From the Section of Microbiology, Division of Biological Sciences, University of California, Davis, California 95616
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ABSTRACT |
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Nuclear hormone receptors are ligand-regulated
transcription factors that modulate the expression of specific target
genes in response to the binding of small, hydrophobic hormone ligands. Many nuclear hormone receptors, such as the retinoic acid receptors, can both repress and activate target gene expression; these bimodal transcription properties are mediated by the ability of these receptors
to tether auxiliary factors, denoted corepressors and coactivators.
Corepressors are typically bound by receptors in the absence of cognate
hormone, whereas binding of an appropriate hormone agonist induces an
allosteric alteration in the receptor resulting in release of the
corepressor and recruitment of coactivator. Structural analysis
indicates that there is a close induced fit between the hormone ligand
and the receptor polypeptide chain. This observation suggests that
different ligands, once bound, may confer distinct conformations on the
receptor that may invoke, in turn, distinct functional consequences. We
report here that different retinoids do differ in the ability to
release corepressor once bound to retinoic acid receptor and suggest
that these differences in corepressor release may manifest as
differences in transcriptional regulation.
Nuclear hormone receptors are hormone-regulated transcription
factors that mediate cellular responses to a diverse set of small
lipophilic hormones; these hormones include the steroids, thyroid
hormones, vitamin D3, and retinoic acid (1-7). Nuclear hormone
receptors function by binding to specific DNA sequences, denoted
hormone response elements, and regulating the transcription of adjacent
target genes (1-7). Many nuclear hormone receptors exhibit bimodal
transcriptional properties and can either repress or activate
expression of their target genes, depending on the hormone status, the
nature of the target promoter, and the cell context (1-7). Thyroid
hormone receptors and retinoic acid receptors (RARs)1 typically repress
transcription in the absence of hormone and activate gene expression in
the presence of hormone (8-15).
The bimodal transcriptional properties of the nuclear hormone receptors
reflect the ability of these receptors to physically recruit auxiliary
proteins, denoted corepressors and coactivators, that help mediate the
actual transcriptional response (15-28). In the absence of cognate
hormone, thyroid hormone receptors and RARs bind to a corepressor
complex composed of SMRT and/or N-CoR, mSin3A or B, histone deacetylase
1 or 2, Rb-associated proteins p46 and p48, and a number of additional
proteins of as-yet unknown function (reviewed in Refs. 29 and 30).
Conversely, addition of hormone leads to release of the corepressor
complex by the receptor and the recruitment of a distinct set of
polypeptides that serve as coactivators (reviewed in Ref. 15). Once
tethered to the receptor, corepressors and coactivators appear to
modulate gene transcription through multiple mechanisms that include
modification of the chromatin template and direct interaction with
components of the general transcriptional machinery (29-34). Many of
these same corepressor and coactivator proteins also participate in transcriptional regulation by an assortment of non-receptor
transcription factors (e.g. Refs. 35-40).
The ability of nuclear hormone receptors to toggle between a
corepressor-bound and a coactivator-bound state is therefore a key
aspect of their function. Hormone ligand plays a critical role in this
switching phenomenon, with the binding of cognate hormone by receptor
leading to the release of corepressor and the recruitment of
coactivator (e.g. Refs. 13-17, 20, and 27). Notably,
binding of hormone ligand is believed to cause a series of
conformational changes in the receptor characterized by a global compacting of the polypeptide chain about the ligand and an alteration in the position of the C-terminal X-ray crystallographic studies indicate that the polypeptide chain in
the liganded receptor is in close approximation to the bound hormone,
with the hormone providing a hydrophobic core that helps mediate the
packing of the receptor about it (41-46). This intimate induced-fit
between receptor and ligand suggests that different hormone ligands may
confer distinct polypeptide conformations, and therefore potentially
distinct properties, once bound to a given receptor (41-46). We wished
to investigate if, as a consequence, different ligands differ in their
ability to modulate the recruitment and release of the corepressor
complex. We report here that different retinoid derivatives do indeed
differ in the ability to displace SMRT corepressor from RAR; most
notably, although both 9-cis-retinoic acid and
all-trans-retinoic acid are bound with high affinity by
RAR In Vitro Receptor/Corepressor Binding Assays--
The
glutathione S-transferase (GST), GST-RAR Mammalian Two-hybrid Assays--
The construction of pSG5
vectors expressing either a GAL4 DNA binding domain (DBD)-SMRT fusion
or a GAL4 activation domain (AD)-RAR Protease Resistance
Assays--
[35S]Methionine-radiolabeled, full-length
RAR Transient Transfections--
CV-1 cell transfections were
performed by the same liposome protocol described for the two-hybrid
assay. Approximately 4 × 105 cells were transfected
with 25 ng of pSG5-RAR Different Retinoids Differ in the Ability to Trigger Release of
Corepressor by RAR
We next evaluated the ability of an assortment of other retinoids to
inhibit the interaction of RAR
Intriguingly, there was one notable exception to this general
correlation between affinity of the receptor for the ligand and the
ability of the ligand to induce corepressor release. The 9-cis-retinoic acid isomer, which has an affinity for RAR
It was conceivable that the in vitro translated RAR
To confirm that the differential release of corepressor noted for the
9-cis- and all-trans-isomers of retinoic acid was
not an artifact of our particular methodology, we repeated the GST protocol but employing the reciprocal approach of using a GST fusion
construct of RAR
Our 9-cis-retinoic acid preparation, obtained commercially,
mediated little or no release of corepressor from the receptor at the 5 nM hormone concentration; however, the same
9-cis-retinoic acid preparation did mediate corepressor
release at very high hormone concentrations (e.g. greater
than 50 nM hormone). This dissociation from corepressor at
the highest concentrations of 9-cis-retinoic acid would
appear to be inconsistent with the presumption of a single binding
site, with a single Kd, for the
9-cis-isomer on the receptor, and inconsistent with our
protease resistance data, which indicated substantial or complete
ligand occupancy at the 5 nM 9-cis-retinoic acid
concentration. Notably, 9-cis-retinoic acid is quite labile
chemically, and a variety of enzymatic and non-enzymatic substances can
readily mediate isomerization of 9-cis-retinoic acid to the
all-trans-isomer, with the equilibrium lying strongly in
favor of the latter (e.g. Refs. 53 and 54). Indeed, high
pressure liquid chromatography revealed the presence of 5-10% of
all-trans-retinoic acid in many of our
9-cis-retinoic acid preparations (data not shown). Although
other explanations are possible, we suggest that the release of
corepressor observed at the highest 9-cis-retinoic acid
concentrations is likely due to contamination by the
all-trans-isomer, either due to pre-existing all-trans-retinoic acid in the 9-cis-retinoic
acid preparation or due to isomerization of the 9-cis-isomer
during the incubation period (see "Discussion").
The Impaired Ability of 9-cis-Retinoic Acid to Release Corepressor
Was Unaltered by Addition of DNA or Mammalian Cell Extracts to the in
Vitro Assay and Was Also Observed in a Mammalian Two-hybrid
Protocol--
Although 9-cis-retinoic acid was impaired in
the ability to release corepressor from RAR
We next employed a mammalian cell two-hybrid protocol to determine if
the isomer-specific differences in corepressor release observed
in vitro were also observed in vivo. For this
assay, a GAL4-DNA binding domain (GAL4-DBD) fusion of SMRT was
cointroduced into CV-1 cells together with a GAL4-activation domain
(GAL4-AD) fusion of RAR The Differential Effects of 9-cis- and All-trans-retinoic Acid Are
Also Observed Using the Oncogenic PML-RAR The Differential Ability of 9-cis- and All-trans-retinoic Acid to
Release Corepressor May Manifest as Differences in the Transcriptional
Activity of RAR
We repeated these experiments but using a native RAR All-trans- and 9-cis-Retinoic Acid Confer Indistinguishable Effects
on the Conformation of the C-terminal Helix 12 of RAR Different Ligands That Bind to RAR
Consistent with our hypothesis, we observed that although both
9-cis- and all-trans-retinoic acid bind to RAR
We have considered a variety of alternative explanations for our
observations that might arise from experimental artifact. As previously
noted, we confirmed that the RAR
Although impaired in corepressor release at moderate (<50
nM) hormone concentrations, 9-cis-retinoic acid
did release corepressor from RAR
Although somewhat unanticipated, our results are not without precedent.
Crystallographic analysis of the estrogen receptor indicates that the
C-terminal helix 12 domain of this receptor assumes a dramatically
different conformation when the receptor is bound to a ligand agonist
versus an antagonist (44). As a consequence, the
antagonist-bound estrogen receptor is unable to efficiently bind
certain coactivators and is impaired in transcriptional activation.
Notably, the 9-cis-retinoic acid isomer displays a significantly different topography, manifested as an orthogonal bend in
the center of the retinoid side chain, from that of the all-trans-isomer. Although the specific conformation of
RAR The Differing Abilities of 9-cis- and All-trans-Retinoic Acid to
Induce Corepressor Release May Reflect Different Roles for These Two
Hormones in the Biology of RAR
INTRODUCTION
Top
Abstract
Introduction
References
-helical domain (helix 12) from an
extended to a sequestered conformation (41-47).
, only the all-trans-isomer efficiently induces
release of SMRT. All-trans- and 9-cis-retinoic
acid are natural products found in normal physiological contexts; our
results suggest that these two retinoids may exert distinct allosteric
effects on RAR
and may therefore mediate distinct aspects of RAR biology.
EXPERIMENTAL PROCEDURES
, and
GST-SMRT/TRAC-2 (codons 751-1495) constructs were created as described previously (20, 27, 37). GST fusion proteins were expressed in
Escherichia coli, isolated, and immobilized by binding to
glutathione agarose. [35S]Methionine-radiolabeled,
full-length RAR
, PML-RAR
, or SMRT proteins were synthesized by a
coupled in vitro transcription and translation protocol
(Promega, Madison, WI). The radiolabeled proteins were incubated with
the immobilized GST fusion proteins in HEMG buffer (20); the agarose
matrix was then extensively washed, and proteins remaining bound to the
matrix were eluted with soluble glutathione and analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The
electrophoretograms were visualized by autoradiography and were
quantified by STORM PhosphorImager analysis (Molecular Dynamics,
Sunnyvale, CA). Mutants of RAR
defective for association with the
SMRT corepressor (denoted RAR
AHT mutants) were created by
site-directed mutagenesis (17, 37).
fusion has been previously
described (37, 48). The RAR
(AHT) mutation was transferred into the
pSG5-GAL4AD background in the form of an EcoRV to
XhoI fragment generated by polymerase chain reaction. The
actual transfections were performed in 12-well tissue culture plates
containing 7 × 104 CV-1 cells per well. Transient
transfections were performed employing a liposome/Lipofectin
methodology (Life Technologies, Inc.), using 25 ng of pSG5 GAL4DBD
vector, 100 ng of pSG5 GAL4AD vector, 100 ng of pGL2-GAL4 (17-mer)
luciferase reporter plasmid, and 100 ng of a
pCMV-promoter-lacZ reporter plasmid (used as an internal control) per well. The cells were subsequently incubated for 48 h
in the presence or absence of all-trans- or
9-cis-retinoic acid and harvested, and the luciferase and
-galactosidase activities were determined as previously detailed
(27, 34, 37, 49).
was synthesized in vitro by the coupled transcription
and translation protocol. For the trypsin and elastase assays, 1 µl
of radiolabeled protein was diluted to 16 µl in 50 mM
Tris-Cl (pH 7.4) buffer containing various concentrations of retinoid
hormone or an equivalent volume of ethanol carrier. Proteolysis was
initiated by adding 4 µl of protease (either 2 µg/µl type IV
elastase or 0.125 µg/µl trypsin pretreated with
tosyl-L-phenylalanine chloromethyl ketone; Sigma). After a
10-min incubation at room temperature, proteolysis was terminated by
addition of 14 µl of SDS-PAGE sample buffer, and the samples were
frozen rapidly by transfer to dry ice. The samples were subsequently
boiled for 10 min and resolved by SDS-PAGE. The electrophoretograms
were visualized by STORM PhosphorImager analysis.
, 100 ng of a reporter construct representing
the thymidine kinase promoter joined to luciferase and driven by a
-RARE, and 100 ng of the pCMV-lacZ plasmid employed as an
internal control. Alternatively, the cells were transfected with 25 ng
of a pSG5-GAL4DBD-RAR
construct, 100 ng of the
pGL2-GAL4(17-mer)-luciferase reporter, and 100 ng of the
pCMV-lacZ internal control. Retinoid hormone was added (or
not) 5 h post-transfection, and the cells were harvested at 48 h, and relative luciferase activity (normalized to
-galactosidase activity) was subsequently determined.
RESULTS
--
The SMRT polypeptide represents a principal
site of contact between the nuclear hormone receptors and the
corepressor complex (16, 17, 19, 20-22, 24, 28). We therefore
investigated the effects of different retinoid derivatives on the
association in vitro between SMRT and RAR
by employing a
"GST-pull down" assay. A glutathione S-transferase (GST)
derivative of SMRT was synthesized in E. coli, purified, and
immobilized on a glutathione-agarose matrix (48). Radiolabeled,
full-length RAR
, synthesized by in vitro transcription
and translation, was then incubated with the GST-SMRT matrix (20, 27).
After incubation, the matrix was extensively washed, and any
radiolabeled RAR
remaining bound to the matrix was subsequently
eluted with soluble glutathione, resolved by SDS-PAGE, and quantified
by PhosphorImager analysis (20, 27). As we and others have previously
reported (16, 17, 20, 22, 27, 34), radiolabeled RAR
bound
efficiently to the GST-SMRT matrix in the absence of hormone but not to
a non-recombinant GST construct employed as a negative control (Fig. 1). Also as previously noted (20), the
ability of RAR
to bind to SMRT was severely inhibited by inclusion
of either 50 or 500 nM all-trans-retinoic acid
in the binding assay (Fig. 1).
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Fig. 1.
Different retinoids have different effects on
the binding of RAR to GST-SMRT.
Radiolabeled, full-length RAR
was synthesized by in vitro
transcription and translation and was incubated with a GST-SMRT (codons
751-1495) construct immobilized on a glutathione-agarose matrix. The
incubation buffer contained either hormone-free ethanol carrier
(none), 50 or 500 nM of the retinoids indicated.
RAR
bound to the GST-SMRT matrix after incubation was extensively
washed, eluted with soluble glutathione, resolved by SDS-PAGE, and
quantified by PhosphorImager analysis. The amount of receptor bound to
the GST-SMRT matrix in the absence of hormone was defined as 100%.
RAR
binding to non-recombinant GST, immobilized to
glutathione-agarose and employed as a negative control, was typically
2% or less under identical conditions.
with SMRT corepressor. In general,
the ability of a given retinoid to mediate release of the corepressor
paralleled the binding affinity of RAR
for that retinoid. For
example, RAR
possesses little or no measurable affinity for the
ret-aldehyde derivatives all-trans-retinal and 9-cis-retinal (49), and consistent with this observation,
neither retinal derivative significantly inhibited binding of RAR
to the GST-SMRT matrix at hormone concentrations up to 500 nM
(Fig. 1). Similarly, all-trans-retinol failed to bind to
RAR
, and 500 nM all-trans-retinol failed to
measurably release the SMRT corepressor from RAR
(Fig. 1). In
contrast, 13-cis-retinoic acid is bound by RAR
, although
with a lower affinity than that for all-trans-retinoic acid
(49), and 13-cis-retinoic acid released SMRT from RAR
at
the 500 nM but not at the 50 nM concentration
of this hormone (Fig. 1). Although not unexpected, these results
support the hypothesis that retinoids mediate release of corepressor
exclusively through the ability to bind to the ligand pocket of the receptor.
virtually equal to that of all-trans-retinoic acid (50), was
markedly inefficient in mediating the release of SMRT (Fig.
2A). Under our in
vitro conditions, 10-fold more of the 9-cis-retinoic
acid preparation was required, relative to
all-trans-retinoic acid, to obtain an equivalent inhibition
of the SMRT/RAR
interaction (Fig. 2B). Both the
9-cis- and all-trans-retinoid preparations were
prepared identically, and the concentrations were confirmed spectrophotometrically to ensure that the amounts of each ligand were
indeed equal.
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Fig. 2.
The all-trans- and
9-cis-isomers of retinoic acid have distinct effects
on the association between RAR and
GST-SMRT. A, an electrophoretogram is depicted
illustrating the ability of RAR
to bind to GST-SMRT under conditions
of increasing retinoid concentration. Radiolabeled, full-length RAR
was incubated with GST-SMRT (codons 751-1495) in the presence of
different concentrations of all-trans- or
9-cis-retinoic acid, as indicated in the panel. RAR
bound
to the immobilized GST-SMRT was subsequently washed extensively, eluted
with soluble glutathione, and visualized by SDS-PAGE and
autoradiography. Binding of radiolabeled RAR
to non-recombinant GST
(GST), employed as a negative control, was also determined
in parallel. B, the binding of RAR
to GST-SMRT under
conditions of increasing retinoid concentration was quantified.
Experiments performed as described for A were quantified by
PhosphorImager analysis, and the percentage of the input RAR
bound
to the GST-SMRT construct at the different hormone concentrations was
determined. The average and standard deviation of two or more
experiments are presented. Both wild-type RAR
(WT) and a
mutant form of RAR
defective for SMRT association (AHT mutant) were
tested. The amount of wild-type RAR
bound by a non-recombinant GST
construct, employed as a negative control, was also determined
(×).
protein, as employed in our assay, might be misfolded or in some other manner selectively impaired in the binding of the
9-cis-isomer. Binding of hormone is known to result in
formation of a more compact conformation of the nuclear hormone
receptors that manifests as an increase in resistance to proteolysis
(47, 51, 52). We employed this acquisition of protease resistance as an
indicator of receptor ligand occupancy, thereby permitting us to
monitor hormone binding by the radiolabeled RAR
preparations
employed in our GST-pull down protocol (Fig.
3). Notably, as little as 5 nM of either retinoic acid isomer clearly protected the
in vitro translated RAR
from proteolytic degradation by
either elastase or trypsin (Fig. 3). These results suggest that RAR
is fully or near-fully occupied by 9-cis-retinoic acid at
concentrations of this hormone that fail to mediate release of SMRT
corepressor in the GST-pull down assay.
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Fig. 3.
The all-trans- and
9-cis-isomers of retinoic acid are bound at comparable
levels by RAR . A protease resistance
assay was employed as a measure of hormone binding by the receptor.
Radiolabeled RAR
, synthesized by in vitro transcription
and translation, was incubated with either hormone-free ethanol carrier
or increasing amounts of 9-cis- or
all-trans-retinoic acid (RA). Protease (either
elastase or trypsin) was then added, and the samples were incubated at
room temperature for 10 min. After the reaction was terminated by
addition of SDS sample buffer, the samples were resolved by SDS-15%
PAGE system, and the RAR
-derived peptides resistant to proteolysis
were visualized by autoradiography. A, treatment with
elastase. B, treatment with trypsin.
(synthesized in E. coli) and an in
vitro transcribed and translated derivative of SMRT (Fig.
4). Despite the reciprocal methodologies
employed, the same pattern was observed as before:
all-trans-retinoic acid efficiently released the
SMRT-polypeptide from the GST-RAR
construct, whereas
9-cis-retinoic acid was comparatively impaired in this
property (Fig. 4).
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Fig. 4.
All-trans- and
9-cis-retinoic acid have distinct effects on the
association between SMRT and GST-RAR . The
same general protocol as described for Fig. 2B was repeated
but in a reciprocal manner, using radiolabeled SMRT protein synthesized
by in vitro transcription and translation and a GST-RAR
fusion protein isolated from E. coli. The amount of SMRT
protein bound to the GST-RAR
construct in the presence of increasing
amounts of hormone was quantified. The ability of SMRT to bind to
non-recombinant GST, employed as a negative control, is also indicated
(×).
in vitro, it
was possible that this ligand might have a different effect if the
RAR
was bound to a DNA response element or if additional factors,
present in vivo, were included in the GST assay (18). We
therefore repeated the in vitro GST-pull down assay in the
presence of a large molar excess of an oligonucleotide containing a
consensus retinoic acid response element or in the presence of nuclear
lysates derived from CV-1 mammalian cells (Fig.
5). Neither the addition of the DNA-binding site nor the presence of mammalian cell extracts altered our initial results; SMRT corepressor was efficiently released by
all-trans-, but not 9-cis-, retinoic acid (Fig.
5).
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Fig. 5.
Addition of DNA or nuclear extracts has no
detectable effect on the 9-cis-retinoic
acid-refractory association of RAR with
SMRT. Radiolabeled, full-length RAR
was incubated with GST-SMRT
(codons 751-1495) in the absence of any additions, in the presence of
50 nM 9-cis-retinoic acid, in the presence of 50 nM 9-cis-retinoic acid plus nuclear extracts
derived from 6 × 105 CV-1 cells, or in the presence
of 9-cis-retinoic acid plus 200 ng of a deoxyoligonucleotide
containing a strong binding site for RAR
(
RARE). The
amount of RAR
bound to the GST-SMRT protein was quantified; the
average of duplicate experiments and the range are presented.
. An interaction between the GAL4-DBD-SMRT
and GAL4AD-RAR
proteins results in reconstitution of a functional
GAL4 transcription factor, which we measured by assaying the expression
of a GAL4 (17-mer) luciferase reporter gene (Fig.
6). Neither of the GAL4-fusion constructs
alone are able to stimulate luciferase reporter expression under these
conditions (Fig. 6 and data not shown); similarly, introduction of
mutations into either SMRT or into RAR
that are known to disrupt the
SMRT/RAR interaction in vitro also impair the two-hybrid
interaction (55). The strong two-hybrid interaction observed for SMRT
and RAR
in the transfected CV-1 cells was severely inhibited by the
addition of all-trans-retinoic acid to the culture medium,
with 5 nM all-trans-retinoic acid reducing the
luciferase activity by more than 50%. By comparison,
9-cis-retinoic acid was significantly impaired in this
inhibition, with 25 nM 9-cis-retinoic acid
required to produce an inhibition equivalent to that produced by 5 nM of the all-trans-isomer. Therefore our
results in vivo appear to parallel those in vitro
and demonstrate a differential release of SMRT corepressor in response
to 9-cis-, versus all-trans-, retinoic
acid.
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Fig. 6.
The distinctive effects of
all-trans- and 9-cis-retinoic acid on
the RAR /SMRT interaction are also observed in
a mammalian two-hybrid assay. A pSG5-GAL4DBD-SMRT fusion vector
was introduced into CV-1 cells together with a pSG5-GAL4AD-RAR
fusion vector and a GAL4 (17-mer) luciferase reporter. The cells were
subsequently incubated in the presence of different concentrations of
either all-trans- or 9-cis-retinoic acid, as
indicated. The cells were harvested, and the relative luciferase
activity was determined and normalized to that of a
pCMV-lacZ plasmid introduced as an internal control. Both a
wild-type RAR
construct (RAR) and a mutant RAR
construct defective for SMRT association (RAR-AHT) were
tested. The activity of an empty pSG5-GAL4AD construct, used in place
of the pSG5-GAL4AD-RAR
fusion as a negative control, was also
determined (×). The data represent the average and range of
duplicate experiments.
Protein--
Human acute
promyelocytic leukemias are associated with chromosomal translocations
that create chimeric polypeptides in which the N terminus of RAR
is
been replaced by novel protein sequences, such as that of PML
(promyelocytic leukemia protein) (56). The ability of these chimeric
RAR
derivatives to tether corepressor has been implicated in their
oncogenic activities; consistent with this hypothesis, treatment with
all-trans-retinoic acid both releases SMRT from PML-RAR
and induces differentiation in leukemic cells arising from PML-RAR
translocations, leading to clinical regression (37-39, 56). We
therefore examined the response of PML-RAR
to
9-cis-retinoic acid. Notably, 9-cis-retinoic acid failed to release SMRT from PML-RAR
under conditions in which the
all-trans-isomer mediated significant release of SMRT (Fig. 7). We conclude that the differential
corepressor release observed for all-trans- and
9-cis-retinoic acid extends to the oncogenic PML-RAR
oncoprotein.
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Fig. 7.
The all-trans- and
9-cis-isomers of retinoic acid also have distinct
effects on the association between SMRT and the
PML-RAR oncoprotein. Radiolabeled
PML-RAR
was incubated with GST-SMRT (codons 751-1495) in the
presence of different concentrations of all-trans- or
9-cis-retinoic acid, as indicated in the panel. PML-RAR
bound to the immobilized GST-SMRT was subsequently washed extensively,
eluted with soluble glutathione, and quantified by SDS-PAGE and
PhosphorImager analysis. Binding of radiolabeled PML-RAR
to
non-recombinant GST, employed as a negative control, was also
determined in parallel (×).
--
Given the different capacities of 9-cis-
and all-trans-retinoic acid to release corepressor, it
was plausible that these two hormone ligands might exhibit distinct
transcriptional activation profiles in cells. We therefore examined the
ability of these different ligands to convert RAR
from a repressor
to an activator. We initially employed a GAL4-DBD fusion of RAR
,
restricted to the hormone binding domain, together with a GAL (17-mer)
reporter plasmid, so as to minimize interference by endogenous RARs and RXRs present in the CV-1 cells (Fig.
8A). As expected, introduction of the GAL4DBD-RAR
fusion in the absence of hormone repressed the
expression of the Gal 17-mer reporter construct (Fig. 8A). Addition of all-trans-retinoic acid converted the
GAL4DBD-RAR
from a repressor into an activator, with approximately
100 nM all-trans-retinoic acid required for
induction of maximal reporter gene activity. In contrast, significantly
more 9-cis-retinoic acid, 1000 nM, was required
for maximal activation under identical circumstances. This differential
response to the two hormone isomers is consistent with our in
vitro data and suggests that an impaired release of corepressor at
low 9-cis-retinoic acid concentrations may attenuate
the ability of the GAL4-RAR
construct to toggle from a repressor to
an activator.
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Fig. 8.
All-trans- and
9-cis-retinoic acid differ in the ability to induce
RAR -mediated transcription. A,
the 9-cis-isomer was impaired in inducing transcriptional
activation by a GAL4DBD-RAR
fusion protein on a GAL4 (17-mer)
reporter. A pSG5-GAL4AD-RAR
fusion vector was introduced into CV-1
cells together with the GAL4 (17-mer) luciferase reporter. The cells
were subsequently incubated in the presence of increasing amounts of
either all-trans- (filled columns) or
9-cis-retinoic (hatched columns) acid, as
indicated. The cells were harvested, and the relative luciferase
(Rel. Luciferase) activity was determined and normalized to
that of a pCMV-lacZ plasmid introduced as an internal
control. The activity of an empty pGAL4DBD construct, used in place of
the pSG5-GAL4DBD-RAR
fusion as a negative control, was also
determined (open columns). The data represent the average
and range of duplicate experiments. B, the
9-cis-isomer was more effective than
all-trans-retinoic acid in inducing transcription activation
by the native RAR
on a
-RARE-luciferase reporter. A pSG5
construct expressing native RAR
was introduced into CV-1 cells
together with a thymidine kinase promoter-luciferase reporter
containing three copies of the
-RARE response element. The cells
were treated with hormone and harvested as in A, and the
relative luciferase activity was determined and normalized to
that of a pCMV-lacZ plasmid. The activity of an empty pSG-5
construct was also determined (open columns). The data
represent the average and range of duplicate experiments.
construct and a
reporter containing a retinoic acid response element (Fig.
8B). In contrast to our results with the GAL4DBD-RAR
derivative, native RAR
actually activated transcription more
efficiently in response to the 9-cis-retinoic acid than to
the all-trans-isomer (Fig. 8B). However, it
should be noted that in the absence of hormone the native RAR
also
failed to function as a transcriptional repressor in the CV-1 cell
background (Fig. 8B; compare the levels of reporter
expression in the absence and presence of receptor at the zero hormone
concentration). This may be due to the differing natures of the two
different reporter constructs or to the presence of an additional
activation domain (denoted AF-1) in the N terminus of the native RAR
that is not present in the GAL4DBD-RAR
fusion (57). Whatever the
precise mechanistic basis of this phenomenon, it appears that the
response of RAR
to 9-cis-retinoic acid can be measurably
different from that of all-trans-retinoic acid, depending on
the target gene and the nature of the receptor construct.
--
A change
in the conformation of the C-terminal helix 12 of RARs and thyroid
hormone receptors has been proposed to play an important role in the
release of corepressor (24, 41-47). To determine if 9-cis-
and all-trans-retinoic acid differ in the ability to invoke
this conformational change in helix 12, we applied an exopeptidase
technique. If, as proposed from crystallographic analysis, the C
terminus of the unliganded RAR
is in an extended conformation, it
might be expected that the unliganded receptor would be relatively
susceptible to degradation by an exopeptidase such as carboxypeptidase
Y. Conversely, if the C terminus of RAR
becomes more sequestered
from solvent on addition of hormone, the liganded receptor would be
expected to be more resistant to carboxypeptidase Y degradation (47).
These predictions were fulfilled in experiments utilizing
all-trans-retinoic acid; RAR
was significantly more
susceptible to carboxypeptidase Y in the absence versus in
the presence of the all-trans-isomer, suggesting that the C
terminus of the receptor is more sequestered in the presence of the
all-trans-isomer (Fig. 9).
Intriguingly, a virtually indistinguishable protection from
carboxypeptidase Y degradation could be observed at analogous
concentrations of 9-cis-retinoic acid (Fig. 9). We conclude
that both all-trans- and 9-cis-retinoic acid
confer detectable sequestration of the receptor C terminus, and that
the differences in corepressor release observed for these two ligands
must therefore reside in alterations to the conformation of the
receptor not visualized by our exopeptidase assay. These differences
may represent alterations in the ligand-induced folding of other
domains of the RAR
or may represent alterations in the conformation
of helix 12 that cannot be resolved by the carboxypeptidase Y
methodology.
View larger version (16K):
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Fig. 9.
Both all-trans- and
9-cis-retinoic acid induce sequestration of the C
terminus of RAR . A carboxypeptidase Y
resistance assay was employed as a measure of the accessibility of the
RAR
C terminus to solvent under different hormone conditions (47).
Radiolabeled RAR
, synthesized by in vitro transcription
and translation, was incubated with either hormone-free ethanol carrier
or increasing amounts of 9-cis- or
all-trans-retinoic acid. Carboxypeptidase Y was then added
(4 µg per reaction), and the samples were incubated for 10 min at
room temperature. After the reaction was terminated by addition of
SDS-sample buffer, the samples were resolved by SDS-PAGE, and the
amount of full-length (undegraded) RAR
remaining was quantified by
PhosphorImager analysis. The results of duplicate experiments, and the
range, are presented.
DISCUSSION
Can Invoke Distinct
Functional Responses--
Binding of hormone by nuclear hormone
receptors is believed to result in a series of conformational changes
in the ligand binding domain of these polypeptides. These
conformational changes consist of a global compaction of the
polypeptide in a tight induced-fit about the hormone ligand, a flip in
the conformation of an "omega loop," and a reorientation in the
position of the C-terminal helix 12 (41-46). Concomitant with these
rearrangements, and presumably mediated by them, the receptor loses its
affinity for the SMRT/N-CoR corepressor complex and acquires instead
the ability to bind certain coactivator polypeptides. In essence,
hormone provides a hydrophobic core, around which the receptor
polypeptide chain assumes its final conformation. This close
approximation between hormone and liganded receptor suggests that
structurally distinct ligands might induce distinct receptor
conformations, which in turn might display different propensities to
recruit or release transcriptional cofactors. To test this hypothesis,
we examined the ability of different retinoid ligands to release the
SMRT corepressor complex from RAR
; we chose this particular
experimental model due to the important role of corepressor in RAR
function, the accessibility of a wide assortment of well characterized
retinoid ligands, and the availability of a high resolution
crystal-derived structure for the liganded RAR
polypeptide (5,
42).
with near equal affinities, only the all-trans-isomer
induces the release of SMRT corepressor efficiently. We could
demonstrate this differential corepressor release both in a GST assay
in vitro and in a mammalian two-hybrid protocol in
vivo. RAR
displays this hormone-specific differential SMRT
release at concentrations of all-trans- and 9-cis-retinoic acid that, by protease assay, result in an
indistinguishable ligand occupancy of the receptor by the two isomers.
We interpret these results to suggest that the differential release of
SMRT corepressor is an allosteric phenomenon and that binding of
all-trans-retinoic acid by RAR
confers a set of
conformational changes in the receptor that result in corepressor
release; binding of 9-cis-retinoic acid, in contrast, fails
to invoke one or more components of this set of conformational changes
and thus fails to release corepressor in our assay.
preparation employed in our
in vitro assays actually binds 9-cis- and
all-trans-retinoic acid with comparable avidities. We also
demonstrated that the impaired release of corepressor observed in
vitro was mimicked in a two-hybrid assay in vivo. We
were concerned that the all-trans- and
9-cis-retinoic acid might be differentially adsorbed by
components of the in vitro translation mix or by the walls
of the assay tubes, thereby lowering the effective concentration of the
9-cis-hormone available for binding by the receptor.
Although our protease protection assay (demonstrating equal occupancy
at nominally equal hormone concentrations) would appear to argue
against both of these scenarios as potential artifacts, we also
repeated our assays using siliconized tubes with no alteration in the
results.2 Similarly, we also
modified the experimental protocol so as to first bind the radiolabeled
RAR
to the GST-SMRT matrix; we then washed the matrix extensively
and subsequently challenged the now-purified GST-SMRT·RAR
complex
with retinoid; once again, the all-trans-retinoic acid
efficiently released receptor from the corepressor, whereas the
9-cis-isomer was significantly impaired in this function. We
conclude that the difference we observed in the ability of
all-trans- and 9-cis-retinoic acid to release corepressor from RAR
is most likely an authentic reflection of an
actual biological difference in the properties of these two hormone isomers.
at high (>50 nM)
hormone concentrations. This may be simply due to isomerization of a
small percentage of the 9-cis-retinoic acid to the
all-trans-form prior to or during the experimental protocol;
notably the chemical equilibrium lies greatly in favor of this
transition and many common chemical reagents can help catalyze this
isomerization (53, 54). Alternatively, although less likely, we cannot
exclude the possibility that there are actually two populations of
RAR
in our preparation as follows: one that binds
9-cis-retinoic acid with a high affinity, but fails to
release corepressor, and one that binds 9-cis-retinoic acid with a lower affinity but does, in response, release corepressor. These
two RAR
populations might represent receptor dimers
versus monomers, for example, or might represent two
conformationally distinct forms of RAR
monomer (e.g.
Refs. 58-60). Whatever the molecular basis for this phenomenon, our
studies indicate that the effects of all-trans- and
9-cis-retinoic acid on RAR
are clearly distinguishable at
physiological concentrations of hormone.
bound to 9-cis-retinoic acid has not been
experimentally determined, molecular modeling and genetic analysis
suggests that the 9-cis-isomer can be accommodated in the
binding pocket of RAR
but that the 9-cis-isomer would
differ in its contacts with receptor helices 3, 11, and 12 relative to
those mediated by the all-trans-isomer (42, 61). Given that
these helices play important roles in the allosteric changes observed
for RAR
on binding of all-trans-retinoic acid (41-46),
it is plausible that binding of the 9-cis-isomer may result
in a distinct, if subtle, reorientation of one or more of these
essential helices, resulting in impaired release of the SMRT corepressor.
--
Although
9-cis-retinoic acid hormone was first identified as a
naturally occurring ligand for the retinoid X receptors (RXRs), 9-cis-retinoic acid is bound by RARs at a substantially
higher affinity than by RXRs, and the affinity of RARs for the
9-cis-isomer is comparable to the affinity of RARs for its
archetypic ligand, all-trans-retinoic acid (50). Therefore,
although 9-cis-retinoic acid has traditionally been thought
of as an inducer of RXR-mediated responses, this same retinoid isomer
also represents an extremely potent mediator of RAR-mediated responses.
What might be the evolutionary rationale of having two distinct,
naturally occurring hormone isomers able to bind to a single given
receptor? Our results suggest that the 9-cis-isomer might
play a previously unanticipated role: that of an allosteric effector
able to bind to RAR
but impaired in the ability to release
corepressor. Thus, the transcriptional response of RAR
to
9-cis-retinoic acid might be anticipated to be different
from that to all-trans-isomer, at least in certain contexts,
and this might allow physiologically distinct roles for the two isomers
in RAR biology. Our work suggests that 9-cis-retinoic acid
exhibits an impaired ability to toggle GAL4DBD-RAR
constructs from
repressors to activators of transcription. However, this impaired
response to 9-cis-retinoic acid is not invariably observed under all conditions. In fact, native RAR
on a retinoic response element exhibited the opposite effect, with the
9-cis-retinoic acid isomer conferring transcriptional
activation at lower hormone levels than required for
all-trans-retinoic acid. There are several plausible
explanations for this apparent discrepancy. For example, the retinoic
response elements employed in the latter experiments are likely to bind
RXR/RAR heterodimers, and the RXR component may mediate the enhanced
response to the 9-cis-retinoic acid. Alternatively, an
N-terminal, hormone-independent transcriptional activation domain,
denoted AF-1, is present in the native RAR
but absent in our
GAL4DBD-RAR
construct and may be responsible for overcoming the
effect of bound corepressor by activating transcription even in the
presence of a bound corepressor. It is also possible that other
differences in the nature of the reporter constructs, or the precise
conditions employed, generate the distinct retinoid responses we report
here. Whatever the mechanistic basis, it appears that distinct
retinoids can invoke distinct transcriptional responses once bound to
RAR
and thus are potentially able to mediate distinct physiological
consequences. We suggest that the multiplicity of retinoids isolated
from nature reflects this multiplicity of allosteric effects that these
retinoids can invoke in their cognate receptors.
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ACKNOWLEDGEMENTS |
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We thank P. Chambon for generously providing
the RAR molecular clone adapted for use in this research and C. Meares for advice and providing access to a high pressure liquid chromatograph.
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FOOTNOTES |
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* This work was supported by Public Health Services/National Institutes of Health Grants R37 CA-53394 and R01 DK-53528.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 530-752-3013;
Fax: 530-752-9014; E-mail: mlprivalsky{at}ucdavis.edu.
The abbreviations used are: RARs, retinoic acid receptors; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; DBD, DNA binding domain; AD, activation domain; RXR, retinoid X receptors; PML, promyelocytic leukemia; RARE, retinoic acid response element.
2 S.-H. Hong and M. L. Privalsky, unpublished data.
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REFERENCES |
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