From the Department of Pharmacology, University of Minnesota
Medical School, Minneapolis, Minnesota 55455
The mouse orphan nuclear receptor TR2-11
functions as a repressor for reporter genes containing a direct
repeat-5 or direct repeat-4 hormone response element. The functional
domains responsible for its suppressive activity are defined, including
the DNA-binding domain and the ligand-binding domain. The C-terminal 30 amino acid residues can be deleted without compromising its suppressive activity, whereas a deletion for 40 amino acids completely abolishes the suppressive activity and receptor dimerization, and reduces the
DNA-binding affinity. Point mutation at three conserved leucine residues located on the predicted dimer interface abolishes the suppressive activity, receptor dimerization and its DNA binding property. However, mutation at two consecutive glutamate residues located within the hinge between the last two helices of the
ligand-binding domain (helix 10 and helix 11 according to the human
retinoid receptor X
structure) drastically reduces its DNA-binding
affinity and abrogates the suppressive activity without compromising
its ability to dimerize, indicating that receptor dimerization property can be functionally uncoupled from its suppressive activity. A transferable, active silencing activity is encoded within the DEF
segment of the receptor molecule, as evidenced by the suppression of a
GAL4 reporter by a chimeric protein containing the DNA-binding domain
of GAL4 and the DEF segment of TR2-11. Moreover, the C-terminal 49 amino acid sequence is required for this trans-suppressive activity. It
is suggested that TR2-11 functions as a repressor, mediated by
mechanisms requiring high affinity DNA binding, receptor dimerization,
and active silencing.
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INTRODUCTION |
Nuclear receptors constitute a large family of transcription
factors that play key roles in gene regulation. Many of these receptors
are transcription regulators that modulate target gene expression by
binding to specific DNA sequences in its promoter region (1-6). These
include the receptors for glucocorticoid, estrogen, progesterone,
vitamin D, thyroid hormone, and retinoic acid, as well as a large
number of orphan members that have no known ligands. The nuclear
receptors share a common modular structure consisting of a variable N
terminus, a DNA-binding domain
(DBD)1 in the middle of the
receptor molecule, and a C-terminal ligand-binding domain (LBD) (1, 2).
The LBD determines the ligand specificity by formation of a highly
specified binding pocket and mediates receptor dimerization by
interacting at the dimerization interface located on the LBD surface.
In their apo-forms, nuclear receptors function as repressors by
interacting with corepressors, such as the nuclear receptor corepressor
(N-CoR) (7) and silencing mediator for retinoid and thyroid hormone
receptors (SMRT) (8). These corepressors interact with the
apo-receptors at their hinge regions between the DBD and LBD. Ligand
binding induces a conformational change of the receptor, resulting in
the release of the corepressor and recruiting of the co-activator,
which binds to the activation function-2 (AF-2) domain located at the C
terminus of the receptor molecule (9-14). Direct interaction with the
components of the basal transcription machinery such as transcription
factor IIB and TATA box-binding protein (TBP) has also been implicated
in the ligand-independent silencing activity of some of these receptors (15, 16).
The mouse TR2-11 is a member of the orphan receptors and is expressed
mainly in the developing germ cells in the testis (17, 18). Several
mRNA isoforms have been identified (19-20), including one that
encodes the full-length receptor and an alternatively spliced isoform
that encodes a truncated receptor deleted in the entire LBD (19). Our
previous studies have shown that the full-length receptor strongly
suppresses reporters containing either a direct repeat-5 (DR5) (18, 19)
derived from the human RAR
gene promoter (21), or a DR4 type
response element (22) derived from the mouse cellular retinoic
acid-binding protein-I gene promoter (23). In contrast, the truncated
receptor exerts no consistent biological activities in similar
transient transfection experiments, despite the presence of an intact
DBD. By comparing the primary structures of the nuclear receptors, the
full-length TR2-11 receptor is much larger than most other nuclear
receptors due to the extraordinary large DEF segment of its coding
region (approximately 435 amino acid residues for TR2-11, as compared
with approximately 200-300 amino acids for most other receptors) (24).
In order to understand how TR2-11 suppresses gene expression as
demonstrated by many previous studies (18, 19, 22, 25, 26), we have set
up to dissect the functional domains responsible for its biological activity and to determine whether TR2-11 employs any unique pathway or
adopts similar mechanisms employed by other receptors for a strongly
suppressive activity.
In this study, the molecular mechanisms underlying the suppressive
activity of TR2-11 were dissected in the established DR5 reporter
system (18). In this system, it has been shown that RA induction of the
reporter was dramatically suppressed by the full-length TR2-11
receptor, but not by the truncated variant. Here, we first examined the
mutant receptors made by deletion and point mutagenesis, in their
dimerizing ability and DNA binding property, and compared these
properties to their suppressive activities in the DR5 reporter system.
We then examined the role of the predicted ninth heptad repeat within
the LBD in receptor dimerization, DNA binding, and suppressive
activity. We further identified one conserved glutamate residue located
between helices 10 and 11, which was critical for the suppressive
activity and DNA-binding affinity, but not for receptor dimerization.
We went on to determine whether its suppressive activity is
transferable by using the GAL4 fusion protein system. Finally, we
explored the possibility of its interaction with the common corepressor
N-CoR as well as heterodimerization with RAR/RXR family.
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MATERIALS AND METHODS |
Plasmid Constructs--
The reporter, RARE-tk-luc, containing a
DR5 was made as described previously (18). The RAR-luc reporter was
constructed by fusion of a promoter fragment between positions
99 and
11 of the hRAR
gene (27) into the Mlu I/XhoI
sites in front of a promoter-less luciferase reporter, pGL3-Basic
vector (Promega, Madison, WI). The cytomegalovirus vector containing
the full-length TR2-11 (590 amino acid residues) for mammalian
expression was as described previously (22). The amino acid numbering
system was according to our previous study (18). To construct the
C-terminal deletions of TR2-11, fragments deleted in various sizes from
the C terminus were generated by polymerase chain reactions (PCRs), flanked by HindIII and XbaI sites and used to
replace the HindIII/XbaI fragment of the wild
type expression vector. TR2
C49, TR2
C40, TR2
C30, TR2
C20, and
TR2
C10 were each deleted for 49, 40, 30, 20, and 10 amino acids,
respectively, from the C terminus. TR2
N1 was deleted in the
N-terminal amino acid residues 1-99 (the N-terminal variable region),
and TR2
N2 was deleted in the N-terminal 166 amino acids (the
N-terminal variable region and the DBD). Point mutations were generated
using a two-step PCR protocol (28). The mutated fragment flanked by
HindIII and XbaI sites were used to replace the
HindIII/XbaI fragment in the wild type vector. The LLL(537-539)YPP mutant was mutated at the consecutive three leucine residues (positions 537-539) by replacing the wild type sequence TTACTTCTC with a mutant sequence TATCCTCCC. The EE(553,554)RG mutant was mutated at the two glutamate residues at positions 553 and
554 by replacing the wild type sequence GAAGAG with a mutant sequence
CGAGGG. Green fluorescent protein (GFP) fusion of TR2-11-f and all
mutants were constructed by ligating the in-frame cDNA cassettes
from the cytomegalovirus expression vectors using the 5'
BglII site in front of the ATG start codon and the 3'
SmaI sites into predigested BglII/SmaI
pEGFP-C1 vector (CLONTECH) downstream of the GFP
coding region.
Electrophoretic Mobility Shift Assay--
The double stranded
DNA fragments containing the DR5 of the human RAR
promoter were
labeled with [
-32P]dCTP using Klenow fragment
(Boehringer Mannheim). TR2-11 and its mutants were synthesized using a
T7-coupled transcription/translation reticulocyte lysate system (TNT
system, Promega, Madison, WI). The reactions were conducted as
described (22). Briefly, the in vitro translated products
were incubated with 32P-labeled probes (1 ng) for 20 min at
room temperature in the presence of 100 mM Tris-HCl, pH
7.5, 1 mM dithiothreitol, 1 mM EDTA, 100 mM KCl, 1 µg of poly(dI-dC), and 10% glycerol.
Protein-DNA complexes were analyzed by electrophoresis in 5%
nondenaturing polyacrylamide gels, followed by autoradiography. To
determine the Kd values of receptor binding to the
DR5, the signals of specifically shifted bands were quantified using a
phosphorimager and used to construct Scatchard plots as described
(22).
Transient Transfection Assay--
COS-1 cells were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum treated with charcoal. To determine the suppressive
activity of TR2-11 and its mutants, the reporters (RARE-tk-luc or
RAR-luc), the receptor expression vector, and an internal control
vector (SV40-lacZ) were co-transfected into COS cells by calcium
phosphate coprecipitation (22). Typically, 0.1 µg of each expression
vector, 0.3 µg of the reporter, and 0.05 µg of the internal control
plasmid were used. Cells were plated at the density of 5 × 104/well in 24-well plates overnight before transfection.
For RA induction, RA (5 × 10
7 M) was
added into the medium 20-24 h following transfection and incubated
for another 20-24 h before collection of the samples. Luciferase and
lacZ activities were determined as described previously (22). The
specific reporter activity was represented as relative luciferase units
by normalizing to the internal control lacZ activity. Each experiment
was carried out in duplicate cultures, and three to four independent
experiments were conducted to obtain the means and the standard error
of the mean (S.E.).
To determine the trans-suppressive activity of TR2-11, GAL4 fusion
proteins were generated and tested on a luciferase reporter containing
five copies of the GAL4-binding site at the 5' of a thymidine kinase
promoter, designated as (GAL)5-tk-luc. The mammalian expression vectors of fusion proteins were generated using the mammalian MATCHMAKER two-hybrid assay kit
(CLONTECH). Transfection procedure and
determination of reporter activities were as described above. To
generate GAL4 fusion, i.e. pBD-TR2
N2, pBD-TR2
N2
C49, and pBD-TR2(518-590), the GAL4-binding domain (amino acids 1-147) was
fused, in frame, to the entire DEF segment, the DEF deleted in the
C-terminal 49 amino acids, and the amino acid residue 518-590, respectively.
The Yeast Two-Hybrid Interaction Assay--
Yeast strain YRG-2
containing a lacZ reporter controlled by three copies of GAL4 binding
sites, the bait plasmid expressing the yeast GAL4 DNA-binding domain
(pBD), and the prey plasmid expressing the yeast activation domain of
yeast GAL4 (pAD) were purchased from Stratagene (La Jolla, CA). A
series of bait and prey constructs were made by fusing various
fragments of the LBD of TR2-11 into the pBD and the pAD vectors. The
fragments were synthesized by PCR or retrieved from the existing
cDNA vectors. The mRAR
prey and bait each contains the
full-length receptor. The mRXR
bait and prey each contains amino
acids 148-410 (29). The N-CoR prey contains amino acids 1843-2453 (7)
fused to the pAD vector. The TR2-11 baits include the full-length
(TR2), the DEF segment (TR2
N2), the DEF deleted in the C-terminal 49 amino acids (TR2
N2
C49), and the two point mutants
(TR2
N2-LLL/YPP and TR2
N2-EE/RG). The TR2-11 preys include the DEF
segment (TR2
N2), a smaller DEF segment (amino acids 238-590)
deleted at its N-terminal portion, TR2-LBD (22), as well as the four
C-terminal deletions of the DEF segment (TR2
N2
C10,
TR2
N2
C20, TR2
N2
C30, TR2
N2
C40). The positive controls
p53 and pSV40 were purchased from Stratagene. The yeast culture,
transformation procedures, and determination of lacZ activity were as
described previously (22).
Pull-down Assay--
The full-length TR2-11 cDNA was cloned
into the pGEX-2T vector (Amersham Pharmacia Biotech) for the production
of GST fusion protein. For in vitro interactions, a total of
5 µg of fusion protein produced in bacteria, GST-TR2 or GST alone,
was bound to a glutathione-agarose column and incubated with
35S-labeled TR2-11 protein made in TNT reactions. The
binding buffer contains 20 mM HEPES-KOH, pH 7.4, 150 mM KCl, 5 mM Mg Cl2, 0.5 mM dithiothreitol, 0.1% Nonidet P-40, 5 mg/ml bovine serum
albumin, 10% glycerol, and a protease inhibitor mixture. Incubation
was conducted for 60 min at 4 C. The unbound protein was removed by five washes with a solution of the binding buffer without bovine serum
albumin and protease inhibitors. The specifically bound protein was
eluted with 50 mM reduced glutathione in 50 mM
Tris, pH 8.0, and resolved by SDS-polyacrylamide gel
electrophoresis.
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RESULTS |
Dissection of Functional Domains for the Suppressive Activity of
TR2-11--
In our previous studies, we have demonstrated that TR2-11
exerted a strongly suppressive activity on the reporters containing either a DR5 type RARE (from the human RAR
promoter) (18) or a DR4
type hormone response element (from the mouse CRABP-I promoter) (22).
In this study, we have utilized two reporter systems to dissect the
mechanisms of the suppressive activity of TR2-11. The first was a DR5
artificial reporter (RARE-tk-luc) that had been widely used to study RA
induction mediated by a DR5 element. In order to confirm the fidelity
of responses observed in this artificial reporter, we also included a
natural promoter from which this DR5 was derived, the RAR
promoter,
a well established gene directly responding to RA induction (27). To
first define the functional domains of TR2-11 that were required for
its suppressive activity, we began by systematic deletions from the N
and C termini of the wild type receptor, and followed by making
specific point mutations in the LBD of TR2-11. Fig.
1A shows the maps of two N-terminal deletions, five C-terminal deletions, and four point mutations used in these studies. The suppressive activity of each receptor on RA induction of the RARE-tk-luc and the RAR-luc reporters was assessed in transient transfection assays and the results are shown
in Fig. 1 (B and C, respectively). As compared
with the transfection with a control expression vector (lane
CMV), expression of the wild type TR2-11 (TR2-11-f) or three small
C-terminal deletions (lanes TR2
C10, TR2
C20, TR2
C30, and data
not shown), resulted in 50-60% inhibition of RA induction on
RARE-tk-luc activity and 75-80% inhibition on RAR-luc activity. In
contrast, deletion of 49 amino acids from the C terminus (TR2
C49)
completely abolished the suppressive effect of TR2-11 on both
reporters. Interestingly, the 40-amino acid deletion (TR2
C40) only
partially abolished the receptor function in the RAR-luc reporter (Fig.
1C), yet it affected the receptor function in the
RARE-tk-luc reporter more dramatically (Fig. 1B). As
expected, further truncations (94 amino acids or larger) also resulted
in the complete loss of the suppressive activity (data not shown). The
N-terminal deletion mutant which retained the intact DBD remained
suppressive (lane TR2
N1), whereas a further deletion into
the DBD completely abolished the suppressive activity (lane
TR2
N2). Collectively, these data demonstrate that both the N-terminal variable region and the C-terminal 30 amino acid
sequence are not required for the suppressive activity of TR2-11 on
either reporter, the region between the 30th and the 40th amino acid
from the C terminus exerts slightly different effects between the two
reporter systems, whereas the DBD and the LBD are important for the
suppressive biological activity in both reporter systems.

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Fig. 1.
Inhibition of RA induction of the
RARE-tk-luciferase reporter by TR2-11, its C- and N-terminal deletion,
and point mutants. A, schematic representation of the
TR2-11 and deletion constructs used in this experiment.
Numbers on the right represent the last amino
acid of each construct. TR2-11-f represents the wild type
protein, while TR2 C denotes the C-terminal deletion
followed by the numbers of amino acid deleted, and TR2 N1
and TR2 N2 represent the N-terminal truncation deleted in
the sequence 1-99 and 1-166, respectively.
LLL(537-539)YPP construct represents a point mutant whose
amino acids at position 537 to 539 (*) has been changed from three
contiguous leucine to tyrosine, proline, and proline, respectively.
EE(553,554)XX represents point mutants whose amino acid
residue 553 and 554 (**) have been mutated from double glutamate to
three different expression vectors, designated as EE(553,554)RE,
EE(553,554)EG, and EE(553,554)RG. B and
C, the folds of RA induction of the RARE-tk-luciferase and
RAR-luc reporters in COS-1 cells. COS-1 cells were co-transfected with
0.3 µg of the reporter, 100 ng of the TR2-11 expression vectors, and
50 ng of the internal control (SV40-lacZ) for 20-24 h. LacZ and
luciferase activities were determined at 40-44 h to obtained the
specific activity. The -fold of RA induction was determined by
comparing the specific activity in the presence of RA to that without
RA addition. More than three independent experiments were performed.
D, localization of GFP fusion of TR2-11 proteins expressed
in COS-1 cells. Wild type, deletion mutants, as well as point mutants
were tagged with GFP expression vector at the N terminus of the
proteins. a, the full-length TR2-11-f nuclear localization
pattern. b and c, examples of the deletion and
point mutant localization patterns, respectively. d,
exclusive cytosolic localization of the TR2- N2 GFP fusion
protein.
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Amino acid comparison of TR2-11 and other receptors revealed a striking
sequence homology in the region containing the heptad repeats 7-9,
which corresponded to helices 9-11 according to the x-ray structure
data (30). Within this region, a contiguous LLL sequence located in the
ninth heptad repeat is most highly conserved. The LLL sequence was
mutated into YPP, designated as LLL(537-539)YPP. As shown in Fig. 1
(B and C), the mutated receptor failed to exert
the suppressive activity in both reporter systems. This result
demonstrates a critical function of these conserved leucine residues in
the suppressive activity of TR2-11.
The observation that deletion of 40 amino acids, but not 30 amino
acids, from the C terminus of TR2-11 resulted in a loss of the
suppressive activity suggested an important role of the 10 amino acids
(between the 30th and the 40th residues from the C terminus) in this
receptor activity. Sequence comparison among various nuclear receptors
revealed that this 10-amino acid sequence constituted the hinge region
between helices 10 and 11, as well as the N-terminal portion of helix
11. Most interestingly, a glutamate residue (Glu-553) in the hinge
region was conserved among TR2-11, RXR
, RAR
, and RAR
. We then
asked whether the hinge region between helices 10 and 11 was important
for the suppressive activity. The two consecutive glutamate residues
were first mutated to arginine and glycine, designated as
EE(553,554)RG, and tested in transient transfection experiments. As
shown in Fig. 1 (B and C), this mutation completely abolished the suppressive activity in both reporter systems.
To determine which glutamate residue was important, single mutation was
generated, designated as EE(553-554)RE (mutated at Glu-553) and
EE(553-554)EG (mutated at Glu-554). We found that mutation at Glu-553
(EE/RE), but not Glu-554 (EE/EG), abolished the suppressive activity.
Therefore, it is concluded that the conserved glutamate residue
(Glu-553) plays an important role in TR2-11-mediated suppression.
The failure of the mutant receptors to exert an effect on these
reporters raised a concern about the possibility of altered mutant
protein stability or defect in protein trafficking; we then examined
whether these mutant receptors were made inside the cells and properly
transported into the nuclei, by using a GFP-tag strategy as shown in
Fig. 1D. The wild type receptor tagged with GFP showed a
distinct nuclear localization pattern (Fig 1D, a), which was also observed for the receptors of C-terminal
deletions (b) and point mutations (c). In
contrast, the receptor deleted in the N terminus and the DBD,
N2,
which was deleted in the putative nuclear localization signal,
exhibited a completely cytosolic distribution (d). The
N-terminal deletion (
N1) also showed a nuclear retention pattern
identical to the wild type pattern (data not shown). In addition,
protein stability of all these mutant proteins remained relatively
constant, as evidenced by the fact that all the nuclear fluorescent
signal could be followed for the same duration. We also tested the
biological activity of the receptors tagged with GFP in the reporter
systems, and a similar pattern of biological activity as that shown in
Fig. 1 (B and C) was observed for these
GFP-tagged receptors (data not shown). From these results, it is
concluded that all the mutant receptors, except
N2, which is deleted
in both the N terminus and the DBD, are made, folded properly and
transported into the nuclei.
TR2-11 Receptor Dimerization--
Dimerization is essential for
the function of most nuclear receptors. Two dimerization interfaces
have been defined, one within the second zinc finger of the DBD and the
other within the LBD (31). We first would like to confirm whether
dimerization of TR2-11 occurred in vivo and if dimerization
was also required for the suppressive activity of TR2-11. The ability
of TR2-11 and its deletion mutants to form homodimers was examined in
the yeast two-hybrid interaction assay. Various portions of TR2-11were cloned in the yeast expression vectors and tested by co-transfection of
the bait and the prey vectors into the yeast. As shown in Fig. 2A, receptors containing the
DEF segment remained capable of dimerization (column 2),
whereas deleting the C-terminal 49 amino acids rendered receptors
unable to dimerize (column 3). Moreover, the interaction between the wild type receptors (column 4) is much stronger
than the positive control, between p53 and SV40 large T-antigen
(column 1), suggesting a rather strong interaction between
the dimer partners. To further dissect the sequence required for this
interaction, small deletions from the C terminus were cloned into the
prey vector and tested in similar interaction assays as shown in Fig. 2B. In consistence with the results of their suppressive
activities (Fig. 1, B and C), receptors deleted
for up to 30 amino acids from the C terminus (columns 3-5)
remained capable of interacting with the wild type receptor, while
further deletion to 40 amino acids (column 6) drastically
affected the interaction between the dimer partners. Therefore, it is
concluded that deletion from the C-terminal 40 amino acids of TR2-11
abolishes both receptor dimerization property and its ability to
suppress target gene expression.

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Fig. 2.
Homodimerization of TR2-11 detected in yeast
two-hybrid interaction assays. A, loss of interaction
of TR2-11 by deleting the C-terminal 49 amino acids, but not the
N-terminal truncations. Growth on triple selection medium was denoted
under Growth on His, and the reporter activity was shown on
the right-hand panel. B, characterization of the
C-terminal domain required for homodimerization. Various C-terminal
deletions (columns 3-6), as well as the two point mutations
(columns 7 and 8), were tested and compared with
the wild type receptor interaction (column 2) as well as the
positive control (column 1).
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In transfection experiments as shown in Fig. 1, the LLL(537-539)YPP
and the EE(553,554)RG mutants completely lost their suppressive activities. We then examined their ability to dimerize in the two-hybrid interaction test. As shown in columns 7 and
8 of Fig. 2, the LLL(537-539)YPP failed to interact with
wild type receptor (column 7). Unexpectedly, EE(553,554)RG
mutant remained capable of interacting with the wild type receptor
(column 8) and the strength of interaction was comparable to
that of the wild type receptor (column 2). Collectively,
these data indicate that while efficient dimerization is positively
correlated with the suppressive activity of TR2-11 (as evidenced by the
results of C-terminal deletion and the leucine mutation), other
mechanisms mediated by the conserved glutamate residues are also
important for the suppressive activity.
To determine whether TR2-11 could form homodimers in a DNA
binding-independent manner, pull-down assays were conducted. TR2-11 was
tagged with the GST at the N terminus and applied to a
glutathione-agarose column. The wild type TR2-11 protein was labeled
with [35S]methionine in a TNT reaction and applied to the
GST-TR2-bound column as described under "Materials and Methods." As
shown in Fig. 3, the
35S-labeled wild type TR2-11 was eluted from the GST-TR2
column (lane 3) but not the GST column control (lane
2). Therefore, it is concluded that TR2-11 is capable of
homodimerization in a DNA binding-independent manner.

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Fig. 3.
TR2-11 homodimerization in
vitro. The full-length TR2-11 was translated in
vitro in the presence of [35S]methionine and
analyzed be affinity pull-down assay with the indicated GST fusion
proteins bound to the glutathione-agarose columns. Lane 1 shows the input control, lane 2 shows the GST alone, and
lane 3 shows the positive interaction.
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DNA Binding Property of TR2-11--
To ask whether the mutant
receptors that lost their dimerization property and suppressive
activity, such as receptors deleted at the C terminus or mutated at
leucine or glutamate residues, behaved differently in the DNA binding
property, we set up the gel retardation experiments using
32P-labeled DR5 fragments as the probes. As shown in Fig.
4A, the wild type receptor
(lanes 2-7) was able to shift the DR5 probes to a distinct
position labeled with an arrow at the left. This band was competed out approximately 50% by unlabeled fragments at
50-fold excess (lane 7) and supershifted by a specific
TR2-11 antibody (lane 3) (22) to a position labeled with a
double arrow. The LLL/YPP mutant completely lost its DNA
binding property (lane 16), whereas the EE/RG (lanes
12-15) or the TR2
C40 (lanes 8-11) mutant remained capable of binding DNA, although at a much
lower affinity as demonstrated by almost complete competition by a
50-fold excess of the unlabeled fragments (lanes 15 and
11, respectively). In order to confirm the negative result
of the LLL(537-539)YPP mutant, we compared the TNT products for the
wild type, EE(553,554)RG, and LLL(537-539)YPP mutants by including
[35S]methionine in the TNT reactions as shown in
panel B. The specific protein products of these TNT
reactions were comparable, indicating that the wild type,
LLL(537-539)YPP, and EE(553,554)RG proteins were made equally
efficiently. We also found that the specifically shifted band could not
be competed by the unlabeled DNA fragments containing either one of the
two half-sites and that probes containing one half-site was not shifted
by TR2-11 (data not shown), suggesting no binding by TR2-11 monomers.
It is apparent that deletion or mutation from the C terminus of TR2-11
drastically affects its DNA-binding affinity.

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Fig. 4.
Binding characteristics of TR2-11 and mutant
proteins. A, in vitro translated TR2-11-f,
TR2 C40, and EE(553,554)RG were incubated with
32P-labeled probe in the absence (lanes 4,
8, and 12) or presence (lanes 5-7,
9-11, and 13-15) of an increasing amount of
unlabeled probes. Lane 16, in vitro translated
LLL(537-539)YPP protein. Lane 1 (F), free probes
incubated with the reticulocyte lysate for a negative control. The
specifically retarded band is indicated with an arrow.
Pre-immune and antibody-mediated supershift are shown on lanes
2 and 3, respectively. The supershifted band was
labeled with double arrows. B, autoradiography of
the three proteins used in A. Each wells was loaded with an
equal amount of the in vitro
[35S]methionine-translated proteins
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In order to determine the magnitude of the effect caused by the
EE(553,554)RG mutation for DNA binding, we tried to determine the
binding affinity of this mutant and the wild type receptors. As shown
in Fig. 5A, a constant amount
of the TNT product was incubated with different amounts of labeled
probes. The intensities of the free probes (free) and the specifically
retarded bands (bound) were quantified using an imaging densitometer
(Bio-Rad, model GS-700). Fig. 5B shows the plot of the data
from the wild type receptors. The ratios of bound/free were plotted
against the concentrations of the bound forms. From these plots, the
Kd was determined as 7.4 nM for the wild
type. However, due to the very weak binding, the Kd
of the mutant receptor could not be calculated reliably.

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Fig. 5.
Binding affinity of TR2-11 to the DR5
sequence. A, binding of the in vitro
expressed TR2-11 and EE(553,554)RG mutant to the indicated amount of
unlabeled fragments in gel retardation assay. Constant amounts of
proteins were incubated with various amount of the probes as indicated.
Lanes 1-5, wild type TR2-11; lanes 6-10,
mutant. B, Scatchard plot analysis. The ratios between the
intensity of the specific DNA-protein complex of each retarded band
(Bound, nM) and free DNA probes
(Free, nM) were plotted against the value of the
bound forms. The dissociation constant (Kd) and
Bmax were determined as 7.4 and 5.76 nM for the wild type receptor. The Kd
value of the mutant receptor could not be determined reliably because
the calculated ratios of bound/free resulted in a large degree of
scatter in the linear least squares plots.
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Therefore, it is concluded that TR2-11 binds DR5 as homodimers,
C-terminal deletion mutants (for up to 49 amino acids) or the
EE(553,554)RG mutant remain capable of binding to DR5, but at a much
lower affinity, whereas mutation at the three leucine residues
(537-539) completely abolishes the DNA binding property.
A Trans-suppressive Activity Encoded within the LBD of
TR2-11--
To determine whether the suppressive activity of TR2-11 is
transferable and to define the modular domain that is responsible for
this activity, we transferred different portions of its DEF segment to
the yeast GAL4-binding domain, resulted in pBD-TR2
N2, pBD-TR2
N2
C49, and pBD-TR2(518-590) (Fig.
6A), and tested their activity
on the GAL4-tk-luc reporter as described under "Materials and
Methods." As shown in Fig. 6B, the expression of
pBD-TR2
N2 resulted in a strong suppression (approximately 70%) of
the basal activity of the luciferase reporter (column 2) as
compared with the control (column 1). Interestingly,
deletion of the C-terminal 49 amino acids abolished this
trans-suppressive activity (column 3), whereas the
C-terminal 73 amino acids alone had no trans-suppressive activity
(column 4). Therefore, it is concluded that the suppressive activity of TR2-11 is transferable and requires the LBD including the
C-terminal segment (the last 49 amino acids). It is still not known
whether this C-terminal domain encodes a ligand-dependent activation function like the AF-2 of other receptors.

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Fig. 6.
Active silencing of transcription of
TR2-11. A, schematic representation of the chimera
proteins used in this experiment. B, trans-suppression of
the GAL4 reporter by the GAL4 fusion. The relative luciferase
activities of the reporter were measured 40-44 h after cotransfection
with 0.3 µg of the reporter, 100 ng of the TR2-11 expression vectors,
and 50 ng of the internal control (SV40-lacZ). Three independent
experiments were performed.
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One mechanism for the transferable, suppressive activities of nuclear
receptors involves receptor interactions with the corepressors such as
N-CoR and SMRT (7, 8). Alternatively, heterodimerization with RXRs or
RARs has also been implicated in the function of nuclear receptors
(32). To determine whether TR2-11 employs such a mechanism for its
suppressive activity, we examined the possibility of TR2-11 interaction
with the known corepressor (N-CoR), RXRs, or RARs using the yeast
two-hybrid interaction assay. As shown in Fig.
7, the DEF region of TR2-11 failed to
interact with the known receptor interacting domain of the N-CoR
(column 4), the RXR
(column 6), and the RAR
(column 8). The positive controls for interactions including
TR2-11, N-CoR, RXR
, and RAR
were provided in columns
2, 3, 5, and 7, respectively.
Therefore, it is concluded that the suppressive activity of TR2-11,
although transferable and requiring the LBD, is not mediated by an
interaction with the common receptor interacting domain of the N-CoR or
heterodimerization with RXRs or RARs.

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Fig. 7.
Two-hybrid interaction tests for other
potential pathways. Yeast were cotransformed with the pair of bait
and prey as indicated. RAR , RXR , and N-CoR fusion of the bait and
the prey each encodes amino acid sequence 1-462, 148-410, and
1848-2453, respectively.
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DISCUSSION |
In this study, we have first dissected the functional domains
involved in the suppressive activity of the wild type TR2-11 using a
deletion strategy. We then produced mutant receptors by point
mutations, and examined their biological activities and biochemical
properties including receptor dimerization and DNA binding. From these
mutation studies, it is concluded that receptor dimerization and DNA
binding are generally important for the suppressive activity of TR2-11
and the C-terminal 49 amino acid sequence is involved in the
suppressive activity, receptor dimerization and DNA binding. Fig.
8 shows the comparison of this region
among TR2-11, RAR
, RAR
, and RXR
. The three leucine residues
(537-539) located on the predicted helix 10 are critical for receptor
dimerization, DNA binding and suppressive activity. A glutamate residue
located between helices 10 and 11, which is conserved among TR2-11,
RXR
, RAR
, and RAR
, is critical for the suppressive activity
and DNA binding property but not for receptor dimerizing activity. This result indicates that the suppressive activity of TR2-11 is DNA binding-dependent but can be functionally uncoupled from
its dimerizing activity. Furthermore, in the GAL4 reporter system, we
demonstrate that TR2-11 encodes a transferable suppressive domain in
its LBD and the C-terminal 49 amino acid residues are required for this trans-suppressive activity.

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Fig. 8.
A summary of the C-terminal sequence
comparison among TR2-11, RAR , RAR , and
RXR . The C-terminal sequences of the TR2-11, RAR ,
RXR , and RAR were aligned to their optimal homology. The
conserved heptad repeat 9, Glu-553, as well as the putative AF-2 domain
were denoted with bold letters. Helices 10 and 11 according
to the reported x-ray data were labeled with hatched boxes
under the sequences.
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The slightly different effect of deleting 40 amino acids (TR2
40)
between the artificial RARE-tk-luc and the native RAR-luc reporter
systems is interesting. It is possible that the conformational change
of this mutant receptor has a more drastic effect on the RARE-tk-luc
than on the RAR-luc reporter. This is also supported by other data
showing that the C-terminal 30-40-amino acid sequence plays an
important role, as evidenced by the consequence of mutating the Glu-553
residue (see more discussion below). Despite this subtle variation, the
overall changing pattern of mutant receptors agrees between the two
reporter systems. Therefore, the physiological relevance of the DR5
used in RARE-tk-luc reporter (Fig. 1B) is strongly supported
by the results using the native RAR
promoter (Fig. 1C)
from which this particular DR5 is derived. RAR
directly responds to
RA induction and has been established as one of the earliest responding
retinoid receptors in eliciting biological responses, such as cell
proliferation, differentiation, and apoptosis (33). In the testis, RA
induction of RAR
expression is also shown to correlate with
spermatogenesis (34). Our results would suggest that TR2 suppression of
RA induction on the RAR
gene probably serves to fine tune the
expression level of RAR
in developing germ cells.
The Kd of TR2-11 to the DR5 element is known to be
much lower than that of the RAR/RXR heterodimers (35). Therefore, it
has been suggested that competing for binding sites is the major
mechanism mediating its suppressive activity on gene expression. In
this current study, we demonstrate the presence of a trans-suppressive activity of TR2-11, in addition to its high affinity DNA binding. It is
suggested that suppression by TR2-11 could occur at two different
levels, i.e. by competition for DNA binding and by a transferable, active silencing activity. Since TR2-11 does not interact
with RAR or RXR and the suppressive activity requires DNA binding, the
sequestering mechanism can be excluded. The RNA polymerase II
transcription component, i.e. TBP, has also been shown to
interact directly with unliganded nuclear receptor (15, 16). However,
we have failed to detect efficient interaction of TR2-11 with this
molecule in the two-hybrid interaction assay (data not shown).
Therefore, it is unlikely that TR2-11 suppresses the target genes by
directly interfering with the basic transcription machinery such as
TBP. However, interactions with other transcription factors cannot yet
be excluded. Because its DEF segment can be transferred to GAL4-binding
domain and remains suppressive, TR2-11 appears to be able to function
in a manner similar to that of the unliganded thyroid hormone receptor
and RAR. However, it does not interact with the known
receptor-interacting domain of N-CoR. Therefore, the suppressive
activity of TR2-11 is not mediated by interaction with these domains of
N-CoR. It remains to be determined whether other portions of N-CoR can
interact with TR2-11.
Efficient dimerization is important for the suppressive activity (as
evidenced by the results of C-terminal deletions and the point
mutations); however, dimerization alone is not sufficient for this
suppressive activity (as evidenced by loss of suppressive activity of
the glutamate mutant, which remains capable of dimer formation). As
demonstrated also in other receptors (36, 37), the ninth heptad, which
spans the conserved leucine residues (537-539), is important for both
DNA-independent dimerization and the suppressive activity. The
C-terminal helix of TR2-11 is important for its suppressive activity,
particularly the C-terminal 30-40 amino acid residues. This region
probably functions to maintain the integrity of the LBD and allows a
specific conformation to be adopted for its high affinity DNA binding
and suppressive activity, since a single Glu-553 mutation in this
region completely abolishes the suppressive activity and drastically
reduces its DNA-binding affinity. This is also supported by the fact
that the chimera of GAL4-binding domain and TR2 C terminus requires
this domain to exhibit the active silencing. It will be interesting to
examine how the EE/RG mutation affects the receptor conformation.
The C-terminal 49-amino acid sequence includes part of the dimerization
interface as well as the homologous region of the AF-2/
4, which is
essential for recruiting co-activators in other receptor systems
(9-14). It remains unknown whether this domain of TR2-11 contains an
AF-2-like ligand-dependent activation domain (as labeled
with a question mark in Fig. 8). In other receptors, this
motif participates in both transcriptional activation and the relief of
silencing, as deletion of this domain results in failure to bind
ligands and the receptor becomes a constitutive silencer due to the
inability to release the corepressor (8, 38). It is suggested that a
specific conformational change at the C terminus is induced by ligand
binding, which subsequently causes the release of corepressors (39). It
is possible that TR2-11 also employs such a mechanism, as a single
glutamate mutation (Glu-553) is sufficient to release the suppressive
activity. However, it remains to be determined whether TR2-11 interacts
with any unknown corepressors and if any specific ligands are present
for TR2-11 to function as an activator.
We thank Core B of a program project (DA08131)
for help in oligonucleotide synthesis.