From the Department of Biochemistry, School of
Medicine, Case Western Reserve University, the Research Institute of
University Hospitals of Cleveland and the Comprehensive Cancer Center
of Case Western Reserve University and University Hospitals of
Cleveland, Cleveland, Ohio 44106 and the ¶ Howard Hughes Medical
Institute, Gene Expression Laboratory, The Salk Institute for
Biological Studies, La Jolla, California 92037
Received for publication, July 26, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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Nuclear hormone receptors coordinately regulate
the activity of genetic networks through the recruitment of
transcriptional co-regulators, including co-repressors and
co-activators. Allosteric modulation of the ligand-binding domain by
hormonal activators shifts the co-factor binding preference by defined
structural changes in overlapping docking sites. We report here that
mutations at conserved residues within the docking motif of the
retinoic acid receptor Members of the steroid hormone receptor superfamily are
hormone-activated transcription factors that control vertebrate
development, differentiation, and homeostasis through coordinate
regulation of complex genetic networks in multiple target cells (1, 2). Unliganded thyroid hormone
(TR)1 and retinoid acid
receptors (RAR) function as potent transcriptional repressors becoming
activators upon hormone binding. The repression activity by unliganded
RAR and TR is mediated through the recruitment of either the silencing
mediator for retinoid and thyroid hormone receptors (SMRT) or nuclear
receptor co-repressor complexes that include mSin3A and a
variety of histone deacetylases (3-7). In contrast, transcriptional
activation by nuclear hormone receptors involves the recruitment of
histone acetyltransferase complexes that include CREB-binding
protein/p300, p300/CBP-associated factor, and members of the
p160 family (SRC-1, GRIP1/TIF2, and ACTR/RAC3/P/CIP) (8-14).
Nuclear receptors contain two conserved modules, the DNA-binding domain
and the carboxyl-terminal ligand-binding domain (LBD). DNA-binding
domains bind hormone response elements and thus direct receptors to
appropriate target genes. LBDs are required for nuclear localization,
homodimerization and/or heterodimerization, co-regulator association
(including co-repressors and co-activators), and most importantly,
ligand binding. The LBDs are composed of 12 helices in which helices
3-5 are the most conserved among receptors and define the nuclear
receptor LBD signature motif for co-regulator recruitment. The
transcriptional switch of the receptors from repressors to activators
involves a ligand-induced conformational change, resulting in an
exchange of co-repressors and co-activators. Using site-directed
mutagenesis, we and others have previously shown that helices 3-5 of
TR RARs and RXRs form heterodimers in solution and on response elements
containing direct repeat spaced by one (DR1) or five base pairs (DR5)
with the half-site sequence AGGTCA (1). Although the LBD
three-dimensional structures of RXRs and RARs are very similar (22,
23), they exhibit distinct ligand binding properties toward
stereoisomers. RXRs bind only to 9-cis-retinoic acid,
whereas RARs can bind both 9-cis-retinoic acid and
all-trans-RA (AT-RA), albeit with different affinities. RXRs
are thought to function as a silent partner whose ligands alone do not
activate transcription activity of the RXR/RAR heterodimer. In
contrast, RARs ligands alone are able to activate transcription.
In an attempt to examine the molecular basis of co-regulator
association with RAR Plasmid Construction--
The plasmids pCMX, pCMX-Gal4,
pCMX-Gal4-RAR Transient Transfection--
CV-1 cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum, 50 units/ml of penicillin G, and 50 µg/ml of streptomycin sulfate at
37 °C in 7% CO2. For Gal4-RAR Electrophoresis Mobility Shift Assays--
GST fusion proteins
were expressed in Escherichia coli DH5 Identification of RAR Helices 3 and 4 of RAR
We next measured the association between RAR Association of Co-activators with RAR Co-regulator Association with RAR Distinct Activities of RAR
To understand how co-regulator association correlates with the
transcriptional activity of K262A, transient transfection assays were
employed. Unliganded wild-type Gal4-RAR
We further examine the activity of the mutants on a DR5-containing
reporter construct (Fig. 5F). In the presence of AT-RA, the
expression of the reporter activity is induced in the absence of
exogenous RAR Site directed mutagenesis was used to dissect the mechanistic
links between co-repression, co-activation, and ligand binding. Our
results indicate that residues within the receptor signature motif
contribute in specific ways to both co-repressor and co-activator binding (Table I and Fig.
6). One unusual mutation is K262A, which
results in increased co-activator and reduced co-repressor binding but
functionally acts as a dominant-negative mutation. This implies an
altered co-repressor off rate leading to the suggestion that
co-repressor release is dominant to co-activator binding. This in turn
implies that the principle role of ligand is to induce co-repressor
release, enabling the signature motif to attract the co-activator.
Taken together, these data strongly suggest that transcriptional
repression and activation are mechanistically linked.
cause defects in dimerization, co-regulator
association, and transcriptional regulation. Furthermore, although a
minimal co-repressor receptor interaction domain is sufficient for
receptor binding, flanking sequences appear to stabilize this
interaction without interfering with ligand sensitivity. However,
ligand sensitivity is changed by the K262A mutation, which requires
much higher concentrations of all-trans-retinoic acid to
promote co-repressor dissociation. Consequently, K262A functions as a
dominant-negative mutant at low concentrations of
all-trans-retinoic acid. As a result, transcriptional activation is mechanistically linked to co-repressor release.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and peroxisome proliferator-activated receptor
play a
pivotal role in binding co-regulators (15-20). Reciprocally, SMRT and
nuclear receptor co-repressor contain two short peptide motifs that are
both necessary and sufficient for mediating co-repressor binding to
unliganded RARs (15-17). Both motifs contain a hydrophobic core
(I/L)XX(I/V)I. These two motifs, termed coreID I and
coreID II (17 and 19 amino acids, respectively), are conserved in both
position and in sequence between nuclear receptor co-repressor and
SMRT. A third TR
binding motif amino-terminal to those previously
mapped in nuclear receptor co-repressor may also exist (21). Helices
3-5 also interact with co-activators through a
LXXLL-containing signature motif. The p160 co-activator proteins contain three putative LXXLL motifs, but the
affinity and specificity of the individual LXXLL motifs
within p160 proteins toward different receptors are not well characterized.
, we have generated mutations within both conserved and diverged residues within RAR
helices 3 and 4 and systematically analyzed the properties of these mutants. We have identified critical residues for RAR
functions including
dimerization, co-regulator association, and transcriptional activity.
Our results suggest that the transcription activity of RAR
depends
primarily on its ability to dissociate from co-repressors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, pCMX-RAR
, pCMX-VP16-RXR
, pCMX-Gal4-RXR
(LBD), CMX-LacZ, and pMH100-TK-Luc have been described (24, 25).
GST-SMRT and GST-ACTR fusion constructs were generated by inserting
SMRT and ACTR receptor interaction domain (ID) PCR fragments into the
vector pGEX4T-1. Site-directed mutagenesis was carried out using the
QuikChange kit according to the manufacturer's protocol (Stratagene).
All of the constructs were verified by double-stranded sequencing to
confirm the identity and reading frame.
, CV-1 cells (60-70%
confluence, 48-well plate) were co-transfected with 16.6-66.6 ng of
pCMX-Gal4 and pCMX-VP16 (for mammalian two-hybrid assays) fusion
constructs, 100 ng of pMH100-TK-Luc, and 100 ng of pCMX-LacZ in 200 µl of Dulbecco's modified Eagle's medium containing 10% fetal
bovine serum by the
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate-mediated procedure (7). For DR5-TK-Luc (see Fig. 5), the
cells were transfected with 0.33 ng of pCMX-RAR
, 100 ng of
-RARE-TK-Luc, and pCMX-LacZ. The amount of DNA in each transfection
was kept constant by the addition of pCMX. The CV-1 cells were
transferred to charcoal-stripped serum after adding DNA. After 24 h, the medium was replaced with or without
all-trans-retinoic acid. The cells were harvested and
assayed for luciferase activity 36-48 h after transfection. The
luciferase activity was normalized to the level of
-galactosidase
activity. Each transfection was performed in triplicate and repeated at
least three times.
strain and
affinity purified by glutathione-Sepharose 4B beads. Immobilized GST
fusion proteins were eluted by 20 µM glutathione and
dialyzed against 1× phosphate-buffered saline. RAR
and RXR
synthesized in vitro (Promega) were incubated with a
32P-labeled probe containing a DR5 element derived from the
RARE promoter containing the sense strand sequence
5'-GGT-AGG-GTT-CAC-CGA-AAG-TTC-ACT-C-3' with or
without 1 µM AT-RA. DNA-protein binding was conducted in
a reaction mixture containing 20 mM Hepes, pH 7.4, 50 mM KCl, 1 mM
-mercaptoethanol, and 10%
glycerol. After 30 min of incubation at 25 °C, the purified GST-SMRT
or GST-ACTR fusion proteins were added followed by an additional 30 min
of incubation. The final reaction mixtures were loaded onto a 5%
polyacrylamide (29.2:0.8) nondenaturing gel followed by electrophoresis
in 0.5× TBE buffer. After electrophoresis, the gel was dried and
subjected to autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Residues Critical for Dimerization with
RXR
--
To determine the residues that are critical for RAR
function, we generated mutations on conserved and diverged residues
within helices 3 and 4. Fig.
1A shows the nuclear receptor
signature motif and the targets for site-specific mutations in this
study. Primarily, alanine substitutions were generated and tested for their ability to dimerize with RXR
(Fig. 1B). The ability
of RAR
mutants to dimerize with wild-type RXR
was evaluated by mammalian two hybrid (M2H) assays and electrophoresis mobility shift
assays (EMSA) on a template containing a DR5. M2H assays were conducted
using Gal4-RXR
and VP-RAR
(LBD) along with a reporter construct
(pMH100) containing multiple copies of Gal4-binding sites upstream of
the thymidine kinase (TK) promoter fused with luciferase gene.
Induction of the luciferase activity is an indicative of interaction of
the partners. As a control, Gal4-RXR
with VP16 alone only gave a
basal activity (Fig. 1B). In the presence of VP16-RAR
,
the reporter activity is induced, suggesting an association between
RXR
and RAR
. In the absence of AT-RA, all of the mutants except
F249A and Q257A retain at least 75% of wild-type activity. As
expected, the addition of 1 µM AT-RA increased the
reporter activity of the wild-type RAR
, probably because of the
recruitment of co-activators, which further increase transcriptional
activation by Gal4-RXR
/RAR
. Interestingly, most of the RAR
mutants gave lower reporter activity than that of the wild type,
indicating that these mutants have lower activation activity. This
result suggests that these mutants might be defective in co-activator binding. Although unliganded F249A binds RXR
much less efficiently than wild type, the reporter activity of G4-RXR
/RAR
(F249A) increased dramatically upon addition of 1 µM AT-RA. To
test the heterodimerization ability of wild-type or mutant RAR
with
RXR
on DNA, in vitro synthesized RAR
and RXR
were
mixed with a radiolabeled DNA duplex containing a DR5 element derived
from the promoter of
RARE gene. Consistent with M2H assays, F249A
and Q257A failed to heterodimerize with RXR
productively on DR5
(data not shown; see below). A likely possibility is that these two
mutants failed to form heterodimer efficiently.
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Fig. 1.
Heterodimerization properties of mutations in
helices 3 and 4 of RAR . A, amino acid
alignment of helices (helices 3 and 4 of RAR
and helices 3-5 of
TR
) of selected nuclear receptors. The conserved LBD signature motif
is boxed. The asterisks indicate the position of
the mutations targeted in this work. The consensus nuclear receptor LBD
signature motif is indicated. B, in vivo
dimerization activity of RAR
helices 3 and 4 mutants with RXR
.
M2H assays were conducted to evaluate the association of RXR
and
RAR
in the presence and absence of 1 µM AT-RA.
Are Critical for Co-repressor
Association--
The ability of the unliganded RAR
mutants to bind
SMRT was tested by M2H assays. M2H assays were conducted using
Gal4-SMRT ID I + II (amino acid 2064-2307 of mouse SMRT, a fragment
containing both ID I and II) and VP-RAR
(Fig.
2A). Our data indicate that unliganded mutants V240A, F249A, Q257A, I258A, and L261A dramatically lost their SMRT binding activity (lanes 3, 6, and
8-10). In the presence of 100 nM AT-RA, RAR
and SMRT interaction is completely abolished in all cases except mutant
K262A (lane 11). We also tested whether these RAR
mutants
bind SMRT in EMSAs. Using the same SMRT ID I + II fragment used in M2H
assays, we constructed pGEX-4T-1-SMRT ID I + II for bacterial
expression and subsequently affinity-purified the GST-SMRT ID I + II
fusion proteins. In vitro synthesized RAR
and RXR
were
mixed with a radiolabeled DNA duplex containing a DR5 element derived
from the promoter of the human
RARE gene. EMSAs were performed with
or without purified GST-SMRT ID I + II (Fig. 2B). Consistent
with M2H assays, RXR
/RAR
heterodimers with RAR
mutants V240A,
F249A, Q257A, I258A, and L261A, significantly lost the ability to bind
SMRT. The SMRT binding activity of mutants S232A, K244A, G248T, I254A,
and K262A is moderately reduced. As with M2H assays, the addition of
100 nM AT-RA completely blocked SMRT ID association with
RAR
except for mutant K262A (data not shown). These data indicate
that the LBD signature motif is critical for co-repressor binding. We
also noted that F249A and Q257A failed to heterodimerize with RXR
productively on DNA. This result is consistent with M2H data. A likely
possibility is that these two mutants failed to bind DNA because they
do not form heterodimers efficiently.
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Fig. 2.
SMRT binding activity of mutants in
RAR helices 3 and 4. A, mammalian
two-hybrid assays of SMRT ID with RAR
helices 3 and 4 mutants.
Association of SMRT ID I + II with RAR
mutants was determined with
or without the presence of 100 nM AT-RA. For A
and C, the reporter activity of Gal4-SMRT with VP16 alone
was defined as 1-fold. Fold activation is calculated as the ratio of
the reporter activity of Gal4-SMRT ID I + II with VP16-RAR
divided
by the reporter activity in the presence of Gal4-SMRT ID I + II with
VP16 alone. B, SMRT binding of RAR
helices 3 and 4 mutants on DR5 element. GST-SMRT ID I + II fusion protein was employed
in EMSA assays to determine RAR
mutant SMRT binding activity.
C, the sequence flanking coreID II stabilizes the
association of SMRT and RAR
LBD. The activities of the wild-type
RAR
to associate with SMRT ID I + II and SMRT coreID II are defined
as 100% binding activity. The percentage of SMRT binding was
determined by luciferase activity (mutant)/luciferase activity (wild
type).
LBD mutants with coreID
II (Fig. 2C) and found that SMRT coreID II domain alone is
highly sensitive to mutations within RAR
LBD. Three patterns of
association were identified. Most of the mutants, with the exception of
I254A and K262A, failed to bind SMRT coreID II. In another group,
S232A, K244A, and G248A bound ID I + II but not the coreID II alone.
Finally, association of coreID II and ID I + II with RAR
LBD was
abrogated in a third group consisting of V240A, F249A, Q257A, I258A,
and L261A. Based on these results, we conclude that these residues are
critical for co-repressor association and that sequences flanking
coreID II may play a role in the association with RAR
LBD.
Mutants--
Because
helices 3 and 4 of TR
and peroxisome proliferator-activated
receptor
have also been shown to be critical for co-activator association (18-20), we tested the interaction between RAR
helices 3 and 4 mutants and p160 co-activator proteins. M2H assays were carried
out to determine in vivo association of p160 co-activator proteins with RAR
using Gal4-ACTR (SRC-1 or GRIP1) ID (receptor interaction domain) and VP16-RAR
(LBD). Among the three known p160
family members, RAR
bound ACTR/RAC3/PCIP the best, with SRC-1 less
well and GRIP1 the least (Fig.
3A), whereas TR
bound SRC-1
better than ACTR and GRIP1. M2H assays were used to determine in
vivo association of ACTR and wild-type or mutant RAR
. The data
in Fig. 3B show that the association between ACTR and RAR
is severely compromised for mutants V240A, K244A, F249A, Q257A, I258A,
and L261A. Furthermore, we found that the interactions between ACTR and
RAR
are ligand concentration-dependent (data not shown; see
below). We then tested the effect of RAR
mutations on ACTR binding
by EMSA. EMSA was conducted as described in Fig. 2 with or without
GST-ATCR in the presence of 100 nM AT-RA. We found that
GST-ACTR RID was able to supershift DNA-bound wild-type heterodimers
RXR
/RAR
efficiently. However, ACTR binding activity is
dramatically impaired for mutants V240A, K244A, F249A, Q257A, I258A,
and L261A, indicating the loss of ACTR binding activity of these
mutants.
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Fig. 3.
Association of RAR
with p160 family proteins. A, RAR
binds ACTR with
highest affinity. The strength of co-activator association was examined
by M2H assays with or without corresponding ligands. B,
association of RAR
with ACTR ID in M2H assays. Gal4-ACTR ID was
co-transfected with VP-RAR
(LBD) for M2H assays. The reporter
activity from Gal4-ACTR ID + VP16 alone was set as 1-fold.
C, association of liganded RAR
with ACTR on DR5. ACTR
binding was assayed by EMSA assays on DR5 in the presence of 100 nM AT-RA. Note that F249A binds weakly to ACTR. Note that
the ratio of GST-ACTR-bound/unbound G248T is comparable with that of
S232A.
Correlates with
Transcriptional Activity--
The ability of these RAR
mutants to
regulate transcription was examined by transient transfection assays.
Wild-type and RAR
mutants were fused to the yeast Gal4 DNA-binding
domain to generate Gal4-RAR
expression plasmids. Transient
transfection assays were conducted in the presence or absence of 100 nM of AT-RA. In the absence of AT-RA, the degree of
transcriptional repression activity of RAR
varies among these
mutants. Notably, V240A, F249A, Q257A, I258A, and L261A lose more than
80% of their repression activity (Fig.
4A). The repression activity
correlates with the ability of these mutants to interact with SMRT.
Furthermore, the activation activity of mutants K244A, F249A, Q257A,
L261A, and K262A was dramatically impaired at 100 nM AT-RA
(Fig. 4B). We noted that liganded V240A, I258A, and L261A
bind ACTR less efficiently than the wild-type RAR
, but their
ligand-dependent transcriptional activation is only
partially inhibited. We propose that this observation may reflect the
loss of SMRT binding activity by V240A, I258A, and L261A. In addition,
although K262A and wild type bound ACTR equally well in the presence of
100 nM, K262A only moderately activated transcription in
transient transfection assays. We interpret this to mean that K262A is
still associated with co-repressor complexes in the presence of 100 nM AT-RA, as shown in Fig. 2 (see "Discussion").
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Fig. 4.
Transcription activity of
RAR mutants. A, transcriptional
repression by unliganded Gal4-RAR
. The activity of Gal4-RAR
(LBD)
was assayed by transient transfection assays. B,
transcriptional activation by liganded Gal4-RAR
at 100 nM AT-RA. Note that at this concentration, F249A only
slightly activates transcription.
Helices 3 and 4 Mutants F249A and
K262A--
During our analyses for ligand
concentration-dependent association/dissociation of RAR
mutants and co-factors, we found that the association/dissociation
profile of K262A with co-factors was distinct from those of other
helices 3 and 4 mutations. Fig. 5A is an EMSA with wild-type
RAR
and mutant K262A in the presence of GST-SMRT with or without
increasing concentration of AT-RA. Similar to other mutants, K262A
acquires reduced SMRT association activity (Fig. 5A,
compared the ratio of SMRT-bound/unbound RAR
). We noted that at 1 µM, residue SMRT still remains associated with DNA-bound
RXR
/RAR
heterodimer. M2H assays were also carried out to
determine ligand-dependent dissociation between SMRT and wild-type RAR
or K262A (Fig. 5B). Consistently, M2H
assays demonstrated that dissociation between SMRT and K262A requires
at least 1 µM AT-RA as opposed to 100 nM for SMRT and wild type RAR
. In contrast, heterodimer
RXR
/RAR
(K262A) binds ACTR in the absence of ligand in
vitro (EMSA), indicating a better ACTR binding activity than that
of the wild-type RAR
(Fig. 5C). However, in
vivo association of K262A with ACTR requires the presence of
AT-RA. Although K262A binds to ACTR equally as well as that of wild
type at 100 nM AT-RA, K262A binds less tightly than wild
type at 1 µM AT-RA in M2H assays (Fig. 5D).
Under the same conditions, F249A can only associate with ACTR in the
presence of 1 µM AT-RA.
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Fig. 5.
A unique association profile of
RAR mutant K262A and F249A with co-regulators.
A, association of SMRT with K262A on DR5. B,
association of SMRT with K262A in M2H assays. C,
ligand-dependent association of ACTR with K262A on DR5.
D, ligand-dependent association of F249A and
K262A with ACTR in M2H. E, transcription activity by
Gal4-RAR
. F, transcription activity of RAR
mutants on
DR5-TK-Luc. Note that K262A functions as a dominant-negative mutant at
low concentrations of AT-RA. WT, wild type.
represses transcription efficiently (Fig. 5E, lane 5), whereas K262A
moderately repressed basal transcription (lane 13). The
reduced repression activity is consistent with the reduced SMRT binding
activity shown in Fig. 5 (A and B). In the
presence of 10 nM AT-RA, wild-type RAR
dramatically
activated transcription (lane 6, 31.3-fold), whereas K262A
only activated transcription 3-fold compared with basal (lane
14). Of note, transcriptional activation by K262A at 1 µM AT-RA was lower than that of the wild type at 10 nM AT-RA (lanes 6 and 16).
Surprisingly, F249A strongly activates transcription at 1 µM AT-RA. This result is consistent with the observation that F249A acquires co-activator association at 1 µM
AT-RA shown in Fig. 5D.
. Presumably, this activity is derived from the endogenous RARs (lanes 2-4). Exogenous expression of the
wild-type RAR
further induced the expression of the reporter
activity (lanes 6-8). Intriguingly, we found that although
F249A binds ACTR weaker than the wild-type RAR
at 1 µM
AT-RA as shown in Fig. 5D, the transcription activity of
F249A is comparable with that of wild-type RAR
. Furthermore, at
lower AT-RA concentration, expression of K262A resulted in lower
reporter activity than that in the absence of AT-RA (lanes
14 and 15 compared with lanes 2 and
3), suggesting that K262A inhibits endogenous RAR activity
and that K262A functions as a dominant-negative mutant of the wild-type
RAR
.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Summary of RAR mutant properties
is shown as +++. The activity of the
mutant RAR
is denoted: +++, 80-100%; +++/++, 50-80%; ++,
30-50%; ++/+, 10-30%; +, 5-10%; +/
, 0-5%;
, 0%. The
percentage is defined as mutant activity/wild-type activity. The assays
were carried out in the absence of AT-RA (SMRT association and
repression) or presence of 100 nM AT-RA (ACTR association
and activation). Note that V240A, I258A, and L261A bind ACTR weakly
(only 5%), but their activation activity is only partially impaired
(50-80%). In contrast, wild-type RAR
and mutant K262A bind ACTR
equally well, but K262A only moderately activates transcription. We
found that at 100 nM, K262A still associates with SMRT.
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Fig. 6.
Ribbon diagrams of RXR
and RAR
based on the crystal structure
of liganded RXR
/RAR
heterodimer (Protein Data Bank code 1DKF) (26).
A, the dimeric arrangement of RXR
/RXR
heterodimer is
viewed perpendicular to the dimer axis. Mutated residues of RAR
are
represented in red and gray (Gly248
and Phe249 depicted in gray are behind
Ile254 in this view) and denoted with residue number (van
der Waals' radii of the side chains are shown). RAR
helix H3 is
depicted in blue, helix H4 is in pink, and helix
H12 is in yellow. RAR
helices H7, H9, H10, and H11
forming in part the heterodimer interface are depicted in light
green. RXR
helices H7, H9, H10, and H11 as well involved in
heterodimer interface formation are depicted in banana
green. Wire frame models of bound ligands are depicted in
gray. Unmodeled in 1DKF region between H1 and H3 is not
shown. B, structural model of RAR
rotated ~90°
relative to A. Mutated residues are denoted as in
A; helix H3 is depicted in blue, helix H4 is in
pink, and helix H12 is in yellow. C,
structural model of RAR
. Orientation is the same as in B.
Only Gln257 and Phe249 are depicted by their
side chain van der Waals' radii and residue number. D,
structural model of RAR
viewed as in C.
Gln257 is mutated to Ala. E, structural model of
RAR
viewed as in C. Phe249 is mutated to Ala.
Molecular simulations were performed using GlaxoSmithKline R & D Deep
View v3.7 Swiss-PdbViewer.
Recent structural studies have demonstrated that helices H7, H9, H10,
and H11 of RAR are involved in heterodimerization with RXR
(Ref.
26 and Fig. 6A). Our results are consistent with their
findings because helices 3 and 4 mutants possessed at least 60% of the
heterodimerization activity in M2H assays of the wild-type RAR
. The
exceptions were F249A and Q257A, which are absolutely conserved within
class II nuclear receptor (Fig. 1A). We also note that both
F249A and Q257A fail to bind SMRT. It is possible that the global
structure of mutants F249A and Q257A is dramatically disturbed, so that
they lose most of the LBD-associated activities. Gln257 is
located at the middle of helix 4, and both of its amide groups can
donate hydrogen bonds, whereas the carbonyls of Leu252,
Thr250, and Phe249 can accept the hydrogen bond
(not shown). Phe249 is positioned in the middle of the loop
connecting helices H3 and H4. Thus, mutations of either
Gln257 to Ala or Phe249 to Ala will abolish
these hydrogen bonds and very likely destabilize helices 3 and 4 and
the whole structure of the molecule. Both F249A and Q257A mutations
create large cavities (Fig. 6, C-E), and as a consequence
the protein structure may tend to relax. This could result in
significant structural rearrangements, indirectly altering the
dimerization interface involving helices H7, H9, H10, and H11. In
addition, mutation of Phe249 to Ala would probably result
in rotation of the Gln257 side chain as modeled by
Swiss-PdbViewer (Fig. 6E) and also very likely destabilize
the loop connecting helices H3 and H4. One might speculate that
Phe249 and Gln257 are part of the folding
nucleus. Folding nuclei are known to include conserved amino acid
residues (29-31). Mutation of the residues critical for the formation
of the nucleation core could also result in folding defects. However,
the exact structural changes caused by Gln257 to Ala as
well as Phe249 to Ala mutations have yet to be determined.
Although M2H assays indicated that F249A did not interact with RXR
,
EMSAs showed that F249A was able to associate with RXR
, albeit with
much less affinity than that of wild type. This discrepancy could be
due to the fact that RAR
LBD was used in M2H assays, whereas
full-length RAR
was used for the EMSA assays. One possibility is
that the RAR
DNA-binding domain may contribute to heterodimerization
with RXR
on DNA.
We have previously shown that SMRT coreID association is highly
sensitive to mutations within the LBD of TR (16). All of the TR
mutants examined failed to bind SMRT ID. Our data show that most RAR
helices 3 and 4 mutants, with the exception of I254A and K262A, failed
to bind SMRT coreID II (Fig. 2C). These results imply the
existence of a common co-repressor-interacting surface between the LBD
of RAR
and TR
. Intriguingly, we found that some of these mutants,
such as S232A, K244A, and G248T, failed to bind coreID II and could
still associate with SMRT ID I + II. These data suggest that the
sequence flanking coreID II contacts these residues and stabilizes the
interaction between SMRT and RAR
. However, mutants including V240A,
F249A, Q257A, I258A, and L261A failed to bind both SMRT coreID II or
SMRT ID I + II. One common characteristic of these residues is that
they are highly conserved among nuclear receptors. These data suggested
that these conserved residues are absolutely essential for co-repressor interaction.
Among all of the mutants analyzed, K262A displays characteristics that
are distinct from others. First, K262A binds SMRT more poorly than
wild-type RAR both in EMSA and M2H assays. Consistently, Gal4-RAR
(K262A) repressed transcription to a lesser extent than the wild-type
RAR
. Second, K262A did not respond to ligand properly. Dissociation
of K262A with SMRT required a high concentration of AT-RA (1 µM). This aberrant ligand responsiveness could be derived
from a differential ligand binding affinity of K262A. However, previous
studies on RAR
and RXR
suggested that ligand binding,
dimerization, and DNA binding of this mutant were not affected (23,
27). Intriguingly, unliganded K262A binds ACTR better than that of
wild-type RAR
in EMSA assays. Similar to the wild-type RAR
,
addition of ligand up to 100 nM enhanced the association of
K262A with ACTR. However, at 1 µM of AT-RA, the association of K262A with ACTR was only slightly enhanced. These results suggest that Lys262 has a dual function, preventing
ligand-independent association of the co-activators and promoting
ligand-dependent dissociation of the co-repressors. The
distinct co-regulator association profiles of K262A are also reflected
in its ability to regulate transcription. In fact, K262A functions as a
dominant-negative mutant at low concentrations (<100 nM)
of AT-RA (Fig. 5F, lanes 2, 3,
14, and 16). At 1 µM AT-RA, K262A
is able to activate transcription, although the level of activation is
much lower that that of the wild-type RAR
. It is apparent that loss
of SMRT binding, as shown in M2H assays and EMSAs, correlates with loss
of repression activity by F249A (Fig. 2). A similar observation has
been reported for mutant F249R (28). Similar to our findings, this
mutant did not activate transcription at 50 nM AT-RA.
However, our results indicate that even though ACTR association of
F249A is not as strong as that of the wild-type RAR
at 1 µM AT-RA (Figs. 3, B and C, and
5D), F249A activated transcription equally well, if not
better (Fig. 5, E and F). This is likely due to
the fact that F249A does not associate with a co-repressor whose
dissociation is a prerequisite for co-activator association and that
the overall transcriptional activation activity depends on both the
dissociation of co-repressors and association of co-activators. Two
additional results suggest that the limiting step for transcriptional
activation by RAR
is the dissociation of the co-repressor. First,
although V240A, I258A, and L261A bind less efficiently to ACTR than the wild-type RAR
, the ligand-dependent transcriptional
activation by these mutants is only partially inhibited. We propose
that this observation is largely due to the loss of SMRT binding
activity. Second, unliganded K262A binds ACTR better than wild type in
EMSAs (Fig. 5C), but unliganded K262A was unable to activate
transcription in transient transfection assays (Fig. 5E). We
interpret this to mean that in M2H assays, K262A is strongly associated
with co-repressor complexes in the absence of ligand. Indeed, at 100 nM, K262A still remained associated with SMRT (Fig. 5,
A and B) and functioned as a dominant-negative
mutant (Fig. 5F). Although the co-regulator association
property is not identical to that of PML-RAR
, the transcription
activity of K262A is reminiscent of PML-RAR
at low concentrations of
AT-RA. In summary, we conclude that helices 3 and 4 of the RAR
LBD
play a critical role in RAR
functions and that co-repressor release
is a prerequisite for co-activator association and consequently
transcriptional activation.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Y.-X. Wang for the reagents; the sequencing facilities at the Salk Institute; Dr. Samols for critical comments on the manuscript; and Lita Ong and Elaine Stevens for administrative assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the start-up fund at Case Western Reserve University (to H.-Y. K.) and National Institutes of Health Grant HD27183 (to R. M. E.).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.
§ Recipient of the James T. Pardee-Carl A. Gerstacker Assistant Professor of Cancer Research Faculty Chair in Cancer Research at Case Western Reserve University Cancer Center. To whom correspondence may be addressed: Case Western Reserve University, Dept. of Biochemistry, 10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-844-7572; Fax: 216-368-3419; E-mail: hxk43@po.cwru.edu.
Investigator of the Howard Hughes Medical Institute at the
Salk Institute for Biological Studies and March of Dimes Chair in
Molecular and Developmental Biology. To whom correspondence may be
addressed: Howard Hughes Medical Institute, Gene Expression Laboratory,
The Salk Institute for Biological Studies, 10010 Torrey Pines Rd., La
Jolla, CA 92037. Tel.: 858-453-4100 (ext. 1585); Fax: 858-455-1349;
E-mail: evans@salk.edu.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M207569200
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ABBREVIATIONS |
---|
The abbreviations used are: TR, thyroid hormone receptor; SMRT, silencing mediator for retinoid and thyroid hormone receptor; ACTR, activator for thyroid hormone and retinoid receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; EMSA, electrophoresis mobility shift assays; M2H, mammalian two-hybrid; LBD, ligand-binding domain(s); coreID, co-repressor receptor interaction domain; AT-RA, all-trans-retinoic acid; GST, glutathione S-transferase; ID, interaction domain(s); DRn, direct repeat spaced by n base pairs; TK, thymidine kinase.
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