From INSERM U459, Faculté de Médecine Henri Warembourg, 1, place de Verdun, 59045 Lille Cedex, France
Received for publication, September 1, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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Heterodimerization of retinoic acid receptors
(RARs) with 9-cis-retinoic receptors (RXRs) is a
prerequisite for binding of RXR·RAR dimers to DNA and for retinoic
acid-induced gene regulation. Whether retinoids control RXR/RAR
solution interaction remains a debated question, and we have used
in vitro and in vivo protein interaction assays
to investigate the role of ligand in modulating RXR/RAR interaction in
the absence of DNA. Two-hybrid assay in mammalian cells demonstrated
that only RAR agonists were able to increase significantly RAR
interaction with RXR, whereas RAR antagonists inhibited RXR binding to
RAR. Quantitative glutathione S-transferase pull-down
assays established that there was a strict correlation between agonist
binding affinity for the RAR monomer and the affinity of RXR for
liganded RAR, but RAR antagonists were inactive in inducing RXR
recruitment to RAR in vitro. Alteration of coactivator- or
corepressor-binding interfaces of RXR or RAR did not alter
ligand-enhanced dimerization. In contrast, preventing the formation of
a stable holoreceptor structure upon agonist binding strongly altered
RXR·RAR dimerization. Finally, we observed that RAR interaction with
RXR silenced RXR ligand-dependent activation function. We
propose that ligand-controlled dimerization of RAR with RXR is an
important step in the RXR·RAR activation process. This interaction is
dependent upon adequate remodeling of the AF-2 structure and amenable
to pharmacological inhibition by structurally modified retinoids.
The ligand-dependent transcriptional activation of
nuclear receptors is a DNA-dependent multistep process in
which the ligand/receptor interaction is the key initiating event.
Ligand binding to nuclear receptors leads to dramatic structural
transitions of the polypeptidic chain that alter several functions of
the ligand-bound receptor. These functions are specified by structural
domains, the organization of which is shared in this gene superfamily.
Another general property of nuclear receptors is their capacity to
engage protein/protein interactions, which serves essentially two
purposes: (i) increasing the specificity and affinity for hormone
response elements through homodimerization or heterodimerization with
other nuclear receptors and (ii) modulating nuclear receptor
transcriptional activity through ligand-modulated recruitment of
nuclear coactivators and corepressors.
Although the role of ligand in inducing conformational alterations of
the receptor that either disrupt (nuclear corepressor) or stabilize
(nuclear coactivator) protein/protein binding interfaces is abundantly
documented (reviewed in Refs. 1 and 2), the biological relevance of
ligand-induced nuclear receptor homodimerization and heterodimerization
in the absence of DNA is more controversial. Retinoic acid receptors
(RARs)1 and
9-cis-retinoic acid receptors (RXRs) have two dimerization interfaces, which are located in the DNA-binding domain and the ligand-binding domain (LBD) (3). These two domains are functionally autonomous, and isolated RAR and RXR DNA-binding domains bind as dimers
specifically and cooperatively to retinoic acid response elements
(RAREs) similarly to wild-type (WT) receptors (4). We have shown that
the RAR LBD contains a strong dimerization interface in helix 9 enabling RXR/RAR interaction in the absence of DNA (3). These
observations and others also demonstrate that receptor dimerization and
DNA binding are, at least in vitro, ligand-independent
events. This consequently questions the role of ligand as a regulator
of receptor dimerization, despite the presence of a strong dimerization
interface in the LBD, which is likely to be altered, directly or
indirectly, upon ligand binding.
Ligand-dependent dimerization of some nuclear receptors
has, however, been reported for various systems. 1,25-Dihydroxyvitamin D3 favors the dissociation of vitamin D3
receptor homodimers into monomers, rendering vitamin D3
receptor molecules available for dimerization with RXR (5). Thyroid
hormone has been shown to promote thyroid hormone receptor dimerization
with RXR prior to DNA binding both in vivo and in
vitro (6), and 9-cis-retinoic acid was shown to induce
RXR homodimerization, providing a molecular basis for the observed
inhibition of vitamin D3 receptor- and thyroid hormone
receptor-enhanced transcription by 9-cis-retinoic acid
(5-7). We have also shown that RAR agonists promote the formation of
hRAR Since the role of ligand in receptor dimerization has not yet been
studied for the RXR/RAR system, we decided to investigate the role of
retinoids in the regulation of RXR·RAR dimerization in
vivo and in vitro using a panel of natural and
synthetic retinoids with distinct biological properties. Results
obtained using quantitative GST pull-down experiments and a mammalian
two-hybrid system demonstrated that RAR agonists stabilized
hRXR Materials--
atRA was obtained from Sigma (Saint-Quentin
Fallavier, France). Synthetic retinoids were a kind gift from Dr. U. Reichert. Ro 41-5253 was a gift from Hoffman-La Roche. Ligand
structures and properties have been described in detail (13). DNA
restriction and modification enzymes were from Promega
(Charbonnières, France). Polyethyleneimine (ExGen 500) was from
EuroMedex (Souffelweyersheim, France).
Plasmids--
pGEX2TKhRXR Cell Culture and Transfections--
HeLa Tet-On cells were
cultured and transfected as previously described (17).
Protein/Protein Interaction Assays--
DNA-independent
protein/protein interactions were performed as described (18).
Restriction Site (SmaI) Accessibility Assay--
Cells were
grown and treated as indicated and collected in ice-cold 1×
phosphate-buffered saline. The cell pellet was resuspended in 5 volumes
of buffer A (20 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 50 mM KCl, 1% Triton X-100, 0.15 mM
spermine, 0.5 mM spermidine, 5 mM sodium
butyrate, and protease mixture inhibitor (1:100 dilution; Sigma)).
Cells were lysed by several strokes in a Dounce with an A-type pestle,
and the homogenate was layered onto a 0.8 M sucrose cushion
in buffer A. Nuclei (~3-5 A260) were digested with 80-100 units of SmaI (Promega) in a final volume of
200 µl in SmaI digestion buffer. The reaction was stopped
by adding an equal volume of stop buffer (20 mM Tris-HCl
(pH 7.5), 1% SDS, 25 mM EDTA, and 5 mg/ml proteinase K
(Promega)) and incubated for 8 h at 37 °C. DNA was purified,
and 50-80 µg was digested with 25 units of PstI. Cleaved
DNAs were analyzed on native 1% agarose gels and stained with ethidium
bromide. Gels were transferred to nylon membranes (Hybond
N+, Amersham Pharmacia Biotech), and DNA was
UV-cross-linked to the membrane. Hybridization was carried out with a
600-bp RAR Reverse Transcription-PCR Analysis of mRAR Statistical Analysis--
All incubations or assays were
performed at least in triplicate. Measured values were used to
calculate mean ± S.D. Calculations were carried out using Prism
software (GraphPAD Inc., San Diego, CA)
hRAR
The ligand-dependent activation paralleled the pattern of
induction observed when using full-length hRAR
These observations thus revealed several features of the two-hybrid
system. First, they suggest that RXR/RAR interaction occurs in the
absence of ligand. In line with this observation, coexpression of RAR
and RXR inactivated RXR responsiveness to ligand, suggesting that RAR
interaction inhibits RXR function. Second, only RAR Tethering of Coactivators and Corepressors to RAR and RXR Does Not
Alter the Ligand-induced Response of the Two-hybrid
System--
Unliganded, heterodimeric hRAR
Another possibility is that the observed luciferase activity results
from the synergistic contribution of the hRAR Antagonists Do Not Inactivate the VP16 Activation Domain--
The
observed inactivity of the two-hybrid system in the presence of
antagonists may stem from the inhibition of the VP16 moiety, as
suggested for progesterone antagonists (10). To test this hypothesis,
we monitored the activity of WT hRAR A RAR Antagonist Restores the RXR Response to a RXR-specific
Ligand--
The inhibition of RXR activity upon expression of WT
hRAR Agonist-mediated Stabilization of hRXR
Retinoids able to bind hRAR Ligand-induced AF-2 AD Folding of hRAR
The RAR
The RXR AF-2 AD motif has been proposed to regulate negatively
nuclear coactivator access to RAR by direct interaction with the LBD
hydrophobic groove (25), suggesting that this domain may be involved in
the stabilization of the RXR·RAR complex. We thus tested whether
truncation of the hRXR Affinity of Liganded hRAR
The results shown above (Fig. 3B) documented the ability of
a retinoid antagonist to inhibit, in a dose-dependent
manner, the interaction of RAR with RXR in intact cells. We therefore carried out an analogous competition experiment using the in
vitro GST pull-down assay (Fig. 5B). The
RAR
Since we were able to observe a dose-dependent increase in
the RXR/RAR interaction in this system, we assayed the affinity of
hRAR atRA, but Not a Retinoid Antagonist, Induces Chromatin Structure
Alterations in the Endogenous RAR
P19 cells were thus treated with atRA or CD3106 (a retinoid
antagonist), and SmaI accessibility to the endogenous
mRAR In this study, we show that RAR agonists promote RXR recruitment
to liganded RAR in solution. A comparative study of amino acid
substitutions in hRAR The agonist-enhanced activity of the two-hybrid system could
potentially be interpreted as a contribution of the AF-2 inherent in
either RXR or RAR, which may synergize with the VP16 AD. Manipulating the level of interaction of either hRAR Very intriguingly, the RXR-specific ligand CD2425 induced a clear RXR
response when this receptor was expressed alone. This ligand-induced
activity was inhibited in the presence of hRAR The effect of ligand on RXR/RAR interaction was similar in both
in vitro and in vivo assays, in which a
full-length hRXR The three-dimensional structure of the heterodimer RXR LBD·RAR LBD
has been published recently (39). As for other nuclear receptors (40),
ligand binding does not promote detectable variation of the structure
of the dimerization interface, which is essentially composed of helices
7-9. However, biochemical studies showed that ligand is a strong
promoter of estrogen (41), progesterone (42), RAR (8), and RXR (7)
homodimerization, and our data, as well as others (5, 6, 43), support
the concept that ligand induces receptor heterodimerization with RXR.
Thus, ligand exerts structural constraint(s) on region(s) of hRAR As the most decisive message, we would like to propose that ligand
plays a critical role in an early step of receptor activation, i.e. receptor dimerization, and that the ability to
interfere with this process is a key feature of retinoid antagonists.
Finally, since several studies documented similar properties for
estrogen antagonists (9, 11) and since all retinoid antagonists tested so far blocked atRA-induced dimerization,2 we would like to
suggest that inhibition of dimerization may be a important feature of
pure nuclear receptor antagonists.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
LBD homodimers (8). Moreover, the importance of ligand
structure in regulating receptor dimerization is poorly understood, and
conclusions drawn from the study of synthetic ligands for sex steroid
receptors such as antiestrogens (9) and antiprogestins (10), which are
clinically important molecules, suggested that transcriptional
inhibition stems either from a repressing activity of antagonist-bound
monomer or from impaired dimerization (11, 12).
·hRAR
heterodimers as a function of their affinity for
monomeric hRAR
, whereas antagonists were unable to promote
heterodimer formation. This lack of activity may result, at least in
part, from an impaired AF-2 (activation function-2) folding since a mutation designed
to prevent the stabilization of the holo-hRAR
structure similarly
decreased the affinity of hRAR
for hRXR
. hRXR
AF-2 did not
display any significant contribution to dimerization, suggesting that
the hRAR
monomer has a predominant role in regulating the
dimerization rate with RXR.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and pGEX2ThRAR
vectors were
obtained by cloning the RXR
and RAR
cDNAs into pGEX2TK and
pGEX2T, respectively. hRXR
was truncated from the first 44 N-terminal amino acids. Constructs containing either the wild-type
hRAR
or hRXR
cDNA subcloned into pSG5 (Stratagene) have been
described (8, 14). The UAS-tk-Luc reporter gene was a gift from
V. K. Chatterjee and contains two 17-mer UAS Gal4 response
elements (6). pCMV-Gal4-hRXR
LBD and pCMV-VP16-hRAR
were obtained
from T. Perlmann (15) (see Fig. 1). The (GRARE)3-tk-Luc
reporter gene is a pGL3-based vector (Promega) containing three repeats
of a composite response element made up of a glucocorticoid response
element half-site separated from a RARE half-site by a 5-bp spacer
(AGAACAccgaaAGGTCA). This response element was cloned upstream of the
thymidine kinase promoter (
105 to +50). This reporter gene was
activable only in the presence of overexpressed RARs and of a mutant
hRXR
, RXGR: mutations introduced into hRXR
converted the P
box sequence scEGckGff to ccGSckVff, hence conferring to hRXR
the
ability to bind specifically glucocorticoid response
elements.2 The VP16-hRAR
AHT mutant was constructed by site-directed mutagenesis (QuickChange, Stratagene) using the following oligonucleotides: 5'-ggtgcgcaaagggggccaggaaacctttcctgccctctgcc-3' and
5'-ggcagagggcaggaaaggtttcctggccccctttgcgcacc-3'. This mutagenesis
procedure led to the introduction of two mutations, converting
Ala194 into Gly and His195 into Gly. The
hRAR
L409A mutant was constructed according to the same protocol
using the following mutagenic primers:
5'-catgccgcctgccatccaggaaatgctcgagaactcagagg-3' and
5'-cctctgagttctcgagcatttcctggatggcaggcggcatg-3'. Other plasmids were constructed using standard subcloning procedures (16). All
constructs were verified by automatic sequencing.
2 32P-labeled probe (Rediprime II, Amersham
Pharmacia Biotech), which was amplified from mouse genomic DNA using
the following set of primers: 5'-ttggccaggaacaggagagc-3' and
5'-tttaccattttccaggctcg-3' (nucleotides 418-437 and 1031 to 1012, respectively, in the mouse retinoic acid receptor-
gene promoter
region; GenBankTM/EBI accession number X56850.1).
After hybridization, blots were washed, and radioactive material was
detected using a Storm 860 Phosphoimager.
2 and Actin
Transcripts in P19 Cells--
Total RNA was prepared using RNAble
reagent (Eurobio, Les Ulis, France) according to the manufacturer's
protocol. Total RNA (50 µg) was then treated with 10 units of
RNase-free DNase I (Genhunter, Nashville, TN) for 1 h at 37 °C
to digest genomic DNA. The purified RNA was adjusted to 1 µg/µl and
checked for integrity by agarose gel electrophoresis. Reverse
transcription was performed using random primers (Promega) as
recommended by the manufacturer. PCR primers were designed as follows:
RAR
, 5'-aagtggtaggaagtgagctg-3' and 5'-ctacattgagcagtatgccg-3; and
actin, 5'-atcatgtttgagaccttcaa-3' and 5'-catctcttgctcgaagtcca-3'. PCR
conditions were 40 cycles of 30 s at 94 °C, 1 min at 58 °C,
and 1.5 min at 72 °C followed by an elongation step at 72 °C for
7 min (RAR
) and 30 cycles of 30 s at 94 °C, 1 min at
56 °C, and 1.5 min at 72 °C followed by an elongation step at
72 °C for 7 min (actin).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Interacts with hRXR
in Vivo in a
Ligand-dependent Manner--
Interaction of hRAR
with
hRXR
was first characterized using a mammalian two-hybrid
interaction assay. In this system, the acidic VP16 activation domain
(AD) is fused to the N terminus of full-length hRAR
, whereas the
Gal4 DNA-binding domain is linked to the N-terminal end of the hRXR
LBD (Fig. 1A). The activity of
a reporter gene containing two Gal4 consensus DNA-binding sites (UAS-tk-Luc) was the biological readout used to monitor the interaction between these two receptors in HeLa cells. Transfection of VP16-hRAR
did not promote a significant increase in reporter gene
activity,2 but Gal4-hRXR
LBD alone turned out to be
responsive to the RXR-specific ligand CD2425, which induced a 4-5-fold
increased activity (Fig. 1B). When WT hRAR
was
overexpressed together with Gal4-hRXR
LBD, a very modest increase
(1.5-fold) in the level of luciferase activity was detected in cell
extracts in response to retinoid treatment, suggesting that the hRAR
AF-2 region is unable to activate this system. Since Gal4-hRXR
LBD,
WT hRAR
, and VP16-hRAR
were expressed at similar levels in HeLa
cells as judged by whole cell ligand binding assays (from 30,000 to
100,000 sites/cell),2 this clearly suggests that WT hRAR
is unable per se to mediate a significant
ligand-dependent activation of the system. Coexpression of
Gal4-hRXR
LBD and VP16-hRAR
led to a detectable
ligand-independent activation of the reporter gene (3-5-fold), and
addition of RAR-specific ligands strongly increased reporter gene
activity (10-15-fold). However, the ligand-mediated activation of the
two-hybrid system appeared to be restricted to RAR
-binding agonists
such as atRA and Am580. As expected, RAR
-selective (CD417) and
RAR
-selective ligands (Fig.
2)2 had no effect in our
system, in agreement with their very poor affinity for hRAR
. The RAR
antagonist Ro 41-5253 did not activate this system, and Ro 41-5253 competitively inhibited atRA-induced activity.2 More
surprisingly, the RXR-specific ligand, which was able to induce a
4-5-fold increase in reporter gene activity when RXR was expressed
alone, was inactive when WT hRAR
or VP16-hRAR
was coexpressed
with Gal4-hRXR
.
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Fig. 1.
hRAR interacts with
hRXR
in HeLa cells. A,
structures of the fusion proteins and reporter genes used in this
study. Major features of each expression vector and reporter genes are
indicated. B, heterodimer formation of hRAR
with hRXR
.
HeLa cells were transfected with the indicated combination of reporter
genes and expression vectors. 24 h post-transfection, cells were
stimulated by RAR ligand (100 nM atRA or 100 nM
Am580), RAR
-specific ligand (70 nM CD417), a RAR
antagonist (1 µM Ro 41-5253), a RXR-specific retinoid (1 µM CD2425), or a combination of agonist and antagonist
(100 nM Am580 + 1 µM Ro 41-5253 or 70 nM CD417 + 1 µM Ro 41-5253). Luciferase
activity was assayed 16 h later as described under "Experimental
Procedures." Results are expressed as the means ± S.D. of at
least three individual experiments, with the basal level of luciferase
activity (usually between 3000 and 10,000 RLU) scaled to 1.
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Fig. 2.
Coactivator and corepressor recruitment to
hRAR does not influence VP16 AD activity.
A, effect of mutations in the hRAR
LBD on the activity of
hRAR
in the two-hybrid system and on its ability to interact with
transcriptional intermediary factors. Left panel, HeLa cells
were transiently transfected with the UAS-tk-Luc reporter gene and the
CMV-Gal4-hRXR
LBD and CMV-VP16-hRAR
(WT or mutants)
expression vectors as indicated. Cells were treated with 0.1 µM atRA, and luciferase activity was assayed as described
in the legend to Fig. 1. Results are expressed as the means ± S.D. of at least three individual experiments, with the basal level of
luciferase activity (usually between 3000 and 10,000 RLU) scaled to 1. Right panel, recruitment of SRC-1 and SMRT by WT and mutant
hRAR
. Bacterially expressed GST fusion proteins (SMRT or SRC-1) were
used to generate affinity matrices with which 35S-labeled
WT or mutant hRAR
was incubated in the absence or presence of 1 µM atRA. SMRT- or SRC-1-bound hRAR
was then resolved
by SDS-polyacrylamide gel electrophoresis and quantified by
autoradiography. B, VP16 transactivating potential in the
presence of retinoid antagonists. HeLa cells were transfected with the
(GRARE)3-tk-Luc reporter gene, the RXGR expression vector,
and the WT hRAR
(left) and VP16-hRAR
(right) expression vectors. 24 h after transfection,
cells were treated with vehicle (dimethyl sulfoxide (DMSO)),
0.1 µM atRA, 1 µM CD3105, 1 µM CD3106, or 0.1 µM atRA + 1 µM CD3105 or CD3106, and luciferase activity was assayed
as described in the legend to Fig. 1. Results are expressed as the
means ± S.D. of at least three individual experiments. Note that
values are expressed here as RLU. C, coexpression of RAR
inhibits RXR activity. HeLa cells were transfected with the UAS-tk-Luc
reporter gene, CMV-Gal4-hRXR
LBD, and CMV-VP16-hRAR
and
challenged with ligands as described above. Results are expressed as
the means ± S.D. of at least three individual experiments, with
the basal level of luciferase activity (usually between 3000 and 10,000 RLU) scaled to 1.
, hRXR
, and a DR5 RARE-driven reporter gene ((GRARE)3-tk-Luc) (Fig.
1B). In the same assay, VP16-hRAR
responded to ligands
its wild-type counterpart, but promoted a much higher
transcriptional activity of the (GRARE)3-tk-Luc reporter,
as expected from the presence of the strong VP16 AD acidic activator.
-specific agonists are able to increase significantly the activity of this system. Third, RAR-dependent activation is enhanced by the
VP16 AD, consistent with the hypothesis of ligand-dependent
dimerization. However, our data do not rule out at this stage that RAR
agonist requirement merely reflects a synergism of RAR AF-2 with the
VP16 AD and/or a repression exerted by unliganded hRAR
on VP16
activation function.
interacts with the
corepressor SMRT, which dissociates upon agonist binding, and this
interaction is stabilized in the presence of antagonists (19).
To test whether the agonist-induced activity of the two-hybrid system
is due to corepressor release, we assayed the activity of a hRAR
mutant containing a double mutation (A194G/H195G, hRAR
AHT) in the
nuclear corepressor box (20), which considerably lessened hRAR
interaction with SMRT (10-12-fold) (Fig. 2A) without
altering the ligand binding activity of the
receptor.3 Interaction with
SRC-1 was also diminished, yet clearly detectable. Such a mutation
would thus predictably lead to an enhanced basal level of reporter gene
activity and/or to a decreased ligand-induced activity. While showing a
low constitutive activity, as already reported for an analogous
mutation in the thyroid hormone receptor (6), hRAR
AHT displayed a
ligand-induced activity similar to that of WT hRAR
, arguing against
a possible role for corepressors and coactivators in VP16 activity
control. In keeping with this, we observed that truncation of the RXR
AF-2 AD region dominant negative (RXR), which strongly increases
corepressor binding to RXR (21),2 did not affect the
response of the system to atRA treatment (see Fig. 4).
AF-2 AD and VP16 AD to
the transcriptional activation of the reporter gene. Leucine 409 of
hRAR
is located in helix 12, and its mutation leads to
transcriptional inactivation of hRAR
while maintaining WT ligand
binding activity (Fig. 3C)
(22).2 This amino acid becomes part of the hydrophobic
groove formed upon agonist-induced AF-2 AD folding to which
coactivators bind through their LXXLL motif. The L409A
substitution abolished ligand-induced SRC-1 recruitment to hRAR
,
whereas receptor binding to SMRT was left unchanged (Fig.
2A). RAR
L409A induced both basal and ligand-induced activities comparable to WT hRAR
in the two-hybrid system (Fig. 2A), suggesting again that atRA-induced activity is indeed
due to receptor heterodimerization and not to the concerted activation of the hRAR
AF-2 AD and VP16 AD.
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Fig. 3.
Ligand biological properties correlate with
ability to induce RXR·RAR dimerization in vivo.
A, ligand-induced activation of the two-hybrid system
in the presence of WT RAR . HeLa cells were transiently transfected
with the UAS-tk-Luc reporter gene and the CMV-Gal4-hRXR
LBD and the
CMV-VP16-hRAR
expression vectors. Cells were treated with retinoids,
and luciferase activity was assayed as described in the legend to Fig.
1. Results are expressed as the means ± S.D. of at least three
individual experiments, with the basal level of luciferase activity
(usually between 3000 and 10,000 RLU) scaled to 1. Biological
activities of retinoids are indicated at the top of the bar
graph. B, ligand-induced activation of the two-hybrid
system in the presence of RAR
L409A. Experiments and analysis
results are presented exactly as described for A using
VP19-RAR
L409A in place of VP16-RAR
. C, limited
proteolytic clipping assays. Assays were carried out exactly as
described (44). 35S-Labeled RAR
(upper panel)
or RAR
L409A (lower panel) was incubated with 1 µM atRA, Am580, or CD3106 for 2 h at 4 °C and
digested with 1, 2.5, 5.0, or 10.0 µg/ml trypsin. Cleavage products
were then resolved by standard 10% SDS-polyacrylamide gel
electrophoresis and autoradiographed. DMSO, dimethyl
sulfoxide; TTNN,
6-(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphtalenyl)-2-naphatale
n-carboxylic acid.
and VP16-hRAR
in a standard
transactivation assay using a DR5-driven reporter gene (Fig.
2B). As expected, atRA promoted a very strong increase (8-15-fold induction) in reporter gene activity in the presence of
overexpressed hRAR
or VP16-hRAR
, whereas a very modest increase (2-fold) was observed when cells were challenged by two antagonists, CD3105 and CD3106 (23). Competitive inhibition of atRA-induced transcription was observed when each antagonist was used together with
atRA. The basal level of transcription was higher (8-10-fold increase)
when VP16-hRAR
was expressed in HeLa cells together with hRXR
, as
expected from the strong transactivating potential of the VP16 AD. The
basal level of transcription was again slightly increased (2-fold) in
the presence of both antagonists and was not decreased, as it could be
predicted if VP16 function was conditioned by RAR activity,
demonstrating that VP16 AD transactivating potential is not altered
upon antagonist binding to hRAR
.
or VP16-hRAR
suggested an allosteric control of RXR function
by RAR and implied that a fraction of RAR and RXR populations is involved in ligand-independent dimerization. We thus tested whether antagonist binding to hRAR
affected RXR response to a specific ligand (Fig. 2C). When Gal4-hRXR
was expressed alone, the
system was responsive to RXR ligand, and this responsiveness was not affected by RAR ligands. Overexpression of hRAR
conferred
responsiveness to a RAR agonist (Am580), and the RXR-induced activity
was repressed, as shown above. Cotreatment of cells with the
RAR-specific agonist Am580 and the RXR-specific agonist CD2425 did not
reveal any synergy between both receptors. Very interestingly, addition
of the RAR antagonist CD3106 together with the RXR-specific agonist
restored a RXR-induced response, similar to that observed in the
presence of Gal4-hRXR
alone, suggesting that RXR is able, under
these conditions, to function autonomously. These results thus support our view that RAR antagonists prevent RAR interaction with RXR in
vivo.
·hRAR
Dimers in
Vivo--
The results shown in Fig. 1 suggested several important
conclusions. (i) Ligand binding is required to stabilize RXR·RAR
dimers in vivo; (ii) antagonists, despite having a high
affinity for RAR, do not induce detectable interactions between these
two receptors; and (iii) dimerization is dependent solely on ligand
occupancy of the hRAR
ligand-binding pocket. We extended our
observation to a panel of synthetic retinoids with distinct biological
properties: isotype-selective RAR agonists, a partial agonist,
antagonists, and dissociated (anti-AP-1) retinoids (Fig.
3A). Comparing the maximal induction of the GAL4
reporter gene in the presence of these retinoids allowed us to
distinguish two classes of molecules: active and inactive compounds. A
closer examination showed that active compounds were referenced, based
on transactivation assays, as RAR
agonists (atRA, CD367, Am580, and
Am80). Inactive compounds were either RAR
-selective (CD417) or
RAR
-selective (CD666) retinoids or RAR antagonists (CD3106, CD2331,
CD2905, and CD2856). The recently described anti-AP-1 retinoid CD2409
(24) was unable to promote RXR·RAR dimerization, and the partial
agonist CD2665 had an intermediate ability to induce RXR·RAR
heterodimerization in vivo. We then monitored the activity
of the VP16-RAR
L409A mutant in response to several retinoids in the
same system (Fig. 3B). This transcriptionally defective
RAR
mutant displayed a similar response profile to agonists and
antagonists compared with WT RAR
, establishing that RAR
AF-2 does
not contribute to the activity of the system. Agonist-induced luciferase activity was fully inhibited, in a
dose-dependent manner, by CD3106, suggesting that the
observed inhibition results from direct competition for the
ligand-binding pocket of RAR
. Moreover, limited proteolytic assays
carried out in parallel using WT RAR
and RAR
L409A (Fig.
3C) showed that both receptors underwent similar structural
transitions upon ligand binding, establishing that the L409A mutation
did not significantly alter RAR
three-dimensional structure. Thus,
our data and others show that the L409A mutation is compatible with
antagonist binding and ligand-induced dimerization.
with high affinity can be classified
according to their ability to induce hRXR
·hRAR
dimerization: agonists and others, this latter category including, most notably, anti-retinoids. The two-hybrid system thus discriminates between agonists and potential antagonists on the basis of ligand ability to
induce dimerization in vivo.
Is Necessary for
RXR·RAR Dimerization--
Major structural transitions occur in the
LBD of nuclear receptors upon ligand binding and lead to the
repositioning of several
-helices. The AF-2 AD region (helix 12),
which is fully exposed to solvent in the unliganded state
(aporeceptor), establishes salt bridges with basic amino acids located
in helices 3 and 4. This repositioning along the LBD creates a
hydrophobic groove to which nuclear coactivators bind via
LXXLL motifs (holoreceptor). RAR AF-2 AD repositioning may
thus have a role in remodeling dimerization interfaces of RAR, and we
tested this hypothesis using both in vitro and in
vivo protein/protein interaction assays (Fig.
4).
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Fig. 4.
Preventing holo-RAR
AF-2 folding upon ligand binding alters agonist-induced
dimerization in intact cells. A, hRAR
K244A/K262A is
transcriptionally inactive and interacts weakly with hRXR
.
Left panel, transactivation assay. HeLa cells were
transfected with the (GRARE)3-tk-Luc reporter gene, the
RXGR expression vector, and the WT hRAR
or hRAR
K244A/K262A
(K244-262) expression vector. 24 h after transfection,
cells were treated with vehicle (dimethyl sulfoxide (DMSO))
or 1 µM atRA. Luciferase activity was assayed 16 h
later and is expressed as the mean ± S.D. of at least three
independent experiments. Middle panel, in vitro
protein interaction assay. The K244A/K262A mutant was synthesized and
radiolabeled by in vitro coupled transcription/translation
and then incubated with increasing concentrations of atRA. The receptor
was incubated with a GSH-GST-hRXR
-Sepharose matrix, and the amount
of RAR bound to the RXR affinity matrix was quantified by 8%
SDS-polyacrylamide gel electrophoresis followed by autoradiography.
Right panel, in vivo protein interaction assay.
HeLa cells were transfected with the UAS-tk-Luc reporter gene and the
CMV-Gal4-hRXR
LBD and CMV-VP16-hRAR
or hRAR
K244A/K262A
expression vectors. 24 h after transfection, cells were treated
with 1 µM for 16 h, and luciferase activity was
assayed. Results are expressed as the means ± S.D. of at least
three independent experiments. B, a C-terminally truncated
hRXR
is transcriptionally inactive, but heterodimerizes with hRAR
as does WT hRXR
. The transcriptional activity and the ability of the
truncated RXR (dnRXR) to interact both in vitro and in
vivo with hRAR
were assayed as described for
A.
holo-LBD three-dimensional structure designates
Lys244 and Lys262 as establishing salt bridges
with acidic residues located in the AF-2 AD region (Glu412
and Glu415), and mutation of each lysine yields receptor
mutants with impaired transactivating properties (17). Mutating both of
these lysines yielded an inactive receptor (hRAR
K244A/K262A) in the
transactivation assay (Fig. 4A). This RAR mutant was unable
to interact with nuclear coactivators and corepressors in
vitro.2 GST pull-down assays revealed that hRAR
K244A/K262A was still able to interact with hRXR
in a
ligand-dependent manner, with an estimated
Kd of 40-60 nM when complexed to atRA.
This double mutation induced a 4-5-fold decrease in the affinity of hRAR
for RXR, which was also reflected in the mammalian two-hybrid system by only a 2-fold increase in the activity of the Gal4/VP16 system, in sharp contrast to WT hRAR
, which promoted a 5-7-fold increase (Fig. 4A). Thus, a 4-fold decreased activity in the
two-hybrid system is correlated with a 4-fold decrease in the hRAR
affinity for hRXR
in vitro.
AF-2 region perturbed RXR/RAR interaction.
Although dnRXR was a strong inhibitor of RAR-mediated transactivation
(Fig. 4B), it displayed normal affinity for WT hRAR
in
the in vitro interaction assay (Kd
20 nM). The two-hybrid assay showed that hRAR
interacted
with a similar efficiency with WT hRXR
and dnRXR (Fig.
4A), leading to the conclusion that the RXR AF-2 AD has no
major influence on RXR/RAR interaction.
for hRXR
Correlates with
Agonist Binding Affinity for Monomeric hRAR
in Vitro--
To
determine which parameter(s) are critical for triggering
hRXR
/hRAR
interaction, we carried out an in vitro
interaction assay in which GST-tagged full-length hRXR
was used as a
bait to trap in vitro translated, 35S-labeled
hRAR
(Fig. 5). Labeled hRAR
was
incubated for 1 h at 4 °C with increasing concentrations of the
indicated ligand, and then the GST-hRXR
affinity matrix was added to
the binding mixture. In the absence of ligand, a weak constitutive
interaction of RAR with RXR was observed (Fig. 5A).
Increasing the atRA concentration from 10
10
to 10
6 M caused a
dose-dependent increase in the RXR/RAR interaction (up to
5-7-fold). A similar pattern was observed with CD367, a synthetic RAR
agonist. In contrast, the antagonist CD3106, which was inactive in the
two-hybrid assay, was unable to stabilize RXR/RAR interaction. We noted
that at concentrations exceeding 50 nM, this ligand even
promoted a clear decrease in the constitutive association of the
heterodimer. Finally, a RAR
-selective ligand 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphtalenyl)-2-naphtale n-carboxylic acid, did not exhibit a significant effect on
RXR/RAR interaction.
View larger version (32K):
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Fig. 5.
In vitro protein interaction
assays establish a correlation between ligand binding affinity for
hRAR and ligand-induced dimerization.
A, DNA-independent heterodimerization in vitro.
hRAR
was translated in vitro as a 35S-labeled
protein by coupled transcription/translation in rabbit reticulocyte and
incubated with increasing concentrations of retinoids. After
incubation, ligand·hRAR
complexes were adsorbed onto a
GSH-GST-hRXR
-Sepharose affinity matrix and resolved by 8%
SDS-polyacrylamide gel electrophoresis. Representative autoradiographs
are shown for the indicated ligands, and quantification of RXR-bound
hRAR
was performed using ImageQuant software (Molecular Dynamics,
Inc.). Data were used to generate saturation curves and to calculate
binding affinity constants (Kd), which are shown
in C. B, a RAR antagonist prevents
agonist-induced heterodimerization. GST pull-down experiments were
carried out as described for A. The Ro 41-5253/Am580
competition experiment was performed in the presence of 25 nM Am580 and increasing concentrations of Ro 41-5253, ranging from 0.1 nM to 0.1 µM
(10
10, 5 × 10
10, 10
9, 5 × 10
9, 10
8,
10
7, and 10
6
M). C, comparison of hRAR
affinity
for hRXR
in the presence of various retinoids. Ki
values were taken from Ref. 13 (a), provided by Galderma
(b), this report and Ref. 19 (c), Ref. 41 (d),
and Ref. 42 (e). Affinity constants of hRAR
for hRXR
were
calculated as described for A and are presented as the
means ± S.D. of at least four individual experiments.
NA, not assayed; NB, Kd > 5 µM.
-selective ligand Am580 was, as expected from its biological
classification, able to promote RXR·RAR dimerization, whereas Ro
41-5253, a RAR antagonist, was inactive in this assay. When RAR
was
incubated simultaneously with 25 nM Am580 and increasing
concentrations of Ro 41-5253 (from 0.1 nM to 0.1 µM), a dose-dependent decrease in the rate of
heterodimer formation was observed, establishing a correlation between
this in vitro assay and the two-hybrid assay.
for hRXR
in the presence of the retinoids used previously. Values are tabulated in Fig. 5C along with relative
affinities of hRAR
for each ligand as determined by in
vitro ligand binding assay using hRAR
overexpressed in
Escherichia coli (13). Quite strikingly, a classification
similar to that established on the basis of the mammalian two-hybrid
system could be proposed: active and inactive compounds. The first
category comprised only high affinity ligands classified as
agonists. Within this category, a clear correlation between ligand
binding affinities and the affinity of liganded hRAR
for hRXR
was
observed. Inactive compounds could be subdivided into two groups: high
and low affinity ligands. The first subgroup included retinoids
classified as antagonists, whereas the second includes
and
isotype-selective retinoids. Thus, RAR antagonists can be identified on
the basis of two criteria: high affinity for monomeric hRAR
and
failure to promote RXR/RAR interaction in vitro and in
vivo.
2 Promoter--
P19 embryonic
carcinoma cells are a well documented cellular model to study molecular
responses to retinoids (reviewed in Refs. 26 and 27). The mRAR
2 gene
is, in this cell line, highly inducible in response to atRA treatment
(28), and the chromatin organization of this promoter undergoes
structural alterations within minutes after retinoid treatment of P19
cells (29, 30). These alterations are correlated with receptor binding
to the DR5 RARE present in this promoter, which is organized around a nucleosome core both in vitro (31) and in
vivo.4 Such alterations
can be monitored by variation of restriction site accessibility, and
this assay has been used to document glucocorticoid receptor-mediated activation of the murine mammary tumor virus promoter (32) and RXR·RAR-mediated activation of the mRAR
2 promoter (29). Since this chromatin remodeling activity is correlated with RARE occupancy as determined by in vivo footprinting
(30), this assay may be considered as an indirect assessment of
receptor binding to DNA in vivo.
2 promoter was assayed in each condition (Fig.
6B). In parallel, the rate of
expression of the mRAR
2 gene was assayed by reverse
transcription-PCR (Fig. 6A), which was, as expected,
strongly induced after a 2-h induction with 1 µM atRA,
but not with 1 µM CD3106. P19 cell DNA was thus extracted
and cleaved by PstI, generating a 1185-bp fragment. atRA
treatment strongly increased SmaI access to its cutting
sites located in the vicinity of the DR5 RARE (608-bp fragment) and in
the 5'-untranslated region (424-bp fragment), whereas the antagonist CD3106 did not induce major structural transitions in the mRAR
2 promoter. We note that the accessibility of both SmaI sites
is increased in this assay, suggesting that retinoid receptor binding triggers a general opening of the chromatin in the vicinity of the DR5
RARE, which may be related to the ordered occupancy of the various
cis-acting elements located along this promoter (30).
View larger version (30K):
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Fig. 6.
Impeded dimerization is correlated with
transcriptional inactivity and chromatin structure stability of the
mRAR 2 promoter. A, reverse
transcription-PCR analysis of mRAR
2 and actin transcripts in P19
cells. P19 cells were treated for 2 h with vehicle (dimethyl
sulfoxide (DMSO)), 1 µM atRA, or 1 µM CD3106, and total RNA was extracted. mRNA
expression levels of the mRAR
2 gene and
-actin were assayed by
semiquantitative reverse transcription-PCR. Amplification products were
resolved on 1% agarose gel and stained with ethidium bromide.
Molecular size markers are also shown (lane M).
B, effect of retinoid treatment on SmaI
accessibility to the mRAR
2 promoter. P19 cells were treated as
described for A, and nuclei were purified. DNA was digested
with 80-100 units of SmaI for 10 min at 25 °C and
purified. After a secondary digestion with PstI, DNAs were
analyzed by Southern blotting and detected using a mRAR
2 probe
abutting the 3'-PstI site. UTR, untranslated
region.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helices 3, 4, and 12 revealed that a mutation
in helix 12 (L409A) that was designed to inactivate the RAR
coactivator-binding interface did not affect agonist-induced RAR
dimerization with RXR
in vivo. In contrast, mutations in helices 3 and 4 (K244A/K262A) that prevent AF-2 AD positioning along
the holo-LBD structure strongly inhibited RXR
recruitment to RAR
.
From this we conclude that RXR·RAR heterodimerization is not
dependent on the integrity of hRXR
and hRAR
AF-2 ADs, but that
proper repositioning of the hRAR
AF-2 AD region is important for
RXR·RAR dimerization.
or hRXR
with corepressors or coactivators by amino acid changes or deletion in LBDs did not
modify the responsiveness of this system to retinoids, arguing against
such a hypothesis. Moreover, the perfect correlation between in
vivo and in vitro data, for which a contribution of
additional modulators cannot be invoked, strongly argues for an
agonist-dependent dimerization in vivo, as
previously demonstrated for the thyroid hormone receptor (6).
, suggesting that RAR
binds to RXR and represses its autonomous activation function. However,
RAR antagonists restored RXR function within this experimental setting,
strongly arguing for a blockade of retinoid receptor heterodimerization
in the presence of a RAR antagonist. RXR has been reported to occur as
a stable tetrameric complex that is destabilized into RXR dimers and
monomers upon agonist binding. However, RXR tetramers did not
dissociate in the presence of unliganded RAR (33). The active fraction
of RXR molecules is thus likely to occur in our system as a monomeric subunit, available for dimerization. RAR-mediated repression of RXR has
been widely documented for RXR·RAR heterodimers bound to DNA, and our
data suggest that DNA binding is not necessary to observe allosteric
regulation of RXR function by RAR.
or an isolated hRXR
LBD was used, respectively,
suggesting that RXR and RAR LBDs are the main contributors to the
measured dimerization activity. We note also that similar results were
obtained when using full-length proteins with RAR used as a bait in the
in vitro assay.2 This is in keeping with our
previous observations showing that amino acids located in helix 9 of
hRAR
control both homodimerization and heterodimerization of RAR
(3). Since dimerization through LBDs is important for heterodimer
stabilization, we conclude that this activity would be of the utmost
importance at physiological levels of expression of both receptors,
mostly by modulating the DNA binding activity of receptors.
Heterodimerization has been shown to increase strongly the DNA binding
affinity of several nuclear receptors (5, 34-37). As a consequence,
one would predict that a decreased affinity for DNA may be caused by
impaired dimerization. The retinoid antagonist CD3106 did not trigger
structural alteration of the chromatin structure of the mRAR
2
promoter in intact cells, in opposition to atRA. This may be
interpreted as an impaired interaction with nucleosomal DNA. This
observation therefore strongly supports our hypothesis and is in
agreement with previous reports showing that the retinoid antagonists
Ro 41-5253, BMS453, and BMS411 did not induce a dimethyl sulfate
genomic footprint over the DR5 RARE region in P19 and NB4 cells (29,
30, 38).
lying outside of the LBD, and further investigations will be aimed at
identifying receptor subdomains involved in the
ligand-dependent enhancement of RXR·RAR heterodimerization.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. U. Reichert and S. Michel (Galderma, Sophia-Antipolis, France) for the gift of retinoids and sharing information and T. Perlmann and V. K. Chatterjee for providing expression vectors and the reporter gene plasmid.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from INSERM, the Ligue Nationale contre le Cancer, and the Association pour la Recherche sur le Cancer. INSERM U459 is part of the Institut Fédératif de Recherches IFR22 (INSERM, Center Hospitalier Régional et Universitaire, Center Oscar Lambret, and University of Lille 2).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 a fellowship from the Fondation pour la Recherche
Médicale.
§ To whom correspondence should be addressed. Tel.: 33-3-20-62-68-87; Fax: 33-3-20-62-68-84; E-mail: p.lefebvre@lille.inserm.fr.
Published, JBC Papers in Press, December 13, 2000, DOI 10.1074/jbc.M008004200
2 C. Depoix, C. Brand, and P. Lefebvre, unpublished observations.
3 A. Mouchon and P. Lefebvre, unpublished observations.
4 P. Lefebvre, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
RARs, retinoic acid
receptors;
hRAR, human retinoic acid receptor-
;
mRAR
2, mouse
retinoic acid receptor-
2;
RXR, 9-cis-retinoic acid
receptor;
hRXR, human 9-cis-retinoic acid receptor;
LBD, ligand-binding domain;
RARE, retinoic acid response element;
WT, wild-type;
GST, glutathione S-transferase;
atRA, all-trans-retinoic acid;
bp, base pair;
PCR, polymerase
chain reaction;
AD, activation domain;
RLU, relative light units;
CMV, cytomegalovirus;
UAS, upstream activating sequence.
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