Evidence for Ligand-Dependent Intramolecular Folding of the AF-2 Domain in Vitamin D Receptor-Activated Transcription and Coactivator Interaction
Hisashi Masuyama,
Cynthia M. Brownfield,
Rene St-Arnaud and
Paul N. MacDonald
Department of Pharmacological and Physiological Science (H.
M., C. M. B., P. N. M.), Saint Louis
University Health Science Center, St. Louis, Missouri
63104, Genetics Unit, Shriners Hospital (R.
S.), Montreal, Quebec H3G 1A6, Canada
Address correspondence to: Paul N. MacDonald, Ph.D., Saint Louis University Health Science Center, Department of Pharmacological and Physiological Science, 1402 South Grand Boulevard, St. Louis, Missouri 63104. Reprints are not available.
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ABSTRACT
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A ligand-dependent transcriptional activation
domain (AF-2) exists in region E of the nuclear receptors. This highly
conserved domain may contact several coactivators that are putatively
involved in nuclear receptor-mediated transcription. In this study, a
panel of vitamin D receptor (VDR) AF-2 mutants was created to examine
the importance of several conserved residues in VDR-activated
transcription. Two AF-2 mutants (L417S and E420Q) exhibited normal
ligand binding, heterodimerization with retinoid X receptor, and
vitamin D-responsive element interaction, but they were
transcriptionally inactive in a VDR-responsive reporter gene assay. All
AF-2 mutations that abolished VDR-mediated transactivation also
eliminated interactions between VDR and several putative
coactivator proteins including suppressor of gal1 (SUG1), steroid
hormone receptor coactivator-1 (SRC-1), or receptor interacting protein
(RIP140), suggesting that coactivator interaction is important for
AF-2-mediated transcription. In support of this concept, the minimal
AF-2 domain [VDR(408427] fused to the gal4 DNA binding domain was
sufficient to mediate transactivation as well as interaction with
putative coactivators. Introducing the L417S and E420Q mutations into
the minimal AF-2 domain abolished this autonomous transactivation and
coactivator interactions. Finally, we demonstrate that the minimal AF-2
domain interacted with an AF-2 deletion mutant of the VDR in a
1,25-(OH)2D3-dependent
manner, suggesting a ligand-induced intramolecular folding of the VDR
AF-2 domain. The L417S mutant of this domain disrupted the interaction
with VDR ligand-binding domain, while the E420Q mutant did not affect
this interaction. These studies suggest that the conserved AF-2 motif
may mediate transactivation through ligand-dependent intermolecular
interaction with coactivators and through ligand-induced intramolecular
contacts with the VDR ligand-binding domain itself.
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INTRODUCTION
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The vitamin D receptor (VDR) is a member of the nuclear receptor
superfamily (1, 2), and it functions as a ligand-induced transcription
factor that mediates the genomic effects of 1
,25-dihydroxyvitamin
D3 (1,25-(OH)2D3) (3, 4, 5). VDR and
other nuclear receptors display a modular structure, with several
regions (A/B, C, D, and E) exhibiting different degrees of evolutionary
conservation (2, 6). The N-terminal A/B region is the most divergent
module in these receptors, and an autonomous activation function,
designated AF-1, is present in the A/B region, which activates
transcription constitutively in the absence of the ligand binding
domain (LBD) (7, 8). The VDR is atypical in this regard since the A/B
region of VDR consists of only 20 amino acids, and deletion of these
residues does not affect VDR function (9). The highly conserved C
domain contains two zinc modules responsible for DNA binding and
sequence-specific recognition of vitamin D-responsive elements or
VDREs. The D region, or hinge domain, is located between the
DNA-binding domain (DBD) and the LBD. The hinge domain is hypothesized
to impart flexibility or a high degree of rotational freedom that
facilitates receptor binding to a variety of response elements (6). The
D region is also implicated in nuclear localization of receptors and in
transactivation (2). Finally, region E is responsible for selective
binding of the individual ligands with high affinity and selectivity.
Moreover, this C-terminal domain contains a dimerization interface and
a ligand-dependent transcriptional activation domain designated AF-2
(8, 10, 11, 12, 13).
The mechanism through which the nuclear receptor-DNA complex regulates
the transcriptional process is largely unknown. Recent data suggest
that protein-protein contacts between the receptor and the basal
transcriptional machinery are important for ligand-mediated
transactivation or repression. Nuclear receptors directly contact
several general transcription factors (GTFs) in the preinitiation
complex (PIC), including TATA-binding protein (TBP) (14, 15),
TBP-associated factors (TAFs) (16, 17), and transcription factor IIB
(TFIIB) (18, 19, 20, 21). The interaction of receptors with these GTFs is
thought to either recruit these limiting factors to PIC assembly or to
stabilize the PIC itself (2). However, other factors in addition to the
GTFs are required. This is based on the observation that one nuclear
receptor interferes with another receptors transcriptional activation
pathway without affecting basal transcription or the transcription of
other promoters (22, 23, 24). Thus, while the interaction between nuclear
receptors and the GTFs may be necessary, it is not sufficient for
nuclear receptor-mediated transcription. Another class of factors that
contact the nuclear receptors are needed, and these are collectively
termed coactivators (review in 25 . Recently described
coactivators, including steroid hormone receptor coactivator-1 (SRC-1)
(26), estrogen receptor-associated protein (ERAP 160) (27), and
receptor interacting protein (RIP140) (28), interact in a
ligand-dependent manner with several members of the nuclear receptor
superfamily to enhance ligand-induced transactivation. In contrast,
several corepressors, such as nuclear receptor corepressor (N-CoR) and
silencing mediator for retinoic acid receptors (RARs) and thyroid
hormone receptors (SMRT), interact with unliganded receptors to inhibit
basal transcription of the associated promoter (6, 25, 29, 30).
Interestingly, the LBD (AF-2 domain) of the nuclear receptor is
required both for the interaction with the majority of coactivators and
for the dissociation of corepressor proteins, suggesting a mechanistic
link between transcriptional suppression and activation (6, 30).
The AF-2 activating domain has been characterized in the C-terminal
part of region E of the RAR, retinoid X receptor (RXR), thyroid hormone
receptor (TR), and estrogen receptor (ER), and this region corresponds
to an amphipathic
-helix motif whose main features are conserved
between all known transcriptionally active members of the nuclear
receptor superfamily (10, 12, 13). In most instances, the AF-2 motif is
transcriptionally silent in the absence of ligand, and ligand binding
activates its enhancer potential (10, 12, 13). In this paper, we
demonstrate that the conserved AF-2 motif of VDR is required both for
1,25-(OH)2D3-dependent transactivation and for
1,25-(OH)2D3-dependent interactions between VDR
and several putative coactivators including SRC-1 and RIP140. Indeed,
the minimal AF-2 domain of VDR was sufficient to mediate
transactivation as well as to mediate interactions between VDR and
SRC-1 or RIP140. Finally, we present evidence for a ligand-dependent
intramolecular interaction of the AF-2 helix with the VDR LBD. These
data support the hypothesis that the ligand promotes the folding of the
AF-2 domain to create a transactivation surface for coactivator
interaction and subsequent transactivation.
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RESULTS
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Characterization of the C-Terminal Mutants of VDR
As illustrated in Fig. 1
, the
C-terminal region of VDR examined in this study contained heptad 9,
which is an important region of VDR for dimerization with RXR (31), and
the AF-2 motif, which is highly conserved throughout the nuclear
receptor superfamily (10, 12, 13). A series of point mutations were
introduced into this C-terminal region of VDR to test the effects of
the mutations on vitamin D-mediated transactivation, ligand binding,
RXR heterodimerization, and VDRE interaction. Transient transfection
assays in COS-7 cells demonstrated that wild type VDR showed a 60-fold
induction of reporter gene expression in the presence of
10-8 M 1,25-(OH)2D3,
whereas the C-terminal deletion mutants [VDR(1386) and
VDR(1403)], which lacked the conserved AF-2 motif, were
transcriptionally inactive (Fig. 2A
).
Introducing more subtle mutations into the AF-2 domain itself also
abolished VDR-mediated transactivation. The glutamic acid residue at
position 420 was changed to glutamine (E420Q) and the leucine residue
at position 417 was changed to serine (L417S), and both mutants lost
the ability to activate transcription. However, mutation of the
glutamic acid located downstream of the AF-2 core motif (E425Q) did not
significantly affect VDR-mediated transactivation compared with wild
type VDR. Similar results were obtained using another reporter
construct (VDRE4-TK-GH) and other cell lines (Hela cells,
ROS17/2.8 cells) (data not shown).

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Figure 1. Amino Acid Sequence of C-Terminal Region of VDR
Heptad 9 of the putative dimerization domain and the conserved AF-2
motif are indicated along with several of the VDR point mutations and
deletion mutations that were examined in this study. Also illustrated
in the box, is an amino acid sequence comparison of the
AF-2 region of several nuclear receptors.
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Figure 2. The Abolishment of VDR-Mediated Transcription by
Introducing Mutations into the Conserved AF-2 Motif of VDR
A, The effect of AF-2 motif mutations in a vitamin D-responsive
transient gene expression system. COS-7 cells were transfected with 2
µg of the VDRE4-TATA-GH reporter gene construct together
with 100 ng of the wild type or mutant VDR expression plasmids. Sixteen
hours after addition of the calcium phosphate-DNA precipitate, the
cells were washed, media were replaced, and the cells were treated with
ethanol vehicle or with 10-8 M
1,25-(OH)2D3. GH secreted into the media was
quantitated 24 h following ligand addition using a RIA kit. The
results represent the mean ± SD of triplicate
determinations. B, High concentrations of
1,25-(OH)2D3 do not rescue transactivation of
the L417S and E420Q mutant VDRs. COS-7 cells were transfected as
described in panel A. The cells were treated with ethanol vehicle or
with 10-10, 10-8, or 10-6
M 1,25-(OH)2D3 for 24 h. GH
secretion was quantitated as described in panel A.
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The loss in transcriptional activation was not due to altered hormone
binding since all of the point mutants examined in this study had
ligand binding affinities that were comparable to wild type VDR (Table 1
). In contrast, the VDR(1386)mutant displayed no
detectable ligand binding, and the apparent dissociation constant
(Kd) for the VDR(1403) mutant was approximately 10-fold
higher than wild type VDR. However, as demonstrated by Whitfield
et al. (32), it is possible that subtle changes in ligand
binding may not be apparent in this in vitro binding assay.
Therefore, we examined whether high concentrations of ligand could
rescue the impairment in transactivation that was observed in the L417S
and E420Q mutants. While wild type VDR was maximally activated at
10-10 M 1,25-(OH)2D3,
the L417S and the E420Q VDR mutants were completely inactive even when
cells were treated with 10,000-fold higher ligand concentrations
(10-6 M) (Fig. 2B
). Thus, the absence of
transactivation in the L417S and E420Q mutants was not due to modest
alterations in ligand binding in vivo or in
vitro.
We also examined the interaction of these VDR mutants with two factors
that are important in VDR-mediated transcription, namely RXR and TFIIB.
Disruption of heptad 9 and elimination of the AF-2 motif in the
VDR(93386) mutant resulted in loss of VDR interaction with both RXR
and TFIIB. However, removing only the AF-2 motif in the VDR(93403)
construct allowed substantial interaction of VDR with RXR and with
TFIIB. Importantly, mutants within the AF-2 domain that are
transcriptionally silent (L417S and E420Q) still show considerable
interaction with RXR and TFIIB, indicating that the loss of activity is
not due to the inability of VDR to interact with these two important
factors (Fig. 3
).

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Figure 3. Interaction of Wild Type and Various Mutant VDRs
with RXR or TFIIB in Two-Hybrid System
Yeast expressing AS1-VDR and pGAD.GH-RXR or pGAD.GH-TFIIB yeast
expression vector were grown for 16 h at 30 C in the absence or
presence of 10-8 M
1,25-(OH)2D3. The interactions were analyzed in
a ß-galactosidase assay. Results are presented as the mean ±
SD of triplicate independent cultures.
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To determine whether these mutations altered the DNA binding properties
of the VDR-RXR complex, electrophoretic mobility shift assays were
performed using overexpressed pSG5-VDR proteins in COS-7 cells and a
[32P]-labeled VDRE. Mutations in the heptad 9 region of
VDR were severely compromised in VDRE interactions in this assay.
Removal of a portion of heptad 9 [pSG5-VDR(1386)] eliminated
VDR-VDRE interaction, and point mutations of heptad 9 (K386Q, R391Q)
resulted in much weaker interactions with the VDRE (Fig. 4A
). Based on previously published data
(31) and on our two-hybrid results (Fig. 3
), this is likely due to
disrupting the ability of VDR to heterodimerize with RXR and subsequent
weaker binding of the heterodimer to the VDRE. Importantly, the L417S
and E420Q mutants retained the ability to heterodimerize with RXR and
bind to the VDRE (lane 6 and 7) compared with wild type VDR (lane 1).
Western analysis showed that all VDR proteins were expressed to similar
levels in each of these extracts (Fig. 4B
).

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Figure 4. Gel Mobility Shift Assay of Wild Type and Mutant
VDRs
A, Cellular extracts of COS-7 cells transfected with wild type VDR or
mutant VDR expression vectors were incubated with a
32P-labeled VDRE, electrophoresed on a 4% nondenaturing
polyacrylamide gel, and autoradiographed as described in
Materials and Methods. B, These same extracts were
analyzed by a Western immunoblot using the 9A7 antibody raised against
VDR to determine whether the mutants were expressed in a similar
fashion.
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Autonomous Transactivation Function by the VDR AF-2 Motif
To test whether the transactivation domain encompassing the
conserved motif could function independently of the ligand
binding/dimerization domain, various deletions and point mutants of the
VDR LBD were fused to the gal4 DBD [gal4(1147)]. Transient reporter
gene expression assays were performed in COS-7 cells using
Gal45-TATA-GH. Expressing the full-length LBD of the VDR
fused to the gal4 DBD [gal4-VDR(93427)] demonstrated no
transcriptional activation in the absence of hormone. However, treating
these cells with 10-8 M
1,25-(OH)2D3 activated reporter gene expression
by 15-fold (Fig. 5A
). The
ligand-dependent transactivation by this heterologous construct was
mediated through the AF-2 domain since the AF-2 deletion mutant
[gal4-VDR(93403)] and the AF-2 point mutants [gal4-VDR(L417S and
E420Q)] were inactive in the presence or absence of
1,25-(OH)2D3. N-Terminal deletions
[gal4-VDR(281427, 374427, and 387427)] that eliminated ligand
binding were transcriptionally inactive in both the presence and
absence of 1,25-(OH)2D3 ligand. However, a
fusion protein consisting of the last 20 amino acids of VDR linked to
the gal4 DBD [gal4-VDR(408427)] showed autonomous transactivation
activity that was unaffected by ligand. The level of reporter gene
expression with this minimal AF-2 domain fusion was approximately 40%
of the activity of the full-length LBD. To examine whether the
ligand-independent transactivation by this minimal AF-2 domain has an
amino acid requirement similar to that of ligand-dependent
transactivation, three-point mutations were introduced into the
gal4-VDR(408427) construct (Fig. 5B
). The L417S and the E420Q mutants
abolished the autonomous transactivation by the minimal AF-2 domain,
and E425Q mutation expressed an activity that was comparable to the
wild type AF-2 motif (Fig. 5B
).

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Figure 5. Autonomous Transactivation Function of the Minimal
AF-2 Domain of VDR
A, The minimal AF-2 domain mediates autonomous, ligand-dependent
transactivation. COS-7 cells were transfected with 2 µg of a reporter
construct (Gal45-TATA-GH) and 0.5 µg of various deletion
mutants or point mutants of pSG5-gal4-VDR. The cells were treated in
the absence or presence of 10-8 M
1,25-(OH)2D3 for 24 h and GH secretion was
determined. Values obtained with gal4(1147) in the absence of
1,25-(OH)2D3 are set arbitrarily as 1.00. B,
The L417S and E420Q mutations ablate autonomous transactivation by the
minimal AF-2 domain. COS-7 cells were transfected with 2 µg of a
reporter construct (Gal45-TATA-GH) and 0.5 µg of
pSG5-gal4, pSG5-gal4-VDR(93427), or wild type or point mutants of
pSG5-gal4-VDR(408427). The cells were treated and GH secretion was
determined.
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Interactions between the AF-2 Domain of VDR and mSUG1, SRC-1, and
RIP140 in the Yeast Two-Hybrid System
Our laboratory has been using two-hybrid strategies to identify
proteins that interact with the LBD of VDR in a ligand-dependent manner
(21, 33). The bait construct used in this screen was AS1-VDR(93427),
which contained the gal4 DBD fused to the LBD of VDR (amino acids
93427) (21). A mouse osteoblastic MC3T3 cell cDNA library was
constructed in the yeast multicopy expression vector pAD-Gal4.
Interaction between AS1-VDR and fusion proteins of pAD-GAL4 expressed
in the library were characterized by monitoring ß-galactosidase
activity and growth on selective media lacking histidine in the
presence of 1,25-(OH)2D3. Several cDNA clones
that interact with VDR in the presence of
1,25-(OH)2D3 were obtained. DNA sequence
analysis identified three clones as full-length mSUG1 (34), the
C-terminal region of mSRC-1 (amino acids 12581465) (35), and mRIP140,
which was 80% identical to human RIP140 (amino acids 867-1158) (28).
As demonstrated previously for the interaction of VDR with SUG1 (34) or
with another putative coactivator, glucocorticoid receptor interacting
protein 1 (36), the interaction between VDR and these three clones in
the two-hybrid system was dependent on the
1,25-(OH)2D3 ligand (data not shown).
To test whether the conserved AF-2 motif mediated the ligand-dependent
interaction between VDR and these putative coactivators, the VDR AF-2
mutants were examined in the two-hybrid interaction assay. Deletion of
the AF-2 motif [VDR(93403)] resulted in no or weak interaction of
this mutant with the cofactors tested here (Fig. 6
). Point mutations within the AF-2 core
(L417S and E420Q) also disrupted the interaction of VDR with mSUG1,
mSRC-1, and mRIP140 whereas mutations flanking the core had little
effect (K386Q, R391Q, and E425Q). It is important to note that most of
the AF-2 mutants still retained strong interactions with both RXR
and TFIIB (Fig. 3
). Interestingly, the minimal AF-2 domain (residues
408427), which demonstrated ligand-independent, autonomous
transactivation (Fig. 5A
), also showed significant interactions with
all of the cofactors tested here. As illustrated in Fig. 7
, AS1-VDR(408427) demonstrated
significant interactions with mSRC-1 and mRIP140 while
pAS1-VDR(281427), which had no autonomous transactivation activity
(Fig. 5
), also did not interact with these putative coactivators in
this system. The introduction of two AF-2 point mutations into pAS1-VDR
(408427) abolished the interaction with all cofactors, while the
mutation located downstream of the core AF-2 motif (E425Q) retained the
interactions. Interestingly, none of these minimal AF-2 fusions
interacted with RXR
(Fig. 7
) or with the pAD-Gal4 parent expression
vector (data not shown). Thus, ß-galactosidase activity determined in
these studies is a reflection of protein-protein interaction and is not
due to reporter gene expression driven by autonomous activation by the
AS-1-VDR(408427) construct alone. In fact, these data demonstrate
that while the gal4-VDR(408427) is an autonomous activator in
mammalian cells, it does not express detectable constitutive
transactivation in this yeast system.

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Figure 6. The Effect of Deletion or Point Mutations in the
AF-2 Activation Domain on the Interaction between VDR and Coactivators
Yeast expressing the wild type, deletion, or point mutations of AF-2
activation domain of AS1-VDR or pAS1 yeast expression vector alone and
pAD-SUG1, pAD-SRC-1, or pAD-RIP140 were grown for 16 h at 30 C in
the presence of 10-8 M
1,25-(OH)2D3. The interactions were assessed in
a ß-galactosidase assay. Results are presented as the mean ±
SD of triplicate independent cultures.
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Figure 7. The AF-2 Domain Is Sufficient to Mediate
Interaction with Putative Coactivator Proteins
Yeast expressing the AS1-VDR(93427, 281427, 408427), mutations of
AS1-VDR(408427) or pAS1 yeast expression vector alone, and pAD-SUG1,
pAD-SRC-1, pAD-RIP140, or pAD-RXR were grown for 16 h at 30 C
in the absence or presence of 10-8 M
1,25-(OH)2D3. The interactions were assessed in
a ß-galactosidase assay. Results are presented as the mean ±
SD of triplicate independent cultures.
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Intramolecular Interaction between the Minimal AF-2 Domain and an
AF-2 Deletion Mutant of VDR
These data suggest that the AF-2 domain functions in
transactivation perhaps by contacting coactivator proteins and that
ligand promotes these interactions by altering the conformation of the
AF-2 region. A comparison of the crystal structures of unliganded RXR
and liganded RAR indicates that, upon binding of ligand, the AF-2
domain folds down on the receptor to close off the ligand-binding
pocket (11, 37). This suggests that the AF-2 domain forms
ligand-dependent intramolecular contacts with residues in the VDR LBD.
To test this hypothesis, we examined the interaction of minimal AF-2
domain [VDR(408427)] with the AF-2 deletion mutant of VDR [VDR
(93403)]. Since VDR(93403) retains ligand binding, albeit with
somewhat reduced affinity, the effect of ligand on the interaction of
AF-2 domain with VDR(93403) was also examined. As illustrated in Fig. 8A
, the minimal AF-2 domain showed weak,
but significant, interactions with VDR(93403) in the presence of
1,25-(OH)2D3. This interaction was markedly
reduced in the absence of ligand and was also not apparent using the
AS1-VDR(93386) bait or the pAS1 parent expression vector.
Interestingly, introducing the E420Q or E425Q mutations into the
minimal AF-2 domain did not affect its interaction with VDR(93403),
while the L417S mutant disrupted this interaction (Fig. 8B
).

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Figure 8. Intramolecular Interaction between Minimal AF-2
Domain and AF-2 Deletion Mutant of VDR
A, Interaction between the minimal AF-2 domain and the AF-2 deletion
mutant of VDR. Yeast expressing the AS1-VDR(93386, 93403) or pAS1
yeast expression vector alone and pGAD.GH-VDR(408427) or pGAD.GH
expression vector alone were grown for 16 h at 30 C in the absence
or presence of 10-8 M
1,25-(OH)2D3. The interactions were assessed in
a ß-galactosidase assay. Results are presented as the mean ±
SD of triplicate independent cultures. B, Interaction
between point mutants of minimal AF-2 domain and AF-2 deletion mutant
of VDR. Yeast expressing the AS1-VDR(93403), and wild type and point
mutations of pGAD.GH-VDR(408427) were grown for 16 h at 30 C in
the absence or presence of 10-8 M
1,25-(OH)2D3. The interactions were assessed in
a ß-galactosidase assay. Results are presented as the mean ±
SD of triplicate independent cultures.
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DISCUSSION
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The C-terminal, 50-amino acid residues of the VDR encompass two
key structural motifs that play a central role in
1,25-(OH)2D3-mediated transcription. One region
corresponds to heptad 9 (residues 383390), which apparently functions
as a protein-protein interaction interface that is essential for VDR
heterodimerization with RXR. This is based on studies of VDR (31) and
related receptors (38, 39), which demonstrate that mutations in this
region disrupt the ability of these receptors to heterodimerize with
RXR and to bind their cognate response elements. Crystallography of the
RXR homodimer clearly established the role of an
-helical region
encompassing heptad 9 (helix 11) as the central dimerization interface
in RXR and strongly implicated similar roles in structurally related
receptors (11). A second important domain in the C terminus of VDR is
the AF-2 motif, which functions as a ligand-dependent transactivation
domain. Mutation of conserved residues in RARs and TRs ablate
hormone-induced transactivation, implicating a predominant role of the
AF-2 domain in nuclear receptor-signaling pathways (10, 13). Although
the precise mechanisms involved in the AF-2 transactivation function
are not well understood, recent data indicate that this
-helical
domain functions as an interaction surface for transcriptional
coactivator proteins that are putatively involved in nuclear
receptor-mediated transcription (6, 25). A detailed functional analysis
of the VDR AF-2 domain and the role of the
1,25-(OH)2D3 ligand in the activity of this
domain have been lacking. In this paper, we demonstrate the requirement
of an intact AF-2 core domain for
1,25-(OH)2D3-activated transcription.
Furthermore, we show that the AF-2 domain alone is sufficient to
mediate transactivation in a heterologous system and that it is also
sufficient to mediate interaction with several putative transcriptional
coactivator or adapter proteins.
Nakajima et al. (31) demonstrated that deletion of the VDR
AF-2 domain abolished VDR-mediated transactivation without compromising
heterodimerization of VDR with RXR and subsequent binding of
the VDR-RXR heterodimer to the VDRE. These data indicated a
selective role of the VDR AF-2 domain in
1,25-(OH)2D3-activated transcription. However,
1,25-(OH)2D3 binding to the VDR(1403) mutant
was reduced by an order of magnitude compared with wild type VDR,
suggesting an additional role for the AF-2 domain in high- affinity
ligand binding (31). Similar results were obtained with VDR(1403) in
the transactivation, two-hybrid interaction, and ligand-binding assays
in the present study (
Figs. 24

and Table 1
). Indeed, the crystal
structures of liganded RAR and TRs show that the AF-2 motif is packed
onto the body of the receptor with a portion of it forming part of the
ligand-binding pocket (37, 40). This dual role of the AF-2 domain in
both transactivation and in high-affinity ligand binding complicates
the functional analysis of this domain in VDR-mediated transactivation.
However, in the present study, we defined point mutations within the
VDR AF-2 core motif (Fig. 1
) that effectively discriminated between
transactivation and ligand-binding effects. While the VDR(E420Q) and
the VDR(L417S) mutants bound 1,25-(OH)2D3 with
equilibrium binding constants similar to wild type VDR (Table 1
), both
point mutants were transcriptionally silent. Furthermore,
transactivation was not evident when the mutant receptors were
incubated with ligand concentrations that were 10,000-fold greater that
the apparent Kds (Fig. 2B
). Altering a glutamic acid
residue that is adjacent to the conserved region (E425) did not
significantly affect transactivation. Consequently, these data
highlight the central importance of the charged and hydrophobic
residues within the AF-2 core motif in VDR-activated transcription.
Fusing the minimal AF-2 domain of VDR (residues 408427) to a
heterologous DBD [Gal4(1147)] produced a hybrid protein that
expressed significant transactivation potential in mammalian cells
(Fig. 5
). These data are consistent with previous findings that similar
regions in TR, RAR, and RXR also function as autonomous transactivation
domains (10, 12, 13). Introducing the E420Q and L417S mutations into
this hybrid construct abolished transactivation by the minimal VDR AF-2
motif. Thus, E420 and L417 are essential for ligand-activated AF-2
activity in the intact receptor and for autonomous transactivation
mediated by the minimal AF-2 domain. Interestingly, Gal4-VDR hybrids
that expressed additional N-terminal sequences along with the minimal
AF-2 domain [i.e. VDR(383427), VDR(373427), and
VDR(281427)] were transcriptionally silent in this assay. Perhaps
additional N-terminal sequence adjacent to the AF-2 core motif may mask
the AF-2 domain and inhibit its transactivation potential.
Alternatively, additional factors such as corepressor proteins may
interact with the VDR constructs containing this additional N-terminal
sequence that may inhibit or suppress AF-2 activity (29, 30).
Regardless, the chimera containing the full-length, intact LBD
[VDR(93427)] was inactive in the absence of ligand, but it
expressed substantial transactivating activity in the presence of
10-8 M 1,25-(OH)2D3.
These data indicate that the AF-2 domain, while active on its own, is
not in the appropriate orientation or conformation for efficient
participation in the transactivation process in the intact receptor in
the absence of ligand. However, in the context of the intact receptor,
binding of ligand may alter the conformation of the AF-2 to promote
transactivation.
Importantly, we demonstrate that the minimal AF-2 domain of VDR
interacts with an AF-2 deletion mutant of VDR [VDR(93403)] in a
ligand-dependent manner. We interpret this trans interaction
between the isolated AF-2 domain and the remainder of the VDR LBD as a
ligand-induced intramolecular folding of the AF-2 domain. Whereas the
AF-2 mutation (E420Q) did not affect this ligand-dependent
intramolecular contact, the L417S mutation severely impaired AF-2
interaction with the VDR LBD. These data imply that these two residues
may, in fact, play distinct roles in the transactivation process. The
hydrophobic residues (e.g. L417) may be involved in forming
the hydrophobic core of the ligand-binding pocket and may be required
for 1,25-(OH)2D3-dependent intramolecular
folding of the AF-2 domain. In contrast, the charged residues
(e.g. E420) may not be involved in intramolecular folding of
the AF-2 domain, but instead this may be surface exposed and required
for coactivator interaction. These observations are consistent with the
putative model of ligand-induced conformational changes in the AF-2
domain based on the crystal structures of liganded RAR compared with
unliganded RXR (11, 37). Interestingly, the minimal AF-2 domain
[gal4-VDR(408427)] expresses only 40% of the transcriptional
activity and coactivator interaction observed with the full-length LBD
in the presence of 1,25-(OH)2D3. This
observation suggests that additional residues outside the AF-2 domain
contribute to the transactivation surface. Additional studies are
required to refine various aspects of this model and identify other
residues that comprise the transactivation surface. Strong candidates
may reside within the two activation domains identified in the VDR LBD
in a yeast-based system (41).
Our ongoing studies to identify putative transcriptional adaptor
proteins that interact with the VDR led to the isolation of several
VDR-interactive clones from a murine osteoblast cDNA library in the
yeast two-hybrid system. Sequence analysis revealed that three of the
cDNA clones encoded putative coactivators implicated in nuclear
receptor-mediated transcription including SRC-1 (26), RIP140 (28), and
SUG1 (34). Importantly, these putative coactivators interacted with the
VDR in a 1,25-(OH)2D3-dependent manner with the
AF-2 domain of the VDR playing an essential role in this interaction.
This is best exemplified in the observation that mutations in the AF-2
core motif disrupt interactions between VDR and SRC-1 or RIP140. These
same mutations that abolish interaction with the coactivators also
abolish transactivation. Moreover, the VDR AF-2 domain alone was
sufficient to mediate interaction with these coactivators and to
mediate autonomous transactivation. Although a direct examination of
the effects of these coactivators on VDR-mediated transcription needs
to be addressed, the strong correlation between coactivator interaction
and transactivation observed in these studies supports a role for these
putative coactivators in the mechanism of
1,25-(OH)2D3-activated transcription mediated
through the VDR AF-2 domain.
In summary, we have analyzed the functional role of the extreme
C-terminal region of VDR. Our data illustrate the distinct roles of
heptad 9 (helix 11) in VDR dimerization with RXR as well as the central
role that the conserved AF-2 motif plays in ligand-dependent
coactivator interaction and transactivation. Importantly, these data
support structural studies in which ligand binding induces a dramatic
conformational change in the AF-2 domain mediated through an
intramolecular folding of the AF-2 domain that creates a
transcriptional surface for coactivator binding.
 |
MATERIALS AND METHODS
|
---|
Transient Transfection Studies
The VDRE4-TATA-GH plasmid contained four copies of
the rat osteocalcin VDRE adjacent to the rat osteocalcin promoter (-40
to +32) driving to a human GH reporter sequence (33). The
Gal45-TATA-GH plasmid contained five copies of the
gal4-responsive element, an Elb promoter fragment, and the human GH
reporter sequence. The pSG5-VDR expression plasmid was described
previously (42, 43). The pSG5-gal4 was constructed by inserting the
gal4 cDNA encoding amino acids 1147 into the pSG5 expression plasmid
(Stratagene, La Jolla, CA). Point mutations were introduced into the
human VDR cDNA using oligonucleotide-directed mutagenesis (44, 45), and
C-terminal deletions were generated by introducing stop codons at the
indicated positions. All of the mutants were confirmed by DNA
sequencing. VDR mutants were subcloned into pSG5 or pSG5-gal4. To
generate pSG5-gal4-VDR(408427), oligonucleotides corresponding to the
last 20 amino acids of human VDR with EcoRI and
BamHI overhangs at the 5'- and the 3'-end, respectively,
were subcloned in frame into pSG5-gal4.
COS-7 cells were cotransfected with reporter gene constructs
(VDRE4-TATA-GH or Gal45-TATA-GH) and with
receptor expression vectors (pSG5-VDR or pSG5-gal4-VDR). In all
transfections, the amount of total DNA was kept constant at 10 µg by
adding pTZ18U (U.S. Biochemical, Cleveland, OH) as a carrier plasmid.
The cells were transfected by standard calcium phosphate
coprecipitation procedures as described previously (43). Transfected
cells were treated with the indicated concentrations of
1,25-(OH)2D3 or ethanol vehicle for 24 h,
and the amount of secreted GH was determined with a RIA kit (Nichols
Institute, San Juan Capistrano, CA).
Ligand-Binding Assay
COS-7 cell lysates expressing wild type or mutant human VDRs
were prepared as described previously (32). The lysates were incubated
with five 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. Bound and free
ligand were separated with dextran-coated charcoal and analyzed by
Scatchard plots to determine the dissociation constant.
Gel Mobility Shift Analysis
The VDRE oligomer corresponding to the rat osteocalcin VDRE was
described previously (46). The VDRE oligomer was labeled to high
specific activity by a fill-in reaction with Klenow fragment of DNA
polymerase I and [
-32P]dCTP (3000 Ci/nmol). Ten
micrograms of the lysates containing wild type or mutant human VDRs
were incubated with 32P-labeled VDRE probe for 30 min at 22
C in 10 mM Tris-HCl, pH 7.6, 100 mM KCl, 1.0
mM dithiothreitol, 15% glycerol, 0.1 µg/ml BSA, and 50
µg/ml poly(deoxyinosinic-deoxycytidylic)acid. Unbound probe and
protein-DNA complexes were separated by nondenaturing electrophoresis
on a 4% polyacrylamide gel in 0.25xTris-borate-EDTA. Gels were dried
and exposed for autoradiography.
Preparation of Two-Hybrid Expression Vectors and cDNA Library
Screening
All two-hybrid plasmids constructs used the pAS1 (47) and the
pGAD.GH (48) or pAD-GAL4 (Stratagene, La Jolla, CA) yeast expression
vectors. AS1-VDR constructs, containing the full-length and deletion
mutants of VDR [(93427), (93386), (281427)], were described
previously (21). Other deletions [(93403), (387427), (408427)]
and point mutations (K386Q, R391Q, L417S, E420Q, E425Q) of VDR were
also subcloned into the pAS1 or the pGAD.GH vector for examination in
the two-hybrid assay. The MC3T3-E1 cell cDNA library was prepared in
the pAD-GAL4 vector. For cDNA library screening, the library was
cotransformed with pAS1-VDR(93427) into the yeast strain Hf7c, which
was made competent with lithium acetate (49). Transformants were plated
on media lacking leucine, tryptophan, and histidine (SC-leu-trp-his)
and containing 10-8 M
1,25-(OH)2D3 and 10 mM
3-amino-1,2,4-triazole. Histidine-positive colonies were assayed for
ß-galactosidase expression using a colony lift filter assay (47).
ß-Galactosidase Assays
The pAD-GAL4-mSUG1, -SRC-1, -RIP140, -RXR
, and the
pGAD.GH-TFIIB, -RXR
, -VDR were cotransformed with wild type and
mutant pAS1-VDR into the yeast strain Hf7c as described above.
Transformants were plated on media lacking leucine and tryptophan
(SC-leu-trp) and were grown for 4 days at 30 C to select for yeast that
had acquired both plasmids. Triplicate independent colonies from each
plate were grown overnight in 2 ml of SC-leu-trp with or without
10-8 M of
1,25-(OH)2D3. Cells were harvested and assayed
for ß-galactosidase activity as described (50).
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank The Endocrine Society and its Student
Affairs Committee for the generous support of C. M. B.
 |
FOOTNOTES
|
---|
This work was supported in part by NIH Grants R29DK-47293 and
R01DK-50348 (to P.N.M.) and an Endocrine Society Student Research
Fellowship (to C.M.B.). R. St-A. is a chercheur-boursier from the Fonds
de la Recherche en Santé du Québec.
Received for publication April 21, 1997.
Revision received June 5, 1997.
Accepted for publication June 9, 1997.
 |
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