
result of competition between the AF-2 helix of RXR and the
LXXLL motif for the coactivator-binding pocket on the
partnering receptor; Westin et al. (27) showed previously that deletion of the RXR AF-2 helix slightly enhanced SRC-1 binding to
RAR/RXR. Consistently, in the crystal structure of the RXR tetramer,
the AF-2 helix of one RXR monomer occupies the coactivator-binding site
of the adjacent monomer of the symmetric dimer (28). We find that this
enhancement is dramatic in the gel shift assay. Therefore, coactivators
may have to compete with the RXR AF-2 helix for the coactivator site of
the partnering receptor. Together, these studies suggest that
coactivators, corepressors, and the RXR AF-2 helix all might interact
with a similar surface with ligand-inducing conformational changes that
serve to dissociate the corepressor and recruit coactivator. The
function of the RXR AF-2 helix may serve to adjust the overall loading
of coactivators and corepressors to the receptor complex bound to
promoter, adding an additional layer of controls to regulate precise
levels of target gene expression.
Additionally, we have characterized the involvement of the RAR
coactivator pocket in the binding of corepressor SMRT. Interestingly, the V240R, F249R, and L261R mutations that affect coactivator binding
also significantly affect corepressor binding. Importantly, these
mutations also disrupt the transcriptional repression activity of RAR8.
Thus, corepressor recruitment correlates well with transcriptional repression by RAR. Furthermore, it seems that structural differences exist between the coactivator- and corepressor-binding surfaces. In
addition to the opposite contributions from the AF-2 helix, the F249R
mutation retained residual binding to RAC3, but disrupts SMRT binding
completely. On the other hand, the V240R mutation disrupts RAC3
binding completely but retained detectable binding to SMRT. These data
are consistent with recent studies suggesting that corepressors
interact with unliganded nuclear receptors via a similar hydrophobic
pocket on the receptor as coactivators (18-20). However, unlike
coactivators, the charge clamp and AF-2 helix are not involved in the
binding of corepressors, because mutation of glutamate 412 from the
AF-2 helix has no effect on the RAR-SMRT interaction, but it disrupts
RAR-RAC3 interaction completely. The differences between the relative
roles of each residue in coactivator versus corepressor
binding may also be involved in the mechanism by which the receptor
discriminates between these cofactors.
In summary, our data suggest that each receptor in a receptor
heterodimer is differentially required in the recruitment of coactivators and corepressors. The recruitment of coactivators and
corepressors by the RXR/RAR heterodimer involves a complex series of
interactions that in turn regulates the transcriptional activity of the
receptor to control gene expression. Multiple RAC3 LXXLL
motifs mediate the binding of coactivator to the liganded receptor,
with different receptors preferring different motifs for interaction.
Both coactivators and corepressors interact with a similar hydrophobic
pocket of the ligand-binding domain, and the AF-2 helix of RXR seems to
be an important regulatory motif in mediating cofactor recruitment.
Overall, this study provides several new insights to the understanding
of the mechanism by which individual coactivator pockets are utilized
in a nuclear receptor dimer to recruit coactivators and corepressors.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical LXXLL motifs, where L
is leucine and X is any amino acid (6, 7). Previous analyses
of these motifs have indicated that motifs i, ii, and iii are critical to ligand-dependent interactions with nuclear receptors. In
contrast, motifs iv, v, and vi are important for transcriptional
activation likely via direct interaction with CREB-binding protein/p300
(6-8). Our laboratory and others have also uncovered a
receptor-specific code of interaction, where different nuclear
receptors prefer different LXXLL motifs of the coactivator
(9-12).
-helical LXXLL motif make direct contacts with this coactivator pocket, and a single LXXLL peptide
interacts with each monomer of the receptor dimer. In addition, a
"charge clamp," consisting of a conserved glutamate in the AF-2
helix (helix 12/H12) and a conserved lysine in helix 3, positions the LXXLL motif into the coactivator pocket of the receptor
(15). Interestingly, in the antagonist-bound structure, helix 12 mimics the LXXLL motif and occludes the coactivator site,
consistent with the inability of coactivators to bind antagonist-bound
receptors (16, 17). Recent studies also suggested a similar mechanism of interaction for the corepressors SMRT and N-CoR with unliganded receptors (18-20). Both proteins contain LXXLL-like motifs
in their respective receptor-interacting domains (ID) (20). These
motifs share a consensus sequence of
LXXI/HIXXXI/L and form
-helices. Mutation of
these motifs blocked the interaction with unliganded receptors and
abolished transcriptional repression. Furthermore, using mutational
analysis and molecular modeling, the corepressor motif seems to contact
the receptor at the same surface that accommodates the LXXLL
motif of the coactivator (18).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase and luciferase activities as described previously (22).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and RXR
--
To determine
the LXXLL motif preferences for RAR and RXR, we mutated the
two consecutive leucines within each of the three motifs in the
RAC3-ID613-752 (Fig.
1A). The mutants were
expressed as GST fusion proteins and tested for their abilities to
interact with RAR, RXR, and VDR via a GST pull-down assay (Fig.
1B). Wild-type RAC3-ID pulled down a significant amount of
[35S]-RAR in the presence of
all-trans-retinoic acid (atRA). Mutation of the
LXXLL motif i reduced this interaction, whereas the mutation of motif ii abolished the binding completely. In contrast, the motif
iii mutation had no effect. With RXR, a different pattern was evident.
Wild-type RAC3-ID interacted strongly with RXR in the presence of
9-cis-RA. Mutation of the LXXLL motifs i or ii each substantially inhibited this binding, reducing it to near background level, whereas the mutation of motif iii had less effect. In
contrast, the LXXLL motif iii mutation significantly
affected RAC3 binding to VDR. These data indicate that the
LXXLL motif ii is most critical for interaction with RAR,
whereas motifs i and ii are equally important for interaction with RXR,
contrasting with the motif iii preference for VDR.
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Fig. 1.
Mutation of RAC3 LXXLL
motifs reveals retinoid receptor-specific motif preferences.
A, schematic illustration of RAC3 and its functional domains
as well as LXXLL motif mutations generated in the context of
the GST-RAC3-ID fusion protein. AD, activation domain,
i-vi, LXXLL motifs; bHLH,
basic helix-loop-helix; PAS-A, per-arnt-sim A;
PAS-B, per-arnt-sim B; Q-rich, glutamine-rich
domain. B, GST pull-down assay of indicated
[35S]methionine-labeled receptors and GST fusion proteins
in the presence of 1 µM atRA (RAR ),
9-cis-RA (RXR
), or 1,25-dihydroxy-vitamin
D3 (VDR). wt, wild type.
C, gel shift assay of the effects of LXXLL motif
mutations on RAC3-ID binding to DR5-bound RXR/RAR in the presence of 1 µM atRA. To resolve the complexes better, free probes
were run out of the gel, and therefore only the receptor/DNA and the
RAC3-ID supershifted complexes are shown. D, gel shift assay
of the effects of LXXLL motif mutations on RAC3-ID binding
to the DR1-bound RXR homodimer. The gel shift assay was performed as in
C except 1 µM 9-cis-RA and a DR1
probe were used. E, gel shift assay performed as in
C except the H12 truncated RXR443 and RAR403 were used where
indicated. *, nonspecific band from lysate. F,
autoradiograph of in vitro transcribed/translated wild-type
and mutant receptors used in this study. G, gel shift assay
performed as in E except the RAR-specific ligand TTNPB (10 nM) was used where indicated.
-selective agonist
(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl] benzoic acid (TTNPB), confirming that the inhibition by the RXR AF-2
helix occurs in the absence of ligand binding to RXR.
and RXR
--
A coactivator-binding pocket, consisting of residues
from helices 3, 4, 5, and 12, that accommodates the LXXLL
motif of coactivators has been identified (13-16). In light of these
findings, we decided to characterize the coactivator-binding pockets of
RAR and RXR, by creating site-directed mutations, and determine the
relative contribution of each coactivator-binding pocket of the RXR/RAR heterodimer to the recruitment of RAC3. We aligned several receptor sequences and identified conserved residues that form direct contacts with the LXXLL motif in the crystal structures of estrogen
receptor
, TR
, and PPAR
(Fig.
2A). These residues were
mutated in the context of full-length RAR
and RXR
. These
mutations are homologous to those made in the TR
, which retained
their ability to bind hormone (13). Each RAR mutant was expressed
in vitro (Fig. 2B) and tested for interactions
with RAC3 in a GST pull-down assay (Fig. 2C). As expected,
GST-RAC3-ID pulled down a significant amount of wild-type RAR in the
presence of atRA, compared with only minimal binding to GST alone.
However, mutation of any of the conserved pocket residues drastically
reduced this interaction. In particular, the V240R, L261R, and E412K
mutations each abolished RAC3 binding completely, whereas F249R
retained slight interaction with RAC3. Therefore, these residues in the
coactivator-binding pocket of RAR are critical for RAC3 binding,
suggesting that the coactivator pocket is conserved among different
nuclear receptors and for interactions with different SRC
coactivators.
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Fig. 2.
Mutation of the coactivator pocket inhibits
interaction between retinoid receptors and RAC3 in
vitro. A, alignment of the coactivator
pocket sequences of nuclear receptors. The underlined
residues in RAR or RXR were mutated based on homologous amino acids (in
bold) of estrogen receptor , TR
, and PPAR
demonstrated to contact the LXXLL motif in the respective
crystal structures. B, autoradiograph of in vitro
translated [35S]methionine-labeled RAR probes
demonstrates equal expression of wild-type (wt) and mutant
proteins. C, GST pull-down assay of the indicated RAR probe
with GST-RAC3-ID in the presence of 1 µM atRA.
D, the autoradiograph of in vitro translated
[35S]methionine-labeled RXR probes. E, GST
pull-down assay of the indicated RXR probe with GST-RAC3-ID in the
presence of 1 µM 9-cis-RA.
for their contributions to RAC3 binding. The leucine
276/valine 280 from helix 3 (m1) and valine 298/leucine 301 from helix
5 (m2) were mutated to alanines. A partial proteolysis assay indicated
that both mutants still bind to 9-cis-RA efficiently (data
not shown). The wild type and mutants were expressed at approximately
equal levels in the in vitro translation reaction (Fig.
2D), and the 35S-labeled receptors were tested
for interactions with RAC3 in the presence of 9-cis-RA (Fig.
2E). The RAC3-ID interacted specifically with wild-type
RXR
as expected. Interestingly, both mutations affected RAC3 binding
significantly, with the m1 mutation abolishing the interaction
completely and the m2 mutation retaining barely detectable binding.
Therefore, we have also identified and disrupted residues in the RXR
coactivator pocket that are critical to RAC3 binding.
eliminated the RAC3-dependent
supershift despite the presence of an intact coactivator pocket in
RXR
(lanes 3-10). It seemed that the F249R mutant
retained weak binding to RAC3-ID. We found that the addition of
9-cis-RA to the reactions had no effect on the binding (data
not shown), suggesting that the RXR coactivator pocket alone is not
sufficient to recruit RAC3 to the RXR/RAR heterodimer in the absence of
a functional RAR coactivator pocket.
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Fig. 3.
Mutation of the RAR or RXR coactivator pocket
differentially affects RAC3 binding to the DNA-bound RXR/RAR
heterodimer. A, gel shift assay of the effects of RAR
mutations on RAC3-ID binding to RXR/RAR. Indicated receptors were added
to a binding reaction containing 1 µM atRA and a DR5
probe in the absence ( ) or presence (+) of GST-RAC3-ID fusion
protein. wt, wild type. B, effects of RXR
mutations on RAC3-ID binding to an RXR homodimer. The gel shift assay
was performed as described in A except 1 µM
9-cis-RA and a DR1 probe were used in the absence (
) or
presence (+) of GST-RAC3-ID fusion protein. C, effects of
RXR mutations on RAC3-ID binding to RXR/RAR. The gel shift assay was
performed as described in A. D, similar results
are obtained when AF-2 H12-truncated RXR443 is substituted for
wild-type RXR.
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Fig. 4.
Transcriptional activity of retinoid
receptors harboring mutations in the coactivator pocket.
A, empty vector, wild type (wt), or coactivator
pocket mutated RAR was co-transfected in HEK293 cells with a DR5-driven
luciferase reporter. Cells were treated with solvent or 50 nM atRA. Those mutants that failed to bind RAC3 in
vitro also fail in activating transcription. B,
mutation of the RXR coactivator pocket inhibits transcriptional
activation by the RXR homodimer. Transfection was performed as
described in A using wild-type or mutant RXR and a
DR1-driven reporter with cells treated with solvent or 100 nM 9-cis-RA (9cRA). C, the
same experiment as described in B was repeated using a
DR5-driven reporter and wild-type RAR in the absence or presence of 100 nM atRA. Note that the RXR mutations markedly affect the
transcriptional activity of the RXR homodimer but not the activity of
the RXR/RAR heterodimer. D, the same experiment as described
in C was repeated comparing the ability of wild-type RXR and
RXR443 in sustaining transcriptional activation from a DR5-driven
promoter and transcriptional coactivation by RAC3.
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Fig. 5.
Mutation of the RAR coactivator pocket
inhibits interaction with the corepressor SMRT in vitro
and transcriptional repression in vivo.
A, GST pull-down assay of the indicated RAR probe with
GST-SMRT-ID (amino acids 982-1291) in the absence of ligand.
wt, wild type. B, gel shift assay with the
indicated receptors and a DR5 probe in the absence ( ) or presence (+)
of GST-SMRT-ID fusion protein. C, empty vector (
), RAR403,
or RAR403 containing each of the indicated coactivator pocket mutations
were co-transfected in HEK293 cells with a DR5-driven luciferase
reporter. The indicated coactivator pocket mutations disrupt the
ability of RAR403 to repress basal transcription.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, TR
, and PPAR
complexed with
LXXLL peptides, highly conserved amino acids from RAR and
RXR were selected for analysis in this study. We find that the mutation
of valine 240, phenylalanine 249, or lysine 261, from helices 3, 4, and
5, respectively, each strikingly inhibits RAR interactions with RAC3
in vitro and transcriptional activation by the receptor
in vivo. Mutation of the charge clamp glutamate, glutamate
412, shows the same effect. These mutations also abolish
recruitment of RAC3 to a DNA-bound RXR/RAR heterodimer despite the
presence of an intact coactivator-binding site in RXR. While it remains
to be demonstrated, these single point mutations are unlikely to affect
the overall structure of the receptor because they retain intact
DNA-binding and RXR heterodimerization activities. These data suggest
that an intact coactivator pocket is essential to RAR interactions with
RAC3 and that coactivator interaction is required for the
transcriptional activation function of RAR.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Amy (Hong-Bing) Chen and Mausumi Bhaumik for technical assistance and members of the Chen laboratory for helpful discussion.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK52888 and DK52542 (to J. D. C.) and a United States Army Breast Cancer Research Program pre-doctoral fellowship (to C. L.).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.
Dept. of Biochemistry and Molecular Pharmacology, University of
Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. Tel.: 508-856-1481; Fax: 508-856-1225; E-mail:
don.chen@umassmed.edu.
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M100462200
2 C. Leo, X. Yang and J. D. Chen unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: SMRT, silencing mediator for retinoid and thyroid hormone receptor; N-CoR, nuclear receptor corepressor; SRC, steroid receptor coactivator; RAC3, receptor-associated coactivator 3; AF-2, activation function 2; ID, receptor-interacting domain; RAR, retinoic acid receptor; TR, thyroid hormone receptor; VDR, vitamin D3 receptor; PPAR, peroxisome proliferator receptor; RXR, retinoid X receptor; GST, glutathione S-transferase; DR, direct repeat; 9-cis-RA, 9-cis-retinoic acid; atRA, all-trans-retinoic acid; TTNPB, (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl] benzoic acid; m1, leucine 276/valine 280 from helix 3; m2, valine 298/leucine 301 from helix 5.
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