Isoform-Specific Transcriptional Regulation by Thyroid Hormone Receptors: Hormone-Independent Activation Operates through a Steroid Receptor Mode of Coactivator Interaction
Zhihong Yang and
Martin L. Privalsky
Section of Microbiology Division of Biological Chemistry
University of California at Davis Davis, California 95616
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ABSTRACT
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Thyroid hormone receptors (T3Rs) are
hormone-regulated transcription factors that play important roles
in vertebrate homeostasis, differentiation, and development. T3Rs are
synthesized as multiple isoforms that display tissue-specific
expression patterns and distinct transcriptional properties. Most T3R
isoforms associate with coactivator proteins and mediate
transcriptional activation only in the presence of thyroid hormone. The
pituitary-specific T3Rß-2 isoform departs from this general rule and
is able to interact with p160 coactivators, and to mediate
transcriptional activation in both the absence and presence of hormone.
We report here that this hormone-independent activation is mediated by
contacts between the unique N terminus of T3Rß-2 and an internal
interaction domain in the SRC-1 (steroid receptor coactivator-1) and
GRIP-1 (glucocorticoid receptor interacting protein 1) coactivators.
These hormone-independent contacts between T3Rß-2 and the p160
coactivators are distinct in sequence and function from the LXXLL
motifs that mediate hormone-dependent transcriptional activation and
resemble instead a mode of coactivator recruitment previously observed
only for the steroid hormone receptors and only in the presence of
steroid hormone. Our results suggest that the transcriptional
properties of the different T3R isoforms represent a combinatorial
mixture of repression, antirepression, and hormone-independent and
hormone-dependent activation functions that operate in
conjunction to determine the ultimate transcriptional outcome.
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INTRODUCTION
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Nuclear hormone receptors are transcription factors that mediate
cellular responses to a variety of small lipophilic ligands, including
the steroids, vitamin D3, retinoids, and thyroid
hormones (1, 2, 3, 4). As such, nuclear hormone receptors play essential
roles in metazoan reproduction, development, differentiation, and
homeostasis. Nuclear hormone receptors function at the molecular level
by binding to specific DNA sequences, denoted response elements, and
regulating the expression of adjacent target genes. Intriguingly, many
nuclear hormone receptors display bimodal transcriptional properties,
with a given receptor able to either repress or activate expression of
its target genes under different conditions (5, 6, 7, 8, 9, 10).
Best characterized of this class of bimodal nuclear receptors are the
thyroid hormone receptors (T3Rs). T3Rs are expressed in virtually all
vertebrate tissues and are involved in regulating such diverse
physiological processes as general metabolic rate, thermogenesis,
central nervous system development, and glucose utilization in response
to T3 and T4 (11, 12, 13, 14). The
- and ß-loci, in turn, can be expressed through differential
splicing to generate three primary T3R protein isoforms: T3R
-1,
T3Rß-0 (in birds, analogous to ß-1 in mammals), and T3Rß-2 (Fig. 1A
). T3R
-1 expression begins in early
embryonic development and continues into the adult, where it is found
in most tissue and cell types; the onset of T3Rß-0/1 and ß-2
expression occurs later in embryonic development and generally
parallels the appearance of circulatory T3 and
T4 hormone (11, 12, 13, 14). Whereas T3Rß-0/1 is
expressed in an assortment of different tissue types, the T3Rß-2
isoform is restricted in its expression primarily to the adult
pituitary and hypothalamus (11, 12, 13, 14). Genetic disruption experiments
confirm that the T3R
and T3Rß isoforms play different, if
overlapping, roles in normal physiology (12, 14).

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Figure 1. Transcriptional Activation by T3Rß-2 in the
Absence of Hormone
A, Schematic of different T3R isoforms. The different T3R isoforms
described in the text are represented from N to C terminus, with the
locations of the DNA binding and hormone binding domains indicated. The
amino acid numbering system for T3Rß-2 is shown beneath the
corresponding schematic. B, Transcriptional regulation by
full-length T3R isoforms. CV-1 cells (left panel) or JEG-3
cells (right panel) were transfected with a
TRE-tk-luciferase reporter, a pCH110-lac Z (CV-1) or
pCMV-lac Z (JEG-3) internal control reporter, and with a
pSG5-expression vector encoding the avian T3R isoforms indicated
beneath the panel. The cells were incubated in the absence
(hatched bars) or presence (solid bars) of 100
nM T3 hormone and were
subsequently harvested, and the relative luciferase activity was
determined as described in Materials and Methods. The
averages of two or more experiments and the standard deviations are
shown. C, Transcriptional regulation by the abstracted
N-terminal domains of different T3R isoforms. CV-1 cells were transfected
with a 5 X Gal 17-mer luciferase reporter, a pCMV-lac Z
internal control reporter, and a pSG5-GAL4DBD expression plasmid
containing the N-terminal 50 amino acids of T3R , the N-terminal 70
amino acids of T3Rß-2, or the N-terminal 107 amino acids of T3Rß-2,
as indicated below the panel. The cells were incubated in
the absence of hormone and were subsequently harvested, and the
relative luciferase activity was determined as described in
Materials and Methods. The averages of two or more
experiments and the SD values are shown.
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The transcriptional properties of the T3Rs reflect the ability of these
receptors to associate with auxiliary protein complexes, denoted
corepressors and coactivators (5, 6, 7, 8, 9, 10). Once tethered to the nuclear
receptor, the corepressors and coactivators mediate the molecular
events that confer the ultimate transcriptional outcome, negative or
positive. Known corepressor proteins include SMRT (silencing mediator
for retinoid and thyroid hormone receptors) and its paralog N-CoR
(nuclear receptor corepressor), mSin3, histone
deacetylases, and an assortment of additional polypeptides, such as
ski, SAP-18 and SAP-30 (5, 6, 7, 8, 9, 10). Coactivator proteins include the p160
polypeptide coactivator family [such as SRC-1 (steroid receptor
coactivator) and GRIP-1 (glucocorticoid receptor interacting protein
1)], the CBP (CREB-binding protein)/p300 proteins, and the
DRIP/TRAP/SMCC/ARC complex. Corepressors and coactivators appear to
modulate transcription both by covalent modification of the
chromatin/nucleosome template and by direct interactions with
components of the general transcriptional machinery (5, 6, 7, 8, 9, 10).
T3Rs typically repress transcription in the absence of hormone and
activate transcription in the presence of cognate
T3 hormone (e.g. Refs. 15, 16, 17, 18). In the
absence of hormone, a hydrophobic groove on the surface of the nuclear
hormone receptor is believed to bind to an
-helical I/LXXII motif on
the surface of the SMRT/N-CoR corepressor, thereby tethering the
corepressor complex to the receptor (19, 20, 21).
Conversely, the binding of hormone by the nuclear hormone receptor is
thought to reorient the C-terminal helix 12 of the receptor so as to
occlude the binding site for corepressor, and to form a new binding
site for LXXLL protein interaction motifs that are present in many
coactivators (22, 23, 24, 25). The interaction surface on the nuclear hormone
receptor that binds coactivator in this hormone-dependent fashion maps
to helices 3, 4, 5, and 12 within the hormone-binding domain and
has been denoted the "activation function-2" (AF-2) domain
(22, 23, 24, 25).
The T3R
-1 and ß-0/1 isoforms conform to this generic model and
operate as transcriptional repressors in the absence of
T3 and as activators in its presence (15, 16, 18, 26, 27, 28). The T3Rß-2 isoform, however, is a notable exception and
fails to repress in the absence of hormone (18, 28). Instead, in the
absence of hormone the T3Rß-2 isoform either is neutral in its
effects or exhibits a moderate activation of target gene expression
that is further enhanced by the presence of hormone (18, 28, 29, 30). We
have reported that the inability of the T3Rß-2 to repress
transcription is due to an antirepression mechanism by which the
unliganded T3Rß-2 recruits the SMRT/N-CoR protein in an inactive form
and prevents the subsequent assembly of a functional corepressor
complex (31). This mechanism accounts for the failure of T3Rß-2 to
repress, but does not fully explain the hormone-independent T3Rß-2
transcriptional activation that is observed with certain promoters or
in certain cell contexts.
Here, we report that there is an isoform-specific transcriptional
activation domain in the N terminus of T3Rß-2 that is able to recruit
the p160 family of coactivators in the absence of hormone. This
N-terminal AF-1 activation domain in T3Rß-2 interacts with an
internal glutamine-rich (Q-rich) p160 domain that is distinct in
sequence and function from the LXXLL motifs that contact the
hormone-dependent AF-2 domain. Therefore, the T3Rß-2 isoform makes at
least two distinct contacts with the p160 coactivators: the AF-1/Q-rich
p160 domain interaction that prevails in the absence of hormone and the
AF-2/LXXLL interaction that dominates in the presence of hormone. This
hormone-independent mode of coactivator recruitment by T3Rß-2 is
reminiscent of the dual contact mode of coactivator interaction
reported for androgen receptor (AR), although for the AR these dual
contacts serve to enhance hormone-dependent activation, rather than to
mediate hormone-independent transcriptional regulation (32, 33, 34, 35, 36). Unlike
T3Rß-2, the T3R
-1 and ß-0 isoforms lack detectable N-terminal
coactivator interaction domains, and interact with p160 coactivator by
a single set of contacts in a strictly hormone-dependent manner. Taken
as a whole, our results indicate that the transcriptional properties of
the different T3R isoforms represent an admixture of repression,
antirepression, and hormone-independent and hormone-dependent
activation functions that operate in conjunction to determine the
ultimate transcriptional outcome.
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RESULTS
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T3Rß-2 Fails to Repress Transcription and Displays
Hormone-Independent Activation in Certain Cell Contexts
We first employed a thymidine kinase promoter-luciferase reporter
construct bearing a direct repeat (DR)-4 response element to
characterize the transcriptional properties of the different T3R
isoforms in CV-1 cells. CV-1 cells express very low levels of
endogenous T3Rs and exhibit little or no reporter gene responsiveness
in the absence of exogenously introduced receptor (Fig. 1B
, left
panel). As previously noted (31), introduction of the avian
T3R
-1 or T3Rß-0 isoforms repressed reporter gene expression in the
absence of hormone, resulting in lower luciferase expression than that
observed without receptor (Fig. 1B
, left panel). Conversely,
addition of T3 hormone to the cells transfected
with T3R
-1 or T3Rß-0 resulted in a strong stimulation of reporter
gene expression above the levels observed in the absence of receptor
(Fig. 1B
). Introduction of the avian T3Rß-2 isoform resulted in an
analogous activation of reporter gene expression in the presence of
hormone, but in contrast to the other two isoforms, T3Rß-2 failed to
repress in the absence of hormone (Fig. 1B
, left panel).
Rather than being truly neutral in the absence of hormone, the
unliganded T3Rß-2 isoform often induced a weak increase in reporter
gene expression above that observed with an empty vector (Fig. 1B
, left panel). This hormone-independent activation by T3Rß-2
could be observed over a range of expression and reporter vector
concentrations (data not shown). Other researchers have also described
this phenomenon and have noted that the magnitude of this
hormone-independent activation differs with different promoters and in
different cell types (18, 28, 29). Consistent with these studies, the
hormone-independent activation function of the T3Rß-2 isoform was
stronger in JEG-3 cells than in CV-1 cells (Fig. 1B
, right
panel). We conclude that T3Rß-2 not only fails to repress, but
can actually activate, gene expression in the absence of hormone.
The N Terminus of T3Rß-2 Possesses a Hormone-Independent
Activation Function
The T3Rß-0 and ß-2 isoforms are expressed by alternative mRNA
splicing and differ only in that the latter contains an extra
N-terminal A/B domain (Fig. 1A
) (18, 37). We therefore examined whether
the N terminus of T3Rß-2 possessed an inherent activation function
that might contribute to the hormone-independent activation properties
of this isoform. An empty GAL4 DNA binding domain (DBD) construct
exhibited little or no ability to modulate the expression of a reporter
gene containing GAL4 17-mer binding motifs (Fig. 1C
). In contrast,
GAL4DBD-fusions bearing the N-terminal 170, or 1107 amino acids of
T3Rß-2 strongly activated expression of the GAL4 17-mer reporter and,
as expected from the nature of the fusion, activation by the T3Rß-2
N-terminus did not require T3 hormone (Fig. 1C
).
The N-terminal A/B domain from the T3R
-1 isoform, when tested as an
analogous GAL4DBD fusion, failed to activate transcription from the
GAL4 17-mer reporter (Fig. 1C
), whereas the T3Rß-0 isoform lacks an
A/B domain of significant length to test. We conclude that the T3Rß-2
isoform possesses an autonomous, hormone-independent transcriptional
activation function within its N terminus that is not present in
T3R
-1 or ß-0.
T3Rß-2 Displays a Hormone-Independent Interaction with the SRC-1
and GRIP-1 Members of the p160 Coactivator Family
SRC-1 and GRIP-1 are members of the p160 class of transcriptional
coactivators and play important roles in hormone-dependent
transcriptional activation by a variety of nuclear hormone receptors
(38, 39). We therefore examined whether T3R
-1, ß-0, and ß-2
displayed distinctive interactions with these p160 coactivators that
paralleled the transcriptional activation properties of these
different isoforms. We first tested whether glutathione
S-transferase (GST)-T3R constructs, immobilized on
glutathione-agarose, were able to bind to the various full-length p160
coactivators, synthesized and radiolabeled by in vitro
transcription and translation. Coactivator bound by the immobilized
receptors was eluted from the agarose matrix, was resolved by SDS-PAGE,
and was visualized by phosphorimager analysis (Fig. 2
). Only low, background levels of
binding of either SRC-1 or GRIP-1 to GST-T3R
-1 or ß-0 were
observed in the absence of hormone (Fig. 2A
, lanes 4 and 6, and
quantified in Fig. 2
, B and C). In contrast, both p160 coactivators
bound at much higher levels to GST-T3R
-1 and to GST-T3Rß-0 in the
presence of 100 nM T3 (Fig. 2A
, lanes 5 and 7, and quantified in Fig. 2
, B and C). Neither
coactivator exhibited appreciable binding to nonrecombinant GST
protein, used as a negative control, in either the absence or the
presence of T3 (Fig. 2A
, lanes 2 and 3, and
quantified in Fig. 2
, B and C). We conclude that the interaction of
T3R
-1 and T3Rß-0 with SRC-1 or with GRIP-1 is strongly dependent
on the presence of hormone.

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Figure 2. Binding of the SRC-1a and GRIP-1 Coactivators to
Different GST-T3R Fusion Proteins in Vitro
A, Binding of radiolabeled GRIP-1 and SRC-1a to immobilized
GST-T3R protein constructs. Radiolabeled GRIP-1 or SRC-1a was incubated
with nonrecombinant GST protein, or with GST-fusion proteins
representing the full-length open reading frames of T3R -1, ß-0, or
ß-2, in the absence or presence of 100 nM
T3 hormone as indicated above the
panels. The GST proteins, immobilized to a glutathione-agarose matrix,
were then extensively washed and any proteins remaining bound to the
glutathione-agarose matrix were eluted with soluble glutathione and
were resolved by SDS-PAGE. Aliquots of the SRC1a and GRIP-1
preparations employed in the binding assays were analyzed in separate
lanes for comparison ("10% input"). A phosphorimager scan of the
resulting electrophoretograms is presented. B,
Quantification of the SRC-1a results. The results from the experiments
depicted in panel A were quantified by phosphorimager analysis; the
data are expressed as the percentage of the input coactivator protein
that remained bound to the different GST fusion proteins after washing.
Different scales are used for the (-) and (+) hormone conditions.
C, Quantification of the GRIP-1 results. The same protocol as in
panel B was employed.
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In contrast to the GST-T3R
-1 or ß-0 isoforms, binding of the
GST-T3Rß-2 protein to the p160 coactivators displayed both a
hormone-independent and a hormone-dependent component (Fig. 2A
, lanes 8
and 9). Approximately 8.4% of the SRC-1 input bound to GST-T3Rß-2 in
the absence of hormone, and this increased to 30.3% in the presence of
100 nM T3 hormone (Fig. 2B
). Although
modest, this interaction of SRC-1 with T3Rß-2 in the absence of
hormone was highly reproducible, was not seen with the nonrecombinant
GST control, and was approximately 6-fold above that seen with the
other receptor isoforms. An analogous, hormone-independent interaction
of GST-T3Rß-2 was also observed with the GRIP-1 coactivator;
approximately 4% of the input GRIP-1 protein bound in the absence of
hormone and this increased to 67% in the presence of 100
nM T3 hormone (Fig. 2B).
The N Terminus of T3Rß-2 Is Responsible for the
Hormone-Independent Recruitment of p160 Coactivators
T3Rß-0 and ß-2 differ only due to the presence of an
extended N-terminal domain in the latter (Fig. 1A
), suggesting that the
T3Rß-2 N terminus was likely responsible for the hormone-independent
interaction of this isoform with the SRC-1 and GRIP-1 coactivators. To
test whether the N terminus of T3Rß-2 alone was sufficient for this
interaction, we examined the ability of a GST-fusion restricted to the
N-terminal 1107 amino acids of T3Rß2 to bind to the full-length,
radiolabeled p160 coactivators. Both SRC-1 and GRIP-1 were able to bind
to the abstracted GST-T3Rß-2 N terminus in
vitro, whereas little or no coactivator bound to a nonrecombinant
GST control or to a corresponding N-terminal domain of T3R
-1 (Fig. 3
). In fact, the hormone-independent
interaction of T3Rß-2 with the p160 coactivators was more readily
detected in this context than when the GST-full-length receptor fusion
was employed; approximately 30% of the input SRC-1 and 24% of the
input GRIP-1 could bind to the abstracted T3Rß-2 N terminus under
these conditions, whereas binding to the nonrecombinant GST control was
less than 0.1% of input (Fig. 3
, bottom panels).

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Figure 3. Binding of the SRC-1a and GRIP-1 Coactivators to a
GST-Construct Limited to the N Terminus of T3Rß-2
An experiment similar to that in Fig. 2 utilizing radiolabeled
GRIP-1 and SRC-1a was performed, but the coactivator proteins were
incubated with nonrecombinant GST, with a GST-fusion polypeptide
representing the N-terminal 107 amino acids of T3R -1, or with a
GST-fusion polypeptide representing the N-terminal 107 amino acids of
T3Rß-2; no hormone was employed. Coactivator proteins remaining bound
to the immobilized GST or GST fusion proteins after washing were
eluted, were resolved by SDS-PAGE, and were visualized and quantified
by phosphorimager analysis. Both a phosphorimager scan and the
quantified results (expressed as the percentage of the input
coactivator protein that remained bound to the different GST fusion
proteins after washing) are presented.
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The T3Rß-2 N Terminus Interacts with a Glutamine-Rich p160
Coactivator Domain That Maps Outside of the LXXLL Motifs Implicated in
Hormone-Dependent Activation
We next examined the sites of contact of the T3Rß-2 N terminus
within the SRC-1 and GRIP-1 coactivators. We employed GST fusions
representing different subdomains of the p160 coactivators and
determined the ability of these GST constructs, isolated from
recombinant Escherichia coli, to bind to the different T3R
isoforms, synthesized as full-length proteins by in vitro
translation (Fig. 4
). A phosphorimager
scan from a representative experiment utilizing a series of GST-SRC-1
constructs is presented (Fig. 4A
); this experiment, together with
others, was also quantified (Fig. 4
, B and C). Both full-length
T3Rß-0 and full-length T3Rß-2 bound in a hormone-enhanced manner
to a GST-SRC-1 fusion construct containing the three internal,
clustered LXXLL repeats, whereas little or no binding of T3Rß-0 was
observed in the absence of hormone (Fig. 4
, A and B). This was also
true of a derivative of SRC-1 limited to the single LXXLL motif at the
extreme C terminus, indicating that this single LXXLL motif alone,
which is not found in the GRIP-1 paralog, is sufficient for a
hormone-dependent recruitment of this coactivator by the T3Rs (Fig. 4
, A and B). As expected, little or no binding of T3Rß-0 to any of the
SRC-1 derivatives was observed in the absence of hormone (Fig. 4
, A and
B, crosshatched bars).

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Figure 4. Mapping of Interaction Domains within the p160
Coactivators
A, Binding of radiolabeled T3Rß-0 and T3Rß-2 to GST fusion
proteins representing different domains of the p160 coactivators.
Radiolabeled T3Rß-0 (upper panel) or T3Rß-2 (lower
panel) protein preparations were incubated with immobilized GST
fusion proteins containing different domains of the SRC-1 protein, as
indicated above the panels. The incubations were performed
in the absence (-) or presence (+) of T3
hormone. The immobilized GST-protein was then washed extensively
(maintaining hormone in the washes where appropriate), and the proteins
remaining bound to the agarose matrix were eluted with glutathione and
were resolved by SDS-PAGE. Aliquots of the T3Rß-0 and ß-2
preparations employed in the binding assays were analyzed in separate
lanes for comparison ("10% input"). The locations of the
radiolabeled T3Rs were visualized by phosphorimager analysis. As noted
in the text, the T3Rß-2 translation products include a polypeptide,
initiated at an internal AUG, that lacks the native T3Rß-2 N
terminus; this truncated protein is denoted T3Rß-2t. B,
Quantification of the SRC-1a results. The results from the experiments
depicted in panel A were quantified by phosphorimager analysis and were
combined with similar quantifications of experiments employing
additional GST-SRC1a fusion constructs. Schematic representations of
the different GST and GST-SRC-1a proteins are depicted on the
left, and the quantified data, expressed as the percentage
of each T3R input preparation that remained bound to the different
GST-fusion proteins, is shown on the right. The
schematics indicate the locations of the LXXLL motifs, and
of the Q-rich internal domain described in the text. C,
Quantification of the GRIP-1 results. A similar protocol as in panel B
was employed.
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Conversely, the hormone-independent interaction of T3Rß-2 with
GST-SRC-1 did not require the LXXLL motifs of the coactivator, but was
dependent instead on a distinct p160 domain mapping between amino acids
977-1172 of SRC-1 (Fig. 4
, A and B). Analogous results were obtained
when the GST pull-down experiments were performed in a reciprocal
manner, using the T3R isoform as the GST fusion and the p160
coactivator as the in vitro translation product (data not
shown). Removal of this central, glutamine-rich (Q-rich) interaction
domain of SRC-1 greatly reduced the ability of T3Rß-2 to bind to
coactivator in the absence of hormone, whereas GST-p160 constructs that
retained this central domain retained the hormone-independent
interaction with T3Rß-2 (Fig. 4
, A and B). Very similar results were
obtained when GST-GRIP-1 fusions were employed in these assays; the
hormone-independent interaction of T3Rß-2 with these GST-GRIP-1
coactivator constructs depended on the presence of amino acids
11211304 of GRIP-1, which encompasses a Q-rich region closely
analogous to that found in the SRC-1 paralog (Fig. 4C
). The C-terminal
region of GRIP-1 lacks the solitary LXXLL motif found in this region of
SRC-1 and therefore did not mediate the hormone-dependent interaction
with T3Rß-0 and ß-2 that was observed for the otherwise equivalent
region of SRC1a (Fig. 4C
).
Of note, our in vitro translation reactions produce both
full-length T3Rß-2 and an artificially truncated T3Rß-2 derivative
that lacks the native N terminus [denoted T3Rß-2t and produced by a
translational initiation on an internal AUG; Fig. 4A
(31)]. The
T3Rß-2t derivative retains the C-terminal AF-2 domain and displays
the expected, hormone-dependent interaction with GST-p160 fusions that
contain one or more LXXLL motifs (Fig. 4
, A and B). Unlike the
full-length T3Rß-2 present in the same assays, however, T3Rß-2t did
not exhibit significant binding to any GST-p160 derivative in the
absence of hormone (Fig. 4
, AC). Conversely, the T3Rß-2 N-terminal
domain alone was sufficient for binding to GST-p160 derivatives
containing the Q-rich interaction domain, but exhibited greatly reduced
or no binding to GST coactivator derivatives lacking this domain (Fig. 5
). These results further support our
assignment of the hormone-independent interaction domain of T3Rß-2 to
the receptors N terminus and confirm that the receptor N
terminus interacts primarily with the central Q-rich domain of the p160
coactivators.

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Figure 5. Interaction of the Abstracted N Terminus of
T3Rß-2 with the Q-Rich Domain of the p160 Coactivators
A similar experiment as in Fig. 4A was performed, but employing a
radiolabeled polypeptide limited to the N-terminal 107 amino acids of
T3Rß-2. Both the phosphorimager scans and the quantified results
(expressed as the percentage of each T3R input preparation that
remained bound to the different GST-fusion proteins) are presented.
A, Interaction of the T3Rß-2 N terminus with the GST-SRC-1a
constructs. B, Interaction of the T3Rß-2 N terminus with the
GST-GRIP-1 constructs.
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The Central Interaction Domain of the p160 Coactivator Can Function
Together with, or Independent of, the LXXLL Motifs to Confer
Interaction with T3Rß-2
Our global deletion studies suggested the existence of two
interaction sites within the p160 coactivators: the LXXLL motifs, which
confer a hormone-dependent interaction with all the T3R isoforms
tested, and the central Q-rich domain, which confers a hormone-
independent interaction with T3Rß-2. To confirm this hypothesis,
we created two, more precisely defined GRIP-1 mutants: a small,
in-frame deletion of the central Q-rich domain (denoted
Q-rich), and
a mutant GRIP-1 in which all three LXXLL motifs were changed to LXXAA
motifs (denoted LXXAA) (Fig. 6A
). As
expected, the T3Rß-0 isoform displayed little or no interaction with
wild-type GRIP-1, or with either GRIP-1 mutant, in the absence of
hormone (Fig. 6B
). In the presence of hormone, T3Rß-0 interacted
strongly with wild-type GRIP-1 (Fig. 6C
); this hormone-dependent
interaction was severely inhibited by the LXXLL to LXXAA GRIP-1
mutation, but not by deletion of the Q-rich coactivator interaction
domain (Fig. 6C
). Conversely, the hormone-independent interaction of
T3Rß-2 with GRIP-1 was eliminated by deletion of the internal Q-rich
interaction domain of the coactivator, but was retained in the LXXLL to
LXXAA GRIP-1 mutant (Fig. 6B
). Intriguingly, the LXXLL to LXXAA
mutation of GRIP-1 impaired, but did not fully eliminate, the
hormone-dependent component of the interaction of T3Rß-2 with this
coactivator (Fig. 6C
). We suggest that the LXXAA substitution mutant of
GRIP-1 retains a residual ability to interact with all T3R isoforms in
the presence of hormone, as can be observed for T3Rß-0, and that this
very modest hormone-dependent interaction is further stabilized for
T3Rß-2 due to the additional interaction surface conferred by the N
terminus of this isoform. Consistent with this proposal, GRIP-1 mutants
that delete, rather than modify, the LXXLL motifs, or that lack both
the LXXLL and central interaction domains, do not display this
residual, hormone-dependent interaction with T3Rß-2 (e.g.
Fig. 4
and data not shown).

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Figure 6. Interaction of the T3Rß-0 and T3Rß-2 Isoforms
with Specific p160 Coactivator Mutants
A, Schematic of the GRIP-1 mutants employed. A schematic
representation of the GRIP-1 coactivator is presented from N to C
termini. The locations of the LXXLL to LXXAA and the Q-rich
mutations, described in the text, are shown. B and C, Binding of
radiolabeled mutant and wild-type GRIP-1 proteins to different,
immobilized GST-T3R fusions. Radiolabeled wild-type or mutant GRIP-1
proteins (as indicated below the panels) were incubated with
nonrecombinant GST protein, or with GST-fusion proteins representing
the full-length open reading frames of T3Rß-0 or T3Rß-2 (as
indicated within each panel). The incubations and washes were performed
either in the absence (panel B) or presence (panel C) of 100
nM T3 hormone. The amount
of each GRIP-1 protein bound to the immobilized GST-T3R polypeptides
after washing was determined as described in Fig. 2 .
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The same central, Q-rich region of the p160 coactivators identified
here as the interaction site for the T3Rß-2 N terminus has also been
reported to be an interaction site for the AF-1 domains present in a
number of steroid receptors, such as the AR (32, 33, 34, 35, 36, 40). Unlike T3Rs,
however, the unliganded steroid receptors typically are cytoplasmic and
do not associate with DNA or activate transcription except on addition
of hormone. The AF-1 domain in the native AR, for example, does not
function in the absence of androgen, but serves instead in the presence
of hormone to stabilize an otherwise weak interaction of the AR AF-2
domain with the coactivators LXXLL motifs (32, 33, 34, 35, 36). We investigated
whether there was a detectable structural relatedness between the
T3Rß-2 and AR N termini. Indeed, several regions of apparent amino
acid relatedness could be discerned (Fig. 7
). The highest level of relatedness was
observed in comparisons of amino acids 1107 of T3Rß-2 with amino
acids 360556 of AR; these two domains shared a global amino acid
identity of 21% and a global similarity of 44% in a four-way
comparison of avian and human T3Rß-2 to human and mouse AR (Fig. 7
).

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Figure 7. Comparison of the N-Terminal Domain of T3Rß-2 to
That of the AR
The amino acid sequences of the N termini of human (h) and chicken (c)
T3Rß-2 s are aligned with a portion of the N termini of the mouse (m)
and human (h) ARs. Gaps (dashes) have been introduced to
maximize matches between the two sequences. Double dots
indicate identical amino acids in equivalent positions in all four
sequences; single dots indicate related amino acids in
equivalent positions in the four sequences. The amino acid numbering
systems are depicted to the left of the corresponding
sequences.
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The Interaction of the N Terminus of T3Rß-2 with p160
Coactivators in Vitro Correlates with Hormone-Independent
Transcriptional Activation in Vivo
We asked whether the interactions we observed between the
T3Rß-2 N terminus and p160 coactivators in vitro
correlated with hormone-independent transcriptional activation in
vivo. We first examined whether mutations in the T3Rß-2 N
terminus that disrupt p160 coactivator interaction in vitro
also impair T3Rß-2 mediated transcriptional activation in transfected
cells. A T3Rß-2 bearing an N-terminal deletion of amino acids 620
retained a hormone-independent interaction with coactivator in
vitro and retained the ability to activate transcription as a
GAL4DBD fusion in vivo (Fig. 8
, A and B). A T3Rß-2 mutant bearing a
larger N-terminal deletion, removing amino acids 640, exhibited a
significant reduction in both the ability to interact with p160
coactivator in vitro, and in the ability to activate GAL4
17-mer reporter gene expression in vivo (Fig. 8
, A and B).
Still larger deletions within the T3Rß-2 N terminus also reduced both
coactivator binding in vitro and transcriptional activation
in transfected cells, although there is some suggestion of an
additional, residual, and relatively weak activation domain mapping
C-terminal to amino acid 70 (Fig. 8
, A and B). A parallel phenomenon
was observed in the native T3Rß-2 context, with a deletion of amino
acids 620 of the T3Rß-2 N terminus having a slightly stimulatory
effect on reporter gene expression in the absence of hormone, whereas
deletions removing amino acids 640 of T3Rß-2 abolished
transcriptional activation in this background (Fig. 8C
). More extreme
deletions of the native T3Rß-2 N terminus, removing amino acids 660
or 670, impinge on the antirepression domain previously described,
and not only abolished hormone-independent activation, but converted
the unliganded T3Rß-2 into a transcriptional repressor (Fig. 8C
).

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Figure 8. Mapping of the p160 Interaction and Transcriptional
Activation Domains within the T3Rß-2 N Terminus
A, The effect of different deletion mutations on the ability of
the T3Rß-2 to interact with SRC1a in the absence of hormone. The
ability of different wild-type and mutant T3Rß constructs, expressed
as radiolabeled in vitro translation products, to bind to
diffferent GST or GST-SRC1a fusions, indicated below the
panel, was determined as in Fig. 4 . B, The effect of different
deletion mutations on the ability of the T3Rß-2 N terminus to
activate transcription when fused to a GAL4DBD. An experiment similar
to that described for Fig. 1C was performed, but utilizing a
series of deletions of the T3Rß-2 (1107 amino acid) N terminus
fused to the GAL4DBD. Each mutant construct, as indicated
below the panel, was tested for the ability to activate
expression of a 5 X GAL 17-mer luciferase reporter when transfected
into CV-1 cells. An empty GAL4DBD construct ("empty") and a GAL4DBD
fused to the intact 1107 amino acid domain of T3Rß-2 ("Full")
were also tested in parallel for comparison. The average and SD values of two or
more repeat experiments are displayed. C, The effect of different
deletion mutations on the ability of the native T3Rß-2 to regulate
transcription from a hormone response element-reporter construct. An
experiment similar to that described for Fig. 1B was performed, but
utilizing a series of otherwise full-length T3Rß-2 constructs bearing
different N-terminal deletion mutations. CV-1 cells were transfected
with a TRE-tk-luciferase reporter, a pCH110-lac Z reporter
(employed as an internal control), and with a pSG5-expression vector
encoding the T3Rß-2 deletion mutants indicated beneath the
panel. The cells were incubated in the absence of
T3 hormone and were subsequently harvested, and
the relative luciferase activity was determined as described in
Materials and Methods. The averages of two or more
experiments and the SD values are shown.
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We next tested the effect of ectopic p160 expression on the ability of
the T3Rß-2 N terminus to activate transcription when fused to a
GAL4DBD. As noted previously, GAL4DBD-fusions bearing the first 170,
or 1107 amino acids of T3Rß-2 were able to activate expression of a
GAL4 17-mer reporter in transfected CV-1 cells (compare panels B and C
in Fig. 9
with panel A).
Cointroduction of ectopic GRIP-1 further enhanced this activation
in a dose-dependent manner (Fig. 9
, B and C). In contrast,
cointroduction of GRIP-1 had no significant effect on transcriptional
activation by an empty GAL4DBD, by a GAL4DBD-T3R
-1 N
terminus construct, or by a constitutive promoter/ß-galactosidase
reporter used as an internal control (Fig. 9
, A and D, and data not
shown). These results suggest that a functional, as well as a physical,
interaction can occur between the T3Rß-2 N terminus and the p160
coactivators. We next tested how mutations in the p160 coactivator
affected the ability of the coactivator to function with the
T3Rß-2 N terminus. A GRIP-1 construct containing the central
interaction domain, but lacking the three LXXLL motifs, retained the
ability to enhance transcriptional activation by the T3Rß-2 N
terminus, whereas a GRIP-1 construct that lacked the internal
interaction domain but contained the LXXLL motifs did not significantly
enhance reporter expression by the T3Rß-2 N terminus (Fig. 9
, B and
C). We conclude that the ability of the T3Rß-2 N terminus to interact
with the internal domain of the p160 coactivators in vitro
correlates closely with the ability of the T3Rß-2 N terminus to
activate transcription in vivo.

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Figure 9. Functional Interactions between the T3Rß-2 N
Terminus and the p160 Coactivators
GAL4DBD constructs either lacking additional sequences (panel A), or
fused to the N-terminal 170 amino acids of T3Rß-2 (panel B),
N-terminal 1107 amino acids of T3Rß-2 (panel C), or N-terminal
150 amino acids of T3R -1 (panel D), were tested. These constructs
were introduced into CV-1 cells together with a 5 X GAL 17-mer
reporter, a pCMV-lac Z reporter (used as an internal
control), and varying amounts of a expression vector for wild-type
GRIP-1, for the Q-rich GRIP-1 mutant, or for the LXXLL to LXXAA
GRIP-1 mutant, as indicated below the panels. The cells
were incubated in the absence of T3 hormone and harvested,
and the relative luciferase activity was determined as described in
Materials and Methods.
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The T3Rß-2 N Terminus Can Interact Simultaneously with p160
Coactivators and with SMRT Corepressor
We have noted previously that the N terminus of the T3Rß-2
isoform makes contacts with the silencing domain of SMRT corepressor,
and that these contacts interfere with assembly of a functional
corepressor complex (31). We have demonstrated here that an
adjacent region of the same T3Rß-2 N-terminal domain can make
contact with the Q-rich region of the p160 coactivators. Can these
interactions occur simultaneously, such that, as suggested by our
model, the N terminus of T3Rß-2 both prevents repression and mediates
activation in the absence of hormone? To test this hypothesis, we
examined the ability of the abstracted T3Rß-2 N terminus, expressed
and purified as a maltose-binding protein (MBP) fusion, to serve as a
bridge to tether a radiolabeled, soluble GRIP-1 protein to an
immobilized GST-SMRT construct. Little or no binding of the
radiolabeled GRIP-1 protein was observed to a nonrecombinant GST
construct employed as a negative control (Fig. 10
, top panel). Addition of
either the MBP-T3Rß-2 (1107) fusion protein, or a native MBP
protein to the binding reaction failed to alter this lack of
interaction between GRIP-1 and nonrecombinant GST (Fig. 10
, top
panel). Similarly, GRIP-1 did not bind to a GST-SMRT silencing
domain fusion either alone or when tested in the presence of the native
MBP (Fig. 10
, bottom panel). However, addition of increasing
amounts of the MBP-T3Rß-2 (1107) fusion protein resulted in a
parallel increase in the ability of the radiolabeled GRIP-1 to bind to
the GST-SMRT silencing domain fusion (Fig. 10
, bottom
panel). We conclude that the T3Rß-2 N terminus has the capacity
to interact concurrently with both SMRT corepressor and p160
coactivators, consistent with a model wherein both antirepression and
hormone-independent activation occur simultaneously.

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Figure 10. Simultaneous Interaction of the T3Rß-2 N
Terminus with SMRT Corepressor and GRIP-1 Coactivator
The ability of a radiolabeled GRIP-1 protein, synthesized by in
vitro transcription and translation, to bind to an immobilized
nonrecombinant GST protein (top panel) or to an
immobilized GST-SMRT (566680) construct (representing a portion of
the SMRT corepressor silencing domain) was tested by the same general
protocol as described for GST-coactivators in Fig. 4 . The binding
reactions were carried out with no further additions (None), in the
presence of the indicated amounts of a native MBP, or in the presence
of the indicated amounts of a MBP fusion protein representing the
N-terminal 1107 amino acids of T3Rß-2 [MBP-T3Rß-2 (NOREF>1107)].
Radiolabeled GRIP-1 remaining bound to the GST constructs after
repeated washings was eluted and was visualized by SDS-PAGE and
phosphorimager analysis.
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DISCUSSION
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The N Terminus of T3Rß-2 Contributes to Transcriptional
Activation in the Absence of Hormone by Interacting with a Central
Domain of the p160 Coactivators
Unlike the T3R
-1 and T3Rß-0/1 isoforms, the T3Rß-2 isoform
is expressed in a highly restrictive tissue pattern limited principally
to the hypothalamus and pituitary (12, 13, 14, 41). The T3Rß-2 isoform is
also unique in its transcriptional regulatory properties. In contrast
to the other T3R isoforms, T3Rß-2 fails to repress transcription in
the absence of hormone. Instead, the T3Rß-2 isoform activates
transcription in the absence of hormone, and this activation is further
stimulated in the presence of hormone (18, 28, 29, 31, 42). We have
previously reported that the inability of the T3Rß-2 isoform to
repress is not due to a failure to recruit the SMRT or N-CoR
corepressors; in common with T3R
-1 and T3Rß-0/1, the T3Rß-2
isoform interacts strongly with SMRT and with N-CoR in the absence of
hormone (31). However, the T3Rß-2 isoform makes additional contacts
with the silencing domains of SMRT and N-CoR that are not observed with
the T3R isoforms that do repress (31). These additional contacts of
T3Rß-2 with SMRT and N-CoR interfere with the subsequent assembly of
a larger, functional corepressor complex, a phenomenon that we have
denoted antirepression (31). Therefore, the unliganded T3Rß-2
interacts with corepressor, but in an abortive fashion that appears to
preclude repression.
Although accounting for the lack of repression by
T3Rß-2, this antirepression model does not explain the additional,
hormone-independent activation observed for this isoform in many cell
lines and promoter contexts. We report here that hormone-independent
transcriptional activation by T3Rß-2 maps to the N-terminal domain of
this receptor, and that the isolated T3Rß-2 N-terminal domain, when
fused to an ectopic GAL4DBD, is capable of autonomous,
hormone-independent transcriptional activation in transfected cells.
Our results map these transcriptional activation properties to the
T3Rß-2 N terminus, with the bulk of this activity requiring amino
acids 20 to 40. Our experiments also suggest the existence of a more
minor, transcriptional activation domain mapping between amino acids 70
and 107 that is only observed on deletion of more N-terminal sequences.
Significantly, both of these activation domains are outside of the
region of the T3Rß-2 N terminus that we have shown to mediate
antirepression, indicating that activation and antirepression are
distinct phenomena. The presence of hormone-independent transcriptional
activation domains within the T3Rß-2 N terminus has been previously
noted, although there has been disagreement as to the precise location
of these activities (18, 28, 30). Our own mapping studies, reported
here, generally agree with and help reconcile these prior reports by
confirming that there are at least two distinct transcriptional
activation domains within the T3Rß-2 N terminus.
How might the hormone-independent T3Rß-2 N-terminal transactivation
domains function? The ability of the nuclear hormone receptors to
activate transcription in the presence of hormone is dependent on a
conformational change in the receptor on binding of hormone agonist,
leading to a reorientation of the C-terminal helix 12 domain (19, 20, 21, 22, 24, 25). The reoriented helix 12, together with portions of helix 3, 4,
and 5 of the hormone binding domain of the receptor (denoted the AF-2
domain), forms a charge-clamped groove on the surface of the
receptor that can interact with LXXLL motifs present in many of the
coactivator polypeptides (22, 24, 25). Thus, for most of the nuclear
hormone receptors characterized, acquisition of coactivator is strongly
dependent on hormone binding. The same mechanism appears to be
operative for all the isoforms of T3R tested here, including T3Rß-2.
However, in addition to these hormone-dependent interactions of the
C-terminal AF-2 domain, the N terminus of T3Rß-2 displays an
additional, hormone-independent interaction with the p160 coactivators
that is not observed for T3R
-1 or ß-0. This hormone-independent
interaction of the T3Rß-2 N terminus with the SRC-1 and GRIP-1
coactivators can be observed in the absence of the receptor AF-2
domain.
The interaction of the T3Rß-2 N terminus with the SRC-1 and GRIP
coactivators closely correlates with the hormone-independent
transcriptional activation mediated by T3Rß-2 in transfected cells.
For example, the ability of the T3Rß-2 N terminus to activate
transcription is significantly enhanced by the cointroduction of
ectopic GRIP-1. Conversely, mutations of the T3Rß-2 N terminus that
impair its interaction with SRC-1 or GRIP-1 also impair
hormone-independent activation without affecting hormone-dependent
activation. These results are indicative of a functional, as well as a
physical, interaction between the T3Rß-2 N terminus and the p160
coactivators.
As detailed above, hormone-dependent transcriptional activation by
nuclear hormone receptors is thought to be primarily mediated by an
agonist- induced formation of a docking surface within the AF-2
domain of the receptor that can then interact with the LXXLL amino acid
motifs present in many coactivators (22, 24, 25). Indeed, in our
experiments the hormone-dependent interaction of the T3R
-1 and ß-0
isoforms with SRC-1 or GRIP-1 required the presence of at least one
LXXLL motif within the p160 coactivators. In contrast to these LXXLL
motifs, which mediate hormone-dependent p160 interaction, the
hormone-independent interaction of T3Rß-2 with these coactivators was
conferred primarily by a distinct set of contacts between the receptor
N terminus and a central, glutamine-rich domain of SRC-1 and GRIP-1.
Deletion of this glutamine-rich coactivator domain, corresponding to
amino acids 11211305 of GRIP-1 and equivalent to amino acids 977-1172
of SRC-1, abolished the hormone-independent interaction of native
T3Rß-2 with these coactivators without impairing the
hormone-dependent interaction of T3Rß-2, T3R
-1, or T3Rß-0.
Consistent with this central coactivator domain being responsible for
the hormone-independent activation mediated by T3Rß-2 in
vivo, a mutant GRIP-1 lacking the central interaction domain did
not enhance transcriptional activation by the T3Rß-2 N terminus,
whereas wild-type GRIP-1, or a GRIP-1 mutant retaining the internal
interaction domain but lacking the LXXLL motifs, did significantly
enhance transcriptional activation by the T3Rß-2 N terminus.
The central glutamine-rich interaction domain of GRIP-1 and SRC-1,
implicated here in hormone-independent activation by T3Rß-2, does not
display detectable sequence relatedness to the LXXLL coactivator
motifs that mediate hormone-dependent activation, nor does the
N-terminal region of T3Rß-2 display significant sequence relatedness
to the hormone-dependent AF-2 region of this receptor. Intriguingly,
however, the N terminus of T3Rß-2 does display detectable sequence
relatedness to an N-terminal domain within the ARs that also interacts
with the central domain of the p160 coactivators (32, 33, 34, 35, 36). The sequence
relatedness between the T3Rß-2 and the AR N termini is modest
overall, but includes several subregions exhibiting near 25% identity
and more than 50% similarity; notably the regions of highest
interrelatedness correspond to the receptor regions necessary for
interaction with the p160 coactivators in our genetic analysis. These
sequence similarities between T3Rß-2 and AR suggest that a common
structural motif, perhaps reflected within the three-dimensional
structure of these N-terminal domains, may be responsible for the
contacts of these receptors with the p160 central interaction
domain.
It is interesting that two nuclear receptors as diverse as T3Rß-2 and
AR share a similar, dual contact mode of interaction with the p160
coactivators, particularly given that these shared coactivator contacts
appear to be used for different purposes by the two different
receptors. The ARs are cytoplasmic in the absence of hormone, and
neither directly activate nor directly repress transcription in the
unliganded state. Instead, the N-terminal domain of the native ARs is
believed to function primarily in the presence of hormone, operating to
stabilize an otherwise weak, hormone-dependent interaction of the AR
AF-2 domain with the LXXLL coactivator motifs (32, 33, 34, 35, 36, 43). Thus, in
AR, the N terminus bolsters the function of the AF-2 domain. In
contrast, in T3Rß-2 the N terminus appears able to operate
independently of the AF-2 domain to confer transcriptional activation
in the absence of hormone. However, our experiments do not preclude an
additional role for the T3Rß-2 N terminus in the presence of hormone.
In fact, we observed that, even in the presence of hormone, the
interaction of T3Rß-2 with mutant coactivators can be stabilized by
the interaction of the T3Rß-2 N terminus with the central coactivator
domain.
Transcriptional Regulation by Different T3R Isoforms Represents a
Mix of Repression, Antirepression, Hormone-Dependent Activation, and
Hormone-Independent Activation Functions
The hormone binding domain of the nuclear hormone receptors serves
as a primary site of receptor interaction with both corepressors and
coactivators. Within the hormone binding domain, portions of helices 3,
5/6, and 11 form overlapping binding sites for corepressors and
coactivators, with a hormone-mediated reorientation of helix 12
adjudicating the recruitment of one or the other cofactor.
However, our own work, together with that of others, indicates
that the N-terminal domain of many of these nuclear receptors makes
important additional contacts with corepressors and coactivators, and
that these additional contacts can play a pivotal role in regulating
transcription. The T3Rß-2 N terminus is a particularly interesting
case in point. The T3Rß-2 N terminus makes contacts with both
corepressors and with coactivators (31, 44). These N-terminal-mediated
contacts act in addition to the corepressor and coactivator contacts
conferred by the hormone binding domain and serve both to prevent
assembly of a functional corepressor complex and to recruit coactivator
in the absence of hormone. In this manner, the intrinsic
transcriptional regulatory properties defined by the hormone binding
domain are overridden, and the T3Rß-2 is able to activate
transcription in both the absence and presence of hormone. Conversely,
the absence of these N-terminal modulatory domains in the otherwise
identical T3Rß-0 isoform permit the unfettered dominance of the
actions of the hormone binding domain, which manifest as repression in
the unliganded state and as activation in the presence of hormone.
The use of alternative mRNA splicing to generate both
hormone-independent and hormone-dependent forms of transcriptional
activator from the same locus appears to be unique to the T3R class of
nuclear hormone receptors. However, many other nuclear hormone
receptors also encode transcriptional regulatory sequences within their
N termini, and a number of these N-terminal sequences appear to operate
by recruiting coactivators of various genres (e.g. Refs.
32, 33, 34, 35, 36, 40, 43). These N-terminal coactivator interaction surfaces
can confer transcriptional activation in the absence of hormone, can
stabilize an otherwise weak coactivator interaction in the presence of
hormone, or can diversify the transcriptional response by recruiting
coactivators of more than one class to a single nuclear receptor
molecule. Taken as a whole, it appears that the nuclear hormone
receptors are modular structures that can incorporate a variety of
hormone-dependent and independent interaction surfaces for
transcriptional cofactors. The contributions of these distinct
interaction surfaces can vary for different receptors and receptor
isoforms, and these differences result in different modes of
coactivator and corepressor recruitment. The particular transcriptional
properties of a particular nuclear hormone receptor therefore are a
combinatorial mixture of activation and repression, resulting in a
transcriptional output that can be carefully customized through
evolution to the most physiologically appropriate phenotype.
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MATERIALS AND METHODS
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Plasmid Constructs
The wild-type pSG5-avian T3R
-1, pSG5-human T3Rß-1, and
pSG5-avian T3Rß-2 constructs were described previously (18, 45, 46).
The pSG5 avian T3Rß-0 construct was created using PCR and
appropriate oligonucleotide primers to add a Kozak
consensus/translational start sequence and the T3Rß-0 N-terminal
amino acid sequence to the C-terminal portion of the T3Rß open
reading frame (47). The pSG5-T3Rß-2 (amino acids 1107) construct
was created by PCR amplification using appropriate primers and standard
recombinant DNA techniques (47). The generation of the
pSG5-Gal4DBD-T3Rß-1 (amino acids 1101) construct and of the
in-frame, N-terminal deletion mutants of T3Rß-2 (
620,
640,
660, and
670) were previously described (31). The
pSG5-GAL4DBD-T3R
-1 (amino acids 150) and the
pSG5-Gal4DBD-T3Rß-2 (amino acids 170 or amino acids 1107)
constructs were created by standard recombinant DNA approaches, using
PCR amplification to introduce appropriate restriction sites where
necessary. The pSG5-Gal4DBD-T3Rß-2 N-terminal deletions were
constructed by a similar approach, but utilizing the pSG5-T3Rß-2
deletion mutants, described above, as templates.
The different T3R isoforms were cloned into the pGEX-2T and
pGEX-KG vectors as full-length constructs by use of PCR amplification,
employing oligonucleotide primers to insert appropriate restriction
sites, followed by standard recombinant DNA techniques to reconstitute
the intact open reading frames (48). The pGEX-KG-avian T3Rß2 (amino
acids 1107) plasmid was constructed by releasing a EcoRI
to BamHI fragment from the pSG5-Gal4DBD-T3Rß2 (amino acids
1107) construct. The pGEX-KG vector was then cleaved with
XbaI. Both the linearized vector and the fragment were
treated with T4 DNA polymerase, and the resulting blunt-ended DNA
fragments were ligated together; the resulting clones were screened for
the correct insert orientation.
The pSG5-GRIP-1 and pCR3.1-SRC1a expression plasmids were previously
described (38, 39). The GRIP-1 (LXXAA) and GRIP-1 (
Q-rich) mutants
were created by a QuikChange site-directed mutagenesis method, using
the protocol recommended by the manufacturer (Stratagene,
La Jolla, CA). The latter represents a deletion of amino acids
11851260 of GRIP-1. The identity of all mutations was subsequently
confirmed by DNA sequence analysis.
The pCR3.1 clones representing subdomains of SRC-1a or GRIP-1 were
constructed by PCR amplification using appropriate oligonucleotide
primers so as to introduce flanking EcoRI and
BamHI restriction sites (for SRC-1a) or EcoRI and
BglII restriction sites (for GRIP-1), as well as appropriate
upstream Kozak translational start sequences; the resulting restriction
fragments were inserted into the EcoRI and BamHI
sites in the pCR3.1 vector. The subclones created in this fashion
included pCR3.1-SRC-1a (amino acids 977-1441), pCR3.1-SRC-1a (amino
acids 11721441), pCR3.1-GRIP-1 (a.a.11221462), and pCR3.1-GRIP-1
(amino acids 13051462). The corresponding pGEX-KG versions of these
subclones were constructed by PCR amplification using pairs of primers
similar to those described above; the PCR products were subsequently
cleaved and ligated into the EcoRI and BamHI
sites in the pGEX-KG vector. The pGEX-KG-SRC-1a (5601136) plasmid was
described previously (49).
Protein-Protein Interaction Assays
GST fusion proteins were isolated from Escherichia
coli (DH5
-strain) transformed by the corresponding pGEX-KG or
pGEX-2T vectors, and the resulting GST fusion proteins were purified
and immobilized by binding to glutathione-conjugated agarose beads as
previously described (48). The pSG5- or pCR3.1-based plasmids were
transcribed and translated into 35S-radiolabeled
proteins in vitro by use of a T7 RNA polymerase-coupled TnT
kit (Promega Corp., Madison WI). The
35S-labeled proteins were subsequently incubated
for 2 h at 4 C with 25 µl of a 50% slurry of the appropriate
immobilized GST fusion protein in 300 µl of HEMG buffer (40
mM HEPES, pH 7.8, 50 mM
KCl, 0.2 mM EDTA, 5 mM
MgCl2, 0.1% Triton X-100, 10% glycerol, and 1.5
mM dithiothreitol) containing 1x Complete
Proteinase Inhibitor (Roche Molecular Biochemicals,
Indianapolis, IN) and 10 mg/ml of BSA. After incubation, the
immobilized GST-proteins, and any polypeptides bound to them, were
washed four times with 500 µl of HEMG buffer. Proteins remaining
bound to the glutathione-agarose matrix were eluted with free
glutathione and were resolved by SDS-PAGE. The electrophoretograms were
visualized and quantified by PhosphorImage analysis (Storm System,
Molecular Dynamics, Inc., Sunnyvale, CA).
Transient Transfections
CV-1 cells were maintained at 37 C in DMEM containing 10% FBS
in a 5% CO2/bicarbonate-buffering system. Cells
were transfected by a Lipofectin-mediated method following the
recommendations of the manufacturer (Life Technologies,
Gaithersburg, MD). For assays of the function of the full-length T3Rs,
approximately 5 x 106 cells were
transfected with 20 ng of a pSG5 -T3R
, -T3Rß-0, -T3Rß-1,
-T3Rß-2, or -T3Rß-2 mutant plasmid, 50 ng of a
pCH110-lac Z plasmid (employed as an internal transfection
control), and 100 ng of a ptk-DR4-luciferase reporter containing a DR-4
thyroid hormone response element (31, 49). pUC18 plasmid was employed
to normalize the total transfected DNA per sample to 500 ng. The
transfected cells were subsequently propagated in DMEM containing 10%
(hormone-depleted) FBS in the presence of either 100
nM of T3-thyronine, or an
equivalent amount of ethanol carrier, and were harvested at 48 h
post transfection. The luciferase activity was measured and was
normalized relative to ß-galactosidase activity as previously
described (31, 49). Transcriptional activation by the pSG5-GAL4DBD
fusions was assayed by a similar Lipofectin protocol, but employing 25
ng of a pSG5-GAL4DBD vector (either empty or containing a
GAL4DBD-N-terminal T3R fusion), 25 ng of a pCMV-lac Z
plasmid as an internal control, and 100 ng of a 5 x GAL 17-mer
luciferase reporter construct (50). Additional pBluescript plasmid was
used to bring the total amount of transfected DNA per assay to 500 ng.
The cells were harvested at 48 h post transfection and assayed for
luciferase and ß-galactosidase activity as described above. In
certain experiments, a pSG5-GRIP-1, pSG5-GRIP-1(LXXLL to LXXAA), or
pSG5-GRIP-1(
Q-rich) expression plasmid was also included in the
transfections, as indicated in the appropriate figure legends.
JEG-3 cells were maintained in DMEM containing 10% FBS. Transfections
of JEG-3 cells were performed using Effectene and the protocol
suggested by the manufacturer (QIAGEN, Valencia, CA).
Approximately 1.5 x 105 cells were seeded
24 h in advance. The cells were then transfected with 20 ng of the
pSG5-T3Rß-2 plasmid, 25 ng of the pCMV-lacZ plasmid, and 100 ng of
the ptk-DR4-luciferase reporter plasmid. Additional pUC18 plasmid was
used to bring the total amount of transfected DNA per assay to 500 ng.
Transfected cells were washed after 6 h and were placed in fresh
medium in the presence of 100 nM of
T3 thyronine, or an equivalent amount of ethanol
carrier. The cells were harvested 24 h post transfection. The
luciferase activity was measured and was normalized to
ß-galactosidase activity as previously described (31, 49).
Pairwise Sequence Alignment
The N-terminal 107 amino acid of T3Rß-2 was compared by a
pairwise sequence alignment to amino acids 360 to 556 of the human AR,
using the GeneStream sequence alignment program of BCM Search Launcher
(The Baylor College of Medicine Search Launcher).
 |
ACKNOWLEDGMENTS
|
---|
We thank R. Evans, B. OMalley, and M. Stallcup for their
generosity in providing molecular clones and Valentina Taryanik for
dedicated technical assistance.
This work was supported by Public Health Services/NIH Grants R37
CA-53394 and R01 DK-53528.
 |
FOOTNOTES
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---|
Address requests for reprints to: Martin L. Privalsky, Section of Microbiology, Division of Biological Chemistry, One Shields Avenue, University of California at Davis, Davis, California 95616. E-mail:
mlprivalsky{at}ucdavis.edu
Received for publication December 27, 2000.
Revision received March 7, 2001.
Accepted for publication March 12, 2001.
 |
REFERENCES
|
---|
-
Chambon P 1996 A decade of molecular biology of retinoic
acid receptors. FASEB J 10:940954[Abstract/Free Full Text]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz
G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Meier CA 1997 Regulation of gene expression by nuclear
hormone receptors. J Recept Signal Transduct Res 17:319335[Medline]
-
Tsai MJ, OMalley BW 1994 Molecular mechanisms of action of
steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451486[CrossRef][Medline]
-
Chen JD, Li H 1998 Coactivation and corepression in
transcriptional regulation by steroid/nuclear hormone receptors. Crit
Rev Eukaryot Gene Expr 8:169190[Medline]
-
Collingwood TN, Urnov FD, Wolffe AP 1999 Nuclear receptors:
coactivators, corepressors and chromatin remodeling in the control of
transcription. J Mol Endocrinol 23:255275[Abstract/Free Full Text]
-
Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung
L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:11671177[Abstract]
-
Shibata H, Spencer TE, Oñate SA, Jenster G, Tsai SY,
Tsai MJ, OMalley BW 1997 Role of co-activators and co-repressors in
the mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 52:141164[Medline]
-
Torchia J, Glass C, Rosenfeld MG 1998 Co-activators and
co-repressors in the integration of transcriptional responses. Curr
Opin Cell Biol 10:373383[CrossRef][Medline]
-
Xu L, Glass CK, Rosenfeld MG 1999 Coactivator and corepressor
complexes in nuclear receptor function. Curr Opin Genet Dev 9:140147[CrossRef][Medline]
-
Forrest D, Vennström B 2000 Functions of thyroid hormone
receptors in mice. Thyroid 10:4152[Medline]
-
Murata Y 1998 Multiple isoforms of thyroid hormone receptor:
an analysis of their relative contribution in mediating thyroid hormone
action. Nagoya J Med Sci 61:103115[Medline]
-
Lazar MA 1993 Thyroid hormone receptors: multiple forms,
multiple possibilities. Endocr Rev 14:184193[Medline]
-
Zhang J, Lazar MA 2000 The mechanism of action of thyroid
hormones. Annu Rev Physiol 62:439466[CrossRef][Medline]
-
Damm K, Thompson CC, Evans RM 1989 Protein encoded by v-erbA
functions as a thyroid-hormone receptor antagonist. Nature 339:593597[CrossRef][Medline]
-
Sap J, Munoz A, Schmitt J, Stunnenberg H, Vennstrom B 1989 Repression of transcription mediated at a thyroid hormone response
element by the v-Erb-A oncogene product. Nature 340:242244[CrossRef][Medline]
-
Schulman IG, Juguilon H, Evans RM 1996 Activation and
repression by nuclear hormone receptors: hormone modulates an
equilibrium between active and repressive states. Mol Cell Biol 16:38073813[Abstract]
-
Sjoberg M, Vennstrom B 1995 Ligand-dependent and -independent
transactivation by thyroid hormone receptor ß-2 is determined by the
structure of the hormone response element. Mol Cell Biol 15:47184726[Abstract]
-
Hu X, Lazar MA 1999 The CoRNR motif controls the recruitment
of corepressors by nuclear hormone receptors. Nature 402:9396[CrossRef][Medline]
-
Nagy L, Kao HY, Love JD, Li C, Banayo E, Gooch JT, Krishna V,
Chatterjee K, Evans RM, Schwabe JWR 1999 Mechanism of corepressor
binding and release from nuclear hormone receptors. Genes Dev 13:32093216[Abstract/Free Full Text]
-
Xu X, Lazar MA 1999 The CoRNR motif controls the recruitment
of corepressors by nuclear hormone receptors. Nature 402:9396[CrossRef][Medline]
-
Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ,
Baxter JD, Fletterick RJ, Yamamoto KR 1998 Structure and specificity of
nuclear receptor-coactivator interactions. Genes Dev 12:33433356[Abstract/Free Full Text]
-
Feng W, Ribeiro RC, Wagner RL, Nguyen H, Apriletti JW,
Fletterick RJ, Baxter JD, Kushner PJ, West BL 1998 Hormone-dependent
coactivator binding to a hydrophobic cleft on nuclear receptors.
Science 280:17471749[Abstract/Free Full Text]
-
Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa
R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding
and co-activator assembly of the peroxisome proliferator-activated
receptor-
. Nature 395:137143[CrossRef][Medline]
-
Westin S, Kurokawa R, Nolte RT, Wisely GB, McInerney EM, Rose
DW, Milburn MV, Rosenfeld MG, Glass CK 1998 Interactions controlling
the assembly of nuclear-receptor heterodimers and co-activators. Nature 395:199202[CrossRef][Medline]
-
Baniahmad A, Kohne AC, Renkawitz R 1992 A transferable
silencing domain is present in the thyroid hormone receptor, in the
v-erbA oncogene product and in the retinoic acid receptor. EMBO J 11:10151023[Abstract]
-
Casanova J, Helmer E, Selmiruby S, Qi JS, Aufliegner M,
Desaiyajnik V, Koudinova N, Yarm F, Raaka BM, Samuels HH 1994 Functional evidence for ligand-dependent dissociation of thyroid
hormone and retinoic acid receptors from an inhibitory cellular factor.
Mol Cell Biol 14:57565765[Abstract]
-
Hollenberg AN, Monden T, Wondisford FE 1995 Ligand-independent
and -dependent functions of thyroid hormone receptor isoforms depend
upon their distinct amino termini. J Biol Chem 270:1427414280[Abstract/Free Full Text]
-
Hollenberg AN, Monden T, Madura JP, Lee K, Wondisford FE 1996 Function of nuclear co-repressor protein on thyroid hormone response
elements is regulated by the receptor A/B domain. J Biol Chem 271:2851628520[Abstract/Free Full Text]
-
Langlois MF, Zanger K, Monden T, Safer JD, Hollenberg AN,
Wondisford FE 1997 A unique role of the ß-2 thyroid hormone receptor
isoform in negative regulation by thyroid hormone. Mapping of a novel
amino-terminal domain important for ligand-independent activation.
J Biol Chem 272:2492724933[Abstract/Free Full Text]
-
Yang Z, Hong SH, Privalsky ML 1999 Transcriptional
anti-repression. Thyroid hormone receptor ß-2 recruits SMRT
corepressor but interferes with subsequent assembly of a functional
corepressor complex. J Biol Chem 274:3713137138[Abstract/Free Full Text]
-
Alen P, Claessens F, Verhoeven G, Rombauts W, Peeters B 1999 The androgen receptor amino-terminal domain plays a key role in p160
coactivator-stimulated gene transcription. Mol Cell Biol 19:60856097[Abstract/Free Full Text]
-
Bevan CL, Hoare S, Claessens F, Heery DM, Parker MG 1999 The
AF1 and AF2 domains of the androgen receptor interact with distinct
regions of SRC1. Mol Cell Biol 19:83838392[Abstract/Free Full Text]
-
He B, Kemppainen JA, Voegel JJ, Gronemeyer H, Wilson EM 1999 Activation function 2 in the human androgen receptor ligand binding
domain mediates interdomain communication with the NH(2)-terminal
domain. J Biol Chem 274:3721937225[Abstract/Free Full Text]
-
Hong H, Darimont BD, Ma H, Yang L, Yamamoto KR, Stallcup MR 1999 An additional region of coactivator GRIP1 required for interaction
with the hormone-binding domains of a subset of nuclear receptors.
J Biol Chem 274:34963502[Abstract/Free Full Text]
-
Ma H, Hong H, Huang SM, Irvine RA, Webb P, Kushner PJ, Coetzee
GA, Stallcup MR 1999 Multiple signal input and output domains of the
160-kilodalton nuclear receptor coactivator proteins. Mol Cell Biol 19:61646173[Abstract/Free Full Text]
-
Forrest D, Sjöberg M, Vennström B 1990 Contrasting
developmental and tissue-specific expression of
and ß thyroid
hormone receptor genes. EMBO J 9:15191528[Abstract]
-
Oñate SA, Tsai SY, Tsai MJ, OMalley BW 1995 Sequence
and characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional
coactivator in yeast for the hormone binding domains of steroid
receptors. Proc Natl Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
Webb P, Nguyen P, Shinsako J, Anderson C, Feng W, Nguyen MP,
Chen D, Huang SM, Subramanian S, McKinerney E, Katzenellenbogen BS,
Stallcup MR, Kushner PJ 1998 Estrogen receptor activation function 1
works by binding p160 coactivator proteins. Mol Endocrinol 12:16051618[Abstract/Free Full Text]
-
Apriletti JW, Ribeiro RC, Wagner RL, Feng W, Webb
P, Kushner PJ, West BL, Nilsson S, Scanlan TS, Fletterick RJ, Baxter JD 1998 Molecular and structural biology of thyroid hormone receptors.
Clin Exp Pharmacol Physiol Suppl 25:S2S11
-
Safer JD, OConnor MG, Colan SD, Srinivasan S, Tollin SR,
Wondisford FE 1999 The thyroid hormone receptor-beta gene mutation
R383H is associated with isolated central resistance to thyroid
hormone. J Clin Endocrinol Metab 84:30993109[Abstract/Free Full Text]
-
Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ,
Stallcup MR 1998 Nuclear receptor-binding sites of coactivators
glucocorticoid receptor interacting protein 1 (GRIP1) and steroid
receptor coactivator 1 (SRC-1): multiple motifs with different binding
specificities. Mol Endocrinol 12:302313[Abstract/Free Full Text]
-
Oberste-Berghaus C, Zanger K, Hashimoto K, Cohen RN,
Hollenberg AN, Wondisford FE 2000 Thyroid hormone-independent
interaction between the thyroid hormone receptor ß 2 amino terminus
and coactivators. J Biol Chem 275:17871792[Abstract/Free Full Text]
-
Sap J, Muñoz A, Damm K, Goldberg Y, Ghysdael J, Leutz A,
Beug H, Vennström B 1986 The c-erb-A protein is a high-affinity
receptor for thyroid hormone. Nature 324:635640[Medline]
-
Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM 1986 The c-erb-A gene encodes a thyroid hormone receptor. Nature 324:641646[Medline]
-
Ausubel FM 1987 Current Protocols in Molecular Biology. Greene
Publishing Associates and Wiley-Interscience, New York
-
Guan KL, Dixon JE 1991 Eukaryotic proteins expressed in
Escherichia colian improved thrombin cleavage and
purification procedure of fusion proteins with glutathione
S-transferase. Anal Biochem 192:262267[Medline]
-
Yoh SM, Privalsky ML 2000 Resistance to thyroid hormone (RTH)
syndrome reveals novel determinants regulating interaction of T3
receptor with corepressor. Mol Cell Endocrinol 159:109124[CrossRef][Medline]
-
Wong CW, Privalsky ML 1998 Transcriptional silencing is
defined by isoform- and heterodimer-specific interactions between
nuclear hormone receptors and corepressors. Mol Cell Biol 18:57245733[Abstract/Free Full Text]