Regulation of Ligand-Induced Heterodimerization and Coactivator Interaction by the Activation Function-2 Domain of the Vitamin D Receptor
Yan-Yun Liu,
Cuong Nguyen and
Sara Peleg
Department of Medical Specialties The University of Texas
M. D. Anderson Cancer Center Houston Texas, 77030
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ABSTRACT
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Twenty-epi analogs of 1
,25-dihydroxyvitamin
D3 (1,25D3) are
100-1000 times more potent transcriptionally than the natural hormone.
To determine whether this enhanced activity is mediated through
modulation of the dimerization process or through interaction with
coactivators, we performed quantitative protein-protein interaction
assays with in vitro translated vitamin D receptor (ivtVDR)
and fusion proteins containing glutathione-S-transferase
(GST) and either the ligand-binding domain of retinoid X receptor
(RXR
), or the nuclear receptor-interacting domain of the steroid
receptor coactivator 1 (SRC-1), or the glucocorticoid
receptor-interacting protein 1 (GRIP-1). We found that
heterodimerization of the ligand-binding domains of RXR
and VDR was
primarily deltanoid dependent as was the interaction of VDR with the
SRC-1 or with GRIP-1. The ED50 for induction of
heterodimerization was 2 nM for
1,25D3 and 0.05 nM for
20-epi-1,25D3. However, the
ED50 for induction of VDR interaction with
SRC-1 was similar for both 1,25D3 and the
20-epi analog (ED50 = 0.71.0
nM) as was the ED50 for
ligand-mediated interaction of VDR with GRIP-1
(ED50 = 0.10.3 nM).
Mutations in heptad 9 diminished both 1,25D3
and the 20-epi analog-mediated dimerization, without changing binding
of these ligands to VDR. Mutations in VDRs activation function 2
(AF-2) domain/helix 12 residues diminished the ability of
1,25D3 to induce heterodimerization and
interaction with SRC-1. These mutations did not change the ability of
20-epi-1,25D3 to induce dimerization but did
diminish its ability to induce interaction with SRC-1. We hypothesize
that both the hormone and the analog stabilize receptor conformations
that expose VDRs functional interfaces. The mechanisms by which the
two ligands expose these functional interfaces differ with respect to
participation of the AF-2 domain.
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INTRODUCTION
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The transcriptional activity of the vitamin D receptor (VDR)
depends on binding of VDR/retinoid X receptor (RXR) heterodimers (1, 2, 3, 4)
to specific DNA sequences called vitamin D-responsive elements (VDREs)
(5) and on interaction of the VDR with transcription coactivators and
bridging factors of the basal transcriptional machinery (6, 7, 8, 9).
Heterodimerization of VDR with RXR, binding of the dimer to DNA, and
interaction of VDR with coactivators of transcription are
deltanoidmodulated processes. Therefore, these processes may be
subjected to differential regulation by the biologically active
metabolite of vitamin D3,
1
,25-dihydroxyvitamin D3
(1,25D3), and natural or synthetic deltanoids.
For example, many analogs of 1,25D3 are
significantly more potent transcriptionally than the natural hormone
(10, 11, 12, 13, 14) but do not bind the VDR with greater affinity. Several
mechanisms have been proposed for this enhanced activity: stability of
the analogs, their catabolism to potent by products, poor interaction
of the analogs with serum-binding protein (15, 16, 17, 18), or direct
potentiation of VDRs transcriptional activation (10, 19, 20). The
steps in VDR activation that have been shown to be differently
modulated by 1,25D3 and its analogs include
dimerization with RXR and binding to DNA, mode of interaction of the
VDR-RXR complex with DNA, and subtle changes in the affinity of VDR for
RXR (10, 13, 19). An additional, recently proposed mechanism for
differential activation of the VDR by 1,25D3 and
its analogs is differential recruitment of coactivators/bridging
factors of transcription (20).
The 20-epi analogs are a group of deltanoids that attracted
significant research interest because they have up to a 1,000 times
greater transcriptional potency than the natural hormone. The
exceptional transcriptional potency of these superagonists has been
proposed to be mediated, in part, through differential modulation of
the VDR (10, 14, 18, 19, 20). The 20-epi analogs enhance dimerization and
DNA binding of VDR (10, 19) and occupy the ligand-binding pocket of VDR
without contacting the C-terminal residues that regulate transcription
activation function 2 (AF-2; Refs. 14, 21). The mechanism that leads
to the enhanced dimerization by the 20-epi analogs and the consequences
of the differential interaction with the AF-2 on their transcriptional
potency are not known. However, a well established role of the AF-2
domain is to contribute contact sites for coactivator interaction (6, 7, 21). Therefore, two questions are raised with respect to the
mechanism of action of 20-epi analogs: does differential interaction of
1,25D3 and its 20-epi analogs with the AF-2
residues affect the ability of VDR to recruit coactivators, and,
alternately, do AF-2 residues have any role in regulating the
dimerization ability of VDR?
The dimerization of nuclear receptors, including VDR and RXR, is
modulated by regions in their DNA-binding (5) and ligand-binding
domains (5, 22, 23, 24). The residues that regulate dimerization in the
ligand-binding domain (LBD) of VDR are in conserved regions called
heptad 9 [residues 383390 (23)] and the E1 domain [residues
244263 (22)], but so far the AF-2 core residues have not been
implicated in contributing to the dimerization interface. However, the
in vitro systems used to examine DNA-dependent dimerization
of full-length VDR with RXR are often independent of ligand binding
(25, 26, 27, 28). Because AF-2 functions are ligand dependent, the
contributions of the ligand and of the AF-2 to receptor activation,
including the dimerization process, may be compromised in
vitro and under conditions of excess receptor protein (28).
Therefore, to assess the effect of 1,25D3 and its
analog on both heterodimerization and coactivator interaction, we
adopted a highly sensitive pull-down assay system to examine the
quantitative and qualitative aspects of ligand regulation of
heterodimerization and the coactivator-interacting properties of VDR
LBD. We show here that 20-epi-1,25D3 enhanced the
dimerization potency of VDR LBD with RXR relative to
1,25D3, but did not enhance steroid receptor
coactivator 1 (SRC-1) or glucocorticoid receptor-interacting protein 1
(GRIP-1) interaction. We also show that heptad 9 and the E1 domain had
a similar role in regulating hormone- and analog-mediated dimerization.
In contrast, the AF-2 residues preferentially regulated
1,25D3-induced dimerization with RXR.
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RESULTS
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Quantification of Ligand-Dependent Interaction of VDR with RXR,
SRC-1, and GRIP-1
To study the ligand-dependent interaction of VDR with RXR
and with transcriptional coactivators of the p160 family, we used four
constructs expressing the following fusion proteins:
glutathione-S-transferase (GST)-RXR, which contained the LBD
of RXR
fused to GST; GST-VDR, which contained the LBD of VDR fused
to GST; GST-SRC-1, which contained the SRC-1 peptide [nuclear
receptor-interacting motifs 1 and 2, amino acid residues 635734 (29, 30)]; and GST-GRIP-1, which contained the three nuclear
receptor-interacting boxes of GRIP-1 (amino acid residues 636766)
(30). These fusion proteins were used to examine ligand-dependent
interaction of the cellular or the in vitro synthesized
(ivt) 35S-labeled VDR by affinity binding to
glutathione-Sepharose beads. Figure 1A
shows that in
the absence of 1,25D3 there was little
dimerization of VDR with RXR
(34% of input receptor), but in the
presence of the hormone, significant heterodimerization of either
in vitro translated VDR (ivtVDR) or cellular VDR was induced
(20- and 17-fold, respectively). The level of dimerization depended on
the ligand concentration and appeared to reach a maximum at 10
nM. Only 1.4% of input ivtVDR dimerized with
GST-VDR, and this binding neither increased nor decreased in the
presence of the hormone. Because this result is in conflict with data
reported by other laboratories (31, 32, 33), we tested whether the
bacterially expressed GST-VDR was functional and could dimerize in a
1,25D3-dependent manner with synthetic RXR.
Figure 1A
shows that 35S-labeled ivtRXR
dimerized with GST-VDR in a ligand-dependent manner. Therefore, the
bacterially expressed GST-VDR had both significant
1,25D3 binding activity and a functional
dimerization interface in its LBD. From these experiments, we concluded
that both cellular and synthetic VDR dimerize effectively with RXR
without the DNA-binding domain. This heterodimerization is ligand
dependent and is preferable to homodimerization. These assays
exclusively tested VDR-RXR interaction through their LBDs, so they did
not exclude the possibility that full-length VDR, containing the
dimerization interfaces from both the DNA-binding and the
ligand-binding domains, forms homodimers in the presence of
DNA.

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Figure 1. Quantification of Ligand-Dependent VDR-RXR and
VDR-Coactivator Interactions
A, Ligand-dependent heterodimerization was examined by incubating
recombinant VDR expressed in COS-1 cells (cellular VDR) or
35S-labeled ivtVDR with bacterially expressed
GST-RXR (58 µg), and the indicated concentrations of
1,25D3 (upper two panels). As a
control, GST-VDR was examined for its ability to dimerize in a
ligand-dependent manner with 35S-labeled ivtRXR
and with 35S-labeled ivtVDR (lower two
panels). The dimers were captured with glutathione-Sepharose
beads, eluted, and analyzed by SDS-PAGE and autoradiography (for
ivtVDR) or by Western blot analysis (for cellular VDR). NS, Nonspecific
binding to the glutathione-Sepharose beads in the presence of GST.
Input, is the amount of cellular or synthetic receptor used per
incubation; NL, no ligand. B, Ligand-dependent interaction of p160
coactivators with VDR was examined by incubating
35S-labeled ivtSRC-1 with either GST or GST-VDR
LBD fusion protein (left panel) or by incubating
35S-ivtVDR with either GST or GST-SRC-1
(middle panel) or with GST-GRIP-1 (right panel),
in the presence or absence of 10 nM ligand. The
VDR-coactivator complexes were captured with glutathione-Sepharose
beads, eluted, and analyzed as described above.
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To test the ligand-dependent interaction of transcription
coactivators with VDR, we took two approaches (Fig. 1B
): we used
GST-VDR LBD to capture the full-length
35S-labeled ivtSRC-1, and we used the GST-SRC-1
or GST-GRIP-1 to capture the 35S-labeled ivtVDR.
Our results showed that in all three assays, the interaction of ivtVDR
and its coactivators was completely ligand dependent. Furthermore, the
nuclear receptor-interacting peptides of these coactivators were
sufficient to confer ligand-dependent binding.
Effect of Side-Chain Stereochemistry on Ligand-Dependent
Heterodimerization and Coactivator Interaction Potencies of VDR
We, and others, have shown that the analog
20epi-1,25D3 is 100 times more potent
transcriptionally than 1,25D3. This enhanced
transcriptional activity is associated with the ability of the analog
to induce binding of VDR to DNA even at very low concentrations (0.01
nM) (10, 14), suggesting that the enhanced transcriptional
activity and DNA binding activity of VDR-20-epi analog complexes are
due to greater dimerization with RXR (14, 19). Other studies from our
laboratory have also shown that 20-epi-1,25D3 and
other 20-epi analogs interact with the VDR in a manner that does not
require the use of the C-terminal residues of the AF-2 domain, whereas
the natural hormone does use these residues (14). That these residues
are essential for coactivator interaction raises the possibility that
the hormone and the 20-epi analogs may also have different potencies or
efficacies to induce coactivator interaction. The assay systems
described here allowed us to test these two aspects of VDR activation
in vitro and to determine whether they are facilitated by
the analog. To that end, we performed protein-protein interaction
experiments with VDR-ligand complexes and either GST-RXR or GST-SRC-1,
or GST-GRIP-1 using ivtVDR and increasing concentrations of the ligands
(Fig. 2
). The dose-response plots for
20-epi-1,25D3-mediated transcription (Fig. 2A
),
dimerization (Fig. 2B
), and coactivator interaction (Fig. 2
, C and D)
were aligned with the dose-response plots for the corresponding
1,25D3-mediated activities. These experiments
showed that the potency of the 20-epi analog to induce transcription
(ED50 = 0.01 nM) was
200-fold greater than that of 1,25D3
(ED50 = 2 nM). Likewise,
the 20-epi analog-dependent heterodimerization potency
(ED50 = 0.05 nM) was
significantly greater than that of 1,25D3 to
promote this activity (ED50 = 2
nM). In contrast, the potencies of the two
compounds to promote interaction with SRC-1 (ED50
= 0.71.0 nM) or with GRIP-1
(ED50 = 0.10.3 nM) were
similar. In experiments not shown here we compared the potencies of
1,25D3 and its 20-epi analog to induce
interaction of full-length SRC-1 with GST-VDR and found them also
similar. Therefore, we conclude that the greater transcriptional
potency of the analog may be attributed primarily to its ability to
enhance the dimerization potency of VDR.

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Figure 2. Comparison of Transcription, Dimerization, and
Coactivator Interaction Potencies of VDR -1,25D3 and VDR-
20-epi Analog
Transcription (A) was measured by transfecting CV1 cells with hVDR
expression plasmid and the osteocalcin VDRE-thymidine kinase/GH fusion
gene. The transfected cells were incubated with the indicated
concentrations of hormone or analog, and reporter gene expression (GH)
was measured 24 h later. Dimerization (B) or coactivator
interaction (C and D) was measured by incubating
35S-labeled ivtVDR with the indicated concentrations of
ligand for 10 min at room temperature and then adding GST-RXR or
GST-SRC-1 or GST-GRIP-1 (58 µg) and 20 µl of
glutathione-Sepharose beads. The complexes were extracted from the
affinity beads with SDS buffer, detected by SDS-PAGE and
autoradiography, and quantified by densitometric scanning. The results
are expressed as percentage of maximal arbitrary densitometric units
induced by 35S-labeled ivtVDR and are representative of
seven to nine experiments. The ED50 for 1,25D3
to induce transcription in CV-1 cells was 2.0 ± 0.4
nM; The ED50 for 1,25D3 to induce
dimerization of the ivtVDR was 1.8 ± 0.6 nM, to
induce interaction with GST-SRC-1 was 0.7 ± 0.2, and to induce
interaction with GRIP-1 was 0.5 ± 0.1 nM. The
ED50s (mean ± SE) for the 20-epi analog to induce
transcription, dimerization, and interaction with SRC-1 or GRIP-1 were
0.01 ± 0.003 nM, 0.05 ± 0.02 nM,
1.0 ± 0.15 nM, and 0.25 ± 0.03 nM,
respectively.
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Different Changes in Binding Properties of
1,25D3 and
20-epi-1,25D3 during Heterodimerization
The experiments described above demonstrated that the 20-epi
analog enhanced the potency of VDR to dimerize with RXR in a cell-free
system but not the potency of VDR to interact with coactivators of the
p160 family (30). Our previous studies have shown that the
ED50 for competition of
[3H]1,25D3 binding to VDR
by 20-epi-1,25D3 in vivo or in homogenates
from VDR-expressing COS-1 cells was 11.5 nM. This is
approximately 100-fold higher than the ED50 for
transcription in vivo or for dimerization in a cell-free
system but similar to the ED50 for interaction of
VDR-20-epi analog complexes with the coactivators used above. These
experiments imply that concentrations at which the analog does not
occupy a detectable number of VDR-binding sites induce significant
dimerization and transcriptional activity. One possible explanation for
this discrepancy is that the apparent affinity of the analog for VDR,
as measured by competition assays with
[3H]1,25D3, may be
significantly lower than its actual affinity. Another possibility is
that the modes of interaction of the analog with VDR monomer and with
the heterodimerized VDR are different.
To examine these possibilities, we used a quantitative
protease-sensitivity assay to assess directly the interaction of the
analog with ivtVDR monomer and with heterodimerized ivtVDR. This assay
has been used by us and others to examine the induction of qualitative
and quantitative changes in VDR conformation by various ligands (10, 34, 35). Here, we used the autoradiographic intensity of
35S-labeled trypsin-resistant fragments induced
by 1,25D3 and 20-epi-1,25D3
to examine three parameters of ligand-receptor interaction: whether
VDR-ligand conformation is changed by the dimerization, whether the
ligand concentrations required to occupy VDR-binding sites are the same
as those required to induce conformational changes in VDR, and whether
the same ligand concentration induces conformational changes in VDR
monomers and heterodimers.
Figure 3A
shows that
1,25D3 induced trypsin-resistant fragments of 34
and 28 kDa in the VDR monomer. The 34-kDa fragment was first detected
at 0.1 nM and reached maximal intensity at 10
nM. The 28-kDa fragment was faint, and its intensity did
not reach maximum even at 1 µM (not shown). Small changes
occurred in the heterodimerized VDR-1,25D3
complexes: the trypsin-resistant 34-kDa fragment was still first
detected at a concentration of 0.1 nM (Fig. 3B
), but the
28-kDa trypsin-resistant fragment was not induced. These experiments
suggest that the conformation of VDR-1,25D3
complexes changes slightly with dimerization. However, the potency of
1,25D3 to induce conformational changes in VDR
monomers and heterodimers remained the same and was well correlated
with the affinity of 1,25D3 for VDR.

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Figure 3. A Comparison of Conformational Changes in
VDR-Ligand Monomers and Heterodimers
A and C, 35S-labeled ivtVDR was incubated with the
indicated concentrations of 1,25D3 or 20-epi
1,25D3 for 10 min at room temperature and then for 1 h
at 4 C and then subjected to trypsin digestion for 10 min; B and D,
35S-labeled ivtVDR was incubated with 1,25D3 or
with 20-epi 1,25D3 for 10 min at room temperature and then
with GST-RXR and glutathione-Sepharose resins for 1 h at 4 C. The
VDR-RXR heterodimers were purified by washing the affinity resins three
times and then subjected to trypsin digestion for 10 min. All
proteolytic products were separated by SDS-PAGE and visualized by
autoradiography of the dried gels. The position of the
trypsin-resistant fragments and the undigested ivtVDR are indicated by
arrows. NL, No ligand added.
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We then examined the effect of dimerization on the binding properties
of the 20-epi analog, again by using ivtVDR and GST-RXR. These
experiments (Fig. 3C
) showed that the analog induced three
trypsin-resistant fragments in VDR monomers. The main 34-kDa fragment
was first detected at a ligand concentration of 0.1 nM. The
two additional protease-resistant fragments, of 32 and 28 kDa, were
significantly less intense than the 34-kDa fragment. Furthermore, the
32-kDa fragment was first detected at 1 nM, and the 28-kDa
fragment was first detected at 10 nM. These results suggest
that the analog simultaneously stabilizes three different conformations
of VDR, possibly by using three alternative contact sites. The analog
did not induce detectable conformational changes at concentrations
lower than 0.1 nM; therefore, the results of this assay did
not support the hypothesis that the actual affinity of the analog for
VDR monomer is greater than that measured by the competition
assays.
When this analysis was repeated with the heterodimerized VDR-analog
complexes (Fig. 3D
), we found several interesting differences. First,
the conformation of the heterodimerized VDR-analog complexes was
significantly different from that of the monomer. In the dimerized
VDR-analog complexes, the 32- and the 34-kDa fragments had identical
intensities at ligand concentration higher or equal to 0.1
nM, but the intensity of the 28-kDa fragment was
diminished. Furthermore, the 34-kDa fragment was now detectable even at
a concentration of 0.01 nM, and the 32-kDa fragment was
detectable at a concentration of 0.1 nM. These results show
that dimerization increased the potency of the analog to stabilize
VDRs conformation by more than 10-fold, suggesting that the affinity
of the 20-epi analog for the dimerized VDR had changed. Furthermore, as
significant levels of heterodimerized VDR-analog complexes were
detected by this assay even at 0.01 nM, it also explains
how these low analog concentrations can induce significant binding of
VDR to DNA (10) and significant transcriptional activity.
Different Roles for the VDRs AF-2 Domain in Dimerization Induced
by 1,25D3 and Its 20-epi Analog
To determine which residues contributed to the differential
dimerization potencies of 1,25D3 and its 20-epi
analog, we considered the possibility that the two ligands shape
differently the functional surface of the VDRs LBD. These differences
may be reflected by changes in the amino acids required to form contact
of VDR with RXR. To examine this possibility we substituted residues
383 and 385 in heptad 9 of hVDR, a region that regulates
heterodimerization of VDR with RXR (23). Figure 4
shows these mutations diminished
1,25D3-mediated (Fig. 4A
) and
20-epi-1,25D3-mediated dimerization (Fig. 4B
) to
the same extent without changing ligand binding affinities (data not
shown and Ref. 23). These results confirm previous studies by Nakajima
et al. (23), showing that these residues were essential for
DNA-dependent heterodimerization and transcriptional activity of VDR.
These results also suggest that the 20-epi analog does not change the
requirement for heptad 9 residues, although additional mutations in
this area are necessary to substantiate these observations.

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Figure 4. Regulation of Ligand-Induced Dimerization by Heptad
9 and by the AF-2 Domain
A, Dimerization assays were performed with 35S-labeled
ivtVDR and GST-RXR as described in Materials and Methods
by using either WT VDR or VDR mutated at AF-2 residues 419 (L419S), 420
(E420A), or 421 and 422 (V421M/F422A), or the heptad 9 mutant
M383G/Q385A and the indicated concentrations of 1,25D3.
Shown are representative plots from three to seven dimerization
experiments. The ED50 values (mean ± SE)
for induction of dimerization were 1.8 ± 0.6 nM (WT
VDR), 110 ± 10 nM (V421M/F422A), 80 ± 7
nM (L419S), and 1.2 ± 0.15 nM (E420A).
ED50 was not reached with the mutant M383G/Q385A. B,
20-epi-1,25D3-dependent dimerization assays were performed
with 35S-labeled ivtVDR and GST-RXR as described above by
using either WT VDR or the four mutant VDR constructs. Shown are
representative plots from five to seven dimerization experiments. The
ED50 (mean ± SE) for induction of
dimerization were 0.05 ± 0.02 nM (WT VDR), 0.10
± 0.02 nM (V421M/F422A), 0.2 ± 0.015 nM
(L419S), and 0.15 ± 0.01 nM (E420A). ED50
was not reached with the mutant M383G/Q385A.
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Another region in the LBD that has been implicated in regulating
heterodimerization of VDR is the E1 domain (22). Two conserved residues
in this domain (L262 and L263) were substituted, with the result of a
significant loss of both 1,25D3- and
20-epi-1,25D3-mediated heterodimerization (data
not shown). There was no evidence that these mutations affected
differently 1,25D3- and analog-mediated
heterodimerization, again suggesting that the 20-epi analog does not
change the requirement for these two E1 residues.
We then considered the possibility that the effect of the 20-epi analog
on dimerization potency is indirect. Instead of changing the shape of
the dimerization interface the analog may have a greater potency to
expose it. X-ray crystallography of LBDs of nuclear receptors show the
AF-2 residues provide contact points for interaction with coactivators
but are also engaged in forming intramolecular interactions that expose
or mask the coactivator binding site (29, 36, 37). Therefore, we
hypothesized that the AF-2 domain may also contribute to forming or
exposing the dimerization interface. For that, we compared the ligand
concentrations required to achieve maximal
1,25D3-dependent dimerization of the wild-type
(WT) VDR and VDR mutated at residues 419 (L419S), 420 (E420A), and 421
and 422 (V421M/F422A). These four residues have been shown to be AF-2
residues because their substitution abolished the transcriptional
activity of VDR (14, 35). Figure 4A
shows that
1,25D3-dependent dimerization of WT VDR with
GST-RXR became detectable at 0.1 nM and reached saturation
at 100 nM. The average ED50 for this
dose response was 1.8 nM. A similar dimerization potency
was induced by 1,25D3 by using the AF-2 mutant
E420A. On the other hand, the 1,25D3-dependent
dimerization of the AF-2 mutants L419S and V421M/F422A became
detectable only at 10 nM, and reached maximal level at
1,000 nM. These results clearly indicate that these mutant
receptors had a reduced 1,25D3-dependent
dimerization potency. This decrease in sensitivity to the ligand cannot
be due to reduced affinity of 1,25D3 because
Scatchard analysis with cellular VDR (14) and competition assays using
the human ivtVDR (data not shown) showed only a 3-fold to 5-fold
decrease in the affinity of these mutants for the hormone. Furthermore,
the synthetic AF-2 mutants E420A and V421M/F422A had similar affinities
for the hormone (data not shown), but the former did not lose
1,25D3-induced dimerization potency, whereas the
latter did (Fig. 4A
). We next compared the ability of
20-epi-1,25D3 to induce dimerization through WT
and AF-2-mutated VDRs. Figure 4B
shows that the AF-2 mutations had
little or no effect on dimerization potency of the 20-epi analog: the
ED50 values for dimerization of WT VDR and the
AF-2 mutants were 0.050.2 nM. None of these mutants had a
reduced affinity for the 20-epi analog (Ref. 14 and data not shown). We
therefore conclude that differential abilities of the AF-2 mutants to
dimerize in response to 1,25D3 or its 20-epi
analog does not correlate with the ligand binding activities of these
mutants.
To determine whether the changes in dimerization potencies of
AF-2-mutated or heptad 9-mutated VDR were also reflected in
ligand-dependent binding of VDR to DNA, we performed electrophoretic
mobility shift assays with VDR expressed by recombinant expression
vectors in COS-1 cells, and the nuclear extracts from the transfected
cells were treated in vitro with the ligands. Figure 5A
shows a prominent induction of DNA
binding activity by 10 nM of either
1,25D3 or the 20-epi analog, using WT VDR.
However, 1,25D3-induced DNA binding activity of
the AF-2 mutant V421M/F422A was diminished, whereas the 20-epi
analog-mediated DNA binding activity of this mutant was similar to that
of WT VDR. Both 1,25D3- and 20-epi analog-induced
DNA binding activities were diminished with the double substitution of
the heptad 9 residues M383G/Q385A. Differences in DNA binding
activities of the three receptor preparations were not due to
differences in the amount of immunoreactive VDR (Fig. 5B
).

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Figure 5. Regulation of DNA Binding Activity by AF-2- and
Heptad 9 Residues
A, Electrophoretic mobility shift assays were performed with the
32P-labeled osteopontin VDRE and nuclear extracts from
COS-1 cells containing WT VDR, or the AF-2-mutant V421/M/F422A, or the
heptad 9 mutant M383G/Q385A. Nuclear extracts were incubated without or
with 10 nM of 1,25D3 or
20-epi-1,25D3 during the DNA binding reaction. B,
Immunoblots of input VDR (3 µl of nuclear extract from COS-1 cells
expressing recombinant VDR). VDR was detected by Western blot using
anti-VDR antibodies 9A7 (Affinity BioReagents, Inc.). Lane
1, WT VDR; lane 2, V421/M/F422A; lane 3, M383G/Q385A.
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Regulation of Coactivator Interaction and Transcriptional Activity
by the AF-2 Residues
To determine whether the differential use of the AF-2 domain for
dimerization affects transcriptional activity induced by
1,25D3 and the 20-epi analog, we cotransfected
CV-1 cells with a reporter gene containing the osteocalcin VDRE and the
VDR expression plasmids described above. The results showed that all
three AF-2 mutations diminished the transcriptional efficacy of VDR
(Fig. 6
and data not
shown). Only two of the mutations (L419S and E420A) had detectable
transcriptional potency whereas the mutant V421M/F422A had none (data
not shown). The 20-epi analog-mediated transcriptional potencies of
L419S and E420A were 1,000-fold and 100-fold greater, respectively,
than that of 1,25D3. These results suggest that
the differences in the analog-mediated and
1,25D3-mediated dimerization abilities of these
mutants could contribute to the differences in their residual
transcriptional potencies. However, it was necessary to examine the
possibility that the greater potency of the 20-epi analog to induce
transcription through the AF-2 mutants was attributed to a differential
use of this domain to contact transcription coactivators of the p160
family. For that, we compared the ability of the two ligands to induce
interaction of GST-SRC-1 with each of the three AF-2 VDR mutants.
Figure 6D
shows that neither 1,25D3 nor
20-epi-1,25D3 was able to induce interaction of
these three mutants with SRC-1. Similar results were obtained when this
experiment was repeated with GST-GRIP-1 (data not shown). These results
clearly demonstrated that while the substituted AF-2 residues had
similar roles in 1,25D3-mediated and 20-epi
analog-mediated interaction with SRC-1 and GRIP-1, they regulated
hormone- and analog-mediated dimerization differently.

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|
Figure 6. AF2-Mediated Regulation of Transcription
and SRC-1 Interaction
AC, Transcriptional activity was assessed by cotransfecting CV1 cells
with the indicated expression plasmids bearing hVDR and the osteocalcin
VDRE-thymidine kinase/GH fusion gene. The transfected cells were
incubated with the hormone or analog, and reporter gene expression (GH)
was measured 24 h later. WT VDR was included in each transfection
experiment, and the amount of reporter gene expression induced by 100
nM 1,25D3 was considered maximal
transcriptional activity. The mean ED50 values
(n = 2) for induction of transcription by
1,25D3 were 1.5 nM for WT VDR, 250
nM for L419S, and 60 nM for E420A. The
ED50 values for induction of transcription by
20-epi-1,25D3 were 0.01 nM for WT
VDR, 0.4 nM for L419S, and 0.1 nM for E420A.
Transcriptional activity of V421M/F422A was undetectable even at 1
µM 1,25D3 or the 20-epi analog. The
maximal induction of transcription with 1,25D3 or
the 20-epi analog by the WT VDR was 15- to 20-fold. Maximal induction
of transcription by the mutants E420A and L419S was 2- to 3-fold and 3-
to 4-fold, respectively. D, SRC-1 interaction was assessed by
incubating WT or AF-2 mutated 35S-labeled ivtVDR
with 10 nM 1,25D3 or
20-epi-1,25D3 and 58 µg of GST-SRC-1, as
described above. The SRC-VDR complexes were captured by
glutathione-Sepharose and visualized after SDS-PAGE and
autoradiography.
|
|
 |
DISCUSSION
|
---|
This study is a continuation of our efforts to determine the
mechanism of action of superagonists with 20-epi side-chain
stereochemistry (10, 14). Our previous studies have shown that the
enhanced transcriptional potency of 20-epi analogs is associated with
changes in the early stages of receptor activation, such as
stabilization of the analog-receptor complexes and increases in
analog-dependent VDR binding to DNA. Here, we used a cell-free system
to investigate directly whether the analog
20-epi-1,25D3 enhanced the ability of VDR to
dimerize and to interact with transcription coactivators and to examine
the nature of the biophysical changes in VDR that lead to that
enhancement. We found that the potency of the analog to induce
interaction of the LBDs of VDR and RXR was 100 times greater than that
induced by 1,25D3. In contrast, there was no
significant difference in the potency of the two compounds to induce
interaction with coactivators of the p160 family (SRC-1 and GRIP-1).
The results of our experiments suggest that this may be because the
mode of interaction of the 20-epi analog in the ligand binding pocket
of VDR changes both qualitatively and quantitatively during
dimerization. Such a change does not occur when the natural hormone
binds to the heterodimerized VDR. It also does not occur when the p160
coactivators interact with the ligand-occupied VDR monomers.
That the increase in potency to induce conformational change
correlated with the greater potency of the VDR-20-epi analog complexes
to dimerize suggests that the 20-epi analog may expose VDRs
dimerization interface more effectively than
1,25D3. However, because the 20-epi analog also
induced qualitative changes in VDRs conformation, we had to consider
the possibility that it modified the functional surface of VDR to
change the profile of interacting proteins or the nature of their
binding sites. Our preliminary results, using site-directed mutagenesis
at a region that is most likely to provide contact sites for RXR in the
VDRs LBD (heptad 9) (23, 28), strongly suggest that the contact of
RXR with VDR through these residues is not significantly altered by the
20-epi analog. Likewise, results from the site-directed mutagenesis of
AF-2 residues that directly provide contact sites for coactivators of
the p160 family (29, 37) also suggest that the analog does not
significantly change the binding site for these coactivators. However,
it still remains to be determined whether the nature of the
dimerization interface exposed by each of these ligands is different
with respect to recruitment of specific dimerization partners. It also
remains to be determined whether the 20-epi analog modifies differently
the interaction site for bridging factors or components of the basal
transcriptional machinery that interact with the VDR through amino acid
residues outside the AF-2 core. For example, a study by Yang and
Freedman (20) confirmed our findings that 1,25D3
and 20-epi-1,25D3 have similar abilities to
induce interaction of VDR with SRC-1, but also suggested that the
20-epi analogs enhanced the interaction of a factor called DRIP 205
with VDRs LBD. Although the binding of this factor to VDR is also
ligand dependent and AF-2 dependent, its nuclear receptor-interacting
domain is significantly different from that of the p160 family members
(39). A detailed mapping of its binding site to
VDR-1,25D3 complexes and VDR-20-epi analog
complexes may reveal whether DRIP 205 binding site in VDR is changed by
the 20-epi analog. Another candidate for such investigations could be
NCoA-62, a coactivator that interacts with the VDR in a
ligand-modulated but AF-2-independent fashion (40).
Our experiments showed for the first time that the AF-2 domain was an
important regulator of 1,25D3-mediated
heterodimerization of VDR. Mutating this domain reduced the ability of
the hormone to induce protease-resistant conformation (data not shown
and Ref. 14) and DNA-independent heterodimerization 100-fold. These
mutations also diminished 1,25D3-mediated binding
of VDR to DNA. These results suggest that the ligand-induced
conformational changes (which for 1,25D3 are
regulated by the AF-2 domain) determine the VDRs ability to form
contact with the LBD of RXR and eventually to bind DNA. On the other
hand, other residues probably determine the 20-epi analogs ability to
stabilize VDR conformation and to induce heterodimerization. That the
AF-2 residues are differently used by 1,25D3 and
20-epi analogs for stabilization of VDR conformation and for regulation
of heterodimerization may be explained by a recent publication,
describing the fine details of 1,25D3-occupied
VDRs LBD by x-ray crystallography (41). That study revealed that the
position of the AF-2 domain is determined by intramolecular
interactions between the AF-2 core residues and other amino acid
residues in the LBD. It also showed that the AF-2 residues V418 and
F422 and the LBD residues V234, I268, H397, and T401 have a double role
in VDR-1,25D3 complexes: they provide contact
points for the hormone and form the intramolecular interactions that
determine the position of the AF-2 core. Therefore, it is likely that
ligand contact with these residues directly modulates the
intramolecular interactions that stabilize VDRs conformation and the
position of the AF-2. We have already shown that the AF-2 core does not
provide contact for 20-epi analogs (14). Furthermore, in recent studies
(data not shown) we have also confirmed that the residue I268 was
essential for 1,25D3 binding but not necessary
for 20-epi analog binding. Taken together, these results explain how
the AF-2 domain is used by 1,25D3 to stabilize
the VDRs conformation, and why it is not used by the 20-epi analog
for this function. These results also support the hypothesis that the
AF-2 is not positioned in the same way in the two receptor-ligand
complexes, and therefore, may regulate differently the availability of
the functional interfaces in the VDR-hormone and VDR-analog complexes.
That the 20-epi analog does not form contact with residues that are
used for intramolecular interactions may contribute to its apparent
flexibility in the ligand binding pocket when VDR dimerizes with
RXR.
In conclusion, we interpret these findings with respect to the
mechanism of ligand-mediated transcriptional activities of VDR to mean
that the stability of the heterodimerized and DNA-bound
VDR-1,25D3 complexes may be susceptible to
AF-2-mediated intermolecular and intramolecular interactions. For
example, because the AF-2 regulates both 1,25D3
binding to VDR and 1,25D3-mediated dimerization,
any interaction of the AF-2 with additional factors in the
transcription apparatus (SRC-1, GRIP-1, etc.) may stabilize or
destabilize 1,25D3-VDR complexes and therefore
immediately affect VDRs DNA binding activity and transcriptional
potencies. On the other hand, in the
VDR-20-epi-1,25D3 complex, ligand binding is
stabilized by dimerization, is not dependent on AF-2 residues, and
therefore is probably not further affected by AF-2 interactions with
transcription coactivators. Overall, these experiments confirmed our
initial hypothesis that the incredible potency of the 20-epi analogs is
due primarily to their ability to form a stable complex with the VDR
monomer (14) and an even more stable complex with the heterodimer,
thereby producing a long-lasting transcriptionally active VDR complex.
That VDR heterodimers are stabilized by 20-epi analogs suggest that
some of these compounds may be useful for treatment of conditions
whereby VDR action is compromised by defective dimerization, such as
hereditary vitamin D-resistant rickets (HVDRR) (42).
 |
MATERIALS AND METHODS
|
---|
Reagents
Anti-VDR antibody 9A7 was obtained from Affinity BioReagents, Inc. (Golden, CO)
[
-32P]dATP was obtained from ICN Biochemicals, Inc. (Irvine, CA) and
[35S]methionine from Amersham Pharmacia Biotech (Arlington Heights, IL). A coupled
transcription/translation kit was obtained from Promega Corp. (Madison, WI). The GST purification system was purchased
from Pharmacia Biotech (Piscataway, NJ).
1,25D3 and its analog
20-epi-1
,25-dihydroxyvitamin D3 (MC 1288) were
a generous gift from Dr. L. Binderup (Leo Pharmaceuticals,
Bellerup, Denmark).
Plasmid Construction
The GST-RXR
-expressing plasmid was prepared by ligating an
NcoI-EcoRI fragment encoding the LBD of the human
RXR
into the SmaI-EcoRI site of pGEX2T
(Pharmacia Biotech). The GST-VDR-expressing plasmid was
prepared by insertion of a BamHI linker into the
StuI site of hVDR cDNA cloned into pGEM4, digestion with
BamHI, isolation of a 1.1-kb fragment encoding the hVDR LBD,
and ligating this fragment in the sense orientation into the
BamHI site of pGEX2T. GST-SRC-1 expression plasmid was
prepared by PCR amplification of the nucleic acid sequence encoding the
NR boxes 1 and 2 (amino acids 635734) (30) and subcloning of the
amplified DNA into BamHI and EcoRI sites of
pGEX2T. The GST-GRIP-1 plasmid was prepared by RT-PCR amplification of
cDNA encoding amino acid residues 636766 (30) and subcloning the
amplified DNA into pGX2T. The preparation of AF-2 mutant VDR plasmids
by site-directed mutagenesis has been described previously (14, 35).
Purification of GST Fusion Proteins
Overnight cultures of the Escherichia coli
strain BL21 (DE3)pLysS carrying the expression plasmids for GST,
GST-VDR, GST-RXR, GST-SRC, and GST-GRIP-1 were inoculated into 250 ml
of LB and grown for 3 h at 37 C. The cells were treated for 3
h with 0.1 mM
isopropyl-1-thio-ß-D-galactoside, harvested by
centrifugation, and resuspended in 10 ml of PBS containing 1
mM dithiothreitol and 1 mM
phenylmethylsulfonyl fluoride (PBSDP buffer). The cells were frozen and
thawed once and sonicated three times for 15 sec each time, and NP-40
was added at a final concentration of 0.5%. The cell homogenate was
then centrifuged for 10 min at 10,000 x g, and the
supernatant was incubated with 0.7 ml of a 50% slurry of
glutathione-Sepharose beads (Pharmacia Biotech) for 1
h at 4 C. The beads were then washed three times in 5 ml of PBSDP, and
the GST fusion protein was eluted by incubating the beads for 15 min
with 1 ml of 5 mM reduced glutathione at room
temperature. The eluate was dialyzed overnight against 800 ml of 20
mM Tris-HCl (pH 8.0), 100
mM NaCl, 10% glycerol, 1
mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride, and 1 mM
dithiothreitol. The purity and yield of the eluted protein were
assessed by SDS-PAGE and Coomassie blue staining.
Expression of hVDR in Mammalian Cells and Assessment of
Transcriptional Activity
For preparation of cellular VDR, the African green monkey kidney
cell line COS-1 was maintained in DMEM supplemented with 10% FBS.
Forty-eight hours before transfection, the cells were plated in 150-mm
dishes at a density of 6 x 105/dish in DMEM
and 10% FBS. Then the cells were transfected by the diethylaminoethyl
(DEAE) dextran method (43) with 20 µg/dish of recombinant hVDR
plasmid and treated for 1 min with 10% dimethyl sulfoxide. Nuclear
extracts (for dimerization and DNA binding assays) were prepared 2 days
after transfection, as described below.
For assessment of the transcriptional activity of WT and mutant VDR,
the African kidney monkey cell line CV-1 was used. These cells were
plated at a density of 105/ml in 35-mm dishes in
DMEM and 10% FBS, 48 h before transfection. The transfection was
performed by the DEAE dextran method with 2 µg/dish of recombinant
VDR plasmid and 2 µg/dish of reporter gene containing the osteocalcin
VDRE attached to the thymidine kinase promoter/GH fusion gene (10).
Twenty-four hours after transfection, the cells were incubated with
1,25D3 or 20-epi-1,25D3 for
24 h, and then the culture medium was collected for measurement of
reporter gene expression (GH production) by a RIA.
Dimerization and Coactivator Interaction Assays
Each binding reaction contained 11 µl of PBSDP buffer, 3 µl
of 35S-labeled ivtVDR or hVDR-containing nuclear extract
(
0.20.4 ng VDR), 1 µl of ethanol without or with various
concentrations of ligand, 35 µl of purified GST fusion protein, and
20 µl of glutathione-Sepharose beads (equilibrated in PBSDP buffer),
and the volume was brought up to 100 µl with NETND buffer [100
mM NaCl, 1 mM EDTA, 20 mM Tris-HCl
(pH 7.8), 0.2% NP-40, and 1 mM dithiothreitol]. This
mixture was incubated at room temperature for 10 min and then for
4 h at 4 C, and then the beads were washed once in 0.2 ml of NETND
and twice in 0.2 ml of PBSDP. The bound proteins were eluted from the
packed beads by boiling in Laemmli buffer for 3 min and were analyzed
by SDS-PAGE. The dimerized VDR was detected by Western blotting (in the
case of cellular VDR) or by autoradiography of the dried gels (in the
case of 35S-labeled ivtVDR).
Preparation of ivtVDR and Assessment of Ligand-Induced Resistance
to Trypsin
ivtVDR, unlabeled or labeled with
[35S]methionine (1,000 Ci/mmol), was prepared
by in vitro coupled transcription/translation in
reticulocyte lysates (Promega Corp.) with the hVDR cDNA in
pGEM4. The receptor (0.20.4 ng) was incubated with various
concentrations of 1,25D3 or analog for 10 min at
room temperature and then on ice for 34 h. Then, 100 µg/ml trypsin
(Sigma, St. Louis, MO) was added, and the mixture was
incubated for 10 min. The digestion products were analyzed by 12%
SDS-PAGE and autoradiography of the dried gels.
Preparation of Nuclear Extracts
To test the ligand-induced DNA binding and dimerization activity
of VDR, nuclear extracts were prepared from COS-1 cells 48 h after
transfection with the hVDR plasmids. The transfected cells were scraped
from individual dishes into PBS, washed twice in that buffer, and
resuspended in 1 ml of buffer A [10 mM HEPES (pH 7.9), 1.5
mM MgCl2, 10 mM KCl, and
0.5 mM dithiothreitol] and incubated on ice for 30 min.
After they had swollen, the cells were homogenized with 510 strokes
of a Dounce homogenizer and centrifuged for 30 sec at 14,000 x
g. The supernatants were then discarded, and the nuclear
pellets were resuspended in 50 µl of buffer C [20
mM HEPES (pH 7.9), 400 mM
KCl, 1.5 mM MgCl2, 0.2
mM EDTA, 25% glycerol, and 0.5
mM dithiothreitol], incubated on ice for 30 min,
homogenized again, and centrifuged for 30 sec at 14,000 x
g. The nuclear extracts were then collected, frozen in dry
ice immediately, and stored at -80 C for further analysis.
Electrophoretic Mobility Shift Assays
A HindIII fragment containing the mouse osteopontin
VDRE (7) was labeled with 32P to a specific
activity of 15 x 108 cpm/µg. Each
binding reaction contained 50 mM KCl, 12
mM HEPES-NaOH (pH 7.9), 1
mM EDTA, 1 mM
dithiothreitol, 12% (vol/vol) glycerol, 0.5 ng of the DNA probe, 10
µg of total nuclear proteins containing 0.20.4 ng VDR, and 1 µg
of poly(dI-dC) as nonspecific competitor DNA. The binding reactions
were performed at room temperature for 30 min in the absence or
presence of 10 nM of ligand. The complexes were
resolved by electrophoresis through 4% polyacrylamide gels at 4
C, and VDR-DNA complexes were detected by autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. L. Binderup (Leo Pharmaceuticals) for
the generous gift 1,25D3 and the 20-epi analog MC
1288, Dr. J. W. Pike for the human vitamin D receptor expression
plasmid, Dr. R. M. Evans, Dr. D. Mangelsdorf, and the Salk
Institute for the RXR-
expression plasmid, Dr. M.-J. Tsai for the
generous gift of the SRC-1 expression plasmid, and Dr. M. Goode for her
helpful comments on this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Sara Peleg, Section of Endocrinology, Box 15, Department of Medical Specialties, 1515 Holcombe Boulevard, Houston, Texas 77030. E-mail: speleg{at}mail.mdanderson.org
This study was supported by NIH Grant DK-50583 to S.P.
Received for publication October 22, 1999.
Revision received August 10, 2000.
Accepted for publication August 14, 2000.
 |
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