Three Amino Acids Specify Coactivator Choice By Retinoid X Receptors
Gang Shao1,
Richard A. Heyman1 and
Ira G. Schulman1
Nuclear Receptor Discovery Ligand Pharmaceuticals, Inc. San Diego, California 92121
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ABSTRACT
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Binding of agonists to nuclear receptors results
in a conformational change in receptor structure that promotes
interaction between activated receptors and coactivators.
Receptor-coactivator interactions are mediated by the agonist-dependent
formation of a hydrophobic pocket on the part of
receptors, and short leucine-rich sequences termed LxxLL motifs or
nuclear receptor boxes present in coactivators. RXR-PPAR
(retinoid X
receptor-peroxisome proliferator-activated receptor-
) heterodimers
play important roles in adipocyte and macrophage differentiation and
have been implicated as therapeutic targets in diabetes,
atherosclerosis, and cancer. Analysis of interactions between
RXR-PPAR
heterodimers and coactivator nuclear receptor boxes
suggests that RXR and PPAR
can distinguish among coactivators by
recognizing distinct structural features of nuclear receptor boxes. The
results also indicate that coactivator choice by RXR is mediated by
three nonconserved amino acids of the nuclear receptor box. The ability
of an optimized seven-amino acid nuclear receptor box to specifically
interact with RXR and function as a selective inhibitor suggests the
coactivator-binding pocket may serve as a new target for drug
discovery.
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INTRODUCTION
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Nuclear receptors are hormone-dependent transcription factors that
play essential roles in development, differentiation, cell
proliferation, and metabolism by controlling the expression of complex
genetic networks. Members of the nuclear receptor superfamily are
characterized by a conserved structural and functional organization
consisting of a heterogeneous amino-terminal domain, a highly conserved
central DNA-binding domain, and a relatively large carboxy-terminal
ligand-binding domain (LBD). The LBD is functionally complex and
mediates ligand binding, receptor homo- and heterodimerization,
repression of transcription in the absence of ligand, and
ligand-dependent activation of transcription (1).
Crystal structures of several LBDs support molecular and biochemical
studies indicating that ligand binding promotes a conformational change
in receptor structure. What appears to be a relatively flexible
conserved helix near the carboxy terminus (helix 12) occupies
unique positions when structures of unliganded, agonist-occupied,
and antagonist-occupied LBDs are compared. Importantly, mutagenesis
experiments indicate that helix 12, referred to as activation function
2 (AF-2), is necessary for ligand-dependent transactivation by nuclear
receptors (2, 3, 4, 5, 6). Recent work indicates the AF-2 helix contributes an
essential surface to the formation of an agonist-dependent hydrophobic
pocket that serves as a binding site for coactivators. The alternative
positions occupied by the AF-2 helix in the unliganded or
antagonist-occupied conformations preclude the formation of this
binding pocket (2, 5, 6, 7, 8).
Three major classes of coactivators have been studied in the context of
ligand-dependent transcription by nuclear receptors. CREB binding
protein (CBP)/p300 and the steroid receptor coactivators (SRCs; also
referred to as p160 proteins) were identified using two-hybrid and
in vitro protein-protein interaction assays and function as
coactivators in transfection and microinjection experiments (9, 10, 11, 12, 13, 14).
The thyroid receptor activating proteins (TRAPs)/vitamin D interacting
proteins (DRIPs) represent identical protein complexes that were
purified based upon their ability to interact with thyroid hormone or
vitamin D receptors and promote ligand-dependent transcription in
vitro (for review see Ref. 15). Interestingly, the ability of
CBP/p300, SRCs, TRAP/DRIP, and several other cofactors to interact with
nuclear receptors is mediated by a common signature motif, LxxLL
(L = leucine, x = any amino acid), present once to several
times within coactivator sequences. Coactivator domains encompassing
LxxLL motifs shown to be necessary for receptor interaction have been
referred to as nuclear receptor (NR) boxes. When receptor-coactivator
interactions are examined, individual receptors exhibit distinct but
overlapping coactivator preferences (16, 17, 18, 19, 20, 21).
Retinoid X receptors (RXRs) occupy a central position in nuclear
receptor-mediated gene expression by functioning as obligate
heterodimeric partners for a large number of receptors including
retinoic acid, thyroid hormone, vitamin D, and peroxisome
proliferator-activated receptors (22). The observation that peroxisome
proliferator-activated receptor-
(PPAR
) plays essential roles in
adipocyte and foam cell differentiation and that a class of
antidiabetic drugs, the thiazolidinediones, are PPAR
agonists has
stimulated great interest in RXR-PPAR
heterodimers (23, 24). In this
regard, we have shown that this dimeric transcription factor can
regulate gene expression in response to agonists that bind either RXR
or PPAR
(25, 26). Interestingly, RXR and PPAR
exhibit distinct
coactivator preferences. RXR makes a relatively strong interaction with
SRC-1 compared with CBP, while PPAR
prefers CBP relative to SRC-1
(26, 27). In this work we show that RXRs preference for SRC-1 is
determined by three amino acids in and around the LxxLL motif and that
RXR and PPAR
utilize different strategies to distinguish between
SRC-1 and CBP. These different modes of NR box recognition suggest it
will be possible to identify receptor-specific small molecule
antagonists that function by blocking access to the coactivator binding
pocket.
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RESULTS
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Interaction of RXR-PPAR
with Single NR Boxes
We and others have shown, using two-hybrid and in vitro
protein-protein interaction assays, that RXR and PPAR
exhibit
opposite preferences for the coactivators SRC-1 and CBP (26, 27). To
determine whether the ability of RXR and PPAR
to distinguish between
coactivators results from the recognition of single NR boxes, mammalian
two-hybrid assays were used to examine interactions between NR boxes
fused to the DNA-binding domain of GAL4 and the two receptors either
individually or as heterodimers (Figs. 1
and 2
). Each NR box used in this study
consisted of 20 amino acids (Fig. 1A
); eight amino acids amino terminal
to the LxxLL motif (position -8 to position
-1), the five-amino acid LxxLL motif
(+1 to +5), and seven
carboxy-terminal amino acids (+6 to
+12). The analyses in Fig. 1
, BD demonstrate
that the RXR-specific agonist LGD1268 (black bars) promotes
interactions between RXR and the three NR boxes derived from SRC-1 with
a slight binding preference for NR box III (compare bars 35 in Fig. 1B
, average of eight independent experiments). Little or no RXR
agonist-dependent interaction is observed with the NR box derived
from the amino terminus of CBP (bar 2). Gel shift analysis of nuclear
extracts from transfected cells using a GAL4 binding site indicates
that all GAL4 fusions are expressed at similar levels (data not shown).
Importantly, the interactions between RXR and the SRC-1 NR
boxes are completely dependent on agonist binding. Little or no
interaction is detected in the absence of the RXR agonist LGD1268
(white bars in Fig. 1B
) or in the presence of an antagonist
(data not shown) compared with the GAL4 DNA-binding domain alone. The
interaction between RXR and the SRC-1 NR boxes also requires a
functional AF-2 domain on the part of RXR. A mutation in the AF-2
domain that destroys transcriptional activity but allows ligand binding
[leucine 451 to alanine (28)] eliminates interactions with the SRC-1
NR boxes (Fig. 1C
). To confirm the results of the two-hybrid analysis,
interactions between RXR and SRC-1 NR box III or the CBP NR box were
examined by far Western blot analysis. Figure 1D
indicates that SRC NR
box III directly interacts with RXR in an agonist-dependent manner that
requires a functional AF-2 domain (compare lanes 6 and 7 with 9 and
10). As expected, little interaction is observed between RXR and CBP
(lanes 12 and 15).

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Figure 1. Interaction between RXR and Individual NR Boxes
A, Amino acid sequences in one-letter code of NR boxes from
CBP, SRC-1, and TRAP220. The LxxLL motif is boxed. The first
leucine of the LxxLL motif is considered position +1. B, Mammalian
two-hybrid assay. CV1 cells were transfected with constructs expressing
the appropriate GAL4-fusion, and VP16-RXR LBD as described in
Materials and Methods. After transfection, cells were
cultured for 36 h in the absence (white bars) or
presence of 100 nM LGD1268 (black
bars). The activity relative to that observed with VP16-RXR LBD +
the empty GAL4 fusion vector is reported. Gel shift analysis of nuclear
extracts from transfected cells using a GAL4 binding site indicates
that all GAL4 fusions are expressed at similar levels. C, Same as panel
B except a transcription-inactive RXR AF-2 mutant (L451A) was used. D,
Far Western blot, 3 µg of purified GST (lanes 2, 5, 8, 11, 14, and
17), GST-RXR LBD wild-type (lanes 3, 6, 9, 12, 15, and 18), or GST-RXR
LBD L451A mutant (lanes 4, 7, 10, 13, 16, and 19) were resolved on 10%
SDS gels, and either stained with Coomassie blue to visualize
proteins (lanes 14) or transferred to polyvinylidene
difluoride (PVDF) membranes, and renatured. Blots were probed
with 35S-in vitro translated
GAL4-SRC-1 NR box III (lanes 510), GAL4-CBP NR box (lanes 1116), or
full-length hRAR (lanes 1719) in the absence (lanes 57, 1113,
and 1719) or presence of (lanes 810 and 1416) 1
µM LGD1268.
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Figure 2. Interaction between RXR-PPAR Heterodimers and
Individual NR Boxes
A, Modified mammalian two-hybrid assay. CV1 cells were transfected
with constructs expressing the appropriate GAL4-fusion, VP16-RXR LBD,
and the PPAR LBD, as described in Materials and Methods.
After transfection, cells were cultured for 36 h in the absence
(white bars) or presence of 5.0 µM
BRL 49653 (PPAR , striped bars), 100
nM LGD1268 (RXR-specific, black bars),
or with the combination of 5.0 µM BRL
49653 +100 nM LGD1268 (dotted
bars). The activity relative to that observed with VP16-RXR LBD +
PPAR LBD + the empty GAL4 fusion vector is reported. B, Same as
panel A except the VP16-RXR LBD construct was omitted from the
transfection. After transfection, cells were cultured for 36 h in
the absence (white bars) or presence of 5.0
µM BRL 49653 (PPAR ,
striped bars). The activity relative to that observed with
PPAR LBD + the empty GAL4 fusion vector is reported. C, Standard
mammalian two-hybrid assay examining interactions between a
VP16-PPAR fusion protein and GAL4-NR box constructs. After
transfection, cells were cultured for 36 h in the absence
(white bars) or presence of 5.0 µM
BRL 49653 (PPAR , striped bars). The activity
relative to that observed with VP16-PPAR + the empty GAL4 fusion
vector is reported. Note that the y-axis differs in panel A, differs
from the y-axes in panel B, and panel C. Gel shift analysis of
nuclear extracts from transfected cells using a GAL4 binding site
indicates that all GAL4 fusions are expressed at similar levels.
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To examine interactions between PPAR
and individual NR boxes,
a modified version of the mammalian two-hybrid assay was used in which
the LBD of PPAR
is coexpressed with a VP16-RXR LBD fusion protein
(Fig. 2A
) (26). In this analysis the PPAR
agonist BRL
49653 (striped bars) promotes a relatively
strong interaction between RXR-PPAR
heterodimers and the CBP NR box
and little or no interaction with the SRC-1 sequences. The advantage of
the modified two-hybrid system is that it ensures that the response to
PPAR
-specific ligands is mediated by a heterodimer of PPAR
with
RXR (compare Figs. 2A
and 2B
, note the difference in the y-axes).
Removing the VP16-RXR LBD from the system (Fig. 2B
) eliminates the
ability to detect interactions between PPAR
and the CBP NR box.
Nonetheless, a similar preference of PPAR
for the CBP NR box is
observed using a VP16-PPAR
fusion in a standard two-hybrid assay
(Fig. 2C
). Consistent with the results of Fig. 2
, PPAR
interacts
only with the CBP NR box in far Western analysis (data not shown).
Taken together, the results of this analysis indicate that RXR and
PPAR
can distinguish among related NR boxes.
The modified two-hybrid system was also used to examine the response of
RXR-PPAR
heterodimers to the RXR-specific agonist LGD1268 (Fig. 2A
, black bars). As observed with RXR alone, binding of agonist
to RXR in heterodimers promotes interaction with the SRC-1 NR boxes
while little interaction with the CBP NR box is promoted by treatment
with LGD1268 alone. Although we cannot exclude the possibility that
forms of RXR other than RXR-PPAR
heterodimers contribute to the
response to LGD1268 in the modified two-hybrid system (e.g.
VP16-RXR homodimers), there are several observations indicating that
heterodimers are a major species. First, the affinity of RXR for
PPAR
is significantly higher than the affinity of RXR for itself
(29). Second, we and others have previously shown that RXR can interact
with SRC-1 when dimerized with PPAR
(26, 30, 31). Third, reversing
the assay system (VP16-PPAR
+ RXR LBD) yields a similar preference
of RXR for SRC-1 and PPAR
for CBP (data not shown). Finally, the
combination of RXR- and PPAR
-specific agonists has a synergistic
effect on interactions with the CBP NR box (Fig. 2A
, dotted
bars). The synergy observed with the two receptor-specific ligands
requires the presence of both RXR and PPAR
and therefore must be
mediated by heterodimers. The synergistic interaction observed between
RXR-PPAR
heterodimers and the CBP NR box is also consistent with our
earlier work using larger fragments of CBP (26).
Structures of cocrystals composed of receptor LBDs complexed to NR
boxes, and mutagenesis experiments have defined an agonist-dependent
coactivator-binding pocket (2, 5, 6, 7, 8). To further demonstrate the
specificity of receptor-NR box interactions, the ability of
overexpressed NR boxes to inhibit transcription were examined. In
agreement with the results of Figs. 1
and 2
, overexpression of
SRC-1 NR box III functions as a dose-dependent inhibitor of
transactivation mediated by RXR homodimers (Fig. 3A
, solid
circles). The CBP NR box, however, does not inhibit RXR
(Fig. 3A, open boxes). The converse is true for PPAR
.
The CBP NR box is a more effective inhibitor of BRL
49653-dependent transcription by RXR-PPAR
heterodimers than
SRC-1 NR box III (Fig. 3B
). The correlation between NR box binding and
inhibition suggests that inhibition of transcription by overexpressed
NR boxes most likely involves competition with endogenous coactivators
for receptor binding.
Recent studies have implicated the TRAP/DRIP complex in the activity of
several transcription factors (15). TRAP220/DRIP205 contains two LxxLL
motifs (Fig. 1A
) and appears to be the member of the TRAP/DRIP complex
that interacts directly with nuclear receptors. Interestingly, both
LGD1268 (RXR-specific) and BRL 49653 (PPAR
-specific)
promote similar interactions between RXR-PPAR
heterodimers and the
two TRAP220/DRIP205 NR boxes (Fig. 4
).
Interestingly, as observed with the CBP NR box, the combination of
receptor-specific ligands has a synergistic effect on interactions
between RXR-PPAR
heterodimers and TRAP220 NR box II. Thus, the
analysis of six NR boxes indicates that RXR and PPAR
exhibit
distinct but overlapping coactivator preferences.
Three Amino Acids Allow RXR to Distinguish SRC-1 NR Box III from
CBP
To decipher how RXR distinguishes SRC-1 NR box III from the CBP NR
box, chimeric NR boxes were constructed and examined in a standard
mammalian two-hybrid assay with VP16-RXR LBD. Replacement of
Arg+2 and Tyr+3 of SRC-1 NR
box III with Ser and Glu, respectively (Fig. 5A
), results in a 92% decrease in
interaction with RXR relative to SRC-1 NR box III (Fig. 5B
, compare
bars 4 and 5). Conversely, making the complementary change in the CBP
NR box (Ser+2 to Arg/Glu+3
to Tyr) increases interaction with RXR 3-fold relative to the CBP NR
box (Fig. 5B
, compare bars 2 and 3). Nevertheless, CBP
Ser+2Arg/Glu+3Tyr has
only 27% the activity of the original SRC-1 NR box III (Fig. 5B
, compare bars 3 and 5) indicating that while the
+2 and +3 positions are
important determinants, other positions play important roles in RXR
recognition.

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Figure 5. Three Amino Acids Determine RXR-NR Box Specificity
A, NR box amino acid sequences from position -1 to
+6. Amino acids necessary and sufficient for interaction
with RXR are shaded. B and C, Two-hybrid assay. CV1
cells were transfected with constructs expressing the appropriate
GAL4-fusion and VP16-RXR LBD as described in Materials And
Methods. After transfection, cells were cultured for 36 h
in the absence or presence of 100 nM LGD1268
(RXR-specific). D, Modified mammalian two-hybrid assay. Cells were
transfected as above; however, a construct expressing the PPAR LBD
was also included. After transfection, cells were cultured for 36
h in the absence or presence of 5 µM BRL
49653 (PPAR -specific). The fold induction relative to the
activity in the absence of ligand is reported. Note that the y-axis
differs in panels B, C, and D. Gel shift analysis of nuclear extracts
from transfected cells using a GAL4 binding site indicates that all
GAL4 fusions are expressed at similar levels.
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To determine the additional RXR-specific determinants in SRC-1 NR box
III, the eight amino-terminal amino acids of SRC-1 NR box III
were replaced with the amino acids in those positions from
the CBP NR box. This chimeric NR box (CBP
(-8)(-1)/SRC-1
(+1)(+12)) decreased interaction with RXR by
89% (Fig. 5B
, compare bars 5 and 6). Additional chimeras and point
mutations identify Leu-1 as the critical
position. Individually replacing Leu-1 of SRC-1
NR box III with Gln, the -1 residue in the CBP NR box, decreases
interaction with RXR by 87% (Fig. 5B
, bar 7). In contrast the
complementary Gln-1 to Leu change in the CBP NR
box produces a 5-fold increase in interaction with RXR (Fig. 5B
, compare bars 2 and 8). To determine whether the
-1, +2, and
+3 positions are sufficient to allow interaction
with RXR, a triple-point mutant with Leu, Arg, and Tyr at these
positions in the context of the CBP NR box was examined (CBP
Q-1L/S+2R/E+3Y,
Fig. 5C
). Indeed, the triple point mutant interacts strongly with RXR
(Fig. 5C
, bar 4), indicating these three amino acids are necessary and
sufficient for specific recognition of SRC-1 NR box III by RXR.
The contribution of the -1,
+2, and +3 positions to
preferential recognition of the CBP NR box by PPAR
was tested by
examining the same triple point mutant. If PPAR
, like RXR, uses
these three positions to discriminate between SRC-1 NR box III and the
CBP NR box, then replacing the -1,
+2, and +3 positions of CBP
with the amino acids in SRC-1 NR box III (CBP
Q-1L/S+2R/E+3Y)
should eliminate interaction with PPAR
. As shown in Fig. 5D
, however, the triple-point mutant has no effect on the PPAR
-CBP NR
box interaction (compare bars 2 and 3). The difference in sensitivity
of RXR and PPAR
to the -1,
+2, and +3 positions
suggests that coactivator preference is determined by the ability of
RXR and PPAR
to recognize distinct structural features of NR
boxes.
The results of Fig. 5
indicate that RXR preference for SRC-1 is
mediated by three amino acids (-1,
+2, and +3). To examine
whether the structural requirements determined for individual NR boxes
are relevant in the context of full-length coactivators, the
-1, +2, and
+3 positions of the CBP NR box were changed to
the preferred SRC-1 NR box III residues. This chimeric NR box (CBP
Q-1L/S+2R/E+3Y)
was then introduced into the full-length CBP protein. Unfortunately,
the strong constitutive activity of GAL4-CBP full length precluded a
two-hybrid analysis. However, Fig. 6
clearly shows that the GAL4-CBP full-length NR box mutant (CBP
Q-1L/S+2R/E+3Y)
acts as a relatively effective inhibitor of RXR transcriptional
activity compared with the wild-type GAL4-CBP full-length fusion
protein (compare bars 2 and 3). As expected, both the wild-type and
mutant full-length fusions are potent inhibitors of PPAR
activity
(data not shown). The ability of the full-length CBP
Q-1L/S+2R/E+3Y
mutant to act as a selective inhibitor of RXR is consistent with the
idea that the -1, +2, and
+3 positions make significant contributions to
the interactions between RXR and coactivators.

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Figure 6. The -1, +2, and
+3 Positions of the NR Box Are Important for Interaction
with RXR in the Context of Full-Length Coactivators
CV1 cells were transfected with a reporter for RXR homodimers
(CRBPII-tk-LUC), an expression construct for human RXR , and 12 ng of
the appropriate GAL4 fusion/well. After transfection, cells were
cultured for 36 h in the absence or presence of 100
nM LGD1268 (see Materials and Methods).
The fold induction observed in the presence of the parental GAL4 fusion
plasmid (8.3-fold) was set at 100%.
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Seven Amino Acids Are Sufficient for Interaction of NR Boxes with
RXR
Interestingly, RXR exhibits a 4-fold stronger interaction with CBP
triple-point mutant
(Q-1L/S+2R/E+3Y)
compared with SRC-1 NR box III (Fig. 5C
, compare bars 3 and 4).
Relatively strong interaction with RXR is also observed for a chimeric
NR box that contains the first 13 amino acids from SRC-1 NR box III and
the last 7 amino acids from the CBP NR box (SRC
(-8)(+5)/CBP (+6)-(+12);
Fig. 7B
, compare bars 2 and 3). To
determine which amino acids of the CBP NR box are responsible for
enhanced interaction with RXR, mutations were constructed that
individually replaced positions +6 through
+12 of SRC-1 NR box III with the corresponding
amino acids from the CBP NR box. Mutation of
Asp+6 to Arg resulted in a 4-fold increase in
interaction with RXR relative to SRC-1 NR box III, while substitutions
at other positions had little or no effect (Fig. 7B
, compare bars 25;
also data not shown). Analysis of the complementary
Arg+6 to Asp change in the CBP NR box indicates
that the identity of the +6 position also
significantly contributes to the PPAR
-CBP NR box interaction (Fig. 7C
, compare lanes 2 and 3). A similar modified two-hybrid assay was
used to examine the influence of the +6 position
on interaction with retinoic acid (RAR) and thyroid hormone (TR)
receptors. Both RAR and TR interact in an agonist-dependent manner with
SRC-1 NR box III, and, like RXR, the Asp+6 to Arg
mutation enhances the interaction (compare bars 2 and 3 in Figs. 7D
and 7E
). Thus, the identity of the +6 position,
immediately carboxy-terminal to the LxxLL motif, significantly
contributes to interaction with multiple receptors.

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Figure 7. The +6 Position of the NR Box
Influences the Affinity of Multiple Receptors
A, NR box amino acid sequences from position -1 to
+7. Amino acids that contribute to high-affinity
interaction with RXR are shaded. B, Two-hybrid assay.
CV1 cells were transfected with constructs expressing the appropriate
GAL4-fusions and VP16-RXR LBD as described in Materials and
Methods. After transfection, cells were cultured for 36 h
in the absence or presence of 100 nM LGD1268
(RXR-specific). C, Modified mammalian two-hybrid assay. Same as panel B
except that a construct expressing the PPAR LBD was also included.
After transfection, cells were cultured for 36 h in the absence or
presence of 5 µM BRL 49653
(PPAR -specific). D and E, Modified mammalian two-hybrid assay. Same
as panel B except that constructs expressing either the RAR LBD (D)
or the TRß LBD (E) were also included. After transfection, cells were
cultured for 36 h in the absence or presence of 100
nM LGD1550 (RAR-specific) or 100 nM
T3 (TR-specific). The fold induction relative to the
activity in the absence of ligand is reported. Note that the y-axis
differs in B, C, D, and E. Gel shift analysis of nuclear extracts from
transfected cells using a GAL4 binding site indicates that all GAL4
fusions are expressed at similar levels.
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The mutagenesis results identify four positions
(-1, +2,
+3, and +6) that contribute
to high-affinity RXR-NR box interaction in addition to the three
leucines of the LxxLL motif. To determine whether these seven amino
acids are sufficient for agonist-dependent interaction with RXR, the
sequence Leu-Leu-Arg-Tyr-Leu-Leu-Arg (Fig. 8A
) was fused to GAL4 and tested in
two-hybrid assays. Figure 8B
demonstrates indeed this minimal NR box
interacts with RXR (compare black bars 2 and 3), although
quantitatively the interaction is only 34% of that observed with the
20-amino acid SRC-1 NR box III (Fig. 8B
, compare black bars
3 and 4). In contrast, little or no interaction is observed between the
minimal NR box and PPAR
(Fig. 8B
, compare white bars 2
and 3), RAR, or TR (data not shown). The minimal NR box also acts as an
inhibitor of RXR agonist-dependent transactivation but has little
effect on PPAR
agonist-dependent transcription (compare bar 3 in
Figs. 8C
and 8D
). Likewise, no inhibition of RAR- or TR-mediated
transcription is observed (data not shown). The ability of this short
peptide sequence (molecular mass < 1000 daltons) to
specifically interact with and inhibit transcription by RXR suggests it
should be possible to identify small molecule receptor-specific
antagonists that function by blocking coactivator recruitment to
activated nuclear receptors.

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Figure 8. RXR Interacts with an Optimized 7 Mer
A, Amino acid sequence (-1-+6) of the CBP NR
box, SRC-1 NR box III, and optimized seven-amino acid minimal NR box.
Amino acids unique to CBP and SRC-1 are in white and
gray, respectively. B, Modified mammalian two-hybrid
assay. CV1 cells were transfected with constructs expressing the
appropriate GAL4-fusion, VP16-RXR LBD, and the PPAR LBD as described
in Materials and Methods. After transfection, cells were
cultured for 36 h in the absence or presence of 100
nM LGD1268 (RXR-specific, black bars)
or 5.0 µM BRL 49653 (PPAR ,
white bars). The fold induction relative to the activity
in the absence of ligand is reported. Gel shift analysis of nuclear
extracts from transfected cells using a GAL4 binding site indicates
that all GAL4 fusions are expressed at similar levels. C, CV1 cells
were transfected with a reporter for RXR homodimers (CRBPII-tk-LUC), an
expression construct for human RXR , and 36 ng of the appropriate
GAL4 fusion/well. After transfection, cells were cultured for 36 h
in the absence or presence of 100 nM LGD1268 (see
Materials and Methods). The fold induction observed in
the presence of the parental GAL4 fusion plasmid (13.6-fold) was set at
100%. D, CV1 cells were transfected with a reporter for RXR-PPAR
heterodimers (PPREx3-tk-LUC), expression constructs for RXR and
PPAR , and 36 ng of the appropriate GAL4 fusion/well. After
transfection, cells were cultured for 36 h in the absence or
presence of 5 µM BRL 49653. The fold
induction observed in the presence of the parental GAL4 fusion plasmid
(10.2-fold) was set at 100%.
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DISCUSSION
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The results of this study demonstrate that RXR and PPAR
can
distinguish among highly related NR box sequences, and they do so by
recognizing different structural features. RXRs preference for SRC-1
NR box III relative to the CBP NR box is determined by three amino
acids. Interestingly, two of the important positions for RXR-NR box
interaction are at +2 and
+3, the nonconserved xx residues of the LxxLL
motif. A role for the +2 and
+3 positions as specificity determinants is
surprising, given that these residues appear to point away from the
coactivator binding pocket in the crystal structures of LxxLL motifs
bound to estrogen receptor (ER), TR, and PPAR
(2, 5, 6).
Nevertheless, a similar observation has been made for the
glucocorticoid receptor (2). Comparison of the available receptor LBD
structures suggests that differences in the depth of the coactivator
binding pocket among receptors could contribute to receptor-specific
roles for the nonconserved +2 and
+3 residues. Further mutagenesis studies will be
needed to address this question.
The identity of the nonconserved -1
position, directly amino terminal to the LxxLL motif, also
significantly influences NR box choice by RXR. RXRs preference for a
hydrophobic amino acid at -1 has been
independently confirmed using phage display in the study of Chang
et al. (32). Crystal structures of several LBDs complexed
with NR boxes show that the -1 position projects
into the coactivator binding pocket and makes direct contact with a
conserved hydrophobic amino acid in helix 12 (Phenylalanine 450 in
hRXR
) (2, 5, 6). The finding that hydrophobic amino acids occupy the
-1 position in approximately 65% of the LxxLL
motifs analyzed (24 of 37) further highlights the critical function of
this position in receptor-coactivator interactions. Taken together, the
results of this study indicate that subtle changes in NR box sequence
can have large and receptor-specific effects on receptor-coactivator
interactions. Additionally, the possibility that different ligands,
trans-acting proteins, or receptor and coactivator
posttranslation modifications influence receptor-NR box affinity must
be noted (33).
In contrast to RXR, the identity of the amino acids at positions
-1, +2, and
+3 has little effect on the ability of PPAR
to
distinguish the CBP NR box from the SRC-1 NR box III. Preliminary
experiments suggest that positions both amino- and carboxy terminal to
the LxxLL motif are required for specific recognition of CBP by
PPAR
. The difference in sensitivity of the two receptors for
substitution at -1, +2,
and +3 suggests that RXR and PPAR
recognize
different structural features of NR boxes. Unique NR box recognition
may contribute to the ability of RXR- and PPAR
-specific agonists to
activate transcription synergistically (26). As discussed below, this
observation also has important implications for the discovery of drugs
that target the coactivator binding pocket.
Although RXR and PPAR
appear to use different strategies to
distinguish among NR boxes, an arginine at +6 is
favored over an aspartic acid by both receptors. A similar preference
for arginine at +6 is observed for RAR and TR.
Analysis of receptor-coactivator structures suggests that the ability
of arginine to act as a hydrogen bond donor to the
+2 carbonyl group may account for its preference
compared with aspartic acid (5). It has also been suggested that the
+6 position may function as a helix capper (2, 5). Additional studies will be needed to dissect the contributions of
hydrogen bonding and helix capping at this position. Interestingly,
McInerney et al. (19) have recently demonstrated that
mutation of the +6 position interferes with the
ability of SRC-1 to support transactivation mediated by RAR or PPAR
.
Thus, the amino acid directly carboxy terminal to the LxxLL motif
(+6) may also be considered a critical element of
the NR box for at least a subset of nuclear receptors.
Recent studies suggest that nuclear receptors interact cooperatively
with multiple LxxLL motifs present in individual coactivators (21, 34).
Our studies, which utilize GAL4-LxxLL fusions harboring the intact
dimerization domain of GAL4, do not directly address the role of
cooperativity. Nonetheless, coactivators appear to function as members
of large multisubunit complexes that contain several proteins capable
of interacting directly with receptors (35, 36). For instance,
SRC-1 and CBP can directly interact with each other (13, 37, 38). As a
result, it is not difficult to imagine cooperative interactions between
RXR-PPAR
heterodimers and coactivator complexes with each receptor
contacting either an independent subunit (e.g. SRC-1 and
CBP) or independent NR boxes in the same subunit. A similar model of
two transcription factors independently contacting the same coactivator
complex has recently been proposed for synergistic activation of
transcription by the transcription factors STAT3 (signal transducer and
activator of transcription 3) and Smad1 (spinal muscular atrophy
mothers against decapentaplegic 1) (1). Since it is possible, however,
to detect interactions between receptors and synthetic peptides
encoding single NR boxes, we cannot exclude models in which each member
of the heterodimer recruits a unique coactivator complex
(e.g. SRC-1 and TRAP/DRIP). In either model, increased
recruitment of coactivators by liganding each subunit may
contribute to synergistic activation of transcription.
An additional model for a synergistic response to RXR- and
PPAR
-specific ligands arises from our previous observations that a
transcriptionally silent (AF-2 mutant) RXR can mediate synergy (26).
The AF-2-independent ability of RXR agonists to synergize with PPAR
ligands suggests that binding of ligand to RXR can indirectly influence
the conformation and activity of PPAR
. It is likely that RXR makes
both AF-2-dependent (direct coactivator recruitment) and AF-2
independent (indirect effects on PPAR
) contributions to synergistic
activation.
We and others have shown that PPAR
makes a relatively stronger
interaction with CBP compared with SRC-1 and other SRC family members
(26, 27, 31). The preference of PPAR
for CBP is seemingly at odds
with experiments that suggest CBP is recruited indirectly to nuclear
receptors via its association with SRC family members and that the CBP
NR box is dispensable for coactivator function (34, 39). There are
several possible explanations to resolve this apparent paradox. First,
the role of the CBP NR box has not been examined in the context of
PPAR
transactivation. Therefore, it is possible that a direct
PPAR
-CBP interaction may be critical for activity. Second, our
data suggest that RXR and PPAR
recognize different structural
features of NR boxes. We cannot conclude, however, which coactivator is
important in a particular physiological context. For instance, the
ability of PPAR
to interact with the TRAP/DRIP complex may be more
important than direct interactions with CBP. Third, it is possible to
detect direct interactions between PPAR
and SRC-1, although this
interaction is relatively weaker than the PPAR
and CBP interaction
(5, 27, 30, 31) (data not shown). In cells, additional factors such as
high local concentrations of receptors and/or coactivators or
posttranslational modifications may act to increase the relative
affinity and functional importance of PPAR
-SRC-1 interactions.
Knowledge of the critical residues for RXR-NR box interaction allowed
construction of a seven-amino acid sequence (LLRYLLR) that interacts
with RXR in an agonist-dependent fashion and inhibits RXR-dependent
transcription. The small size of this peptide suggests it may be
possible to identify small molecules that mimic this peptide and
function as receptor antagonists by blocking the access of endogenous
factors to the coactivator binding pocket. Given the structural
conservation of the coactivator binding pocket among nuclear receptors
(2, 5, 6, 7, 8), one might predict that such drugs would have pleiotropic
effects by binding to a wide range of receptors. The results of this
and other studies (16, 17, 19, 21), however, indicate that there is
significant specificity built into receptor-NR box interactions.
Therefore, it should be possible to exploit this specificity in the
identification and design of small molecule antagonists. Since many
transcription factor-coactivator interactions are dependent on small
hydrophobic patches, it may also be possible to more generally apply
these same principles to other gene regulation systems.
Antagonists of the coactivator binding pocket would be expected to have
properties that may be advantageous when compared with traditional
antagonists that competitively bind to the ligand binding pocket.
First, coactivator pocket antagonists should recognize a particular
receptor conformation and may provide remarkable tissue and/or gene
selectivity. Second, coactivator pocket antagonists would be predicted
to bind only to receptors that are in an active conformation
(i.e. the ligand-dependent coactivator pocket has formed).
Specificity for activated receptors predicts that coactivator pocket
antagonists would lack the undesirable partial agonist activity that
has limited the use of drugs such as the estrogen receptor antagonist
tamoxifen. Exploiting the uniqueness and specificity of the coactivator
binding pocket opens the door to a new receptor pharmacology that
should have both scientific and therapeutic benefits.
 |
MATERIALS AND METHODS
|
---|
Plasmids
The receptor and reporter vectors used in this study have been
previously described (26, 40, 41). GAL4-NR box fusions were made by
synthesizing oligonucleotides with the appropriate sequences utilizing
mammalian codon preferences followed by cloning into pCMX-GAL4 (40).
The full-length GAL4-CBP construct was provided by D. Chakravarti
(University of Pennsylvania. Philadelphia, PA). The full length
GAL4-CBP
(Q-1L/S+2R/E+3Y)
mutant was made by site-directed mutagenesis using the
Quickchange mutagenesis system (Stratagene, La
Jolla, CA). All clones were confirmed by DNA sequencing.
Cell Culture and Transfection
CV1 cells were cultured in DMEM supplemented with 10% FBS.
Before transfection, cells were seeded in 48-well plates (1.5 x
104 cells per well) in DMEM supplemented with
10% charcoal resin-split FBS. After 1216 h growth at 37 C, cells
were transfected with the DOTAP transfection reagent following the
manufacturers instructions (Roche Molecular Biochemicals,
Indianapolis, IN). For two-hybrid assays each well was transfected with
36 ng of luciferase reporter, 36 ng of the appropriate GAL4 fusion, 36
ng of VP16-RXR LBD, and as an internal control 60 ng of pCMX-ßgal.
Where noted in the figure legends, 72 ng of PPAR
LBD, 36 ng of RAR
LBD, or 36 ng of TR LBD were also transfected. For inhibition
experiments, cells were transfected as described above with 12 ng of
luciferase reporter, 12 ng of the appropriate receptor expression
vectors, and increasing concentrations of GAL4 fusion expression
vectors. As an internal control, 60 ng of pCMX-ßgal were also
included. When necessary, the parental expression plasmid pCMX was
added to ensure that equal amounts of DNA were transfected in each
well. After 5 h at 37 C, the media were removed, the cells washed
once, and 200 µl of fresh media were added with or without the
ligands described in the legend to each figure. Cells were harvested
after an additional 36 h of growth at 37 C. Luciferase activity of
each sample was normalized by the level of ß-galactosidase activity.
Each transfection was carried out in duplicate and repeated at least
four times.
Far Western Blotting
Far Western blots were carried out as previously described (26).
Each blot contained 3 µg of purified
glutathione-S-transferase (GST), GST-RXR, and GST-RXR
LBD.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank M. Manchester and D. Chakravarti for
comments on the manuscript and the medicinal chemistry department at
Ligand Pharmaceuticals, Inc. for providing LGD1268 and
BRL 49653.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Ira G. Schulman, X-Ceptor Therapeutics, 4757 Nexus Center Drive, Suite 200, San Diego, California 92121. E-mail: ischulman{at}x-ceptor.com
1 Present address: X-Ceptor Therapeutics, 4757 Nexus Center Drive,
Suite 200, San Diego, California 92121. 
Received for publication October 8, 1999.
Revision received March 28, 2000.
Accepted for publication April 13, 2000.
 |
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