From the Bone and Mineral Research Program and ¶ Molecular Modeling Facility, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney NSW 2010, Australia
Received for publication, December 31, 2002, and in revised form, January 15, 2003
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ABSTRACT |
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The vitamin D receptor (VDR) is a
ligand-dependent transcription factor that heterodimerizes
with retinoid X receptor (RXR) and interacts with the basal
transcription machinery and transcriptional cofactors to regulate
target gene activity. The p160 coactivator GRIP1 and the distinct
coregulator Ski-interacting protein (SKIP)/NCoA-62 synergistically
enhance ligand-dependent VDR transcriptional activity. Both
coregulators bind directly to and form a ternary complex with VDR, with
GRIP1 contacting the activation function-2 (AF-2) domain and
SKIP/NCoA-62 interacting through an AF-2 independent interface. It was
previously reported that SKIP/NCoA-62 interaction with VDR was
independent of the heterodimerization interface (specifically, helices
H10/H11). In contrast, the present study defines specific residues
within a conserved and surface-exposed region of VDR helix H10 that are
required for interaction with SKIP/NCoA-62 and for full
ligand-dependent transactivation activity. SKIP/NCoA-62, the basal transcription factor TFIIB, and RXR all interacted with VDR
helix H10 mutants at reduced levels compared with wild type in the
absence of ligand and exhibited different degrees of increased interaction upon ligand addition. Thus, SKIP/NCoA-62 interacts with VDR
at a highly conserved region not previously associated with coregulator
binding to regulate transactivation by a molecular mechanism distinct
from that of p160 coactivators.
The vitamin D receptor
(VDR)1 is a
ligand-dependent transcription factor important in the
regulation of calcium homeostasis, development, cell growth, and
differentiation. VDR belongs to the nuclear receptor superfamily,
members of which share a common modular structure including a highly
conserved DNA binding domain (DBD) and a conserved ligand binding
domain (LBD). The LBD has a predominantly The novel coregulator Ski-interacting protein (SKIP)/NCoA-62, which is
structurally distinct from the p160 coactivators, was isolated through
its interactions with both the Ski oncoprotein and VDR (7, 8). It
also interacts with other nuclear proteins including the repressor
CBF-1 and the HPV-16 E7 oncoprotein, can bind coregulators such as
SMRT, Sin3A, HDAC2, and SRC-1, and interacts with Smad proteins to
augment transforming growth factor- SKIP/NCoA-62 augments transactivation by the vitamin D, retinoic acid,
estrogen, and glucocorticoid receptors (7), selectively binding the
VDR:RXR heterodimer in a ligand-enhanced manner (13). SKIP/NCoA-62
forms a ternary complex and acts synergistically with the p160
coactivator GRIP1 to augment VDR transactivation (13). Consistent with
such complex formation, p160 coactivators contact VDR through the AF-2
domain, whereas SKIP/NCoA-62 interactions with VDR are
AF-2-independent. SKIP/NCoA-62 reportedly contacts helix H1 of the VDR
LBD (residues 116-164 of human VDR) and the helix H10/H11 region
(residues 373-403) but not helix H12 (7). It was subsequently shown,
however, that SKIP/NCoA-62 could bind to the VDR:RXR heterodimer,
leading to the conclusion that the heterodimerization interface
(including helix H10/H11) could be excluded as a SKIP/NCoA-62
interaction surface (13).
In the present study, we further investigated structural aspects of
VDR-SKIP/NCoA-62 interaction to determine how structure may mediate
SKIP/NCoA-62 function. An interaction site was mapped to helix H10 of
mouse VDR by mutational analysis. Alteration of structurally exposed
amino acids of this helix markedly reduced 1,25(OH)2D3-independent interactions with
SKIP/NCoA-62 but not ligand-dependent interactions with the
p160 coactivators GRIP1 and RAC3. These same VDR residues were required
for interaction with RXR regardless of ligand status and for
ligand-independent interaction with TFIIB. The results indicate that a
transcriptional coactivator, a basal transcription factor, and the VDR
heterodimerization partner each interact with VDR at the same helix H10
interface, with varying ligand sensitivities. As SKIP/NCoA-62 interacts
with both unliganded and liganded VDR, the data also suggest that it may modulate the ligand-regulated occupancy of this VDR surface by
TFIIB and RXR.
Plasmids and Mutagenesis--
Mouse VDR (mVDR) was amplified
from full-length cDNA (T. Kawada, Kyoto University) using wild type
or mutant primers containing stop codons (for truncation mutants
Molecular Modeling--
A model of the mouse VDR LBD was built
using the 1.8-Å resolution crystal structure of the liganded human VDR
LBD as a template (Protein Data Bank accession code 1DB1; Ref. 2).
Initial CLUSTALW (16) alignments were performed using the LBD sequences of hRAR Yeast Two-hybrid Assays--
pAS2-1mVDRWT or mutant constructs
were transformed into SFY526 cells and tryptophan-selected. pACTIISKIP
(amino acids 145-536), pACTIITFIIB, pACTII Immunoblot Analysis--
Lysates were prepared from diploid
yeast strains (14) and mammalian cells (17). Equal amounts of protein
were resolved by SDS-PAGE (10%), blotted, and probed with 9A7 antibody
(Affinity Bioreagents Inc.) to detect VDR and anti-HA tag antibody
(Roche Molecular Biochemicals) to detect HA-SKIP.
GST Binding Assays--
GST-SKIP (12), GST-TFIIB (18),
GST-mRXR Coimmunoprecipitation Assays--
pSG5WT and mutant VDR
constructs, pSG5GRIP1 and pSG5RAC3, were transcribed and translated
in vitro using the TNTTM T7 quick coupled system
(Promega). [35S]Methionine was incorporated into GRIP1
and RAC3 polypeptide products. Binding reactions were performed (21) in
the presence of 10 Cell Culture and Transfections--
COS-1 cells were transfected
for 6 h using FuGENETM (Roche Molecular
Biochemicals) with 30 ng of pSG5WT or mutant, 450 ng of pGL3-24-hydroxylase-LUC reporter (3), and 60 ng of pRSV VDR Helix H10 Residues Form SKIP/NCoA-62 Interaction
Interface--
SKIP/NCoA-62 has been reported to interact with human
VDR through residues 373-403 in the helix H10/H11 region, excluding the AF-2 domain (7). In the present study a series of truncation mutants in this region of the highly conserved mouse VDR were generated, deleting helices H12 (
A model of the mVDR LBD was built based on the highly homologous (89%
identity) human VDR LBD (2). Residues 380-389 lie on helix H10 at the
receptor surface (Fig. 1C), forming part of an exposed
ridge, with the polar or charged side chains of the conserved residues
Gln-380, Asp-384, and Arg-386 particularly prominent midway
along the helix. The guanidinium group of Arg-386 appears in a highly
conserved bridge with Asp-337, stacked on Arg-338, making the aliphatic
component of the side chain more prominent in the model structure.
Based on the accessibility of the side chains to the exterior,
flexibility in Gln-380, Asp-384, and Arg-386 is anticipated. To
investigate their potential involvement in SKIP/NCoA-62 interaction,
these three residues were individually mutated to retain similar bulk
but altered charge in the context of VDR Helix H10 Not Required for p160 Coactivator
Interaction--
In contrast to SKIP/NCoA-62, interactions of the p160
coactivators GRIP1 and RAC3 with wild type and AX3 mutant VDR were
entirely ligand-dependent (Fig.
3A). Interestingly, p160
coactivator interactions with AX3 were elevated by ~50%, suggesting
that residues Gln-380, Asp-384, and Arg-386 may negatively affect
access of p160 coactivators to the hydrophobic cleft on the LBD.
Neither VDR Helix H10 Mutations Inhibit Transactivation
Function--
Effects of these helix H10 mutations on VDR function
were assessed by transient transfection. Transactivation by either wild type or AX3 mutant VDR was 1,25(OH)2D3
dose-responsive, but AX3 activity was limited to about 40% of wild
type at the highest dose tested, despite comparable VDR protein levels
(Fig. 3C). Thus, the integrity of helix H10 residues
Gln-380, Asp-384, and Arg-386 is necessary for efficient VDR transactivation.
Other Interactions at Helix H10 Interface--
In addition to
SKIP/NCoA-62, TFIIB and RXR also interact with this region of VDR (5,
22-24), so their interactions with the helix H10 mutants were
investigated. Two-hybrid interaction of wild type VDR with TFIIB was
inhibited by 30% upon addition of 1,25(OH)2D3
(Fig. 4A), consistent with a
previous report that VDR-TFIIB contact is inhibited by ligand (5).
Interaction of AX3 with TFIIB in the absence of ligand was ~25% of
wild type, but reinstated to wild type level with
1,25(OH)2D3. Interaction of
Unliganded wild type VDR interacted with RXR and was augmented
~3-fold by 1,25(OH)2D3 (Fig. 4A).
In contrast, unliganded AX3 did not interact with RXR, and
1,25(OH)2D3 induced interaction to only 30% of
wild type level. Mutant SKIP/NCoA-62 is unique among nuclear receptor coregulators in that
its interactions with VDR appear to be independent of the hydrophobic
cleft/AF-2 domain interface, relying instead on an undefined LBD site.
In this study we defined the structural nature of this interaction
interface and highlighted its importance in VDR function and protein
interactions. COOH-terminal mutagenesis of mVDR identified three helix
H10 residues, Gln-380, Asp-384, and Arg-386, required for full
interaction with SKIP/NCoA-62, but not with p160 coactivators. Distinct
differences in ligand-responsive interactions between helix H10 mutants
and SKIP/NCoA-62, TFIIB, or RXR were observed. Helix H10 mutation also
attenuated VDR transactivation function, presumably as a result of
disrupted interactions not only with TFIIB and RXR, but also with
SKIP/NCoA-62. Wild type VDR interacted with SKIP/NCoA-62 in both the
absence and presence of 1,25(OH)2D3, whereas
its interaction with TFIIB was partially inhibited and with RXR was
augmented by 1,25(OH)2D3, consistent with
earlier reports (5, 7, 13).
Mutagenesis of mVDR H10 residues Gln-380, Asp-384, and Arg-386 markedly
reduced ligand-independent interactions with SKIP/NCoA-62 and TFIIB and
completely abrogated RXR interaction. Ligand treatment restored
interactions with TFIIB fully, and with SKIP/NCoA-62 and RXR partially.
Thus, in the absence of ligand the bulky side chains of these helix H10
residues appear to present a prominent interaction site, as their
removal in AX3 attenuated protein-protein interactions. The
reinstatement of interactions upon ligand treatment may result from a
conformational change that brings additional interacting regions into
play. This proposal is consistent with a requirement for hVDR helix H1
residues 116-165 for SKIP/NCoA-62 interaction (7), and for sites in
the DNA binding and ligand binding domains of VDR for TFIIB and RXR
interactions (22-26).
Interaction of SKIP/NCoA-62 with the VDR helix H10 interface would be
compatible with concurrent binding of GRIP-1 to the AF-2-dependent hydrophobic cleft and thus consistent with
ternary complex formation in the presence of ligand (13). SKIP/NCoA-62 interaction with helix H10 of unliganded VDR would also be compatible with formation of a repressive ternary complex in the absence of
ligand, as SKIP/NCoA-62 and unliganded VDR both interact with NCoR and
SMRT (3, 4, 10). Furthermore, by analogy with its tethering function in
the alternate recruitment of corepressors and coactivators in Notch
pathway regulation (10), SKIP/NCoA-62 may perform a similar function in
VDR signaling, i.e. coordinating interactions with
unliganded VDR to mediate transcriptional repression and with liganded
receptor to mediate a switch to transcriptional activation. This
proposal is supported by the finding that SKIP/NCoA-62 can augment or
repress VDR transactivation in a cell-specific manner (27).
Modulation of VDR protein interactions by SKIP/NCoA-62 may also help
regulate its interactions with TFIIB and RXR. It has been proposed that
ligand-induced disruption of VDR-TFIIB interactions releases TFIIB for
incorporation into the transcription complex (5). SKIP/NCoA-62 may
regulate this transition through dynamic helix H10 interactions, as it
can interact directly with TFIIB (data not shown) in addition to both
unliganded and liganded VDR. Similarly, SKIP/NCoA-62 may facilitate RXR
interaction with VDR when ligand is present, consistent with formation
of a ternary complex between SKIP/NCoA-62 and the liganded VDR:RXR
heterodimer (13).
Such a modulator role for SKIP/NCoA-62 may also be important for
nuclear receptors other than VDR. Residues Gln-380, Asp-384, and
Arg-386 are in the ninth heptad repeat region, which is highly conserved among the nuclear receptors. Consistent with this
conservation, TFIIB interacts with RXR In summary we have found that the coregulator SKIP/NCoA-62 interacts
with VDR via a helix H10 interface that is functionally and physically
distinct from the region bound by the AF-2-dependent p160
coactivators. This previously unidentified coactivator interaction surface is required for full VDR transactivation activity and comprises
conserved residues from helix H10 that are also involved in TFIIB
interaction and heterodimerization with RXR. Moreover, the interactions
of SKIP/NCoA-62, TFIIB, and RXR with the VDR helix H10 interface are
dynamic, with varying responses to ligand. This study elucidates the
structural basis of VDR-SKIP/NCoA-62 interactions and suggests that
SKIP/NCoA-62 may regulate the exchange between TFIIB and RXR at the VDR
helix H10 interface. Furthermore, it highlights the novel role of
SKIP/NCoA-62 in VDR transcriptional regulation, a role that may also
extend to other nuclear receptors.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-helical structure with an
activation function-2 (AF-2) domain in the COOH-terminal helix (H12).
The LBD is the main site of VDR interaction with its heterodimer
partner retinoid X receptor (RXR) and basal transcription factors, and
the AF-2 domain in combination with the hydrophobic cleft forms an
interaction surface for transcriptional corepressors and coactivators
(1, 2). In the absence of its ligand 1,25-dihydroxyvitamin
D3 (1,25(OH)2D3), VDR
transcriptional activity is low due to interaction with corepressors such as N-CoR and SMRT and potentially through sequestration of the
basal transcription factor TFIIB. Addition of
1,25(OH)2D3 causes dissociation of
VDR-corepressor and VDR-TFIIB complexes, thereby relieving
repression (3-5). VDR ligand-dependent activation is
facilitated by recruitment of at least one coactivator protein with
intrinsic histone acetyl transferase activity, such as p160 coactivators. Recruitment of multiprotein cointegrator complexes including CBP/p300, p/CAF, and DRIP provide further regulation, coordinating interactions of the receptor with other proteins including
basal transcription factors, RNA polymerase II, and the p160
coactivators (6).
-dependent transactivation (7-12). SKIP/NCoA-62 functions in cell fate
determination, alternatively facilitating gene repression by tethering
CBF-1 to the corepressor SMRT and gene activation by tethering CBF-1 to
the activator NotchIC (10). Such mutually exclusive interactions with
coactivators and corepressors suggest that SKIP/NCoA-62 may facilitate
coregulator switching.
EXPERIMENTAL PROCEDURES
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DISCUSSION
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397,
389, and
380) or point mutations and cloned into pSG5
(Stratagene) and pAS2-1 (Clontech). Mutant
389
was further mutated to create
389Q380E,
389D384K,
389R386E,
and
389AX3, the last being a triple point mutant with coordinate
Q380A, D384A, and R386A changes. Full-length wild type cDNA was
similarly mutated to produce mutant AX3. Constructs pACTIISKIP (amino
acids 145-536) (12), pACTIITFIIB (14), pACTII
-RXR
, pGAD424-GRIP1 (nucleotides 204-4878), pGAD10-RAC3 (nucleotides 1289-3698), pSG5GRIP1, and pSG5RAC3 (15) have been described.
, -
, and -
; hRXR
, -
, and -
; hTR
and -
;
hPPAR
, -
, and -
; and hVDR, rVDR, and mVDR, with subsequent
analyses performed using hRAR
, hVDR, rVDR, and mVDR sequences.
Modeling was performed using InsightII and Homology version 98 software (Molecular Simulations Inc., San Diego, CA). The model of the LBD was
built from residue 124 and required no insertions or deletions. Side
chain rotamer positions were searched by retaining identical residues
in their original positions, and automatically placing others based on
their size and flexibility, placing large hydrophobic residues before
small side chains. Species-related changes in the structure of mVDR due
to the excised insertion domain or sequence differences, which are away
from the ligand binding and interaction sites, are anticipated to be minor.
-RXR
GAD424-GRIP1, and pGAD10-RAC3 were individually transformed into Y187
cells and leucine-selected. Haploid SFY526 and Y187 strains were mated
and diploid strains selected with tryptophan and leucine. Six
independent colonies from each diploid strain were tested in ligand
assays as described previously (14). Cell lysate protein content was
determined (Bio-Rad), and
-galactosidase activity was assayed by
chemiluminescence (Tropix).
(19), and GST-O (Amersham Biosciences) fusion
proteins were expressed in Escherichia coli BL21, purified,
and coupled to glutathione-Sepharose as described (Amersham
Biosciences). Whole cell lysates containing VDR wild type and mutant
proteins were prepared (20) from COS-1 cells transfected with pSG5 mVDR
expression constructs. GST binding reactions were performed (14) in the
presence of 10
7 M
1,25(OH)2D3 or vehicle. Bound proteins were
eluted, electrophoresed, and immunoblotted with 9A7 antibody.
7 M
1,25(OH)2D3 or vehicle. Complexes were
immunoprecipitated with 9A7 antibody and anti-rat IgG-Sepharose 4B
beads (Zymed Laboratories Inc.. Bound complexes were
eluted, separated by SDS-PAGE (10%), and dried gels were autoradiographed.
gal control
plasmid. Cells were then treated with vehicle or 10
10,
10
8, or 10
6 M
1,25(OH)2D3 for 16 h, harvested, and
assayed for luciferase (Promega) and
-galactosidase activities.
Luciferase activity was corrected for transfection efficiency using
-galactosidase results.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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397), H11, and H12 (
389) or part
of H10 plus H11 and H12 (
380), the last mutation removing the highly
conserved ninth heptad repeat (1) (Fig.
1, A and B). Wild
type VDR interacted with SKIP/NCoA-62 in the absence and presence of
ligand in yeast two-hybrid assays, as did the
397 and
389 mutants
(Fig. 2A). The
380 mutant
failed to interact with SKIP/NCoA-62, implicating mVDR residues
380-389 in this interaction.
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Fig. 1.
VDR COOH-terminal mutant structure and LBD
modeling. A, alignment of human and mouse VDR
COOH-terminal amino acid sequences. -Helical regions are indicated
by overlines. Truncation points of mutants
397,
389,
and
380 are indicated by horizontal arrows. Point
mutations indicated by unfilled vertical arrows; coordinate
mutation of residues 380, 384, and 386 in AX3 mutant is indicated by
filled vertical arrows enclosed in dashed box.
B, alignment of helix H10 amino acid residues. Residues
analogous to mVDR Gln-380, Asp-384, and Arg-386 are boxed.
Residues within the ninth heptad repeat are indicated. C,
ribbon diagrams of mVDR LBD model. Left panel shows the
prominent helix H10 residues Gln-380, Asp-384, and Arg-386 in profile,
with the helix oriented so that the right side represents the most
exposed region. The right panel is rotated 90 degrees
clockwise. The figure was prepared with MOLSCRIPT (31).
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Fig. 2.
SKIP/NCoA-62 interactions with VDR
COOH-terminal mutants. Yeast two-hybrid assay of strains
expressing HA-tagged SKIP (amino acids 145-536), with mVDR wild type
and mutants: A, 397,
389, or
380; B,
389,
389Q380E,
389D384K,
389R386E, and
389AX3;
C, AX3 and treated with vehicle (open bars) or
10
8 M 1,25(OH)2D3
(solid bars). Western blots confirm VDR and HA-SKIP
expression. D, in vitro binding assay of mVDR
wild type or AX3 with GST-SKIP or GST-0, with or without
10
7 M 1,25(OH)2D3.
Input was 10% of VDR added. Electrophoresed products were probed for
VDR protein levels.
389 (Q380E, D384K, or R386E;
Fig. 1A). Each point mutation diminished VDR interaction
with SKIP/NCoA-62 by 60-80% compared with wild type and
389
controls (Fig. 2B). Similarly, coordinate mutation of these
residues to alanine in the
389 context (
389AX3 mutant) decreased
this interaction by 80%. Interaction of the full-length VDR AX3 mutant
was limited to ~25% of wild type in the absence of ligand, but
approached wild type levels with 1,25(OH)2D3 (Fig. 2C). Wild type VDR interaction with GST-SKIP in
vitro was also comparable both with and without ligand (Fig.
2D). Mutant AX3 bound less strongly, although without the
ligand-induced increase in SKIP/NCoA-62 interaction seen in yeast.
389 nor
389AX3 interacted with GRIP1 or RAC3, consistent
with the AF-2 deletion. In vitro interactions between the
p160 coactivators and VDR mutants were consistent with the yeast data
(Fig. 3B). Thus, VDR helix H10 is important for interaction
with SKIP/NCoA-62 but not p160 coactivators.
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Fig. 3.
VDR H10 mutant interactions with p160
coactivators and transactivation function. A, yeast
two-hybrid interactions of mVDR wild type, AX3, 389, or
389AX3
mutant, and GRIP1 or RAC3. Treatment with vehicle (open
bars) or 10
8 M
1,25(OH)2D3 (solid bars).
B, coimmunoprecipitations of in vitro translated
VDR and GRIP1 or RAC3 (35S-labeled). Complexes were
precipitated using the VDR-specific 9A7 antibody. Input was 10% of
coactivator added; "no VDR" was coactivator immunoprecipitated
without VDR. C, AX3 mutant transactivation function. Wild
type or AX3 mutant VDR was transfected into COS-1 cells with the
24-hydroxylase promoter-luciferase reporter and treated with increasing
concentrations of 1,25(OH)2D3. VDR expression
in transfected lysates was confirmed by immunoblotting.
389 with TFIIB
was independent of ligand and at least as strong as wild type. In
contrast, interaction of
389AX3 with TFIIB was notably diminished,
with and without ligand. Binding of wild type VDR with GST-TFIIB
in vitro was also inhibited by ligand (Fig. 4B).
AX3-TFIIB interaction was weaker than wildtype, but with no
ligand-induced increase as in yeast.
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Fig. 4.
VDR H10 mutant interactions with TFIIB and
RXR. A, yeast two-hybrid interactions of mVDR wild
type, AX3, 389, or
389AX3 mutant with TFIIB or RXR, with vehicle
(open bars) or 10
8 M
1,25(OH)2D3 (solid bars) treatment.
B, in vitro binding of mVDR wild type or AX3 with
GST-TFIIB, GST-RXR, or GST-0, with or without 10
7
M 1,25(OH)2D3. Input was 10% of
VDR used in binding reactions. Electrophoresed products were probed for
VDR protein levels.
389-RXR interaction was comparable
with wild type VDR without ligand and was unaffected by ligand
treatment, as
389 does not bind 1,25(OH)2D3
(data not shown). Coordinate alanine mutation completely abrogated
389AX3 interactions with RXR. In vitro binding data of
VDR with RXR correlated with yeast results, as binding of wild type and
AX3 to GST-RXR increased with ligand, with AX3 binding lower than wild
type (Fig. 4B). Together these data indicate that mVDR helix
H10 residues 380, 384, and 386 are important for interactions with
TFIIB and RXR as well as SKIP/NCoA-62. Ligand effects on these
interactions of wild type VDR differed, as VDR-SKIP/NCoA-62
interactions were ligand-independent, whereas VDR-TFIIB
interactions were inhibited by 1,25(OH)2D3 and
those for VDR:RXR were augmented.
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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and TR
(14, 28), and
RXR
contacts PPAR
and RAR
(29, 30) through heptad 9 residues
analogous to mVDR Gln-380, Asp-384, and Arg-386. By extension,
therefore, SKIP/NCoA-62 may also interact with nuclear receptors other
than VDR via the helix H10 interface.
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FOOTNOTES |
---|
* This work was supported in part by Aza Research Pty. Ltd. and by a block grant from the Australian National Health & Medical Research Council.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.
Recipient of an Australian Postgraduate Award. Current address:
Molecular Oncology Group, McGill University, Montreal, Quebec H3A
1A1, Canada.
§ Recipient of an Australian National Health & Medical Research Council Postgraduate Medical Scholarship during this study. Current address: Pituitary Research Unit, Garvan Inst. of Medical Research, Sydney 2010, Australia.
Current address: School of Molecular and Microbial
Biosciences, Dept. of Biochemistry, University of Sydney, Sydney
2006, Australia.
** Recipient of an Australian Postgraduate Award. Current address: Gene Regulation Unit, Victor Chang Cardiac Research Inst., Sydney 2010, Australia.
To whom correspondence should be addressed: Bone and Mineral
Research Program, Garvan Inst. of Medical Research, 384 Victoria St.,
Darlinghurst, Sydney NSW 2010, Australia. Tel.: 61-2-9295-8248; Fax:
61-2-9295-8241; E-mail: e.gardiner@garvan.org.au.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.C200712200
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ABBREVIATIONS |
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The abbreviations used are: VDR, vitamin D receptor; RXR, retinoid X receptor; SKIP, Ski-interacting protein; DBD, DNA binding domain; LBD, ligand binding domain; AF-2, activation function 2; 1, 25(OH)2D3, 1,25-dihydroxyvitamin D3; RAR, retinoic acid receptor; PPAR, peroxisome proliferator-activated receptor; HA, hemagglutinin; GST, glutathione S-transferase.
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