From the St. Louis University Health Sciences Center, Department of Pharmacological and Physiological Science, St. Louis, Missouri 63104
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ABSTRACT |
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A ligand-inducible transactivation function
(AF-2) exists in the extreme carboxyl terminus of the vitamin D
receptor (VDR) that is essential for 1 The vitamin D receptor is a ligand-activated transcription factor
that mediates the biological effects of 1 Important aspects of this mechanism are the
ligand-dependent nature of the nuclear receptor-coactivator
interaction and the effect of ligand on the AF-2 motif. Structural
analyses of related nuclear receptors indicate that the AF-2 domain
undergoes a dramatic movement upon binding ligand (13-18). In the
unliganded RXR, helix H12 (which contains the AF-2 motif) projects out
from the globular LBD core (13). In the liganded receptor
(e.g., the estrogen receptor, the retinoic acid receptor, or
the thyroid hormone receptor), the AF-2 helix is folded down onto the
surface of the LBD where it is thought to close off the hydrophobic
ligand binding pocket (14-16). Interestingly, p160 coactivator
proteins do not interact appreciably with unliganded receptors in which
the AF-2 domain may, in fact, be more surface exposed (8, 10). High
affinity coactivator interaction with nuclear receptors apparently
requires a ligand-induced repositioning of the AF-2 helix against the
core of the LBD. This suggests that the AF-2 helix alone is required but is not completely sufficient to mediate coactivator interaction and
that an interaction surface comprised of the AF-2 domain and other
surface-exposed domains is needed for efficient coactivator interaction
and transactivation. Candidate domains surrounding the AF-2 core that
may form a part of a coactivator interaction surface include exposed
residues on the surfaces of helices H3 and H4, the loop between H11 and
H12, and the region between H1 and H3 comprising the Recently, we identified a novel autonomous activation function in helix
H3 of the VDR that is distinct from the AF-2 activation domain.2 Here, we show that
select residues within the helix H3 activation domain are also required
for ligand-dependent transcription and interaction of VDR
with the p160 family of nuclear receptor coactivators including SRC-1
and GRIP-1. Specifically, a Y236A helix H3 mutation in the VDR
selectively disrupted 1,25-(OH)2D3-induced
interactions between VDR and SRC-1 or GRIP-1. These data support a
model for VDR in which ligand binding creates a transactivation surface or platform for the binding of p160 coactivator proteins. Important components of this transactivation platform for the VDR are helices H3
and H12.
Oligonucleotide-directed Mutagenesis--
Single-stranded DNA
for the mammalian expression vector pSG5-VDR (4-427) was isolated as
described.2 Mutations were introduced using the GeneEditor
in vitro oligonucleotide-directed mutagenesis system
(Promega, Madison, WI). All mutations were confirmed by DNA sequencing.
Plasmids and Transient Transfection Studies--
The reporter
constructs, (VDRE)4-TATA-GH and (GAL4)5-TATA-GH
were described previously (6, 19). The expression plasmids for pSG5
GAL4-VDR-(195-238) wild-type and helix H3 point mutants were
described.2 The pSG5-VDR-(4-427) expression plasmid was
described previously (20, 21). The pCR3.1 full-length hSRC-1a
expression plasmid was kindly provided by Drs. Ming-Jer Tsai and Bert
W. O'Malley. The pSG5 full-length GRIP-1 expression plasmid was kindly
provided by Dr. Michael R. Stallcup and has been described previously
(22).
COS-7 cells were transfected by standard calcium phosphate
precipitation procedures as described previously (21). In all transfections, the amount of total DNA was kept constant at 15 µg by
adding pBluescript II KS+ (Stratagene, La Jolla, CA) as a
carrier plasmid. Transfected cells were treated with 10 Ligand-binding Assay--
GST-VDR wild-type and helix H3 point
mutants (H229A, D232A, and Y236A) were expressed in the DH5 Yeast Two-hybrid Expression Vectors, Transformation, and
In Vitro Protein Interaction Assay--
GST-VDR-(116-427)
wild-type or Y236A mutant and GST-RXR Specific Point Mutations within Helix H3 Reduce Both the Autonomous
Activity of a Minimal Transactivation Domain and the
Ligand-dependent Activity of Full-length VDR--
We have
identified a distinct transactivation function within helix H3 of the
human vitamin D receptor that resides between residues
Asp195 and Ile238.2 Fusion of this
helix H3 domain to a heterologous DNA-binding domain such as
GAL4-(1-147) was sufficient to mediate potent transactivation of a
GAL4-responsive reporter gene construct (Fig.
1). Moreover, expression of this minimal
domain (Asp195-Ile238 of the VDR) interfered
with 1,25-(OH)2D3-stimulated transcription by
intact VDR, indicating a central role for this minimal domain in
VDR-mediated transactivation.2 Three important residues
within the minimal domain were identified as His229,
Asp232, and Tyr236. Altering these amino acids
individually to alanine residues reduced (H229A or Y236A) or abolished
(D232A) this autonomous transactivation activity (Fig. 1).
To investigate the functional importance of amino acid residues within
helix H3 for 1,25-(OH)2D3-activated
transcription by full-length VDR, point mutations were introduced into
the full-length pSG5-VDR-(4-427) plasmid, and the ability of these
mutants (S225A, H229A, D232A, S235A, Y236A, and K240A) to activate
transcription of a (VDRE)4-TATA-GH reporter gene in COS-7
cells was examined. As illustrated in Fig.
2, the S225A and K240A mutations showed
only modest effects (20-30% decrease compared with wild-type VDR),
whereas the mutation of Ser235 led to a greater than 55%
reduction in the ligand-dependent transcription of a
vitamin D-responsive reporter. However, most striking were the
mutations of His229, Asp232, and
Tyr236 which individually eliminated VDR-activated
transcription. As shown in the lower panel of Fig. 2,
Western blot analysis of whole cell lysates from duplicate transfection
plates revealed nearly equivalent expression of VDR for the mutants
examined in this assay, with the exception being the D232A mutant which
showed somewhat reduced levels of expression or stability.
Consequently, His229, Asp232, and
Tyr236 are three residues within helix H3 that are
determined to be important for both the autonomous activity of the
minimal domain (Fig. 1) and for ligand-activated transcription of
full-length VDR (Fig. 2).
Characterization of the Helix H3 Mutants of VDR--
To determine
the precise molecular basis for the loss of
ligand-dependent transactivation in these mutant VDRs, a
number of important functional parameters were analyzed including RXR
heterodimerization, ligand-binding, and coactivator interaction. To
determine whether these mutations altered VDR heterodimerization with
RXR, in vitro protein-protein interaction assays were
performed (Fig. 3A), and a
quantitation of these interactions is presented in Fig. 3B. A 5-fold increase in the amount of 35S-labeled VDR
(wild-type) binding to GST-RXR was observed in the presence of
10
Several helix H3 VDR mutants were then examined in an in
vitro ligand-binding assay. Specific, saturable binding curves
were generated for the binding of
1,25-(OH)2-[3H]D3 by purified
wild-type VDR and mutants. As suggested from the previous experiment,
the VDR mutants H229A and D232A exhibited impaired abilities to bind
the 1,25-(OH)2D3 ligand (Fig.
4). The Y236A mutation appeared to bind
ligand with an affinity comparable with wild-type VDR. Dissociation
constants for the binding of 1,25-(OH)2-[3H]D3 by the
wild-type VDR and mutants were determined from Scatchard plots of the
saturable binding curves (Fig. 4, inset). The Y236A mutant
had a KD similar to wild-type VDR (0.2-0.3
nM), whereas the H229A and D232A mutants had 4.5- and
8-fold lower affinities, respectively. Cumulatively, these data
indicate that the H229A and D232A mutations were inactive in
hormone-mediated VDR transcription, presumably because of impaired
ligand binding and subsequent inability to heterodimerize with RXR. In
contrast, the loss of 1,25-(OH)2D3-mediated transactivation by the Y236A mutation could not be explained by this defect because it bound ligand with an affinity similar to that of
wild-type VDR, and it interacted in a
ligand-dependent manner with RXR.
Interactions between the Wild-Type VDR or Helix H3 Mutant Y236A and
Coactivator Proteins--
These data suggested that the Y236A mutant
must be defective in some other aspect of receptor action that did not
include hormone binding or RXR heterodimerization. Therefore, the yeast two-hybrid system was used to assess the ability of wild-type VDR and
mutant Y236A to interact with nuclear receptor coactivator proteins
(Fig. 5). In this system,
ligand-dependent interactions were observed between the
wild-type VDR (pAS1-VDR-(116-427)) and fusion proteins of SRC-1 and
RIP-140 (pAD-mSRC-1-(1169-1465) and pAD-mRIP-140-(867-1158),
respectively). These
1,25-(OH)2D3-dependent interactions
between wild-type VDR and SRC-1 or RIP-140 were observed previously
(6). Interestingly, the helix H3 point mutant Y236A (pAS1-VDR-(116-427) Y236A) showed markedly reduced interaction with
RIP-140 and essentially no interaction with SRC-1. Both wild-type and
mutant Y236A retained strong interaction with RXR
To confirm these in vivo yeast two-hybrid data, interactions
between VDR (Y236A) and p160 coactivator proteins were examined in vitro by GST pull-down analysis. As shown in Fig.
6A, 35S-labeled
full-length SRC-1 showed strong in vitro interaction with
purified wild-type GST-VDR-(116-427) in a
1,25-(OH)2D3-dependent manner
(lanes 3-4). Only minimal interaction was detected with the
liganded GST-VDR Y236A mutant (compare lanes 4 and
6). No interaction was observed with purified GST
(lane 2). Similarly, 35S-labeled full-length
GRIP-1 showed strong interaction with purified wild-type
GST-VDR-(116-427) in the presence of 10 Effect of SRC-1 and GRIP-1 Coactivators on
1,25-(OH)2D3-Activated Transcription by
Wild-type VDR and VDR (Y236A)--
Transient reporter gene expression
assays in COS-7 cells were used to determine the effects of SRC-1 or
GRIP-1 on 1,25-(OH)2D3-activated transcriptional activity by VDR and the Y236A mutant. As shown in Fig.
7, cells were cotransfected with the
full-length wild-type VDR or mutant Y236A and full-length SRC-1 or the
pCR 3.1 parent vector using a (VDRE)4-TATA-GH reporter
construct. Expressing the wild-type VDR showed weak transcriptional
activity in the absence of hormone. However, treating these cells with
10 The AF-2 domain is a small Helix H3 is an intriguing candidate region outside the AF-2 motif of
the VDR that may be important for coactivator contact and
transactivation. Based on the structural analyses of related nuclear
receptors, helix H3 of the VDR is a putative This hypothesis is strongly supported by crystallographic data that
examined the ligand-binding domain of related nuclear receptors
(13-17). These structural data indicate that ligand induces a dramatic
structural change that involves a folding or packing of the helix H12
(AF-2 domain) onto the surface of the globular LBD core with a
juxtapositioning of helix H12 and helices H3/H4. Moreover, scanning
surface mutagenesis of the thyroid hormone receptor
(hTR Asp232 of hVDR is a critical residue for transactivation
mediated by the minimal helix H3 domain (Fig. 1).2 However,
its role in mediating the transcriptional activity of the full-length
VDR is unknown. The role of Asp232 in the intact VDR could
not be tested directly because mutation of Asp232 in the
full-length VDR disrupts the ligand-dependent interaction of VDR with RXR (Fig. 3) and binding of
1,25-(OH)2D3 (Fig. 4). Whether
Asp232 directly contacts the ligand or if it is responsible
for maintaining an important structural role required for high order
binding or RXR heterodimerization remains to be determined. However,
some insight may be gained from modeling studies of the ligand-binding domain of the VDR (32). Based on the crystal structure of holo-RAR An intriguing paradox arises from our studies of the VDR activation
domains as well as previous work of others on related nuclear
receptors. The AF-2 domain of most nuclear receptors is autonomously
active when assayed outside the context of the native receptor. The
same is true for the AF-1 (33-37) and AF-2a (38, 39) motifs of other
nuclear receptors and the helix H3 activation domain that we identified
in the VDR.2 Yet mutations that inactivate the VDR AF-2
core in the intact receptor completely abolish
1,25-(OH)2D3-activated transcription, indicating that, in the context of the full-length VDR with an inactive
AF-2 domain, other resident activation functions such as the helix H3
domain are also nonfunctional. However, our data also demonstrate that
inactivation of the helix H3 domain through the Y236A mutation
abolishes 1,25-(OH)2D3-mediated
transactivation, indicating that the AF-2 domain is also nonfunctional
in this context. Thus, additional structural complexities likely exist in the liganded intact receptors that preclude observation of the
resident activities of the AF-2 or helix H3 activation domains, both of
which are readily apparent when assayed outside the context of the
full-length VDR. Moreover, the co-dependence of the AF-2 and helix H3
domains is not consistent with a mechanism that involves distinct
coactivator proteins functioning exclusively through either the AF-2 or
the helix H3 domains.
In conclusion, although it is difficult to predict the
three-dimensional positioning of specific amino acids in the VDR in the
absence of any structural data, our studies suggest that
Tyr236 is an important residue that may comprise a portion
of the interaction surface through which p160 coactivator proteins such
as SRC-1 or GRIP-1 contact the VDR. Taken together, the findings
described in this manuscript and structural data of related nuclear
receptors suggest that helix H3 of the VDR is a transactivation domain
that functions in concert with the AF-2 domain to form a
transactivation surface that mediates
1,25-(OH)2D3-/VDR-mediated transcription.
,25-dihydroxyvitamin
D3 (1,25-(OH)2D3)-activated transcription and p160 coactivator interaction. Crystallographic data
of related nuclear receptors suggest that binding of
1,25-(OH)2D3 by VDR induces conformational
changes in the ligand-binding domain (LBD), the most striking of which
is a packing of the AF-2 helix onto the LBD adjacent to helices H3 and
H4. In this study, a panel of VDR helix H3 mutants was generated, and
residues in helix H3 that are important for ligand-activated
transcription by the full-length VDR were identified. In particular,
one mutant (VDR (Y236A)) exhibited normal ligand binding and
heterodimerization with the retinoid X receptor (RXR) but was
transcriptionally inactive. Yeast two-hybrid studies and in
vitro protein interaction assays demonstrated that VDR (Y236A)
was selectively impaired in interaction with AF-2-interacting coactivator proteins such as SRC-1 and GRIP-1. These data indicate an
importance of helix H3 in the mechanism of VDR-mediated transcription, and they support the concept that helix H3 functions in concert with
the AF-2 domain to form a transactivation surface for binding the p160
class of nuclear receptor coactivators.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,25-dihydroxyvitamin D3
(1,25-(OH)2D3).1
The mechanism involves a 1,25-(OH)2D3-induced
heterodimerization of VDR with the retinoid X receptor (RXR), VDR-RXR
binding to vitamin D-responsive elements (VDREs) in the promoter
regions of specific genes, and the subsequent impact of the VDR-RXR
heterodimer on transcriptional control of vitamin D responsive genes
(1, 2). Discrete domains of the VDR that are important to this
mechanism are the 90-residue zinc finger DNA-binding module in the
NH2-terminal portion of the VDR, the large COOH-terminal
ligand-binding domain (LBD), an RXR heterodimerization interface
located within the COOH-terminal portion of the LBD, and the activation
function 2 (AF-2) domain. The AF-2 domain is a small
-helical region
located at the extreme COOH terminus of the VDR. The precise role of
the AF-2 domain in VDR-activated transcription is not fully understood. Recent studies show that it plays a central role in mediating ligand-dependent protein-protein interactions with the p160
family of nuclear receptor coactivator proteins such as SRC-1 and
GRIP-1 (3-6). Coactivator interaction with nuclear receptors,
including the VDR, is thought to be necessary for the ligand-activated
transcriptional process (6-11). Indeed, the deletion of the AF-2
domain or mutations within the AF-2 motif of the VDR eliminate
coactivator interaction and ligand-activated transcription (6, 12).
loop
(13-18).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
8
M 1,25-(OH)2D3 or ethanol vehicle
for 24 h, and an immunoassay was used to determine the amount of
secreted GH (Nichols Institute, San Juan Capistrano, CA).
strain
of Escherichia coli and purified by glutathione-agarose
affinity as described previously (23, 24). Purified proteins (0.5 µg)
were each combined with 4.5 µg of nuclear extracts of HeLa cells and
incubated with four different concentrations of
1,25-(OH)2-[3H]D3 (18 Ci/mmol)
overnight at 4 °C in the presence or absence of a 400-fold molar
excess of unlabeled 1,25-(OH)2D3. Nuclear extracts were added because previous studies have shown that highly purified VDR obtained from bacterial and baculovirus expression systems
requires additional nuclear proteins to facilitate high-affinity ligand
binding (25). GST alone or HeLa cell extracts alone generally showed
less than 1% specific binding in this assay. Unbound ligand was
removed with dextran-coated charcoal, and specific binding in the bound
fraction was determined by scintillation counting. Binding curves were
analyzed by Scatchard plots to determine the dissociation constant.
-Galactosidase Assays--
All two-hybrid plasmid constructs used
the pAS1 (26) and pAD-GAL4 (Stratagene, La Jolla, CA) yeast expression
vectors. The pAS1-VDR-(116-427) wild-type and Y236A mutant each
contained the GAL4 DNA-binding domain (amino acids 1-147) and the
carboxyl-terminal region of VDR (amino acids 116-427). The
pAD-mSRC-1-(1169-1465), pAD-mRIP-140-(867-1158),
pAD-GAL4-RXR
-(235-493), and pAD-GAL4 were identical to those
formerly described by our lab (6). Yeast transformations and
-galactosidase assays were performed as described previously (6,
27).
-(235-493) were each
individually expressed in the DH5
strain of E. coli and
purified by glutathione-agarose affinity chromatography as described
previously (23, 24). 35S-Labeled VDR-(4-427) wild-type or
helix H3 point mutants (S225A, H229A, D232A, S235A, Y236A, and K240A)
and SRC-1 or GRIP-1 were each generated using the TNT-coupled
transcription-translation system according to the manufacturer
(Promega, Madison, WI). Interactions between the purified GST fusion
proteins and [35S]methionine-labeled proteins were
assessed as described (23). Autoradiographic images were scanned and
densitometrically quantitated using ImageQuant, Version 3.0, software
from Molecular Dynamics.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Point mutations within helix H3 impair the
ligand-independent activity of a minimal transactivation domain in the
hVDR LBD. Several point mutations were generated in the minimal
transactivation domain of SG5 GAL4-VDR-(195-238) by site-directed
mutagenesis, and these mutants were tested in a GAL4-responsive
reporter gene assay in mammalian cells. COS-7 cells were transfected
with 5 µg of a (GAL4)5-TATA-GH reporter construct and
increasing amounts (0.05, 0.10, or 0.25 µg) of pSG5 GAL4, pSG5
GAL4-VDR-(195-238) wild-type, or the H229A, D232A, or Y236A mutants.
GH secreted into the media was quantitated 24 h
post-transfection.
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Fig. 2.
Point mutations within helix H3 in the
full-length VDR disrupt ligand-activated transcription. A series
of point mutations were generated within helix H3 in the full-length
pSG5-VDR-(4-427) by site-directed mutagenesis, and these mutants were
tested in COS-7 cells. Each transfection included 3 µg of a
(VDRE)4-TATA-GH reporter construct and either 10 ng of
pSG5-VDR-(4-427) wild-type or a mutant version (S225A, H229A,
D232A, S235A, Y236A, or K240A). The cells were treated in the
absence or presence of 10 8 M
1,25-(OH)2D3 as indicated for 24 h, and GH
secretion was determined by an immunoassay. A western immunoblot
analysis of pSG5-VDR-(4-427) wild-type and mutants using the
anti-VDR-9A7 antibody is shown in the bottom panel. Extracts
were prepared from duplicate plates of COS-7 cells transfected
with the wild-type pSG5-VDR-(4-427) and the helix H3 mutants (S225A,
H229A, D232A, S235A, Y236A, and K240A) in
pSG5-VDR-(4-427).
8 M 1,25-(OH)2D3
(compare lanes 3-4). Similarly, 35S-labeled
full-length VDR mutants (S225A, S235A, Y236A, and K240A) showed
ligand-dependent interaction with RXR that was comparable with wild-type (lanes 7-8, 19-20,
23-24, and 27-28). However, 35S-labeled full-length VDR mutants H229A and D232A were
impaired in their ability to interact with RXR in a
ligand-dependent manner (lanes 11-12 and
15-16). Neither wild-type nor any mutant showed interaction
with GST alone (lanes 2, 6, 10, 14, 18, 22, and
26). These data suggest that the H229A and D232A mutations
in full-length VDR compromised the ability of the receptor to bind
ligand or to heterodimerize with RXR.
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Fig. 3.
In vitro interaction of full-length wild-type
hVDR and helix H3 point mutants with RXR .
A, interactions of 35S-labeled wild-type
VDR-(4-427) or S225A, H229A, D232A, S235A, Y236A, and K240A mutants
with purified GST-RXR
were analyzed in a GST pull-down assay.
In vitro transcribed and translated 35S-labeled
wild-type VDR and helix H3 mutants were each incubated with 5 µg of
GST (lanes 2, 6, 10, 14, 18, 22, and 26) or with
5 µg of GST-RXR
in the absence (lanes 3, 7, 11, 15, 19, 23, and 27) or presence (lanes 4, 8, 12, 16, 20, 24, and 28) of 10
8 M
1,25-(OH)2D3. Protein-protein complexes were
washed, analyzed by SDS-PAGE, and visualized by autoradiography. The
input lanes represent 10% of the protein in the binding
assay. B, the data were plotted as relative
densitometric units from a representative experiment.
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Fig. 4.
Specific, saturable binding of
1,25-(OH)2-[3H]D3
by purified wild-type VDR and helix H3 mutants. The ability
of purified wild-type VDR and mutants to bind the
1,25-(OH)2D3 hormonal ligand was determined in
binding assays. Bacterial-expressed and -purified GST-VDR-(116-427)
wild-type ( ) and mutants (
, Y236A;
, H229A;
, D232A), or
GST (
) (0.5 µg of protein) were combined with 4.5 µg of nuclear
extract obtained from HeLa cells and incubated with increasing
concentrations of
1,25-(OH)2-[3H]D3.
Following the removal of free ligand with dextran-coated
charcoal, specific counts in the bound fraction were determined by
scintillation counting. Nonspecific binding was determined by
competition with 400-fold molar excess of unlabeled
1,25-(OH)2D3 and was generally less than 10%
of specific binding values. As shown in the inset,
dissociation constants for the binding of
1,25-(OH)2D3 by the wild-type and mutant VDRs
were determined from Scatchard plots of the saturable binding
curves.
in this system
(data not shown). No interaction was observed with the AD-GAL4 parent
plasmid for either wild-type VDR or mutant Y236A. Thus,
1,25-(OH)2D3-dependent interactions
between VDR (Y236A) and AF-2 coactivator proteins were dramatically
impaired compared with wild-type VDR as determined by the yeast
two-hybrid system.
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Fig. 5.
Interaction of wild-type VDR and VDR (Y236A)
with SRC-1 and RIP-140 in a two-hybrid system. Yeast expressing
the AS1-VDR-(116-427) wild-type or mutant pAS1-VDR-(116-427) Y236A
and pAD-SRC-1, pAD-RIP-140, or pAD-GAL4 were grown for 16 h at
30 °C in the absence or presence of 10 8 M
1,25-(OH)2D3. The interactions were monitored
using a
-galactosidase assay. Results are presented as the mean ± S.D. of triplicate independent cultures.
8 M
1,25-(OH)2D3 which was not apparent in the
absence of hormone (lanes 3-4). Purified GST-VDR-(116-427)
Y236A showed only very weak interaction in the presence of hormone
(lane 6) (Fig. 6B). These findings indicate that
mutant Y236A, despite retaining the ability to bind hormone and to
heterodimerize with RXR, is selectively impaired in interaction with
coactivator proteins both in yeast and in in vitro binding
assays.
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Fig. 6.
In vitro interaction of wild-type VDR and VDR
(Y236A) with full-length SRC-1 or GRIP-1. A,
interactions of 35S-labeled full-length SRC-1 with purified
GST-VDR-(116-427) wild-type or GST-VDR-(116-427) mutant Y236A were
determined in a GST pull-down assay. In vitro transcribed
and translated 35S-labeled SRC-1 was incubated with either
5 µg of GST (lane 2), with 5 µg of GST-VDR-(116-427)
wild-type (lanes 3-4), or with 5 µg of GST-VDR-(116-427)
mutant Y236A (lanes 5-6) in the absence (lanes
3 and 5) or presence (lanes
4 and 6) of 10 8 M
1,25-(OH)2D3. Protein-protein complexes were
washed, analyzed by SDS-PAGE, and visualized by autoradiography. The
input lane represents 10% of the protein in the binding
assay. B, interactions of 35S-labeled
full-length GRIP-1 with purified GST-VDR-(116-427) wild-type or
GST-VDR-(116-427) mutant Y236A was determined in a GST pull-down
assay. In vitro transcribed and translated
35S-labeled GRIP-1 was incubated with either 5 µg of GST
(lane 2), with 5 µg of GST-VDR-(116-427) wild-type
(lanes 3-4), or with 5 µg of GST-VDR-(116-427) mutant
Y236A (lanes 5-6) in the absence (lanes
3 and 5) or presence (lanes
4 and 6) of 10
8 M
1,25-(OH)2D3. Protein-protein complexes were
analyzed as described for panel A.
8 M 1,25-(OH)2D3
activated reporter gene expression by about 9-fold, which was further
augmented nearly 3-fold by coexpression of SRC-1. The transcriptional
enhancement observed with wild-type VDR was not apparent with mutant
Y236A even in the presence of excess SRC-1. Similar results were
obtained for both wild-type VDR and mutant Y236A in cells cotransfected
with additional exogenous full-length GRIP-1. Additionally, although
the wild-type VDR was maximally activated at 10
8
M 1,25-(OH)2D3, the Y236A mutant
was inactive even when the cells were treated with higher
concentrations of ligand (10
6 M) ruling out
the possibility of a partial ligand-binding defect (data not shown).
The overexpression of SRC-1 was previously shown to restore the
transcriptional activity of a helix H3 point mutant (T277A in TR
)
(29); however, we found that neither the overexpression of full-length
SRC-1 or GRIP-1 could promote transactivation by the VDR mutant Y236A.
These findings demonstrate that tyrosine 236 is a critical residue in
the VDR for mediating activated transcription through interaction with
AF-2 coactivators of the p160 family.
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Fig. 7.
Expression of SRC-1 or GRIP-1 augments
ligand-activated transcription by wild-type VDR but not by the VDR
(Y236A) mutant. COS-7 cells were cotransfected with 3 µg of a
(VDRE)4-TATA-GH reporter construct and 10 ng of
pSG5-VDR-(4-427) wild-type or 10 ng of pSG5-VDR-(4-427) mutant Y236A
and 1 µg of either pCR 3.1, pCR 3.1 SRC-1, pSG5, or pSG5 GRIP-1. The
cells were treated in the absence (open bars) or presence
(solid bars) of 10 8 M
1,25-(OH)2D3, and GH secretion was
quantitated after 24 h by an immunoassay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix at the COOH terminus of the
nuclear receptors that is required for ligand-activated transcription. The AF-2 core sequence is highly conserved throughout the nuclear receptor superfamily consisting of a centrally conserved glutamic acid
residue flanked by two pairs of hydrophobic residues (6, 29). Recently,
the AF-2 domain has been shown to be the central motif through which
coactivator proteins such as SRC-1 and GRIP-1 interact in a
ligand-dependent manner with the nuclear receptors (3-6).
The ligand-dependent interaction between coactivators and
the AF-2 helix is thought to be critical for transcriptional regulation
mediated by the nuclear receptor family members. For example, in the
VDR, the deletion of the AF-2 helix or subtle point mutations within
its conserved residues selectively abrogates coactivator interaction
(6) as well as ligand-activated transcription (6, 12). Consistent with
the properties of other activation domains identified in unrelated
transactivating proteins, the AF-2 helix is active when assayed outside
of the context of the nuclear receptor, meaning that it alone confers
transactivation capacity to a heterologous DNA binding domain such as
GAL4-(1-147). Specifically, for the VDR, our laboratory demonstrated
that a fusion protein consisting of the VDR AF-2 helix and
GAL4-(1-147) was sufficient for both transactivation and for
interaction with coactivator proteins including SRC-1 (6). Importantly,
in this heterologous context, both transactivation and coactivator
interaction mediated through the minimal AF-2 motif were substantially
weaker compared with the liganded, intact VDR. These findings suggested that other residues outside the AF-2 core domain may be required for
optimal coactivator interaction and for the full transcriptional activity of the VDR.
-helix that resides
between Leu224 and Lys246 (18). Recently, we
identified a distinct transactivation domain in the VDR that
encompasses a major portion of helix H3.2 This domain alone
was sufficient for transactivation, suggesting that it may serve an
important role in VDR function, perhaps forming a part of a binding
interface for other transcriptional regulatory proteins. Indeed,
expression of the minimal helix H3 activation domain in COS-7 cells
interfered with or squelched VDR-mediated transactivation, indicating
that this domain interacted with limiting factors in the cell which are
essential for VDR-activated transcription.2 It is possible
that the helix H3 interacting proteins are distinct from the AF-2
coactivators SRC-1 and GRIP-1 of the p160 family and are selective for
the helix H3 transactivation domain of the VDR. Alternatively, helices
H3 and H12 in the VDR may fold in a manner that creates a single
transactivation surface in which both helices are required for optimal
interaction with the SRC-1/GRIP-1 family of coactivator proteins. Such
a model was proposed by Barettino et al. (30) who suggested
that the activity of the AF-2 domain of nuclear receptors depends on
the cooperation of a number of activation domains that are dispersed
throughout the hormone binding domain and that are brought together
upon ligand binding.
1) recently identified a hydrophobic cleft formed by
helices H3, H5, H6, and H12 that is crucial for thyroid
hormone-dependent binding of the coactivator proteins SRC-1
and GRIP-1 and for ligand-activated transcription (31). Cumulatively,
these data suggest that ligand binding induces a repositioning of helix
H12 to create a coactivator interaction surface composed of helix H12
and surrounding residues within helices H3, H5, and H6. Our current
data strongly support a similar paradigm for the VDR. This was most
strikingly apparent in the Y236A mutation within the VDR helix H3
activation domain. While the Y236A mutation did not affect the ability
of VDR to bind ligand or to heterodimerize with RXR, alteration of this single tyrosine residue selectively impaired both
1,25-(OH)2D3-dependent interaction
with the AF-2 coactivators SRC-1 and GRIP-1 as well as
1,25-(OH)2D3-activated transcription. The
essential nature of this helix H3 residue in coactivator function is
apparent in Fig. 7, wherein overexpression of SRC-1 or GRIP-1
dramatically augments 1,25-(OH)2D3-activated
transcription mediated by wild-type VDR, but each exhibited absolutely
no effect on transactivation mediated by the VDR (Y236A) mutant. Thus,
in addition to the AF-2 domain, Y236 located within the helix H3
activation domain of the VDR is required for efficient interaction with
coactivators of the SRC-1/GRIP-1 family and the subsequent
transcriptional regulatory effects of the VDR.
, this study strongly suggests that Asp232 of hVDR
(corresponding to Glu232 in RAR
) is spatially distant
from the ligand (>5 Å) and does not directly contact the ligand (32).
In fact, Glu232 of RAR
(corresponding to
Asp232 of hVDR) is solvent-exposed and projects out away
from the ligand, residing on the outer face of helix H3 (data not
shown). These structural modeling data suggest that Asp232
of the VDR may not be directly involved in ligand binding, and its
mutation in the full-length VDR may affect the positioning of other
nearby residues that do directly contact ligand. Regardless, its
putative surface localization and its key role in transactivation by
the minimal domain strongly suggest that it also may play a role in the
transactivation surface of the full-length receptor.
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ACKNOWLEDGEMENTS |
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We thank Drs. Ming-Jer Tsai and Bert W. O'Malley for kindly providing the pCR 3.1 hSRC-1a expression plasmid and Dr. Michael R. Stallcup for the pSG5 GRIP-1 expression plasmid.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant R01DK50348 (to P. N. M.).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 Pre-doctoral training grant from the National
Institutes of Health (T32-GM08306).
§ To whom all correspondence should be addressed: St. Louis University School of Medicine, Dept. of Pharmacological and Physiological Science, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314-268-5318; Fax: 314-577-8233; E-mail: macdonal{at}slu.edu. Reprints are not available.
2 Kraichely, D. M., Nakai, Y. D., and MacDonald, P. N., (1999) J. Cell. Biochem., in press.
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ABBREVIATIONS |
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The abbreviations used are:
1, 25-(OH)2D3, 1,25-dihydroxyvitamin
D3;
VDR, vitamin D receptor;
AF-2, activation function 2;
LBD, ligand-binding domain;
RXR, retinoid X receptor;
SRC-1, steroid
receptor coactivator 1;
GRIP-1, glucocorticoid receptor-interacting
protein 1;
GST, glutathione S-transferase;
VDRE, vitamin D
response element;
AF-1 activation function 1, AF-2a, activation
function 2a;
TR, thyroid hormone receptor;
GH, growth hormone.
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