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Molecular and Cellular Biology, April 1999, p. 3073-3085, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Allosteric Regulation of the Discriminative Responsiveness of
Retinoic Acid Receptor to Natural and Synthetic Ligands by Retinoid
X Receptor and DNA
Arnaud
Mouchon,
Marie-Hélène
Delmotte,
Pierre
Formstecher, and
Philippe
Lefebvre*
INSERM U459, Faculté de Médecine
Henri Warembourg, 59045 Lille Cedex, France
Received 9 October 1998/Returned for modification 10 November
1998/Accepted 12 January 1999
 |
ABSTRACT |
Transcriptional activation by retinoids is mediated through two
families of nuclear receptors, all-trans-retinoic acid
(RARs) and 9-cis retinoic acid receptors (RXRs).
Conformationally restricted retinoids are used to achieve selective
activation of RAR isotype
,
or
, which reduces side effects
in therapeutical applications. Synthetic retinoids mimic some of
all-trans retinoic acid biological effects in vivo but
interact differently with the ligand binding domain of RAR
and
induce distinct structural transitions of the receptor. In this report,
we demonstrate that RAR-selective ligands have distinct quantitative
activation properties which are reflected by their abilities to promote
interaction of DNA-bound human RXR
(hRXR
)-hRAR
heterodimers
with the nuclear receptor coactivator (NCoA) SRC-1 in vitro. The
hormone response element core motifs spacing defined the relative
affinity of liganded heterodimers for two NCoAs, SRC-1 and RIP140.
hRXR
activating function 2 was critical to confer hRAR
full responsiveness but not differential sensitivity of hRAR
to
natural or synthetic retinoids. We also provide evidence showing
that lysines located in helices 3 and 4, which define part of hRAR
NCoA binding surface, contribute differently to (i) the transcriptional
activity and (ii) the interaction of RXR-RAR heterodimers with SRC-1,
when challenged by either natural or RAR-selective retinoids.
Thus, ligand structure, DNA, and RXR exert allosteric regulations on
hRAR
conformation organized as a DNA-bound heterodimer. We
suggest that the use of physically distinct NCoA binding interfaces may
be important in controlling specific genes by conformationally
restricted ligands.
 |
INTRODUCTION |
Analysis of the mechanisms by which
cognate ligands activates nuclear receptors (NRs) identified structural
transitions occurring in the ligand-bound receptor (holo receptor)
compared to the unliganded form (apo receptor). Considerable progress
has been made upon resolution of crystal structures of the apo form of
9-cis retinoid acid receptor (retinoid X receptor)
isotype
(apo-RXR
), the halo form of
all-trans-retinoic acid receptor isotype
(holo-RAR
), thyroid hormone receptor
(T3R-
), estrogen receptor
(ER), and progesterone receptor bound either to natural and
synthetic agonists or to antagonists (4, 5, 48, 59, 61, 62).
The ligand binding domains (LBDs) of these NRs share a common
structural fold defining a ligand binding pocket (LBP) and functional
regions, among which the activating function 2 (AF-2) activating domain (AF2-AD), located in helix 12 of RARs, is the most conserved. Upon
ligand binding, the LBD adopts a more compact structure in which
the AF2-AD is folded against the LBD, creating a new interface (ligand-dependent AF-2) suitable for NR coactivator (NCoA)
binding (41). Additional structural alterations lead to the
disruption of the nuclear corepressor (SMRT/TRAC, N-CoR/RIP13) binding
site, located at the N terminus of the LBD, allowing p/CAF binding to the DNA binding domain of RAR (3). Agonist-bound NRs
bind p160 nuclear factors (SRC-1/NCoA1, p/CIP/AIB1/ACTR/TRAM1/RAC3,
TIF2/GRIP1/NCoA2), other unrelated coactivators (CBP [CREB
binding protein]/p300, TIF1, RIP140) and multicomponent complexes
(TRAPs, DRIPs) in vitro and/or in vivo (reviewed in reference
58). Spacing of core consensus sequences LXXLL found
in p160-receptor interacting domains is critical for the specific
ligand-dependent interaction of these coactivators with the
AF-2 of distinct NRs (10, 36, 44). p160 coactivators
interact in turn with CBP/p300, generating a large complex with
histone acetyltransferase activity, catalyzing a
posttranslational modification of histone tails which is
important for regulating RXR-RAR heterodimers to nucleosomal DNA
(31). Thus, the assembly of a transcriptionally active
multimeric complex sensitive to combinatorial regulation is dependent,
at least in part, on the fit between the receptor AF-2 interaction
surface and coactivator hydrophobic motifs.
How ligand structure specifies the use of a given nuclear corepressor
and coactivator repertoire remains an open question. Comparison of
crystal structures of RARy LBD bound to either all-trans retinoic acid (atRA) or 9-cis retinoic acid
(9-cis RA) or a synthetic retinoid highlighted very
similar structures, showing that ligands positioned in the LBP
identically and induced similar structural changes in the LBD
(19). This finding suggests that similar structural
transitions might confer identical transcriptional properties to the
holo receptor. Conversely, distinct structural arrangements would
generate receptors with distinct activating, and transrepressive,
activities. However, these structural data provide little
information on how ligands with different structures may control the
transcriptional activity of heterodimeric complexes bound to DNA, which
is the transcriptionally active conformation of RARs. The dimerization
partners for RAR, RXR, and DNA binding have been indeed shown to be
allosteric effectors which modulate the transcriptional activity of
RARs (21, 24).
Using a simple, homogenous system to compare the transactivating and
transrepressive properties of synthetic and natural retinoids, we
reported recently that mutating amino acids in the loop between helices
11 and 12 (L box) of human RAR
(hRAR
) could impinge on receptor
function in a ligand structure-dependent manner (26). We
showed that a single mutation in the L box not only led to distinct
transactivating and transrepressive properties in the presence of atRA
or CD367, a synthetic agonist with receptor binding properties similar
to those of atRA, but also to different capacities to interact with
NCoAs and SMRT, a nuclear corepressor (26). Ligand binding
studies also suggested that retinoids were positioned differently
in the LBP (27, 28), leading to the conclusion that
retinoids could bind to hRAR
with similar affinities but generated holo receptors with different transcriptional properties.
In this report, we were particularly interested in evaluating this
latter possibility, i.e., that retinoid binding to the same
receptor can elicit distinct transcriptional responses, and to
elucidate the molecular basis for these differences. We first evaluated
the relative efficiency and potency of several natural and synthetic
retinoids to activate a simple promoter through hRXR
-hRAR
heterodimers. While binding to hRAR
with similar affinities, these
ligands displayed different abilities to activate transcription. We
compared the ability of DNA-bound hRXR
-hRAR
heterodimers to
recruit two transcriptional coactivators, SRC-1 and RIP140. Both
proteins bind to RARs and RXRs in a ligand-dependent manner but differ
in structural organization (reference 16 and references therein). SRC-1 contains four LXXLL motifs, whereas RIP140 harbors nine of these signature sequences required for coactivator binding to NRs. In addition, spacing between LXXLL motifs
located in receptor interacting domains (RIDs) of these coactivators,
as well as flanking sequences, are different and may therefore
introduce some specificity in binding to receptors. A correlation was
found with transcriptional potency and the ability of ligands to
promote recruitment of SRC-1 RID, but not RIP140 RID, to
hRXR
-hRAR
heterodimers in vitro. Retinoic acid response elements
(RAREs) acted as allosteric modulators which altered the ability
of hRAR
to interact with NCoAs in response to ligand binding.
Mutations designed to alter hRAR
AF-2 and thus its interaction with NCoAs had distinct effects depending on the activating ligand. Finally, we demonstrate that the hRXR
AF2-AD is required for maximal
activation of hRAR
by natural or synthetic retinoids. These
results suggest that RAR transcriptional activity is controlled through
ligand structure and that both DNA and its obligate dimerization partner RXR act as major allosteric effectors.
 |
MATERIALS AND METHODS |
Materials.
atRA was obtained from Sigma (Saint Quentin
Fallavier, France). Other synthetic retinoids were a gift from U. Reichert, CIRD-Galderma, Valbonne, France.
[35S]methionine was purchased from Amersham (Les Ulis,
France). Radioinert atRA as well as antiproteases were purchased from
Sigma (St. Louis, Mo.). Acrylamide-bisacrylamide mix (Protogel) was
from National Diagnostics (Atlanta, Ga.). Ampicillin and kanamycin were
from Appligene (Strasbourg, France). Restriction and DNA modification enzymes were from Promega (Madison, Wis.). Oligonucleotides were purchased from Eurogentec (Le Sart-Tilman, Belgium). Site-directed mutagenesis reactions were carried out with the Promega GeneEditor system. Polyethyleneimine (ExGen 500) was from EuroMedex
(Souffelweyersheim, France).
Plasmids.
Constructs containing either the wild-type (wt) or
mutated hRAR
and wt hRXR
cDNAs subcloned into pSG5 (Stratagene)
have been described elsewhere (26, 30, 47). The pGDX-hRAR
vector was obtained by subcloning the hRAR
cDNA as an
EcoRI fragment into pGEM3Z. Mutations at K244 and K262 were
generated by using the appropriate oligonucleotide containing the
desired mutation and an additional silent mutation either introducing a
new restriction site or inactivating a restriction site present in the
wt sequence. The mutagenic primers used were K224A
(5'-CATTAAGACTGTGGAATTCGCCGCGCAGCTGCCCGGC-3' [new EcoRI site]) and K262A
(5'-GATCACCCTCCTCGCGGCTGCCTGCCTGG-3' [EcoNI site lost]) (mutations are indicated in
boldface). The pGDX-hRAR
vector was used as a template in these
experiments. The AF-2-deleted hRXR
(dnRXR) expression vector was
created by isolating the truncated hRXR
cDNA from cdmRXR
19C
(64) which was subcloned into the pSG5-hRXR
backbone.
Glutathione S-transferase (GST)-RIP140 was a kind gift from
V. Cavailles and contained a fragment of the human cDNA coding for
amino acids 752 to 1158 (6). GST-SRC-1 was obtained from
M. J. Tsai and B. O'Malley and encoded a fusion protein of GST
with human SRC-1 spanning amino acids 382 to 842 (45).
GST-CBP was obtained from T. Kouzarides and encodes a fusion protein of
GST to the N-terminal domain of CBP (amino acids 1 to 1099 [2]). The reporter gene
p(TREpal)3 Luc was constructed by inserting
three repeats of the synthetic thyroid response element
AGGTCATGACCT (TREpal motif) upstream of the adenovirus major
late promoter TATA box present in the pTATA Luc vector (26).
All sequences were checked by restriction analysis and automatic sequencing.
Cell culture and transfections.
HeLa cells were cultured as
monolayers in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum (both from BioWhittaker, Verviers, Belgium).
Cells were treated with retinoids at the indicated concentrations
for 16 h. Retinoids were solubilized in dimethyl sulfoxide (DMSO);
vehicle final concentration was 0.1%. Transfections were carried out
by the polyethyleneimine coprecipitation method as described elsewhere
(26). The luciferase assay was performed with the LucLite
system (Packard Instruments, Rungis, France) according to the
manufacturer's guidelines, and activity (as relative luciferase units
was measured with a LumiCount plate reader (Packard).
GST pull-down experiments.
The protocol used has been
published elsewhere (26). DNA-dependent protein-protein
interactions were tested as follows. hRAR
and
35S-labeled hRXR
were synthesized by using a Quick T7
TnT kit (Promega). The amount of receptors was quantified by
trichloroacetic acid precipitation of labeled samples, and volumes were
adjusted to reach identical concentrations of both receptors in the
translation mix. The integrity and the ability of receptors to form
heterodimers were assessed by size exclusion chromatography on a
Superdex 200 HR 10/30 column (Amersham Pharmacia BioTech, Les Ulis,
France). In a typical binding reaction, 5 to 10 pmol (5 µl) of
hRAR
and 35S-labeled hRXR
were incubated with vehicle
(DMSO) or 1 µM atRA for 60 min at 4°C in a 150-µl final volume.
The binding buffer consisted of 20 mM HEPES (pH 7.4), 150 mM KCl, 5 mM
MgCl2, 0.1% Triton X-100, 0.1% Nonidet P-40, and 0.1%
gelatin (Bio-Rad). A 20-mer double-stranded oligonucleotide containing
the TREpal response element AGGTCATGACCT was added
to the binding mix to a final concentration of 0.1 µM or was
substituted by other response elements when indicated in the text.
Direct repeat response element sequences were as follows: DR1,
cggtagGGTTCAaAGGTCActcg; DR2,
cggtagGGTTCAgaAGGTCActcg; DR3,
cggtagGGTTCAcgaAGGTCActcg; DR4,
cggtagGGTTCAcgaaAGGTCActcg; and DR5,
cggtagGGTTCAccgaaAGGTCActcg (half site sequences are indicated in uppercase). The TREpal sequence is
cggtagAGGTCATGACCTctcg. In these conditions, more than 98%
of receptors formed heterodimers, irrespective of the presence of the
ligand (28). After a 2-h incubation on ice, 40 µl of a
Sepharose-glutathione-GST-SRC-1 slurry was added to the mix and
agitated slowly on a rotating wheel for 2 h at 4°C. Unbound
material was removed by three successive washes of Sepharose beads by
10 volumes of ice-cold 1× phosphate-buffered saline-2 mM
dithiothreitol. Resin-bound receptors were then resolved by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on an 8%
gel and detected by autoradiography or quantified with a PhosphorImager
(Molecular Dynamics). Values were averaged from at least four
independent experiments carried out with two different bacterial extracts.
 |
RESULTS |
Retinoids exhibit distinct hRAR
-activating properties.
The
retinoids used in this study belong to distinct structural classes,
differ in the ability to activate selectively RAR isotypes (
,
,
and
[Table 1]), but share a common
feature, which is the ability to bind hRAR
with dissociation
constants (Kd) in the nanomolar range. The
natural derivatives of vitamin A (atRA and 9-cis RA) and
synthetic retinoids CD367 and TTNPB bind to and activate
RAR
, -
and -
. 9-cis RA is a panagonist which
activates RARs and RXRs. Replacing the propenyl linker in the TTNPB
structure by a carboxamide yields the RAR
-selective ligand Am580.
These synthetic compounds are characterized, compared to natural
retinoids, by the aromatization of the polyenic chain, restricting
their flexibility. Chemical structures and biological activities of
these retinoids have been described in detail elsewhere (11, 38; reviewed in reference
55). CD3106 (AGN 193109) is a C-1-substituted
acetylenic RAR antagonist (20), and CD2425 (AGN 190701) is
an RXR-selective ligand with high affinity for
,
, and
isoforms. A luciferase reporter construct carrying the TREpal
response element was cotransfected in HeLa cells together with hRAR
and hRXR
expression vectors. In these conditions, the activity of
endogenous receptors was not detected (29), whereas all
other reporter genes containing a direct repeat RARE were found to be
sensitive to endogenous receptor activity. We note that the TREpal
response element is activable by both RXR homodimers and RAR-RXR
heterodimers (65). CD2425, an RXR-specific retinoid, induced reporter gene activity only weakly, and
simultaneous treatment of target cells with CD2425 and Am580 led to a
reporter gene activity equivalent to that observed in the presence of
9-cis RA (29). The residual activity detected in
the presence of CD2425 may be attributed to RXR homodimerization, since
RXR-RAR heterodimers are not responsive to RXR ligands. We conclude
that our system reflects hRXR
-hRAR
dimer transcriptional activity
and that the TREpal response element provides allosteric regulations
similar to those observed with DR2 and DR5 response elements.
Dose-response curves carried out with selected retinoids from a
concentration of 10
10 to 10
6 M demonstrated
that no correlation could be established between the affinity of the
ligand for hRAR
and its ability to activate transcription from
this minimal promoter, containing the TREpal response element and a
TATA box. As shown in Table 1, ligand concentrations required for
half-maximal activation of the reporter gene by the wt hRXR
-hRAR
pair (50% effective concentration [EC50]) varied for
each retinoid compared to atRA, which was chosen throughout this
study as the reference ligand. Remarkably, synthetic agonists binding
with a high affinity for hRAR
(TTNPB, CD367, and Am580) were the
most efficient at reaching 50% maximal activation, whereas atRA and
9-cis RA displayed EC50s one order of magnitude
higher than those of synthetic retinoids. In contrast, these two
natural stereoisomers induced higher maximal levels of reporter gene
activity at 1 µM. The highest rate of activation in the presence of
synthetic agonists varied from 44 to 90% compared to atRA, suggesting
that maximal occupancy of hRAR
by these ligands still reveals
different abilities of retinoids to promote transcriptional
activation. Increasing further retinoid concentrations in some
cases led to a slightly higher level of reporter gene activity (10 to
15% for CD367) but in other cases inhibited cell proliferation and
triggered cell death in variable ratio, resulting in an apparently
decreased reporter gene activity (atRA and other synthetic
retinoids). In conclusion, no clear correlation between
Kd and EC50 or between EC50 and relative potency can be established. These
observations showing that a given retinoid exhibits distinct
quantitative activating properties (compared to other RAR-selective
ligands binding to the receptor with a similar affinity) suggest that
each ligand could modulate differently one or several receptor
functions. Ligand binding can potentially modify multiple
protein-protein interaction interfaces. First, the dimerization
interface can be altered, and thus ligands might display distinct
abilities to promote hRAR
binding to hRXR
. Although we
observed, using quantitative in vitro (GST pull-down) assays and in
vivo protein-protein interaction assays (two-hybrid assay in mammalian
cells), that ligand binding triggers dimerization of both
partners in a DNA-independent manner, these assays did not provide
evidence for a regulation at this level (11a). Second,
either corepressor displacement from or coactivator recruitment to
hRXR
-hRAR
heterodimers bound to DNA could be altered, and we
thus compared the ligands for the ability to modulate these
interactions.
RAR-selective agonists are equally efficient with respect to
triggering SMRT release from monomeric or DNA-bound heterodimeric
hRAR
.
We used a quantitative protein-protein interaction assay
to test retinoids for the ability to promote either SRC-1 or RIP140 binding to, or SMRT release from, RXR-RAR heterodimers synthesized by
coupled in vitro transcription-translation. For this purpose, we used
conditions in which the hRXR
-to-hRAR
and DNA-to-receptor ratios
yielded 98 to 100% heterodimeric complexes bound to DNA, as
demonstrated by size exclusion chromatography and electrophoretic mobility shift assay (29). Importantly, no receptor monomer could be detected in these conditions. Thus, we generated affinity matrices with SMRT, RIP140 or SRC-1 RIDs fused to GST, which were incubated with either monomeric hRAR
when indicated or performed hRXR
-hRAR
dimers bound to the TREpal RARE. In preliminary
experiments, the formation of heterodimers was monitored by using
labeled hRAR
and hRXR
(29). Note that in experiments
presented below, receptor complexes in which only hRXR
was labeled
were incubated with 10 µM retinoid for 2 h before adsorption
to GST fusion proteins.
We first compared natural and synthetic retinoids for the ability
to release SMRT RID fused to GST from monomeric hRAR

(Fig.
1A), from
hRXR

-hRAR

dimers (Fig.
1B), or from
heterodimers bound
to the TREpal response element (Fig.
1C). As
previously reported
(
7,
26), the monomeric, wt hRAR

bound
strongly to SMRT in
the absence of ligand, and all retinoids
induced SMRT release
with similar efficiencies except CD2425, an
RXR-selective retinoid.
Interestingly, we note that CD3106, an RAR
antagonist, was able
to displace SMRT from hRAR

as efficiently as
agonist compounds.
On the opposite, hRXR

interaction with this
corepressor was not
detectable. When SMRT was complexed to
hRXR

-hRAR

heterodimeric
complexes, the ability of these ligands,
except for CD2425, to
mediate SMRT release was unaffected (Fig.
1B).
The RXR-specific
ligand was in this configuration able to promote SMRT
release,
suggesting that liganded RXR exerts allosteric regulation on
hRAR

-SMRT
binding interface. The interaction of TREpal-bound
heterodimers
with SMRT was modulated in a manner similar to that
observed with
heterodimeric receptors when synthetic RAR agonists
are involved
(Fig.
1C). Remarkably, the RXR-selective ligand and
to a lesser
extent the RAR antagonist displayed decreased
abilities to induce
SMRT release, showing that DNA binding clearly
exerts allosteric
regulation on RAR nuclear corepressor binding
surface. Since similar
results were obtained for DR1, DR2, and DR5
RAREs (
29), we conclude
that whatever their structure,
retinoids have similar SMRT binding
interface remodelling
properties and that no significant allosteric
regulation can be
detected in the presence of DNA and hRXR

.

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FIG. 1.
Retinoids display similar efficiencies with respect to
displacing SMRT from monomeric hRAR and DNA-bound hRXR -hRAR
heterodimers. (A) 35S-labeled hRAR or hRXR was
incubated in the presence of the GST-SMRT (982-1495) fusion protein
for 2 h and then challenged with the indicated retinoids.
SMRT-receptor complexes were isolated by adsorption of the SMRT moiety
on agarose-coupled glutathione. After the beads were washed, complexes
were analyzed by SDS-PAGE (8% gel), and receptor content was assayed
by autoradiography. (B) 35S-labeled hRXR and unlabeled
hRAR were incubated for 2 h with GST-SMRT and then for an
additional 2 h in the presence of the indicated ligand. Complexes
were treated and analyzed as described above. (C) TREpal-bound
heterodimers were bound to GST-SMRT and further activated by the
indicated retinoids. Complexes bound to SMRT were isolated and
analyzed by adsorption to agarose-linked glutathione SDS-PAGE (8%
gel), and autoradiography as described above. The TREpal sequence is
cggtagAGGTCATGACCTctcg. Numbers below gel lanes indicate the
amount of radiolabeled receptor bound to SMRT in the presence of the
indicated ligand relative to that measured in the presence of vehicle
(DMSO), defined as 100%. Values represent mean data from four
independent experiments carried out with two different bacterial
extracts. ND, not determined. Standard errors never exceeded 8.7%.
Representative autoradiograms are shown.
|
|
DNA binding modulates the ability of structurally distinct
retinoids to promote SRC-1 or RIP140 recruitment by
hRXR
-hRAR
heterodimers.
In view of the lack of
difference in the abilities of natural and synthetic retinoids to
promote SMRT release from hRAR
, we analyzed their effects on the
ligand-induced recruitment of several NCoA, in the context both of the
monomeric receptor and of the DNA-bound hRXR
-hRAR
heterodimer (Fig. 2). As expected, monomeric hRAR
complexed to atRA recruited SRC-1 very
efficiently. Similar activities were observed for its natural
stereoisomer, 9-cis RA, and all RAR-selective ligands
(TTNPB, CD367, and Am580). On the contrary, neither the RAR antagonist
CD3106 (AGN 193109) nor the RXR-selective ligand CD2425 (AGN 190701)
was able to induce SRC-1 binding to hRAR
(Fig. 2A). When
RIP140 RID was used as a bait, identical results were
obtained, although it appeared clearly that this putative
coactivator bound to hRAR
with a lower affinity than SRC-1 (Fig.
2B). When similar assays were carried out with preassembled
hRXR
-hRAR
heterodimers on the TREpal RARE, several
observations could be made. First, heterodimer binding to DNA
induced a strong modulation of the capacity of ligands to bind to
SRC-1 and RIP140 (Fig. 2C and D). atRA was twofold less efficient than
9-cis RA at promoting NCoA binding to DNA-bound heterodimers. 9-cis RA turned out to be the most
efficient ligand, whereas synthetic RAR agonists had a
clearly lower efficiency. CD3106 was unable to promote SRC-1
recruitment, in keeping with its antagonistic properties, whereas
the RXR-specific ligand CD2425 enhanced only weakly SRC-1 binding to
the complex. When RIP140 was used in this assay, similar results were
obtained (Fig. 2D). However, we consistently observed that the RAR
antagonist was in this context able to promote detectable binding of
the heterodimer to RIP140, thereby excluding this protein as a
potential coactivator in our system. Thus, DNA binding introduced
dramatic changes in ligand capacity to promote SRC-1 recruitment,
establishing a semiquantitative correlation between DNA-dependent
protein-protein interaction assays and transcriptional activities in
which natural retinoids were the most efficient ligands. Indeed,
the 2-fold higher activity of 9-cis RA in transactivation
assay is clearly correlated to its ~2-fold higher ability to promote
SRC-1 recruitment. This may obviously relate to its ability to activate
both components of the dimer and thus allow binding of SRC-1 to both
receptor AF-2 domains.

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FIG. 2.
Assembly of hRAR into a DNA-bound heterodimer induces
ligand-selective recruitment of SRC-1 and RIP140. (A) Interaction of
hRAR or hRXR with SRC-1 in the presence of various retinoids.
35S-labeled hRAR or hRXR was incubated with the
indicated activating ligand (10 µM) or vehicle (DMSO) for 2 h
and then with the fusion protein GST-SRC1(382-842). Complexes were
precipitated with glutathione-Sepharose beads and extensively washed.
Samples were analyzed by SDS-PAGE (8% gel), and receptors were
detected by autoradiography. (B) Interaction of hRAR or hRXR with
RIP140. Receptors were synthesized in vitro as described in the text
and incubated in the presence of retinoids and a
GST-RIP140(752-1158) fusion protein. Following washing, proteins were
resolved by SDS-PAGE and revealed by autoradiography. In input lanes,
1/10 of the input from in vitro-coupled transcription-translation mix
was analyzed in parallel. (C) Interaction of DNA-bound hRXR -hRAR
heterodimers with SRC-1. 35S-labeled hRXR , hRAR , and
the DNA probe TREpal were incubated as described in Materials and
Methods to ensure >95% heterodimer formation. These complexes were
then incubated in the presence of the activating ligand for 2 h
(10 µM) and then with the GST-SRC-1 fusion protein for an additional
2 h. Complexes were precipitated and analyzed as described above.
(D) Interaction of DNA-bound hRXR -hRAR heterodimers with RIP140.
The procedure was similar to that described for panel C except that
SRC-1 was substituted for RIP140. The TREpal sequence is
cggtagAGGTCATGACCTctcg. Numbers below gel lanes indicate the
amount of radiolabeled receptor bound to SRC-1 or RIP140 in the
presence of the indicated ligand relative to that measured in the
presence of 9-cis RA, defined as 100%. Values represent
mean data from four independent experiments carried out with two
different bacterial extracts. ND, not determined. Standard errors never
exceeded 5.3%. Representative autoradiograms are shown.
|
|
Direct repeats of the PuGG/TTCA motifs are recognition sequences for
RXR-RAR heterodimers present in a number of natural promoters.
They
define a consensus response element to which RAR heterodimers
bind
strongly. Direct repeats of the PuGGTCA motifs separated
by a spacer of
variable length ranging from one to five nucleotides
allow
high-affinity binding of RXR-RAR heterodimers (
37). DR1,
DR2, and DR5 are the most commonly described natural RAREs, but
DR3 and
DR4, which mediate vitamin D3 and thyroid hormone responses,
have been
shown to behave as RAR-responsive elements depending
on the promoter
context (
50). In addition, RAREs containing
the AGGTCA
motif as the second half site displayed significant
affinities
for RXR-RAR heterodimers in vitro, and cooperative
bindings of RXR-RAR
heterodimers to these RAREs involve distinct
dimerization interfaces
(
37). Since DNA has been shown to be
a major allosteric
effector for a variety of transcription factors
and NRs (reviewed in
reference
32), we therefore evaluated the
possible
influence of various spacer lengths (and therefore of
various
dimerization interfaces) on the ability of retinoids to
promote
SRC-1 recruitment to hRXR

-hRAR

heterodimers (Fig.
3).
In the absence of DNA, binding of
heterodimers to SRC-1 was barely
detectable in the presence of
RAR-specific ligands, whereas control
experiments showed again a
very efficient recruitment of this
coactivator by monomeric hRAR

(Fig.
3A). This finding confirms
the specificity of our assay for
receptor heterodimers. Note that
only 9-
cis RA was able
to trigger significant SRC-1 binding, a
property that could be related
to its ability to bind both receptors,
since coincubation of dimers
with the RAR

-specific ligand Am580
and the RXR-specific ligand
CD2425 yielded identical results (
29).
hRXR

-hRAR

heterodimers were then assembled on a DR1 response
element on which
heterodimers have a binding polarity opposite
that observed with other
direct repeat response elements; i.e.,
RAR is the 5'-bound receptor. In
this configuration, SRC-1 recruitment
was observed in the presence of
atRA and was two- to threefold
more pronounced with 9-
cis
RA. The RXR-specific ligand CD2425
was as efficient as atRA,
whereas RAR-specific ligands displayed
no activity in this system
(Fig.
3B). Additional control experiments
showed that heterodimers
formed in these conditions, and therefore
we conclude that
apo-hRXR

formed, when bound to the 3' half motif
from the DR1 RARE,
a high-affinity binding interface for SRC-1.
We observed, however, that
in experiments carried out with labeled
RXR or RAR, and with
labeled RAR and RXR, that the SRC-1-bound
material appeared to be a
mixed population consisting of about
60% RXR homodimers and 40%
RXR-RAR heterodimers in the presence
of CD2425 (
n = 3)
(
29). In the presence of DR2 RARE, atRA- and
9-
cis RA-complexed heterodimers bound avidly to SRC-1, and
RAR-specific
ligands also induced the recruitment of this coactivator,
albeit
with a lower efficiency (Fig.
3C). Increasing the spacer length
to four nucleotides allowed stronger binding of SRC-1 to hRAR
complexed to synthetic retinoids (Fig.
3E), and use of the
high-affinity
DR5 RARE yielded complexes that bound SRC-1 avidly (Fig.
3F).
Although remaining weaker than in the presence of natural
retinoids,
binding of DR5-bound heterodimers to SRC-1 was the
most efficient
and revealed variations between ligands. Am580 turned
out to be
the least efficient in this configuration, whereas it was
the
strongest inducer in the presence of the DR2 RARE. Finally, we
note
that the RAR antagonist CD3106 prevented, in all configurations
tested,
heterodimer binding to SRC-1. This inhibition was also
observed in
competition experiments (
29) in which heterodimers
were
challenged by atRA (100 nM) and CD3106 (1 µM).

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FIG. 3.
The spacing between the two direct repeats of RAREs
modulates ligand ability to promote SRC-1 recruitment to
heterodimerized hRAR . (A) 35S-labeled hRXR and
hRAR produced by coupled in vitro transcription-translation were
incubated in the presence of the indicated retinoids at 10 µM
(final concentration), and complexes were bound to a GST-SRC-1 fusion
protein. After a 2-h incubation, complexes were isolated by adsorption
on a glutathione-linked agarose matrix and analyzed by SDS-PAGE (8%
gel). (B to F) hRXR -hRAR heterodimers associated to the indicated
DRng RARE were analyzed for the ability to interact with
SRC-1 in the presence of natural or synthetic retinoids as
described above. The nature of the response element is indicated below
each panel. Numbers below gel lanes indicate the amount of radiolabeled
receptor bound to SRC-1 in the presence of the indicated ligand
relative to that measured in the presence of 9-cis RA,
defined as 100%. Values represent mean data from four independent
experiments carried out with two different bacterial extracts. Standard
errors never exceeded 4.3%. Representative autoradiograms are shown.
|
|
In summary, ligand capacity to promote SRC-1 recruitment in vitro was
found to be dramatically modulated by the response element
structure to
which receptor heterodimers are bound. The strongest
binding always
occurred with 9-
cis RA and atRA, and we noted that
triggering hRXR

AF-2 remodelling with CD2425 also induced SRC-1
recruitment for some response elements. This is in agreement with
the
weak but constant agonist activity of CD2425 detected in transient
transfection assays using TREpal (Table
1) DR2 or DR5-driven
tk Luc reporter genes (
29). The DR5 RARE appeared
to be the
most efficient DNA template in that it favored a strong
binding
of SRC-1 to heterodimers that could relate to its higher
inducibility
in transcriptional activation
assays.
We then extended this DNA-dependent protein-protein interaction assay
to RIP140 (Fig.
4), for which we have
observed distinct
requirement for binding to heterodimeric complexes
compared to
SRC-1 (see below). Such differences were also reported for
the
estrogen receptor (
17). The lower affinity of RIP140 for
hRXR

-hRAR
dimers was again noted in these experiments, and
receptor complexes
exhibited the highest affinity for RIP140 when bound
to atRA and
9-
cis RA. More strikingly, the DR2 response
element was the DNA
template that allowed the most efficient binding of
RIP140: atRA
efficiently promoted SRC-1 recruitment to heterodimers
bound to
either the DR5, DR4, or DR1 probe (Fig.
3), whereas RIP140
bound
more efficiently to atRA-complexed dimers associated to the DR2
or DR3 RARE. For other retinoids displayed, as observed for SRC-1,
the ability to recruit RIP140 depended on the nature of the response
element, with TTNPB being the most active synthetic ligand in
most
configurations. Note that CD2425 did not promote RIP140 binding
to
heterodimers associated with DR3 and DR5 RAREs. For other data,
it is
also clear that activation of both components by 9-
cis RA
led to a threefold potentiation of RIP140 binding on DR4 and DR5
RAREs,
in opposition to SRC-1 recruitment, for which the synergy
was only
additive (compare Fig.
3E and F to Fig.
4E and F). Finally,
we note
that the antagonist CD3106 is the most efficient ligand
in inducing
RIP140 recruitment to heterodimers in the absence
of DNA. This
property may be related to the recently described
repressive activity
of RIP140 (
25).

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FIG. 4.
The nature of the RARE determines the relative affinity
of liganded heterodimers for nuclear coactivators. (A)
35S-labeled hRXR and hRAR produced by coupled in
vitro transcription-translation were incubated in the presence of the
indicated retinoids at 10 µM (final concentration), and complexes
were bound to a GST-RIP140 fusion protein. After a 2-h incubation,
complexes were isolated by adsorption on a glutathione-linked agarose
matrix and analyzed by SDS-PAGE (8% gel). (B to F) hRXR -hRAR
heterodimers associated with the indicated DRng RARE were
analyzed for the ability to interact with RIP140 in the presence of
natural or synthetic retinoids as described above. The nature of
the response element is indicated below each panel. Numbers below gel
lanes indicate the amount of radiolabeled receptor bound to RIP140 in
the presence of the indicated ligand relative to that measured in the
presence of 9-cis RA, defined as 100%. Values represent
mean data from four independent experiments carried out with two
different bacterial extracts. Standard errors never exceeded 6.2%.
Representative autoradiograms are shown.
|
|
Thus, we conclude that the strength of protein-protein interaction may
differ, for a given ligand and response element, from
one coactivator
to another, suggesting that transcriptional activation
could be
mediated by distinct NCoAs according to the type of RARE
tethering
hRXR

-hRAR

dimers to retinoid-regulated
promoters.
Transcriptional activation of hRAR
by synthetic retinoids is
compromised by mutations in helix 3 or helix 4.
Previous work has
established that the LBD of NRs undergoes structural transitions upon
ligand binding. A lysine residue (K264) from helix 3 has been shown to
establish salt bridges with glutamic acid side chains in the AF2-AD
region of the monomeric holo-RARy LBD (48) which are
essential for transcriptional activation by this receptor (Fig.
5A). The proposed stabilization of the active structure may, however, not be a feature common to all NRs, and
could reflect the need for a hydrophilic region to which coactivators
could bind. Such a role has been proposed for the highly conserved K366
of ER (17) and K288 of TR-
1 (14), equivalent to K244 of hRAR
. Alternatively, it may also provide an alternative anchoring point for AF2-AD acidic residues (Fig. 5A). Whatever their
actual role may be, we reasoned that mutations at K262 or K244 of
hRAR
could impinge differently on hRAR
function according to the
ligand used and therefore reflect a different architecture of the
holo-HBD. We thus mutated K244 or K262 in hRAR
and evaluated first
the impact of these mutations on the transcriptional activity of
hRXR
-hRAR
heterodimers stimulated by natural and synthetic retinoids. When stimulated with atRA, both mutants displayed a diminished response to this ligand (60% of the wt hRAR
activity). In the presence of 9-cis RA, the reporter gene activity was
decreased to 50% of wt hRAR
activity. More surprisingly,
responsiveness to RAR-selective retinoids was abolished for the
K244A receptor, whereas a lower but significant responsiveness was
still observed for the K262A derivative (Fig. 5B). Thus, these amino
acids, which define part of the AF-2 region of hRAR
, are necessary
to confer full responsiveness of hRAR
to retinoids but are
differently required for responsiveness to synthetic RAR-selective
ligands, which displayed a strict requirement for K244.

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FIG. 5.
Alteration of the nuclear coactivator binding interface
inactivates selectively hRAR responsiveness to synthetic
retinoids. (A) Three-dimensional modelling of the hRAR LBD
showing the backbone of the polypeptide, helices 3, 4, and 12, and
lysyl residues mentioned in the text (space-filling mode). The computer
graphic was prepared with RasMol software. (B) Transcriptional activity
of hRAR mutants K244A and K262A in the presence of natural and
synthetic retinoids. HeLa cells were transfected with appropriate
hRAR and hRXR expression vectors and the TREpal-TATA Luc reporter
gene (23) and stimulated with 1 µM retinoid.
Luciferase activities represent induction values over the observed
basal level (~5,000 relative luciferase units in our conditions).
Results represent the average values of six measurements ± standard error of the mean. Each assay was carried out six times.
|
|
Mutation of K244 or K262 abrogates SRC-1 recruitment in vitro to
hRAR
complexed to synthetic retinoids.
Assessing the
effects of these mutations on various receptor functions, we found that
both hRAR
mutants displayed wt affinities for retinoids, were
able to dimerize with hRXR
, and could bind as heterodimers to DNA in
vitro. Furthermore, structural transitions upon ligand binding probed
by limited proteolysis were identical to those observed with wt
hRAR
, and abilities of mutants to release SMRT were unaffected
(29). Since our prediction was that these mutations could
prevent NCoA recruitment, we tested first the capacity of monomeric RAR
mutants to interact with RIP140, SRC-1, and CBP. Both mutations
abolished RIP140 recruitment upon binding to atRA, whereas the
K244A mutation selectively impaired SRC-1 binding. Interaction with the
N-terminal portion of CBP was decreased by 50% for both mutants (Fig.
6A). As shown in Fig. 6B,
hRXR
-hRAR
dimers strongly interacted with SRC-1 in presence
of natural or synthetic retinoids, whereas the
hRXR
-K244A heterodimer did not recruit this coactivator
regardless of the ligand used. hRXR
-K262A heterodimers recruited
SRC-1 with a moderate affinity in the presence of RAR agonists, with no
significant variation among ligands (Fig. 6B). Since we noted that the
monomeric configuration did not reflect the transcriptional activity of
receptors, we tested these mutants in the DNA-dependent protein-protein
interaction assay described above, using SRC-1 as a bait (Fig. 6C).
DNA-bound hRXR
-K244A as well as the hRXR
-K262A dimers interacted
with SRC-1 when complexed to either atRA or 9-cis RA, as
expected from transient transfections experiments (Fig. 5B). Note that
the K262A derivative displayed a higher affinity for SRC-1 than did
K244A. In the presence of synthetic ligands, SRC-1 did not bind
to the K244A-containing heterodimer, whereas a fair level
of activity was still detected with the K262A-containing heterodimers.
Thus, in vitro protein-protein interaction assays established a
correlation between transcriptional responsiveness and ability to
recruit SRC-1. We conclude that K244 is an amino acid
critical for synthetic retinoid-induced SRC-1 recruitment to
hRAR
.

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FIG. 6.
Lysine 244 and lysine 262 are differentially involved in
NCoA binding by liganded hRAR . (A) Interaction of monomeric hRAR
and of K244A and K262A derivatives with RIP140, SRC-1, and CBP(1-1099).
GST fusion proteins corresponding to the indicated coactivator were
incubated in the presence of 35S-labeled, atRA-bound
receptors. Complexes were isolated by adsorption on a
glutathione-linked agarose beads and analyzed by SDS-PAGE (8% gel) as
described for Fig. 1. (B) Interaction of monomeric wt and receptor
mutants with SRC-1 in the presence of natural and synthetic
retinoids. 35S-labeled hRAR derivatives were
incubated with or without 10 µM ligand, and SRC-1-associated
receptors were isolated and analyzed as described above. (C) Mutation
of K244 disrupts synthetic retinoid-induced SRC-1 recruitment by
DNA-bound heterodimers. The ability of each hRAR derivative to bind
to SRC-1 when incorporated into a TREpal-bound heterodimer was assessed
as described for Fig. 1 in the presence of the indicated ligand. In the
control lanes, 1/10 of the coupled transcription-translation mix used
to produce radioinert hRAR was used to assess the efficiency of
synthesis by label incorporation. Products were analyzed in parallel
with other samples containing labeled hRXR . Numbers below gel lanes
indicate the amount of radiolabeled receptor bound to the indicated
coactivator RID in the presence of the indicated ligand relative to
total labeled receptor input, defined as 100%. Values represent mean
data from four independent experiments carried out with two different
bacterial extracts. Standard errors never exceeded 7.2%.
Representative autoradiograms are shown.
|
|
The RXR AF2-AD is required for full responsiveness of
hRAR
to RAR-selective ligands.
Although initially described as
a silent partner whose sole function is to increase the DNA binding
affinity of RARs (21, 22), RXRs appeared to act
synergistically with RARs (8, 12, 40). This synergy is
dependent on the RXR AF-2 (Tc) region (8), but
its contribution varies according to the structure of the response
element (12, 13, 24). We were interested in testing whether
the hRXR
AF-2 region participates differentially to hRAR
activation by RAR-specific ligands. We therefore examined the ability of wt hRAR
to activate the TREpal reporter gene in the presence of dnRXR. This hRXR
mutant, with a deletion of the 19 C-terminal amino acids (448 to 462), has been shown to bind DNA as
either a homodimer or a heterodimer in vitro (65) but to display reduced transcriptional activity. The 9-cis
RA-induced transcriptional activity of the wt hRAR
-dnRXR was indeed
much lower than that of the wt hRAR
-wt hRXR
, reflecting the
contribution of the liganded hRXR
to the transcriptional activity of
the promoter. More surprisingly, hRXR
AF-2 deletion affected
differentially the transcriptional activity induced by atRA or
RAR-selective synthetic retinoids (Table
2). Indeed, atRA- and TTNPB-induced activities were the most severely affected by this mutation
(
40% and
56%, respectively), whereas CD367 and Am580
displayed an 20 to 25% lower activity. However, we noted that
EC50s were not affected, suggesting that this domain of
RXR potentiates RAR AF2 activity without altering its sensitivity to
retinoids.
Deletion of the hRXR
AF2-AD equalizes the capacity of
retinoids to recruit SRC-1 by DNA-bound heterodimers.
The
ability of dnRXR-hRAR
heterodimers to recruit SRC-1 was
characterized in different configurations. In the absence of DNA,
heterodimer-SRC-1 interaction was weak and demonstrated no evidence of
differences between retinoids (Fig.
7A), as shown for the wt hRXR
(Fig.
3). 9-cis RA lost its higher efficiency in this assay, as
expected from the deletion of the hRXR
AF2-AD region. Upon assembly
on a DR1 RARE, in contrast to its wt counterpart (Fig. 3), the
dnRXR-hRAR
dimer became able to recruit SRC-1 in the presence of
RAR-specific retinoids. Again, no difference in the affinity for
SRC-1 was noted with different retinoids, and as was also observed
in the presence of DR2 (Fig. 7C), DR5 (Fig. 7D), TREpal (Fig. 7E), and
DR3 and DR4 (29). This establishes a correlation between
transcriptional responsiveness of dnRXR-hRAR
heterodimers and their
abilities to recruit SRC-1 in vitro, and so we conclude that the
hRXR
AF-2 region is required for maximal activation of hRAR
.

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FIG. 7.
Deletion of RXR AF-2 abolishes selective
retinoid-induced SRC-1 binding to hRXR -hRAR heterodimers. (A)
35S-labeled dnRXR and radioinert hRAR produced by
coupled in vitro transcription-translation were incubated in the
presence of the indicated retinoids at 10 µM (final
concentration), and complexes were bound to a GST-SRC-1 fusion
protein. After a 2-h incubation, complexes were isolated by adsorption
on a glutathione-linked agarose matrix and analyzed by SDS-PAGE (8%
gel). (B to E) dnRXR -hRAR heterodimers associated to the
indicated RARE were analyzed for the ability to interact with SRC-1 in
the presence of natural or synthetic retinoids as described above.
The nature of the response element is indicated below each panel.
Numbers below gel lanes indicate the amount of radiolabeled receptor
bound to SRC-1 in the presence of the indicated ligand relative to that
measured in the presence of 9-cis RA, defined as 100%.
Values represent mean data from four independent experiments carried
out with two different bacterial extracts. Standard errors never
exceeded 4.1%. Representative autoradiograms are shown.
|
|
 |
DISCUSSION |
Due to the highly pleiotropic effects of natural retinoids,
which govern many important biological processes such as
morphogenesis and cell growth, proliferation, differentiation, and
death, much efforts have been devoted to the development of synthetic
retinoids with the aim of improving the therapeutic properties of
such compounds. The identification of RAR isotypes allowed the design
of isotype-specific retinoids with improved clinical potential, and
indeed targeting RAR isotypes hold great promises in the treatment of
skin diseases (53, 54), and cancer chemoprevention and
chemotherapy (35). Importantly, these conformationally
restricted retinoids greatly facilitated the study of molecular
mechanisms by which these ligands activate transcription through their
cognate receptors (23, 46, 51). Transcriptional regulation
by retinoid receptors is thought to be largely dependent on the
ligand-dependent (AF-2), which undergoes major structural alterations
upon ligand binding (reviewed in reference 41).
Several lines of evidence demonstrated that these transitions are
required to permit the interaction of the liganded receptor with
nuclear coactivators (58). Yet, despite intense
investigations, it remains unclear how ligand binding triggers these
structural transitions and, importantly, whether these changes are
affected by the shape of the ligand.
In this study, we demonstrated that natural and synthetic retinoids
differ in the ability to activate a simple promoter, despite similar receptor binding properties. These results showed that ligands
belonging to structurally distinct classes contribute differently
to RAR-mediated transcription. These observations extend our previous
conclusions drawn from the use of mutants of hRAR
and synthetic
retinoids (26), for which mutating amino acids in the
helix 11-helix 12 loop had a different impact on the structure and
transcriptional activity of hRAR
according to the ligand used.
Moreover, we (27, 28) and others (23, 56, 57)
showed that structural requirements for agonist and antagonist binding
to RAR
were different and thus that LBPs for each ligand overlap but
are not totally coincident. This body of data led us to speculate that
different agonists could induce significantly different structural
transitions that in turn would affect protein-protein interaction
surfaces. While we observed no significant effect of the ligand
structure on either heterodimerization properties or corepressor
binding to hRAR
, dramatic alterations of the ability of the receptor
to bind to coactivators were noted. Data from transcriptional assays
and DNA-dependent protein-protein interaction assays led us to several
important conclusions.
First, the DNA-dependent protein-protein interaction assay established
a clear correlation between transcriptional activity and binding of
SRC-1 to DNA-bound heterodimers. Importantly, the hormone response
element (HRE) was found to be critical (i) for establishing
differential NCoA recruitment in response to ligand binding and (ii)
according to the spacing between the two core motifs of the RARE, for
modulating the relative affinity of heterodimers for NCoAs. Thus, the
nature of the HRE to which hRXR
-hRAR
dimers are bound, which has
been shown to modulate the activity of the AF-2s of both partners
(12, 24, 42), affects the capacity of RAR AF-2 to bind to
coactivators and modify the relative affinity of RAR-containing
heterodimers for these nuclear proteins. These observations may be of
importance since we observed that putative or bona fide coactivators
(RIP140, SRC-1, SRC-2, and SRC-3 [see reference 34
for nomenclature]) are coexpressed in a single cell type
(29). These data also lead to the prediction that distinct
direct repeat response elements would respond differently to
retinoids. This hypothesis is currently under investigation but
requires an experimental system in which no contribution of endogenous
receptors to transcriptional activation is detectable. We indeed
observed that DR1, DR2, and DR5 RARE-driven reporter genes are
sensitive to endogenous receptors activity (29, 30), and all
cell lines that we have tested so far coexpress several RAR and RXR
isotypes (29). RAR coexpression implies that the transcriptional activity detected in response to high concentrations of
retinoids (above 10
7 M) would reflect the
contribution of several receptor isoforms in varying ratio, since
ligands are selective only in a defined concentration range. However,
we note that such differences were also described for CD367 and TTNPB
in other systems using different receptor combinations and other
reporter genes (1, 43, 63). Moreover, we and others also
observed this differential response to natural and synthetic
retinoids when studying the expression of endogenous genes driven
either by a DR5 RARE (RAR
[29, 49]) or a DR2 RARE
(CRABPII [49]). These observations strengthen the view
that the phenomenon reported here is of general significance and can be
extended to the expression of endogenous RA-responsive genes. However,
it should be kept in mind that additional factors play a role in
defining the ability of the receptor to elicit a transcriptional
response: the actual sequence of the RARE alters the RXR-RAR DNA
binding repertoire (37), and the relative ratio of
corepressors, coactivators, and receptors may induce a differential response to RAR- and RXR-restricted ligands (50).
We consistently observed that SRC-1 bound more avidly to heterodimers
than RIP140 and that synergistic binding triggered by the panagonist
9-cis RA was more than additive for the latter polypeptide
compared to atRA-induced recruitment on DR1, DR4, and DR5 RAREs. While
the molecular basis for these differences is not clear yet, we note
that the SRC-1 RID used in these assays (amino acids 382 to 842)
contains three LXXLL motifs separated by a 49-amino-acid spacer, while
the RIP140 RID (amino acids 752 to 1158) contains only two LXXLL motifs
with a spacer of 107 amino acids. In light of the recently reported
three-dimensional structure of apo-peroxisome proliferator-activated
receptor
LBD complexed to a region of SRC-1 from amino acids 623 to
710 containing two LXXLL motifs (44) and of the proposed
model of interaction of these two motifs with RXR-RAR heterodimers
(60), one possible explanation could be that the RIP140
RAR-interacting LXXLL motif has a very low affinity for apo-RAR AF-2,
whereas the RXR binding motif has a high affinity for RXR AF-2. In
contrast, SRC-1 motifs would bind to both receptors with similar
affinities, leading only to the observed additive effect on 9 cis-RA binding to both receptors. It is worth noting that
the 9-cis RA-induced potentiation of RIP140 recruitment is
not observed when heterodimers are assembled on DR2 and DR3 RAREs,
arguing for an allosteric regulation through RARE half-site spacing.
Finally, note that such a potentiation was observed in most cases
when 9-cis RA was compared to synthetic retinoid-induced
RIP140 and SRC-1 recruitment, strengthening our view that the apo-RAR
structure is different in the presence of natural or synthetic agonists.
Second, the hRXR
AF2-AD appeared to be critical for maximal
activation of RAR AF-2 by retinoids. The dependency of
hRAR
activity on the hRXR
AF2-AD was reflected by a decreased
interaction of dnRXR-hRAR
dimers with SRC-1, which suggests that
AF2s of both partners, liganded or not, do not function autonomously. Allosteric regulation imposed by dimerization with hRXR
is thus likely to alter the structure of the RAR coactivator binding interface. Alternatively, it may suggest that liganded RAR activates RXR AF-2,
in analogy with the reported allosteric regulation of the RAR AF-2
region by the RXR homodimer antagonist LG100754 (52). Such a
dependency of the RAR signalling pathway on the RXR AF-2 has been
documented in vivo (15), and our data therefore demonstrate a direct role for RXR AF-2 in RAR-mediated recruitment of nuclear coactivators.
Third, synthetic RAR-selective retinoids displayed differing
abilities to recruit SRC-1 in vitro to DNA-bound heterodimers. The
relative ability of a given ligand to recruit a coactivator could be
altered by the nature of the HRE. This is indicative of the modulation
of the NCoA binding interface in a ligand structure-dependent manner, a
hypothesis consistent with the absolute requirement for K244 to observe
synthetic retinoid-induced transcriptional activity. The K244A
mutation also impaired SRC-1 recruitment, in contrast to the K262A
mutant, which was found to impinge moderately and nonselectively on
receptor transactivating properties. These mutations were also found to
discriminate between different coactivators, showing that their
interaction interfaces are not identical, as already reported for ER
(17). We note that the K262A mutation in RARy yielded an
inactive receptor derivative (46), while mutating the
corresponding lysine in hRAR
(K262 [our results]) and TR-
1
(14) showed residual activities (~50 and 30%,
respectively, of wt receptor activity). While the reason(s) for
this discrepancy is not clear, the use of distinct RARs and
RXRs isotypes, as well as different cellular backgrounds (simian versus
human) might account for these differences.
Previous studies have demonstrated that synthetic retinoids can not
only be isotype selective but also display a certain degree of
selectivity toward defined receptor-RARE combinations (24). The role of the ligand structure is emphasized by our observations, which suggest that further refinement in gene selectivity could be achieved by altering NCoA interaction surfaces. Selective
recruitment of p300 or CBP has indeed been shown to be required
for selective activation of the gene encoding p21Cip1 and
p27Kip1, respectively (18). Since
transcriptional activation is the end result of multiple interactions
between the receptor, its dimerization partner, DNA, and ligand, one
may speculate that conformationally restricted retinoids with
highly selective biological activities can be designed (24).
Beside the tremendous interest for therapeutical applications, this
raises the possibility that such retinoids display distinctive
abilities to activate endogenous target genes, a hypothesis currently
under investigation in our laboratory.
 |
ACKNOWLEDGMENTS |
We thank R. M. Evans (Salk Institute), J. D. Chen
(University of Massachusetts), V. Cavailles (INSERM U148, Montpellier,
France), D. D. Moore and B. W. O'Malley (Baylor College of
Medicine), and B. Shroot and U. Reichert (CIRD-Galderma) for providing
plasmids and ligands, and we thank E. Thoreau (CIRD-Galderma) for
hRAR
LBD coordinates, constant interest, and input. We are grateful to Hoffmann-La Roche for the gift of 9-cis RA. We thank C. Brand and B. Lefebvre for critical reading of the manuscript and B. Masselot for technical help.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: INSERM U459,
Faculté de Médecine Henri Warembourg 1, place de Verdun,
59045 Lille Cedex, France. Phone: 33.3.20.62.68.87. Fax:
33.3.20.62.68.84. E-mail:
p.lefebvre{at}lille.inserm.fr.
 |
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Retinoic acid and synthetic analogs differentially activate retinoic acid receptor dependent transcription.
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173:339-345[Medline].
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