From the Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom
Received for publication, December 19, 2002
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ABSTRACT |
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The interaction of coactivators with the
ligand-binding domain of nuclear receptors (NRs) is mediated by
amphipathic The nuclear hormone receptors
(NRs)1 are a family of
structurally related, ligand-regulated transcription factors that exert both positive and negative control of gene expression in metazoans (1).
The NRs are subdivided into classes depending on their DNA binding and
dimerization properties. Class I comprises the steroid hormone
receptors, including the estrogen (ER), androgen (AR), and progesterone
(PR) receptors, which function as homodimers. The largest group (Class
II) function as heterodimers with the 9-cis retinoic acid
receptor (RXR), and includes receptors for retinoic acid (RAR), vitamin
D (VDR), thyroid hormone (TR), and peroxisome proliferators (PPARs).
Binding of cognate ligand induces a conformational change in the ligand
binding domain (LBD) of NRs, which influences their function with
respect to subcellular localization, dimerization, cofactor binding,
and transcriptional activity (2).
A wide range of NR cofactors have been identified which perform
distinct functions at target promoters, including chromatin modification and remodeling and recruitment of the RNA polymerase II
holoenzyme (3). A number of cofactors have been shown to bind the LBD
directly via short amphipathic Different cofactors have been shown to have variable numbers of
functional LXXLL motifs. The p160 coactivators SRC1,
TIF2/GRIP1, and ACTR/AIB1/pCIP have homologous NR interaction domains
(NID) containing three LXXLL motifs (4, 5, 15). These motifs are highly conserved both in sequence and spacing, and it has been
shown that at least two (preferably adjacent) motifs are required for
high affinity binding of SRC1 to Class I receptors (4, 5, 15, 16).
Thus, the presence of multiple LXXLL motifs in coactivators
facilitates cooperative binding to the AF2 surfaces of NR dimers. The
mammalian mediator complex TRAP/DRIP/SMCC/ARC/CRSP contains a single
subunit capable of binding to NR LBDs (17, 18). This protein, termed
TRAP220, DRIP205, or PBP, contains two LXXLL motifs within
the NID that are required for ligand-dependent binding to
NRs (19-21). Other cofactors including TIP60 (22), TIF1 Several cofactors have been found to display preferences for NR
subclasses. For example, TIP60 has been reported to bind the Class I
receptors, but displayed little interaction with VDR, TR, or RXR (22).
The p160s interact with a wide range of NRs, although individual
LXXLL motifs derived from these proteins display differential binding to NRs (14, 38). In addition, we have shown that
LXXLL sequences derived from CBP and RIP140 show selectivity that is at least partly determined by the LXXLL core
sequences (14). Similarly, LXXLL motifs (or variants such as
LXXIL and FXXLL) from other cofactors, such as
PERC (31), NRIF3 (32), and NSD1 (33) have been reported to display
selectivity in their interactions with NRs.
TRAP220 was isolated on the basis of its interaction with Class II NRs
such as TR, VDR, and PPARs, and it has been shown that ablation of
either of its two LXXLL motifs had differential effects on
binding to NRs. RXR binding was disrupted by mutation of LXM1, whereas
TR, RAR, and VDR showed a preference for binding the distal motif, LXM2
(20, 28). TRAP220 has also been reported to interact with ER In this study we assessed the interaction of TRAP220 NID,
LXXLL peptides, and full-length proteins with a panel of NR
LBDs. We show that TRAP220 displays stronger interactions with certain Class II receptors in comparison to steroid receptors. We describe mutational analyses and mapping experiments that shed light on the
molecular basis of the NR binding specificity of TRAP220.
Plasmid Constructions--
The following plasmids used in
transient transfection experiments have been described previously:
pSG5-SRC1e (16) and p3ERE-TATA-LUC (40). The reporter plasmid
pMAL-TK-LUC (41) and hTR
For yeast two-hybrid interaction assays, the p3ERE-lacZ reporter (46),
and vectors pBL1 and modified pASV3 (43) expressing the human ER
For in vitro glutathione S-transferase (GST)
pull-down assays, the control GST construct was a modified version of
pGEX-2TK vector (Amersham Biosciences). The constructs GST-TR Cell Culture and Transient Transfections--
Maintenance of
HeLa cells and transient transfection protocols were as described (45).
The transfected DNA included pJ7-lacZ internal control plasmid (500 ng/well), p3ERE-TATA-LUC (1 µg), or pMAL-TK-LUC (2 µg) luciferase
reporter plasmids, with either pMT-MOR (100 ng) or pRSV-TR Yeast Two-hybrid Interaction Assays--
Saccharomyces
cerevisiae W303-1b (HML GST Pull-down Assays--
Recombinant cDNAs in pSG5 or
pSG5(PT) expression vectors were transcribed and translated in
vitro in the presence of [35S]methionine in
reticulocyte lysate (Promega) according to the manufacturer's
instructions. GST fusion proteins were expressed in Escherichia
coli DH5 TRAP220 Enhances the Transcriptional Activity of TR
We also assessed the ability of TRAP220 to enhance the activity of the
Class I receptor ER TRAP220 NID Exhibits Nuclear Receptor Binding
Specificity--
The ability of TRAP220 to enhance transcription
by TR
To determine whether both LXXLL motifs of TRAP220 are
required for binding the panel of LBDs used in this study, we generated LexA-TRAP220 NID constructs in which either LXM1 or LXM2 was mutated, by replacing the leucines +4 and +5 with alanines. As shown in Fig.
2C, interaction of the TRAP220 NID with TR The NID and Core LXXLL Motifs of TRAP220 Exhibit Distinct NR
Binding Specificities--
It has been shown previously that 8-10
amino acid sequences encompassing the signature motif LXXLL
are sufficient to bind to liganded NR LBDs, and this has been referred
to as the core LXXLL motif (4, 14). To explore the nature of
the specificity exhibited by the TRAP220 NID in more detail, we
assessed the ability of LXXLL core motifs derived from SRC1
and TRAP220 to bind NRs. We generated ER-DBD fusion proteins containing
sequences corresponding to amino acids
To investigate the potential influence of amino acids flanking TRAP220
core LXXLL motifs in determining NR binding specificity, we
generated an additional ER-DBD fusion protein containing the sequence
The NR Binding Specificity of TRAP220 NID Can Be Altered by
Exchange of Extended LXXLL Motifs--
The crystal structures of NR
LBDs in complex with LXXLL peptides (12, 34) or a
polypeptide containing part of the SRC1 NID (13) revealed that the
conserved leucine residues make intimate contacts with a hydrophobic
groove on the LBD surface, whereas amino acids +2, +3, +6, and +7 are
solvent-exposed. Sequences flanking the core motif were undefined in
these structures (12,13), thus it remains unclear as to how
the LBD makes contacts with NID sequence outside the core motif.
However, there is evidence that sequences immediately flanking core
LXXLL motifs play a role in determining NR binding
specificity (14, 28, 30, 34-39). For example, a number of LXMs contain
a cluster of positively charged residues at positions
The spacing between LXM core motifs in the p160 family is highly
conserved, with 51 amino acids between LXM1 and LXM2, and between LXM2
and LXM3, of SRC1 (4). By comparison, the spacing between LXM1 and LXM2
of TRAP220 is 36 amino acids. Previous studies have shown that reducing
the spacing between the GRIP1 or TRAP220 motifs can negatively
influence NR binding properties (28, 35), suggesting there may be an
optimal spacing requirement for specific NR interactions. To examine
whether the spacing between LXM1 and LXM2 is a determinant of the
TRAP220 preference for NR classes, we generated the mutant
(LexA-TRAP220 NID spacer) in which the spacing between TRAP220 LXM1 and
LXM2 core motifs was increased to 51 amino acids, as in p160 proteins.
To achieve this, a 15 amino acid sequence taken from a corresponding
region of SRC1 (amino acids 710-724, located between LXM2 and LXM3)
was inserted between LXM1 and LXM2 of TRAP220 (Fig. 5A,
spacer). As shown in Fig. 5C, this mutation did
not significantly alter the binding of TRAP220 NID to PR or ER Combinatorial Effects of Mutations in LXM1 on the NR Binding
Specificity of the TRAP220 NID--
Having determined that exchange of
the 13 amino acids comprising LXM1 can alter the NR binding specificity
of the TRAP220 NID (mutant F), we used an expanded panel of TRAP220 NID
mutants (A-D) to investigate the specificity determinants
in more detail (Fig. 6A). Yeast two hybrid experiments were
carried out to assess the ability of these mutants to interact with the
ER
To examine whether mutation of the distal LXXLL motif would
also alter TRAP220 NR binding specificity, we generated mutant H, which
replaces the LXM2 sequence Altering the NR Binding Specificity of Full-length
TRAP220--
To support the conclusions from our yeast two-hybrid
data, we introduced mutations similar to that in mutant F into the LXM1 motif of full-length TRAP220 protein, and assessed its ability to bind
NRs in vitro (Fig. 8).
Bacterially expressed GST, GST-ER TRAP220/DRIP205/PBP was identified as a consequence of its strong
ligand-dependent binding to Class II NRs such as TR The presence of multiple LXXLL motifs in p160s and other
coactivators is thought to facilitate co-operative binding to NRs and
may be important for selective interactions through differential usage
of LXXLL motifs (15, 16, 35, 52). Similarly, mutagenesis of
TRAP220/DRIP205 LXXLL motifs revealed preferential
interaction of RXR To investigate whether the LXXLL motifs from TRAP220 display
the same NR specificity as demonstrated by its NID, we examined their
NR binding properties. Remarkably, TRAP220 LXM1 and LXM2 core motifs
displayed strong interactions with all NR LBDs tested (Fig. 3,
C and D), in contrast to the NID (Fig.
2B). Previous studies have shown that the NR binding
specificity of other coactivators is determined in part by
LXXLL core motifs (4, 14, 35, 52) but also involves
sequences immediately flanking the core motif (35, 38). As shown in
Fig. 4, an extended LXM1 sequence, containing additional amino acids on
the N- and C-terminal flanks, showed increased interaction (3.5-fold)
with TR To determine which residues in the extended LXM1 are important for its
NR subclass selectivity, we used a panel of TRAP220 NID mutants (A-H).
Initially, we confirmed that the extended LXM1 sequence is a major
determinant of TRAP220 NR binding specificity, by exchange of the LXM1
for the corresponding sequence of SRC1 LXM2 (Mutant F, Fig.
5A). This resulted in strongly enhanced interaction of the
TRAP220 NID with ER Other studies have shown that a reduction in the spacing between
LXXLL motifs in p160s (35) or TRAP220 (28) has adverse affects on NID/NR interactions. This suggest that a minimal sequence length is required to generate a folded or flexible domain which can
permit docking of both LXM1 and LXM2 with both AF2 surfaces on NR
dimers. However, we noted that the spacing between p160 LXMs (51 amino
acids) is highly conserved, even across species, and differs from that
found between TRAP220 LXMs (36 amino acids). To investigate whether
this differential spacing is a feature of the NR selectivity of
TRAP220, we generated the mutant designated spacer in which the
distance between LXM1 and LXM2 in the TRAP220 NID is increased to 51 amino acids, using a sequence derived from the SRC1 LXM2/LXM3 spacer
region (Fig. 5A). However, the spacer mutant showed no
enhanced interaction with steroid receptors (Fig. 5C), nor
was binding to Class II NRs adversely affected (Fig. 5D).
Thus, while a minimal spacer sequence may be required to allow contact
with both AF2 surfaces, the exact spacing does not appear to be
critical. Moreover, the absence of any effect of the spacer mutation on
binding to Class II NRs is consistent with the hypothesis that rather
than folding into a rigid domain, the NID may be a flexible or largely
unstructured sequence, accommodating interactions with AF2 surfaces on
different NR dimers.
Other TRAP220 NID mutants were used to examine the importance of
different residues within the extended LXM1 sequence for NR binding
specificity. A previous study used phage display to identify subclasses
of LXXLL sequences that show differential interactions with
ER Several studies have highlighted the importance of the +2 and +3 amino
acids in NR/cofactor interactions. Mutation of the +2 and +3 amino
acids of the variant FXXLL motif of NSD1 to alanines (Ser-Thr to Ala-Ala) abolished binding to both Class I and Class II NRs
(33). Similarly, a +3 mutation in TIF2 LXM3 has been reported to reduce
its interaction with ER Mutants A-D (Fig. 6A) were used in NR binding assays to
allow us to investigate the effect of combinatorial changes in the extended LXM1 sequence. All of these mutants displayed enhanced interaction with ER The LXXLL core motif forms a two-turn Purified TRAP/DRIP/mediator complexes have been shown to enhance the
activity of NRs in cell-free or purified in vitro
transcription assays (17, 50, 54). In comparison, only very modest
enhancement of TR In conclusion, we have demonstrated that TRAP220 exhibits preferential
binding to Class II NRs and only weak interactions with steroid
receptors. This binding specificity is determined by an extended
LXXLL motifs of 13 amino acids in length, which when
exchanged is sufficient to alter the specificity of full-length TRAP220
protein. It will be of interest to undertake mutagenesis of NR LBDs to
further explore the molecular mechanism of selective interactions
between NRs and their cofactors.
-helices containing the signature motif
LXXLL. TRAP220 contains two LXXLL motifs
(LXM1 and LXM2) that are required for its interaction with NRs. Here we
show that the nuclear receptor interaction domain (NID) of TRAP220
interacts weakly with Class I NRs. In contrast, SRC1 NID binds strongly
to both Class I and Class II NRs. Interaction assays using nine amino
acid LXXLL core motifs derived from SRC1 and TRAP220
revealed no discriminatory NR binding preferences. However, an extended
LXM1 sequence containing amino acids
4 to +9, (where the first
conserved leucine is +1) showed selective binding to thyroid hormone
receptor and reduced binding to estrogen receptor. Replacement of
either TRAP220 LXXLL motif with the corresponding 13 amino
acids of SRC1 LXM2 strongly enhanced the interaction of the TRAP220 NID
with the estrogen receptor. Mutational analysis revealed combinatorial
effects of the LXM1 core and flanking sequences in the determination of
the NR binding specificity of the TRAP220 NID. In contrast, a mutation
that increased the spacing between TRAP220 LXM1 and LXM2 had little
effect on the binding properties of this domain. Thus, a 13-amino acid
sequence comprising an extended LXXLL motif acts as the key
determinant of the NR binding specificity of TRAP220. Finally,
we show that the NR binding specificity of full-length TRAP220 can be
altered by swapping extended LXM sequences.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helices containing the
LXXLL sequence (4, 5). Structural studies have demonstrated that a hydrophobic channel (AF2) is exposed on the surface of the LBD
as a consequence of ligand binding (6-11). This channel accommodates
the LXXLL
-helix, which is held in place by hydrophobic interactions and a charged clamp involving two amino acids (lysine and
glutamate) that are conserved throughout the NR family (12, 13). The
minimal sequence that can bind the AF2 surface (the LXXLL
core motif) is contained within 8 amino acids (
1 to +7) (14).
(23), PGC1
(24), Fushi tarazu (25), and ASC-2/RAP250/TRBP/PRIP/NRC/AIB3 (26), contain a single functional LXXLL motif, although the stoichiometry of binding of these proteins to NR dimers has yet to be
determined. The transcriptional repressor RIP140 contains nine
functional LXXLL motifs (4), and a similar number have been
identified in the ER
-binding protein PELP1 (27).
,
although this appears to be relatively weak (18, 19, 29, 30). Recent
reports have demonstrated that TRAP220 interacts more strongly with
ER
(30, 50). In addition, data from other studies have suggested
that sequences flanking the core motif also influence NR binding
specificity (30, 34-39).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-RSV (42) were gifts from K. Chatterjee. The
expression construct pCIN4-TRAP220 was a gift from R. Roeder. A
cDNA-encoding full-length TRAP220 with an N-terminal HA tag was
subcloned into the modified vector pSG5(PT) using unique
XmaI and NotI sites.
DNA
binding domain (DBD) and the VP16 acidic activation domain (AAD),
respectively, were gifts from R. Losson and P. Chambon. The constructs
AAD-AR-LBD-(625-919), AAD-PR-LBD-(633-933) (44), AAD-ER
-LBD-(282-595) (4), AAD-RAR
-LBD-(200-462), and
AAD-RXR
-LBD-(230-467) (14) have been described previously. The
constructs AAD-PPAR
-LBD-(173-475) and AAD-TR
-(1-456) were
generated by cloning PCR fragments into the modified pASV3. The
ER-DBD-LXXLL core motif fusion proteins including TRAP220
LXM1-(603-611) and TRAP220 LXM2-(644-652) and the TRAP220 LXM1
extended (600-612), were generated by ligation of phosphorylated,
annealed oligonucleotide pairs into the pBL1 vector. ER-DBD-SRC1 LXM2
(formerly referred to as DBD-SRC1 motif 2) has been described
previously (4). TRAP220 NID-(335-667) and SRC1 NID-(431-761) were
produced by PCR and cloned into a modified pBTM116 vector generating
LexA-TRAP220 NID and LexA-SRC1 NID, respectively. The LexA-TRAP220
NID-(335-667) mutants (A-H, spacer, mut1 and mut2) were generated
using recombinant PCR techniques. All constructs generated by PCR were
sequenced. The expression of fusion proteins in yeast was monitored by
Western blotting using antibodies recognizing VP16, LexA (Autogen
Bioclear), or the ER
F domain epitope tag at the N terminus of the
ER-DBD fusion proteins, as described previously (43).
-LBD
and GST-RXR
-LBD were gifts from K. Chatterjee and E. Kalkhoven,
respectively. GST-ER
-LBD has been described previously (4).
pSG5(PT)-TRAP220 LXM1 mutant F was generated by replacing a
XhoI/KpnI fragment of pSG5(PT)-TRAP220 with the
corresponding fragment from pBTM116-TRAP220 NID mutant F.
(200 ng)
expression vectors and varying amounts of pSG5-SRC1e or pSG5-TRAP220,
as indicated. Empty pSG5 expression vector was used to standardize the
amount of transfected DNA. The ligands used were 10
8
M 17
-estradiol (E2 (for ER
)) and
10
7 M 3,3',5-triiodo-L-thyronine
(T3) (for TR
).
MAT
HMR
his3-11, 15 trp1-1 ade2-1 can1-100 leu2-3, 11 ura3) was transformed
sequentially with p3ERE-LacZ reporter plasmid, ER-DBD fusion protein
expression plasmids and AAD-NR-LBD expression plasmids using the
lithium acetate chemical transformation method (43). L40 (trp1
leu2 his3 ade2
LYS2::(lexAop)4×-HIS3
URA3::(LexAop)8×-LacZ) was
transformed sequentially with LexA-fusion protein expression vectors
and AAD-NR-LBD expression vectors using electroporation (43).
Transformants containing the desired plasmids were selected on
appropriate media and grown to late log phase in 15 ml of selective medium (yeast nitrogen base containing 2% w/v glucose and appropriate supplements) in the presence of 10
6 M
receptor cognate ligand (E2, T3, promegestone
(R5020), 9-cis-retinoic acid (9c-RA),
all-trans-retinoic acid (AT-RA), rosiglitazone, mibolerone)
or an equivalent amount of vehicle. Preparation of cell-free extracts
was by the glass bead method and
-galactosidase assay of the
extracts were performed as described (43, 46). Reporter
-galactosidase activities in the presence or absence of ligand were
determined for two transformants for each condition, in replicated
experiments, as stated. Ligands were purchased from Sigma with the
exception of rosiglitazone, which was a generous gift from GlaxoSmithKline.
using
isopropyl-
-D-thiogalactopyranoside induction, purified
on glutathione-Sepharose beads (Amersham Biosciences), and normalized
amounts were incubated with 35S-radiolabeled protein in
NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 10 mM
dithiothreitol) containing complete protease inhibitors (Roche
Molecular Biochemicals) in the presence or absence of 10
6
M cognate ligand (E2, 9c-RA or T3),
as described previously (16). Samples were washed three times, and
bound proteins were separated on SDS-polyacrylamide gels. Radiolabeled
proteins were visualized by autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
but Not
ER
--
Previous studies have shown that ectopic expression of the
mammalian mediator complex protein TRAP220/DRIP205/PBP modestly enhances TR
/
, VDR, and PPAR
-mediated transcription in a
ligand-dependent manner (19-21). We assessed the ability
of TRAP220 to stimulate TR
-mediated activation of the
T3-responsive reporter gene, MAL-TK-LUC. This reporter
contains a single thyroid response element (TRE) consisting of a direct
repeat spaced by four nucleotides (DR4) within a thymidine kinase
promoter and driving a firefly luciferase gene (41). As shown in Fig.
1A, increasing amounts of
transiently transfected TRAP220 expression vector enhanced
TR
-mediated transcription of the MAL-TK-LUC reporter ~2-fold in
the presence of ligand. This is comparable to previously observed
levels of TRAP220 enhancement of TR
-driven expression of
T3-responsive reporters (19). Under similar conditions, the
p160 nuclear receptor coactivator, SRC1e, also modestly enhanced TR
activation of MAL-TK-LUC (Fig. 1A). Co-expression with
RXR
did not significantly increase the levels of reporter activity
achieved in the presence or absence of coactivators (data not
shown).
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Fig. 1.
Effects of ectopically expressed TRAP220 on
ligand-induced reporter activation by TR and
ER
. A, HeLa cells were transiently transfected as
described under "Materials and Methods" with pMAL-TK-LUC reporter
plasmid (2 µg) and expression vectors for TR
(200 ng), SRC1e (500 ng), or TRAP220 (2, 3, and 4 µg) as indicated. Luciferase activity
was measured 24 h post addition of ligand (10
7
M T3, black bars) or vehicle
(white bars), and the data were normalized to a
-galactosidase internal control. Reporter activity was expressed
relative to that obtained for reporter in the absence of ligand (set at
1). The data represent the mean of triplicate samples, and error
bars are shown to indicate S.D. Similar results were obtained in
replicate experiments. B, reporter assays as in A
except that HeLa cells were transiently co-transfected with the
p3ERE-TATA-LUC reporter (1 µg), together with expression vectors for
ER
(100 ng), SRC1e (500 ng), or TRAP220 (0.25, 0.5, 0.75, 1, 2, and
3 µg) as indicated. In these experiments the ligand was
E2 (10
8 M).
. As shown in Fig. 1B, TRAP220 did not
enhance ER
-mediated transcription of the 3ERE-TATA-LUC reporter gene
(40), under similar transfection conditions to those used for TR
. In
contrast, co-transfection of SRC1e strongly enhanced ER
-mediated
transcription both in the presence and absence of ligand, as reported
previously (4, 16, 44, 45). The expression and nuclear localization of
both TRAP220 and p160 coactivators in transiently transfected cells was
confirmed by indirect immunofluorescence using anti-HA antibodies (data
not shown).
but not ER
suggested that TRAP220 exhibits NR binding
preferences. We therefore decided to compare the ability of SRC1 and
TRAP220 NIDs to bind to a panel of NR LBDs in yeast two-hybrid
experiments. Fusion proteins (shown schematically in Fig.
2), consisting of the LexA-DNA binding
domain (DBD) fused to amino acids 431-761 of SRC1 encompassing LXM1,
LXM2, and LXM3 (SRC1 NID; Fig. 2A), or amino acids 335-667
of TRAP220, encompassing LXM1 and LXM2 (TRAP220 NID; Fig.
2B), were assessed for binding to AAD-NR-LBD fusion
proteins. Western blots using an antibody recognizing LexA confirmed
that these constructs were expressed to similar levels in L40 (see Fig.
6B), and neither LexA-SRC1 NID nor LexA-TRAP220 NID was able
to activate transcription of the reporter either alone or in the
presence of VP16-AAD (data not shown). As shown in Fig. 2A,
strong ligand-dependent reporter activation was observed between SRC1 NID and the LBDs of the PR, ER
, RAR
, RXR
, and full-length TR
, (ranging from 80-160 units of reporter activity) and also a weaker but significant interaction with the AR LBD (25 units
reporter activity). We also observed a significant binding to retinoid
receptors in the absence of ligand (50 and 30 units of reporter
activity for RAR
and RXR
, respectively). The TRAP220 NID fusion
protein also displayed strong ligand-dependent reporter activation when co-expressed with RXR
LBD, TR
, and PPAR
LBD (140, 110 and 300 units of reporter activity respectively, Fig. 2B), and an intermediate level of reporter activation when
co-expressed with RAR
-LBD (40 units of reporter activity). Although
no ligand-independent interactions were observed between TRAP220 NID
and retinoid or thyroid receptors, the reporter activity indicated a
strong interaction with PPAR
-LBD in the absence of exogenous ligand
(120 units of reporter activity), consistent with our previous
observation that yeast cells may contain endogenous ligands for
PPAR
.2 In contrast to
SRC1, the TRAP220 NID showed a greatly reduced ability to interact with
Class I receptors, showing relatively weak ligand-dependent
reporter activities when co-expressed with ER
LBD (20 units), PR LBD
(3 units), and no detectable interaction with AR LBD. Thus, our results
indicate that while the SRC1 NID interacts strongly with both steroid
and Class II NRs, the TRAP220 NID shows a marked preference for binding
to the Class II NRs such as TR
, RXR
, and PPAR
.
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Fig. 2.
Interaction of SRC1 and TRAP220 NIDs with NR
subclasses. A, yeast two-hybrid interactions of the SRC1 NID
with NR LBDs. Schematic representation of the LexA-SRC1 NID bait
protein in which the LexA-DBD is fused in-frame with amino acids
431-761 of human SRC1. The LXM1, LXM2, and LXM3 sequences are
indicated. Prey vectors consisted of the acidic activation domain of
VP16 (AAD) fused in-frame with full-length TR , or the LBDs of AR,
PR, ER
, RAR
, RXR
, and PPAR
. Transformants of the yeast
reporter strain L40 co-expressing bait and prey proteins were cultured
overnight in the presence or absence of 10
6 M
cognate ligand; T3, mibolerone, R5020, E2,
AT-RA, 9C-RA, or rosiglitazone. Reporter activity in cell-free extracts
is expressed as units of
-galactosidase activity. Shaded
bars and black bars represent reporter activity in the
absence or presence of cognate ligand, respectively. A representative
experiment is shown, and similar results were obtained in replicate
experiments. B, yeast two-hybrid interactions of the TRAP220
NID with NR LBDs. The LexA-TRAP220 NID fusion protein is shown
schematically, and consisted of LexA-DBD fused to amino acids 335-667
of TRAP220. The LXM1 and LXM2 sequences are indicated. Reporter assays
to assess interactions with VP16-NR proteins were performed as in
A. C, yeast two-hybrid interactions of wild-type
and mutant TRAP220 NID constructs with VP16-NR-LBDs. LexA-TRAP220 NID
wild-type and mutant constructs are shown schematically. Mut 1 contains
the mutation LL-607/8-AA, which inactivates the LXM1 motif, whereas Mut
2 contains the mutation LL-648/9-AA resulting in loss of LXM2 binding
to NRs. Reporter activation was determined as in A.
and PPAR
LBD was dependent on the presence of two functional LXXLL
motifs, as mutation of either motif resulted in a significant reduction in reporter activation. Similarly, the weak interaction with ER
LBD
was abrogated by mutation of either LXM1 or LXM2, indicating a
requirement for both motifs. In contrast, interaction of TRAP220 NID
with RXR
LBD was severely reduced by mutation of LXM1 but largely
unaffected by mutation of LXM2. This indicates that the RXR
LBD
preferentially interacts with LXM1, consistent with a previous study
that showed selective binding of RXR
LBD (albeit weak) to TRAP220
LXM1 in GST pull-down assays (28).
1 to +8 of TRAP220 LXM1 and
LXM2, and SRC1 LXM2 (Fig. 3A)
and confirmed their expression in W303-1b cells by Western blots (Fig.
3B). As shown in Fig. 3C, TRAP220 LXM1 core motif was able to bind all NR LBDs tested in a ligand-inducible manner. Notably, similar levels of reporter activation were achieved due to
interactions with Class I and Class II NRs, with the exception of the
RXR
LBD, for which the reporter activity was consistently 2-3-fold
higher. This potentially reflects the preference of RXR
for TRAP220
LXM1 shown here (Fig. 2C) and in other studies (28). Similarly, the TRAP220 LXM2 core motif was also able to bind all NR
LBDs tested in a ligand-dependent manner. Binding of
TRAP220 LXM2 to the Class I receptors ER
and PR was 2-4-fold
greater than binding to AR and the Class II receptors TR
, PPAR
,
RAR
, and RXR
. In contrast to TRAP220 LXM1, LXM2 did not exhibit
any enhanced ligand-induced interaction with RXR
. As expected and shown for comparison, the SRC1 LXM2 core motif induced high levels of
ligand-dependent reporter activity with all NR LBDs tested (Fig. 3D). The three core motifs displayed considerable
ligand-independent interaction with PPAR
and RAR
LBDs (and to a
lesser degree with RXR
LBD in the case of TRAP220 LXM2). Taken
together, our results show that while the TRAP220 and SRC1 NIDs appear
to have quite distinct NR binding preferences, LXXLL core
motifs derived from these domains show little selectivity for NRs. This
suggests that LXXLL core motifs are only partial
determinants of the specificity of coactivator interactions with NRs,
and that other sequences in the NID contribute to specificity.
View larger version (19K):
[in a new window]
Fig. 3.
Interaction of TRAP220 and SRC1
LXXLL core motifs with NRs. A, schematic
representation of LXXLL core sequences from SRC1 or TRAP220
fused in-frame with the ER DBD, including TRAP220 LXM1 (amino acids
603-611), TRAP220 LXM2 (amino acids 644-652), and SRC1 LXM2 (amino
acids 689-697). B, Western blot detection of the DBD fusion
proteins in equivalent amounts of cell free extracts from yeast
transformants, using the antibody recognizing the ER
F domain
epitope. C-E, yeast two hybrid experiments assessing
interaction of DBD-TRAP220 LXM1, DBD-TRAP220 LXM2 or DBD-SRC1 LXM2 with
VP16-AAD-NR fusion proteins. Reporter activation assays were carried
out on cell-free extracts of W303-1b clones carrying the 3ERE-lacZ
reporter and co-expressing bait and prey fusion proteins, as described
in the legend to Fig. 2. Shaded and black bars
represent reporter activity in the absence or presence of cognate
ligand, respectively. A representative experiment is shown, and similar
results were obtained in triplicate experiments.
4 to +9 of TRAP220 LXM1 (extended, Fig.
4A). As shown in Fig.
4B, in the presence of AAD-TR
, the TRAP220-extended motif was able to activate the reporter 3.5-fold above the level observed for
the TRAP220 LXM1 core motif, in a ligand-dependent manner, suggesting that flanking sequences stabilize the interaction with TR
. In contrast, when co-expressed with ER
LBD the reporter activation by TRAP220 extended motif was ~10-fold lower than that achieved by the TRAP220 LXM1 core motif, in the presence of ligand. These results indicate that amino acids immediately flanking TRAP220 LXM1 core motif stabilize TR
interaction and reduce ER
binding, demonstrating that residues flanking LXXLL core motifs are
key determinants of NR binding specificity.
View larger version (19K):
[in a new window]
Fig. 4.
The extended TRAP220 LXM1 core motif exhibits
selective NR binding. Yeast two-hybrid experiments as in the
legend to Fig. 3 showing the interaction of TRAP220 LXM1 core and
extended motifs with TR and the ER
LBD. Reporter activities were
determined as described in the legend to Fig. 2. Shaded and
black bars represent reporter activity in the absence and
presence of 10
6 M receptor cognate ligand,
respectively.
4,
3, and
2, that are potentially involved in contacting the conserved glutamic
acid in helix 12 of the LBD, which has been proposed to be part of a
charged clamp (13). To investigate the
contribution of amino acids within extended LXXLL motifs to NR binding specificity, we made a
series of TRAP220 NID mutants (A-H; Figs. 5A and
6A). All of the mutations involve an exchange of amino acids in the extended TRAP220 LXM1 sequence for the corresponding amino acids of SRC1 LXM2. We chose SRC1
LXM2 as this sequence shows strong interactions with both Class I and
Class II NRs (14). Western blotting confirmed that wild type and mutant
proteins were expressed at similar levels in the yeast reporter strain
(Fig. 5B). Exchange of amino acids +2 and +3 of TRAP220 LXM1
for those of SRC1 LXM2 (mutant E) had little effect (<2-fold) on
TRAP220 NID interaction with the Class I receptors PR and ER
(Fig.
5C) or the Class II receptors RAR
, TR
and RXR
(Fig.
5D). Similarly, the replacement of a proline at position
2
that has been suggested to define subclasses of coactivators (37) with
a lysine residue as found in SRC1 LXM2 (mutant G), had little effect on
TRAP220 NID interactions with the Class I receptors, PR and ER
(Fig.
5C) or the Class II receptor TR
(Fig. 5D).
However the ability of mutant G to bind RAR
and RXR
LBDs in the
presence of ligand was slightly reduced (~2-fold), and curiously we
observed an increase in ligand-independent interaction with RXR
LBD
(Fig. 5D). We also generated a mutant in which the entire
TRAP220 extended LXM1 sequence (
4 to +9) was replaced with the
corresponding SRC1 LXM2 sequence (mutant F), incorporating a total of
eight amino acid exchanges at positions
4,
3,
2, +2, +3, +7, +8,
and +9. Remarkably, this mutant displayed a strongly enhanced binding
to Class I receptors PR and ER
(4- and 10-fold respectively, Fig.
5C). Importantly, the interaction of TRAP220 mutant F with
the Class II receptors RAR
, TR
, and RXR
was unaffected and was
similar to that displayed by wild-type TRAP220 (Fig. 5D). Taken together, our results indicate that replacement of the 13 amino
acids incorporating TRAP220 LXM1 with the corresponding SRC1 LXM2
sequence is sufficient to change the NR binding properties of the
TRAP220 NID. In contrast, amino acid exchange at position
2 or +2 and
+3 is not in itself sufficient to permit strong interactions with Class
I receptors.
View larger version (24K):
[in a new window]
Fig. 5.
Effects of mutations altering LXM sequence
and spacing on binding of the TRAP220 NID to NRs. A,
schematic representation of the LexA-TRAP220 NID mutants. Amino acid
exchanges between TRAP220 and SRC1 sequences are highlighted by the
shaded boxes. The additional SRC1-derived sequence used to
generate the TRAP220 spacer mutant is indicated. B, Western
blot showing the expression of the LexA-TRAP220 NID fusion proteins in
cell-free extracts used in reporter assays. The antibody used was a
mouse monoclonal antibody recognizing the LexA DBD. C, yeast
two-hybrid experiments, performed as in Fig. 2, showing the interaction
of LexA-TRAP220 NID wt and mutant proteins with AAD-PR and AAA-ER
fusion proteins, in the absence (white bars) or presence
(black bars) of cognate ligand (R5020 and E2,
respectively). D, yeast two-hybrid experiment as in
C using AAD-RAR
, AAD-TR
, and AAD-RXR
constructs
with or without AT-RA, T3, and 9C-RA, respectively. The
data represent the mean reporter activity of two transformants, and
error bars indicate S.D.
View larger version (27K):
[in a new window]
Fig. 6.
Combinatorial effects of mutations in the
extended LXM1 on the binding of the TRAP220 NID to ER . A,
schematic representation of LexA-TRAP220 NID mutants. The shaded
boxes highlight amino acids from SRC1 LXM2 used to replace the
corresponding amino acids in TRAP220 LXM1. B, Western blot
as in Fig. 5 showing the expression levels of the LexA-DBD fusion
proteins. C, yeast two-hybrid experiment showing interaction
of LexA-SRC1 NID and LexA-TRAP220 NID wt and mutant proteins with
AAD-ER
as described in the legend to Fig. 2.
,
suggesting that increased spacing between the TRAP220 LXXLL
motifs is not sufficient to allow a strong interaction with Class I
receptors. Moreover, this sequence insertion did not adversely affect
the interaction of the NID with TR
or RXR
(Fig. 5D),
although reporter activation due to interaction with AAD-RAR
was
slightly reduced (Fig 5D). Thus, the difference in spacing
between LXMs in TRAP220 and SRC1 does not appear to be a critical
determinant of the distinct NR binding preferences of these proteins.
LBD. Western blots confirmed that the wild-type and mutant
proteins were expressed at similar levels in yeast (Fig.
6B). Mutant A is similar to mutant F with the exception that
amino acids +2, +3, are wild type. Mutant B contains SRC1 sequence at
positions +2, +3, +7, +8, and +9 and thus assesses the contribution of
N-terminal flanking sequence. Mutant C contains SRC1 sequence at
positions
4,
3,
2, +2, and +3, to assess the contribution of the
C-terminal flanking sequence. Finally, mutant D contains SRC1 sequence
at positions
2, +2, and +3. As shown in Fig. 6C, all these
TRAP220 NID mutants showed increased ER
binding compared with wild
type NID. However, none were as efficient at binding ER
-LBD as
mutant F, which contained the entire SRC1 LXM2 extended motif. Mutant A
displayed 8-fold enhanced reporter activation due to binding ER
-LBD,
compared with a 19-fold enhancement seen for mutant F under similar
conditions (Fig. 5B). This suggests that the amino acids at
positions +2 and +3 are important in the context of extended
LXXLL motif, although alone (mutant E) they have only a
minimal effect on NR binding (Fig. 5C). Similarly, enhanced ER
-LBD
interaction was observed for mutant C (6-fold) (Fig. 6C),
suggesting that the N-terminal flanking sequences (
4,
3,
2) in
combination with the +2, +3 amino acids, have an important influence on
TRAP220 NID NR binding specificity. Moreover, a similar increase was
observed for mutant D (7-fold), which has a single amino acid exchange
at the
2 position (Pro to Lys) in combination with the +2 and +3
amino acid exchange. This is in contrast to the results obtained for
mutant G, which contained the Pro to Lys change only, and mutant E,
which has the +2, +3 change only, neither of which showed a strong
increase in binding to ER
(Fig. 5C). Thus, in combination
these mutations influence the NR binding specificity of TRAP220 LXM1.
The relatively weak interaction of AAD-ER
with mutant B, which
contained SRC1 LXM2 sequence at the C-terminal flank (+7, +8, +9),
coupled with the change at the +2 and +3 position suggests that this
combination of amino acids is less important for interaction with
ER
. However, the difference in ER
binding of mutants F and C
(Fig. 6C) suggests that the LXM1 C-terminal flanking
sequence does influence NR binding specificity of the TRAP220 NID.
4 to +9 with the corresponding SRC1 LXM2
sequence (Fig. 7A). As shown
in Fig. 7B, this mutation resulted in a 8-fold increase in
reporter activity due to ER
binding, but had little effect on the
ability of the TRAP220 NID to bind TR
. Thus replacement of either
LXM1 or LXM2 in the TRAP220 NID with the 13 amino acid sequence,
RHKILHRLLQEGS, results in a strong increase in binding to the Class I
receptors ER
and PR, without affecting binding to Class II NRs. In
contrast, altering spacing between LXM1 and LXM2 to that seen in SRC1,
did not alter NR binding. Thus, we conclude the extended
LXXLL motif is the principle NR binding specificity
determinant of TRAP220.
View larger version (31K):
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Fig. 7.
Mutation of the extended LXM2 sequence, and
its effect on the interaction of TRAP220 NID with
ER . A, schematic representation of
highlighting sequence exchanges in LexA-TRAP220 mutant H. B,
yeast two-hybrid experiment showing the binding of LexA-SRC1 NID and
LexA-TRAP220 NID wt and mutant H proteins to AAD-ER
and AAD-TR
as
in Fig. 2.
-LBD, GST-TR
-LBD, and
GST-RXR
-LBD fusion proteins were tested for binding to in
vitro translated, radiolabeled full-length SRC1e and TRAP220
wild-type proteins, or a TRAP220 LXM1 mutant F protein. Equal amounts
of GST fusion proteins and radiolabeled proteins were used in each
experiment. As expected, SRC1e showed strong ligand-dependent binding to ER
, TR
, and RXR
LBDs.
Under the same conditions, TRAP220 wild-type protein showed strong
ligand-dependent or ligand-stimulated binding to RXR
and
TR
, respectively. In contrast, TRAP220 wild-type protein showed
little, if any, detectable binding to ER
LBD, consistent with our
yeast two-hybrid experiments. Remarkably, the TRAP220 LXM1 mutant F
displayed strong interactions with TR
, RXR
LBDs, and also the
ER
LBD in the presence of ligand. Thus, swapping of a single
extended LXM sequence is sufficient to change of specificity of the
TRAP220 coactivator, allowing it to bind the Class I receptor
ER
.
View larger version (24K):
[in a new window]
Fig. 8.
Interaction of wild type and mutant TRAP220
proteins with GST-NRs in vitro. Normalized amounts of GST,
GST-ER -LBD, GST-RXR
-LBD, and GST-TR
-LBD proteins were
immobilized on glutathione-Sepharose beads and incubated with
35S-labeled full-length TRAP220 wild-type (wt),
TRAP220 LXM1 mutant F, or SRC1e wild-type protein (as control), in the
presence or absence of 10
6 M cognate ligand
as indicated (E2, 9C-RA, or T3, respectively).
Bound proteins were analyzed by SDS-PAGE and autoradiography, as
described under "Materials and Methods." One-tenth of the total
35S-labeled protein used in the pull-down is shown for
comparison (10% input).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
, VDR, and PPAR
. In contrast, several groups have shown that the interaction of TRAP220 with ER
is comparatively weak (18, 19, 30,
51). In this study we compared the interactions of TRAP220 proteins
with Class I and Class II NRs using yeast two-hybrid and GST pull-down
experiments. Consistent with this, our results show that TRAP220 NID
(Figs. 2, 5, 6, and 7) or full-length TRAP220 proteins (Fig. 8)
displayed only weak ligand-induced interactions with ER
(or other
Class I NRs) in yeast two-hybrid and GST pull-down assays. While the
SRC1 NID showed no apparent preference for binding to the panel of NRs
used here (Fig. 2A), TRAP220 NID bound preferentially to
TR
, RXR
, PPAR
, and to a lesser extent RAR
, whereas its binding to ER
, AR, and PR was dramatically reduced (Fig.
2B). To understand the molecular basis of these differential
interactions, we undertook an indepth analysis of TRAP220/NR binding.
LBD with LXM1, whereas LXM2 showed preferential
binding to the LBDs of the heterodimeric partners of RXR such as TR
, VDR, and PPAR
(28). Our yeast two-hybrid experiments indicate that
mutation of the conserved leucines in either LXM1 or LXM2 results in
greatly reduced binding to Class II receptors (Fig. 2C),
suggesting that NR LBD dimers contact both TRAP220 motifs. This is
consistent with the crystal structures of agonist-bound LBD homodimers
or RXR
LBD heterodimers in combination with short LXXLL
peptides, or a partial SRC1 NID, which show that both LBDs in the NR
dimer are occupied with a LXXLL core
-helix (12, 13, 34,
53). An exception in our experiments was the RXR
LBD, which required
only a functional LXM1 for strong binding (Fig. 2C).
, but a 10-fold reduced interaction with ER
LBD. Thus, the
extended 13 amino acid LXM1 sequence displays selective NR binding
properties similar to the TRAP220 NID (Fig. 2B), or
full-length protein (Fig. 8).
and PR (Fig. 5C) but had little or no
effect on binding to Class II NRs (Fig. 5D). A similar
result was recently reported in which replacement of TRAP220 LXM1
(residues
5 to +9) with the corresponding sequence from TIF2 LXM2,
enhanced binding to ER
, in contrast to wild-type TRAP220
(30). However, this effect is not restricted to LXM1, as our
data show that exchange of the extended TRAP220 LXM2 sequence for SRC1
LXM2, (Mutant H) also results in enhanced binding to ER
LBD, without
affecting binding to TR
(Fig. 7B).
LBD (37). The LXMs of TRAP220 fall into the subclass typified by
having a conserved proline residue at the
2 position, which shows
relatively weak interactions with ER
. In p160s, three lysine
residues flanking the LXM1 motif, including a lysine at the
2
position, have been shown to be targets for acetylation by CBP/p300,
and this event assists the dissociation of NR/p160 complexes (47). This
lysine residue at the
2 position potentially makes electrostatic
contact with Glu-380 in helix 5 of the ER
LBD (47). Our data show
that exchange of the proline at
2 in LXM1 for lysine (Mutant G) did
not significantly alter the binding properties of the TRAP220 NID (Fig.
5, C and D). Similarly, substitution of proline
for alanine at position
2 of LXM2 had little effect on TRAP220
binding to TR
, VDR, or PPAR
(28). Thus, mutation of this residue
in LXM1 is not sufficient on its own to enhance TRAP220 binding to
steroid receptors.
(30). However, in our experiments, exchange
of the +2 and +3 amino acids of TRAP220 LXM1 for the positively charged
equivalents in SRC1 LXM2 (Mutant E) did not by itself result in a
strong increase in binding of TRAP220 NID to Class I NRs (Fig.
5C), or alter interaction with Class II NRs (Fig.
5D).
compared with wild-type NID (Fig.
6C). For example, ER
binding to Mutant F was greater than
to Mutant A, indicating that the +2, +3 positions are important for
optimal binding. Similarly, exchange of residues +2, +3 resulted in
increased ER
binding only when combined with exchange of N-terminal
(
4,
3,
2), or to a lesser extent the C-terminal (+7, +8, +9)
flanking sequences (compare Mutant E in Fig. 5C with Mutants
B and C in Fig. 6C). The similar extent of ER
interaction
with Mutants C and D also suggests that the
2 position plays a key
role in the interaction of ER
with the N-terminal flank, possibly
via the Glu-380 residue in helix 5 of ER
(47). Taken together, our results indicate that exchange of the entire extended LXM1 motif for
the SRC1 LXM2 sequence is required for optimally enhanced binding to
ER
, and that residues within the core motif and flanking sequence
cooperate to determine NR binding specificity.
-helix that is
clamped in position on the LBD surface by electrostatic interactions with conserved lysine and glutamate residues (13). Unfortunately, the
crystal structures available to date offer little structural insight
into how flanking sequences in extended LXXLL motifs might determine receptor specific contacts with LBDs. A recent study has
revealed the existence of a second charged clamp in the GR LBD that
appears to anchor the third LXXLL motif of TIF2 (LXM3). This
involves interactions with the +2 and +6 amino acids, further highlighting the importance of the +2 position amino acid and C-terminal flank in coactivator interactions with Class I NRs (58).
Further structural studies involving extended LXXLL
peptides, and preferably stably folded NIDs will be required to
visualize selective interactions of different NRs with TRAP220 and
other cofactors such as TIP60, NRIF3, and ASC-2.
/
, VDR, and PPAR
-mediated transcription has
been observed due to ectopic expression of the NR binding subunit
TRAP220/DRIP205/PBP in transiently transfected cells (17, 19-21).
Consistent with this, we observed that exogenously expressed TRAP220
induced a very moderate enhancement of TR
activation of a DR4-driven
reporter gene (Fig. 1A). The relatively weak coactivator
activity of TRAP220 in transfection experiments might be due to
inefficient assembly of exogenous TRAP220 proteins into functional
mediator complexes. By contrast, TRAP220 failed to enhance
ER
-mediated transcription of the 3ERE-TATA-LUC reporter gene (Fig
1B). Nonetheless, chromatin immunoprecipitation experiments
have suggested that TRAP/DRIP proteins are recruited to NR-regulated
promoters, including ER
- and AR-responsive promoters (51, 55, 56),
and microinjection of HeLa cell nuclei with anti-TRAP220/PBP antibodies
down-regulated ER
activation of a reporter gene (57). In addition, a
recent report has shown that the TRAP complex can be purified from HeLa cell nuclear extracts using GST-ER
LBD in the presence of agonist ligand (50). In contrast to reports suggesting that TRAP170 interacts
with steroid receptors, GST-ER
AF1 did not retain TRAP complex in
HeLa cell nuclear extracts (50). Thus, recruitment of TRAP complexes to
the promoters of estrogen-regulated genes may involve additional
interactions with other components of this multiprotein complex.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the following colleagues for gifts of materials: C. Bevan, K. Chatterjee, P. Chambon, E. Kalkhoven, M. Parker, R. Losson, and R. Roeder. We also thank GlaxoSmithKline for providing rosiglitazone.
![]() |
FOOTNOTES |
---|
* This work was supported by the Wellcome Trust.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.
Supported by a Wellcome Prize Studentship.
§ To whom correspondence should be addressed. Tel.: 44-116-252-3474; Fax: 44-116-252-3369; E-mail: dh37@le.ac.uk.
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.M212950200
2 V. H. Coulthard and D. Heery, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NRs, nuclear hormone receptors; LBD, ligand binding domain; NID, nuclear receptor interaction domain; DBD, DNA binding domain; ER, estrogen receptor; E2, estradiol; HA, hemagglutinin; GST, glutathione S-transferase; VDR, vitamin D receptor; PPAR, peroxisome proliferator-activated receptor; RXR, 9-cis retinoic acid receptor; RAR, retinoic acid receptor; T3, 3,3',5-triiodo-L-thyronine; TR, thyroid receptor; TRAP, thyroid hormone receptor-associated protein.
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