From the Division of Molecular Endocrinology, Departments of
Medicine and Pharmacology, and the Department of Cell
Biology, New York University Medical Center,
New York, New York 10016
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ABSTRACT |
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We reported previously that deletion
of the 50-amino acid NH2-terminal A/B domain of the
chicken (c) or rat thyroid hormone (T3) receptor-
(T3R
) decreased the T3-dependent
stimulation of genes regulated by native thyroid hormone response
elements (TREs). This requirement of the NH2-terminal A/B
domain for transcriptional activation was mapped to amino acids 21-30
of cT3R
. Expression of transcription factor IIB (TFIIB)
in cells was shown to enhance T3-dependent
transcriptional activation by cT3R
, and this enhancement by TFIIB was dependent on the same 10-amino acid sequence. In vitro binding studies indicated that cT3R
interacts efficiently with TFIIB, and this interaction requires amino
acids 23KRKRK27 in the A/B domain. In this
study we document the functional importance of these five basic
residues in transcriptional activation by cT3R
, further
supporting the biological significance of these residues and their
interaction with TFIIB. Interestingly, we also find that the same amino
acids also affect DNA binding and dimerization of cT3R
.
Gel mobility shift assays reveal that a cT3R
mutant that
has all five basic amino acids changed from
23KRKRK27 to 23TITIT27
binds to a palindromic TRE predominantly as a homodimer, whereas cT3R
with the wild-type
23KRKRK27 sequence binds predominantly as a
monomer. This results from both a marked decrease in the ability of the
cT3R
mutant to bind as a monomer and from an enhanced
ability to dimerize as reflected by an increase in DNA-bound
T3R-retinoic X receptor heterodimers. These effects of
23KRKRK27 on DNA binding, dimerization,
transcriptional activation, and the association of T3R
with TFIIB support the notion that this basic amino acid motif may
influence the overall structure and function of T3R
and,
thus, play a role in determining the distinct context-dependent transactivation potentials of the
individual T3R isoforms.
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INTRODUCTION |
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Steroid, retinoid, and thyroid hormone nuclear receptors are
ligand-dependent transcription factors that couple
extracellular signals directly to transcriptional responses. These
receptors activate or repress transcription of target genes by binding
to specific DNA sequences referred to as hormone response elements (HREs)1 (1). The nuclear
receptor superfamily can be divided into the steroid hormone receptor
family and the thyroid hormone/retinoid receptor family (1, 2), which
includes receptors that mediate the effects of thyroid hormone
[L-triiodothyronine (T3) (the
T3Rs), all-trans-retinoic acid (the RARs),
9-cis-retinoic acid (the RARs and RXRs), and
1,25-dihydroxyvitamin D3 as well as several orphan receptors (e.g. COUP-TF, c-erbA2) whose ligand(s), if
any, are unknown (3-5).
The T3Rs are encoded by two distinct but closely related
genes ( and
) which, in humans (h), map to chromosomes 17 and 3, respectively (6). Each gene expresses several alternatively spliced
isoforms. The T3R
gene in the rat (r) and man expresses the T3-binding isoform T3R
1 along with
c-erbA
2, which does not bind T3 because of alternative
splicing at the COOH terminus (3, 7, 8). The closely related chicken
(c)
gene expresses only cT3R
, which is more than
90% similar at the amino acid level to rT3R
1 and
hT3R
1 (6, 9, 10). The T3R
gene expresses T3R
1 and T3R
2 that differ only in their
NH2-terminal A/B regions, which are distinct from the A/B
region of T3R
1 (3, 11). Except for the A/B domains, the
T3R
and T3R
receptors are more than 90%
similar at the amino acid level. Thus, three T3Rs are expressed which differ primarily in the A/B domain, suggesting that
this region may play a role in mediating different effects of these
receptors.
One of the central issues in understanding the actions of the T3Rs and other nuclear receptors is elucidation of the details by which target genes are recognized. The T3Rs and certain other members of thyroid hormone/retinoid receptor family bind to their HREs as monomers, homodimers (12-16), or as heterodimers with the RXRs (17-24). In particular, the T3Rs bind to and activate transcription from a wide variety of response elements organized as direct repeats (DR), inverted repeats (IR), or everted repeats (ER) of the optimized AGGTCA hexanucleotide half-site (25-27) and from native half-site motifs that diverge from the AGGTCA core binding sequence (28).
Recognition of specific base pairs within the half-site core binding
motif is mediated by the highly conserved DNA binding domain (DBD),
which defines the nuclear receptor superfamily. This highly conserved
DBD contains 66-68 amino acids that are organized into two zinc finger
structures that include 9 perfectly conserved cysteines followed by a
carboxyl-terminal extension (29, 30). A helix in the carboxyl-terminal
extension, with its extensive minor groove contacts, effectively
extends the contact surface of the DBD beyond the consensus 6-base pair
half-site (31). The ability of nuclear hormone receptors to distinguish among specific HREs is conferred by 3 amino acids at the base of the
first zinc finger in the DBD (the P box) (32). This region is organized
into an -helix that penetrates the major groove and recognizes the
specific nucleotide sequence of the HRE.
Amino acids at the base of the second zinc finger (the D box) are
thought to provide a dimerization interface for protein-protein interactions on certain HREs (32). Structural studies indicate that the
DBDs of certain thyroid hormone/retinoid family members form a
cooperative dimerization interface, when bound to DRs but not to IRs
and ERs (31). For IRs and ERs, binding of homodimers or heterodimers is
thought to result from a dimerization interface located within the
ligand binding domain. In addition to homodimer and heterodimer
binding, the T3Rs also bind IRs, DRs, and other DNA
configurations as monomers (14). In the absence of RXR, T3R binds more efficiently as monomers to these
elements, and T3R
isoforms bind more efficiently as
homodimers (33).
We reported previously that a 10-amino acid sequence within the A/B
domain of cT3R or rT3R
1 was essential for
ligand-dependent activation of native HREs and for
interaction of T3R
with TFIIB (34). Interestingly,
deletion of the 50-amino acid A/B domain of cT3R
markedly reduced monomer binding and increased homodimer binding of the
receptor, suggesting that the A/B domain of T3R
imposes
preferential monomer binding of the receptor (34). In this study we
show that the same 5 basic amino acids
23KRKRK27 which are necessary for efficient
binding to TFIIB are required for transcriptional activity of
cT3R
. These same amino acids are also responsible for
imposing preferential monomer binding and influencing the efficiency of
heterodimer formation with RXR. To our knowledge this is the first
identification of specific NH2-terminal residues involved
in the differential binding of T3R isoforms to DNA.
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EXPERIMENTAL PROCEDURES |
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Plasmids--
MTV-TREp-CAT (14, 25),
MTV-TRE-GH-CAT (25),
and
MTV-TRE-Mal-CAT (35) have been described previously. These CAT reporter genes contain a single copy of each TRE cloned into the HindIII site at
88 of
MTV-CAT, a mouse mammary tumor
viral LTR-CAT reporter that lacks glucocorticoid response elements (25,
32). The TREp (also known as the TRE-IR) is an inverted repeat of
optimized AGGTCA half-sites (AGGTCA TGACCT). TRE-GH and TRE-Mal are
from the rat growth hormone (13) and rat malic enzyme genes (36), respectively, and contain direct and inverted repeats. The
TRE1/2 is the same as the TREp except that it contains a
single G to C change in one of the half-sites (AGGTCA to ACGTCA).
Cell Transfections and CAT Assays-- HeLa cells were transfected by electroporation (14, 34). After transfection, cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 15 mM Hepes, 0.1 mg/ml pyruvate, 50 µg/ml streptomycin sulfate, 50 µg/ml penicillin (DHAP medium) containing 10% (v/v) hormone-depleted calf serum with and without 1 µM T3 for 48 h (as indicated) before harvesting for CAT assay. CAT expression was measured by determining the extent of [14C]chloramphenicol (50 mCi/mmol; NEN Life Science Products) acetylation as described previously (41, 42). Protein concentration was determined (43), and amounts of cell protein assayed for CAT activity were varied to maintain the percentage of [14C]chloramphenicol acetylated in the linear range (<30%) during a 16-h incubation. CAT activity values were normalized to represent the percent of [14C]chloramphenicol acetylated by a specific amount of cell protein in 16 h at 37 °C. All experiments were performed in duplicate, and the results represent the means of at least three independent transfections. Within each transfection, duplicate samples varied less than 10%.
Gel Mobility Shift Assays--
cT3R,
cT3R
(21-408), cT3R
(21-408, 7/8),
cT3R
(51-408), and the various cT3R
NH2-terminal amino acid mutants were expressed by in
vitro translation in reticulocyte lysates (24). A fraction of each
translation received L-[35S]cysteine in the
incubation, and the amount of each 35S-synthesized protein
was analyzed by SDS-gel electrophoresis and quantitated using a
Molecular Dynamics PhosphorImager with ImageQuant software. The number
of fmol of each cT3R
protein synthesized in the lysate
was estimated by the relative amounts of the individual
35S-proteins and quantitation of reticulocyte
lysate-synthesized cT3R
by binding with
L-[125I]T3 (24). Reticulocyte
lysate-translated cT3R
proteins (about 10 fmol/µl of
lysate) were incubated with 5 fmol (30,000 dpm) of the
32P-labeled TREp or the TRE1/2 element. The
30-µl incubation mixture contained 25 mM Tris (pH 7.8),
0.5 mM EDTA, 75 mM KCl, 1 mM
dithiothreitol, 0.2 µg of poly(dI-dC), 0.05% Triton X-100 (v/v), 30 µg of ovalbumin, 0.3 µM ZnCl2, 0.2 µg of
RNase A, and 10% glycerol (v/v) and either 0.5, 1.5, 3.0, or 4.5 µl
of reticulocyte lysate (24). Control reticulocyte lysate was added to
the 0.5-, 1.5-, or 3.0-µl receptor preparations to adjust the final
amount of lysate in each sample to 4.5 µl. Unless indicated
otherwise, a comparison of the different cT3R
proteins
used the same number of fmol of receptor. Samples were analyzed by
electrophoresis at 4 °C for 50 min in nondenaturing 6%
polyacrylamide gels (acrylamide:bisacrylamide, 37.5:1) in buffer containing 10 mM Tris, 7.5 mM acetic acid, 40 µM EDTA (pH 7.8) (12, 14). The gels were then dried and
autoradiographed. The assignment of receptor monomers and homodimers is
based on the mobility of purified cT3R
(14). Certain gel
shift studies were quantitated using a PhosphorImager as indicated.
Influence of the NH2 Terminus of cT3R
on the Formation of cT3R
·RXR Heterodimers in Solution
in the Absence of DNA--
The binding of
35S-cT3R
and mutants to GST and GST-RXR
in vitro were carried out as described previously (34).
L-[35S]Cysteine-labeled wild-type
cT3R
, cT3R
(21-408), and the
cT3R
(21-408, 7/8) NH2-terminal mutant were
prepared using TNT reticulocyte lysates (Promega). 25,000 dpm of
35S-labeled protein was incubated with 25 ng of GST-RXR or
10 ng of GST protein immobilized on glutathione-agarose beads in 300 µl of Buffer A for 1 h at 4 °C on an Orbitron rotator. Buffer A consists of 50 mM KCl, 25 mM Hepes (pH 7.9),
6% glycerol, 5 mM EDTA, 5 mM
MgCl2, 1 mM dithiothreitol, and 0.05% Triton
X-100 (34). The amount of GST protein used (10 ng) was equivalent to
the amount of GST in the GST-RXR fusion protein (25 ng). Beads were
collected by centrifugation at 4 °C for 5 min at ~500 × g and washed three times with 1 ml of Buffer A. The bound
proteins were eluted with SDS-gel loading buffer and analyzed by
SDS-gel electrophoresis followed by autoradiography.
35S-Labeled wild-type or mutant proteins in the binding
assays were analyzed by electrophoresis and autoradiography to ensure
that equal amounts of input radioactivity of the labeled protein were used.
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RESULTS |
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Residues 23KRKRK27 of the
NH2-terminal A/B Domain of cT3R Are
Necessary for Maximal Transcriptional Activation of Native
TREs--
Our previous studies aimed at elucidating the functional
role of the NH2-terminal A/B region of cT3R
indicated that amino acids 21-30 of cT3R
, which are
also conserved in the NH2-terminal A/B domains of
rT3R
1 and hT3R
1, are essential for both
transcriptional activation and for efficient binding to TFIIB (34).
More detailed in vitro binding studies revealed that
residues 23KRKRK27 centered within amino acids
21-30 are required for efficient binding of cT3R
with
TFIIB (34). This suggests that the functional activity that we
originally mapped to amino acids 21-30 may depend solely on these 5 basic residues. To test this possibility we compared the functional
activities of cT3R
(21-408) and
cT3R
(21-408, 7/8) in which
23KRKRK27 was changed to
23TITIT27. In transfection experiments with a
reporter gene regulated by a single idealized TRE organized as an
inverted repeat (TREp) of the optimized AGGTCA half-site
(
MTV-TREp-CAT), cT3R
(21-408) and cT3R
(21-408, 7/8) showed similar activity (Fig.
1A). In contrast, with
MTV-TRE-GH-CAT or
MTV-TRE-Mal-CAT, which contain native TREs,
cT3R
(21-408) was much more active (Fig. 1, B
and C). These results are similar to our previous findings
that showed that the functional effect of the NH2 terminus
is much more prominent on reporters containing lower affinity native
TREs compared with reporters containing the idealized higher affinity
TREp (34). Finally, coexpression of TFIIB, although enhancing the
activity of both receptors, results in a much higher level of
T3-dependent stimulation by
cT3R
(21-408) (Fig. 1C). The ability of TFIIB
to enhance the activity of cT3R
(21-408, 7/8) partially
is not altogether unexpected and is similar to our previous results
with cT3R
(51-408). This most likely stems from the low
affinity of amino acids 119-154 of the cT3R
D region
for TFIIB (34).
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Residues 23KRKRK27 Affect DNA Binding of
cT3R as Monomers and Homodimers--
The
NH2 terminus of T3R isoforms may confer cell
type and promoter specificity not just through divergent,
isotype-distinct, interactions with other transcription factors (34,
44) but also through conferring distinct DNA binding properties to
receptor isotypes (45, 46). For example, the DNA binding properties of
v-erbA differ from those of c-erbA (cT3R
) and more
closely resemble those of the RARs. The structural basis behind this
difference in DNA recognition by v-erbA results from one or more
changes within the v-erbA NH2-terminal domain (45, 47).
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T3 Inhibits Both Monomer and Homodimer DNA Binding of
cT3R NH2-terminal Mutants--
Ligand
binding to DNA-bound T3R is accompanied by a conformational
change of the receptor (48) which may alter the T3R-DNA interaction (49). For example, T3 inhibits homodimer
formation in vitro on various direct and everted, but not
inverted, TREs (49, 50). In addition, T3 increases the
electrophoretic mobility of T3R monomers and homodimers and
of T3R-RXR heterodimers on inverted repeat TREs without
changing their apparent stability (14, 24).
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NH2-terminal Mutants of cT3R Bind Poorly
to a TRE1/2--
The preferential binding of
cT3R
(21-408, 7/8) or cT3R
(51-408) to
the TREp as a homodimer compared with the predominant monomeric binding
of cT3R
(21-408) or wild-type cT3R
may
result from an increased potential to homodimerize and/or a decreased
potential to bind DNA as a monomer. This latter instance might result
in an increased amount of this mutant receptor available to bind as a
homodimer. To assess effects of the 23KRKRK27
sequence in the NH2 terminus on monomer binding, we
compared the binding of cT3R
(21-408) and
cT3R
(21-408, 7/8) with a TRE1/2 in the
absence and presence of T3 (Fig.
4). The TRE1/2 is the same as
the TREp except that it contains a single G to C change in one of the
half-sites (AGGTCA to ACGTCA). Whereas cT3R
(21-408) binds to the TRE1/2 in the absence or presence of
T3 (lanes 1 and 2),
cT3R
(21-408, 7/8) does not bind to this element without or with T3 (lanes 3 and 4). This
suggests that NH2-terminal amino acids
23KRKRK27 impose preferential monomer DNA
binding on cT3R
.
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The DNA Binding Properties of cT3R Are Not Directly
Related to the Number of Basic Residues Contained within
23KRKRK27--
To assess whether all or some
of the basic residues within 23KRKRK27 are
necessary for preferential monomer binding of wild-type
cT3R
, we performed gel mobility shift assays with
NH2-terminal mutants containing different numbers and
combinations of basic and neutral amino acid residues. As shown in Fig.
5A, wild-type
cT3R
and cT3R
(21-408) bind the TREp
predominantly as monomers (lanes 1 and 3),
whereas cT3R
(51-408) and cT3R
(21-408,
7/8) bind predominantly as homodimers (lanes 2 and
4). Interestingly, mutant cT3R
(21-408, 9/10), which has only the first 3 basic amino acid residues changed (23TITRK27), binds the TREp very poorly as a
monomer and not at all as homodimer (lane 5). In contrast,
mutant cT3R
(21-408, 11/12, lane 6), which has the last 3 basic amino acid residues changed
(23KRTIT27), binds the TREp almost as
efficiently as cT3R
(21-408, 7/8) or
cT3R
(51-408) with slightly more monomer than homodimer.
Finally, mutant cT3R
(21-408, 13/14) (lane
7), which has only the middle basic amino acid preserved
(23TIKIT27), binds in a way similar that of
cT3R
(21-408, 11/12) but less efficiently. These data
indicate that the affinity and mode of binding of these mutants to the
TREp are not related directly to the number of the basic amino acid
residues within amino acids 21-30. However, all 5 basic amino acid
residues 23KRKRK27 are necessary for
predominant high affinity monomer binding to TREp.
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Amino acids 23KRKRK27 Affect the Efficiency
of T3R·RXR Heterodimer Formation--
Although amino
acids 23KRKRK27 influence monomer binding of
receptor to DNA, these residues may also affect the ability of the receptor to dimerize. We examined this possibility by assessing the
ability of cT3R(21-408) or cT3R
(21-408,
7/8) to bind as heterodimers with RXR on the TREp (Fig.
6). Incubations were performed either in
the absence or the presence of T3 and/or
9-cis-RA. As shown previously, cT3R
(21-408)
alone binds to the TREp predominantly as a monomer, and the
electrophoretic mobility of this complex is increased by T3
(Fig. 6, lanes 1 and 2). In contrast,
cT3R
(21-408, 7/8) binds the TREp predominantly as a
homodimer, and this complex is abolished by T3 (lanes
7 and 8). Interestingly, in the absence of
T3 cT3R
(21-408) forms heterodimeric
complexes with RXR less efficiently than cT3R
(21-408,
7/8) (lanes 3 and 9), and T3
increases the electrophoretic mobility of both complexes (lanes
4 and 10). 9-cis-RA alone does not affect
either complex (lanes 5 and 11), whereas the
combination of T3 and 9-cis-RA affects the
mobility of both complexes as with T3 alone (lanes
6 and 12). Similar results were also found using a
32P-DR+4 containing AGGTCA half-sites instead of the TREp
(data not shown). Importantly, these results indicate that amino acids 23KRKRK27 affect both monomer DNA binding and
dimerization.
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DISCUSSION |
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Amino Acids 23KRKRK27 of the A/B Domain Are
Required for Maximal Transcriptional Activation of Native TREs by
cT3R--
A conserved sequence of 10 amino acids from
NH2-terminal A/B domain of cT3R
,
rT3R
1, and hT3R
1 is essential both for
transcriptional activation and for efficient binding to TFIIB (34).
Within this sequence, 5 basic amino acid residues,
23KRKRK27, proved absolutely necessary for
efficient receptor binding to TFIIB (34). A sequence similar to the
23KRKRK27 sequence found in T3R
is not found in the A/B domains of T3R
1 or
T3R
2. Indeed, we have found that removal of the A/B
domain of rat T3R
1 does not alter its ability to
interact with TFIIB or to activate the rat TRE-GH or TRE-Mal in
MTV-CAT (data not shown). However, all nuclear receptors contain a
conserved basic sequence that follows their DBD, which appears to play
a role in TFIIB binding (34).
Amino Acids 23KRKRK27 Affect DNA Binding of
cT3R as Monomers and Homodimers--
Previous studies
from our laboratory indicated that the NH2-terminal region
of cT3R
might affect DNA binding of receptor to the TREp
and the native TRE from the rat growth hormone gene promoter by
altering the extent of monomer binding and dimerization (34).
Hollenberg et al. (51) also found that the NH2
terminus of hT3R
1 reduced the ability of the receptor to
dimerize. Our current study documents that the basic amino acid
sequence 23KRKRK27 plays a key role in both DNA
binding and dimerization of cT3R
in addition to its role
in transcriptional activation and TFIIB binding. First, both
cT3R
(21-408, 7/8), in which amino acids 23KRKRK27 were changed to
23TITIT27, and cT3R
(51-408)
bind to the TREp more efficiently as homodimers than
cT3R
(Fig. 2A). More direct evidence for the
involvement of these residues in the DNA binding of receptor is
provided by the finding that cT3R
(21-408) binds
predominantly as a monomer even at high concentrations, whereas
cT3R
(21-408, 7/8) binds efficiently as a homodimer even
at low concentrations (Fig. 2A). We obtained results in gel
shift studies using a DR+4 containing AGGTCA half-sites similar to
those with the TREp (not shown). Interestingly, although the binding of
cT3R
and cT3R
(21-408) to the TREp is not
affected significantly by T3, the binding of cT3R
(21-408, 7/8) and cT3R
(51-408) to
this element is inhibited strongly (Fig. 3). Hence, residues
23KRKRK27 may either stabilize a "proper"
conformation of the DBD, or they may ensure optimal positioning of the
DBD with respect to the TREp half-site. These alternative mechanisms
may occur either through a direct interaction between amino acids
23KRKRK27 and the DBD or through interaction of
these basic residues with some other region of the receptor such as the
ligand binding domain, which may affect the structure of the DBD.
Alternatively, residues 23KRKRK27 may affect
both the integrity and positioning of the DBD and in addition may
contact DNA directly. Irrespective of the actual mechanism by which
amino acids 23KRKRK27 affect receptor DNA
binding, they seem absolutely necessary in the presence of
T3 for the receptor to bind to the TREp (Fig. 3).
DNA Binding of cT3R as Monomers and Homodimers Is
Influenced by Different Basic Amino Acids within the
23KRKRK27 Sequence--
The basic residues
23KRKRK27 are not equally important for
preferential monomer binding of wild-type
-receptor. For example, cT3R
(21-408, 9/10), which has amino acids
23TITRK27, binds to the TREp very poorly and
only as a monomer (Fig. 5). In contrast, cT3R
(21-408,
11/12), which has amino acids 23KRTIT27, binds
to the TREp in a way similar to that of cT3R
(51-408) with slightly more monomer than homodimer. Finally,
cT3R
(21-408, 13/14), which has amino acids
23TIKIT27, binds to the TREp in a way similar
to that of cT3R
(21-408, 11/12) but less efficiently.
Hence, the affinity and the mode of binding of these mutants to the
TREp are not related directly to the number of the basic amino acid
residues within the sequence 23-27, and therefore these residues do
not contribute equally to the receptor DNA binding. However, all 5 basic amino acid residues 23KRKRK27 are
necessary for predominant high affinity monomer binding to TREp. In
contrast, the affinity of cT3R
for TFIIB does correlate directly with the number of these basic amino acids (data not shown).
The NH2-terminal 23KRKRK27
Sequence Influences the Extent of cT3R·RXR Heterodimer
Formation--
In addition to the effect of residues
23KRKRK27 on the binding of receptor monomers
to DNA, these residues also influence the intrinsic dimerization
potential of T3R
. Fig. 6 indicates that cT3R
(21-408, 7/8) binds as a heterodimer with RXR to
the TREp element much more efficiently than
cT3R
(21-408). Thus, ~90% of the total TREp-bound
cT3R
(21-408, 7/8) is bound as a heterodimer with RXR,
whereas only ~20% cT3R
(21-408) participates in such complexes (Fig. 7A, lanes 2 and 7).
Both complexes showed similar dissociation rates in the presence of a
1,000-fold excess of unlabeled TREp (Fig. 7A, lanes
2-5 and 7-10, and Fig. 7B), suggesting
that the increased amount of heterodimers found with the
cT3R
mutant results from the more efficient formation
and/or binding cT3R
(21-408, 7/8)·RXR heterodimers to
DNA. GST binding studies (Fig. 8) suggest that this increase results
from the more efficient formation of heterodimers in solution, which
then bind to DNA. The increased dimerization potential of the
cT3R
(21-408, 7/8) on the TREp does not result in
enhanced activation of the
MTV-TREp-CAT reporter compared with
cT3R
(1-408) or cT3R
(21-408) (Fig.
1A). This discrepancy may reflect the weaker interaction of
cT3R
(21-408, 7/8) with TFIIB, which could result in a
number of transcriptionally active cT3R
(21-408,
7/8)·RXR·TFIIB complexes on the optimized element similar to those
with cT3R
(21-408) or wild-type
cT3R
(1-408).
Influence of the NH2-terminal A/B Domain on DNA Binding
of Other Members of the Thyroid Hormone/Retinoid Receptor
Subfamily--
Several additional reports have provided evidence for
involvement of the NH2 termini of related members of the
nuclear hormone receptor family in DNA binding. Two
NH2-terminal amino acids of v-erbA, His12 and
Cys32 (which correspond to Arg24 and
Tyr44 of cT3R) have been shown, in
conjunction with amino acid changes in the zinc finger domain, to
contribute to a restricted half-site DNA binding specificity (45, 47,
52). RXR
and RXR
, but not RXR
, have been suggested to activate
transcription by forming tetrameric complexes on DNA elements
consisting of four reiterated weak half-sites (53). These
isoform-specific DNA binding properties mapped to the
NH2-terminal A/B domains. Replacing the RXR
A/B domain
with that of RXR
resulted in both tetramer binding to DNA and
transcriptional activation by the chimeric protein. That the
NH2-terminal domain and the zinc finger region of nuclear hormone receptors may functionally cooperate was also shown by a study
of the DNA binding properties of the RORs (46, 54). Thus, the
differential DNA binding activities of ROR
1, ROR
2, and ROR
3
depend on their distinct NH2 termini, which, when fused to
heterologous nuclear hormone receptors (e.g.
hT3R
1), may impose novel DNA binding specificities.
Finally, T3R
and T3R
bind differently to
the same DNA element (33). Whereas T3R
binds
predominantly as a monomer to the TREp (14), T3R
1 binds
predominantly as a homodimer, which is thought to result in part from
its increased ability to dimerize (33, 51, 55). Importantly,
T3R
1 and T3R
show no homology in their
NH2-terminal A/B domains. In particular, T3R
1 does not contain a sequence in its A/B domain
similar to the T3R
-specific KRKRK, which may account for
the finding that mutant cT3R
(21-408, 7/8) exhibits
certain DNA binding properties more like T3R
1 than
wild-type cT3R
.
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FOOTNOTES |
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* This research was supported in part by National Institutes of Health Grant DK16636 (to H. H. S.). Oligonucleotide synthesis was provided by the New York University Medical Center (NYUMC) General Clinical Research Center (National Institutes of Health Grant M01RR00096). Sequence analysis and data base searches were through the NYUMC Research Computing Resource, which received support from National Science Foundation Grant DIR-8908095). This study is in partial fulfillment of a Ph.D. degree from the Sackler Institute for Graduate Biomedical Sciences (for E. H.) and for the Clinical and Molecular Endocrinology Training Program, New York University School of Medicine (for I. H.).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.
§ Present address: Laboratory of Molecular Cell Biology, Rockefeller University, 1230 York Ave., New York, NY 10021.
¶ Member of the NYUMC Cancer Center (Grant CA16087). To whom correspondence should be addressed: Dept. of Medicine and Pharmacology, TH-454, New York University Medical Center, 550 First Ave., New York, NY 10016. Tel.: 212-263-6279; Fax: 212-263-7701; E-mail: samueh01{at}mcrcr.med.nyu.edu.
1 The abbreviations used are: HRE(s), hormone response element(s); T3, triiodothyronine; T3R, triiodothyronine receptor; RAR, retinoic acid receptor; RXR, retinoid X receptor; h, human; r, rat; c, chicken; DR, direct repeat; IR, inverted repeat; ER, everted repeat; DBD, DNA binding domain; TFIIB, transcription factor IIB; MTV, mammary tumor virus; TREp, palindromic thyroid hormone response element; CAT, chloramphenicol acetyltransferase; GH, growth hormone; LTR, long terminal repeat; Mal, malic enzyme; TRE1/2, TREp with single G to C change in one of the half-sites; pEX, pEXPRESS; RSV, Rous sarcoma virus; GST, glutathione S-transferase; 9-cis-RA, 9-cis retinoic acid; ROR, retinoid orphan receptor.
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