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INTRODUCTION |
The vitamin D receptor
(VDR)1 is a member of the
superfamily of nuclear receptors that act as ligand-inducible
transcription factors (1, 2). VDR binds, as a heterodimer with the
retinoid X receptor (RXR), to hormone response elements (VDREs) in
target genes (3-7). Transcriptional actions of nuclear receptors can result not only from direct modulation of target gene expression but
also from cross-talk with other nuclear receptors and signal transduction pathways (e.g. AP-1 activity) (8).
Nuclear receptors display a modular structure with an N-terminal region
A/B, followed by region C (the DNA-binding domain), a hinge region D,
and the C-terminal E/F region containing the ligand-binding domain
(LBD) and the dimerization domain (1, 2). An autonomous
ligand-dependent transcriptional activation function (AF-2)
is located in the LBD (9). The core AF-2 domain has been characterized
in the C terminus of different nuclear receptors (10-15) and consists
of a well conserved amphipathic
-helix motif. The crystal structure
of five different nuclear receptor LBDs has been solved (16-20).
Although some variability exists, the ligand-binding domains contain a
similar helical fold with twelve
-helices (numbered helix 1 to helix
12) (21). The most striking difference between unoccupied (apo) and
ligand-bound (holo-) receptors is the position of the C-terminal helix
12, which contains the core AF-2 domain. This helix projects away from
the LBD in the apo-receptors but is tightly packed against helix 3 of
the LBD upon ligand binding. In addition, the LBDs of the
holo-receptors are more compact that those of the unliganded receptors,
indicating that the ligands may reconfigure other surface features of
the LBD. The conformational change in helix 12, together with other
changes that occur after ligand binding such as the bending of helix 3, are believed to create a surface that allows binding of coactivator
proteins required to transduce the signal to the basal transcriptional
apparatus (9). Three related family members, including SRC-1/NCoA1,
TIF-2/GRIP-1, and ACTR/pCIP, that interact with the nuclear receptors
in a ligand- and AF-2-dependent manner and act as as bona
fide coactivators have been identified (22-27). In addition,
ligand-dependent transcription by different nuclear
receptors appears to require CBP/p300, which also exerts a coactivator
role for other transcription factors and interacts directly with the
nuclear receptors and the coactivators (28-30). It has been proposed
that binding of coactivators such as SRC-1 or ACTR to the liganded
receptors recruits at least two other nuclear factors, CBP and p/CAF
creating a multisubunit activation complex (26, 27). Several of these
proteins have histone acetylase activity, which can disrupt nucleosomes
(26, 27, 31, 32). Therefore, the current model for transcriptional
regulation by nuclear receptors suggests that the AF-2 of nuclear
receptors serves to recruit a multicomponent coactivator complex that
can open up chromatin (33, 34).
By analyzing the transcriptional activity of mutant receptors, it has
been confirmed that residues located not only in helix 12 but also in
helix 3 are required for ligand-dependent transcriptional activity. In particular, mutation of a highly conserved lysine residue
at the C terminus of helix 3 strongly impairs transactivation by
estrogen receptor (35), thyroid hormone receptor (36-38), and VDR
(39). This mutation, which could disrupt interaction between helices
helix 12 and helix 3, has been very recently shown to reduce binding of
the coactivators SRC-1 and TIF-2 to estrogen receptor (35). In this
study we have analyzed the role of mutations in the conserved VDR
residues Leu417 and Glu420 (in helix 12) and
Lys246 (in helix 3) on transcriptional responses to vitamin
D. Our data confirm that these residues are required for
ligand-dependent activation and exert a dominant negative
effect on the response of a VDRE-containing promoter to the native
receptor. Furthermore, the native receptor is able to suppress retinoic
acid-dependent transactivation of the RAR
2 promoter in a
vitamin D-dependent manner, and mutation of these residues
also abolishes this inhibitory effect. In addition, the VDR mutants
increased the activity of the AP-1 containing collagenase promoter in
the presence or phorbol esters or c-Jun, whereas the wild type VDR did
not affect this response. Mutation of the Glu420,
Leu417, and Lys246 residues strongly reduced
the ligand-induced binding of the coactivators SRC-1, ACTR, and CBP.
These data indicate that both the residues in helix 12 and the
conserved lysine residue of the predicted helix 3 of VDR participate in
the recruitment of coactivators and, therefore, in AF-2 activity.
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EXPERIMENTAL PROCEDURES |
Plasmids--
In the plasmid Spp-1-tk-CAT, the
oligonucleotide 5'-AGCTTGACCAACAAGGTTCACGAGGTTCACGTCTCT-3' conforming
the VDRE of the osteopontin-1 (Spp-1) promoter was cloned into the
HindIII and XbaI sites of pBLCAT8+ in front of
the thymidine kinase promoter. The R-140 CAT construct containing the
fragment
124 to +14 of the human RAR
2 promoter was obtained from
the previously described R-140 Luc (40). The promoter fragment was
obtained by PCR with the oligonucleotides
5'-GGGAAGCTTGGATCCTGGGAGTTGGT-3' and 5'-GCTCTAGAG CTCACTTCCTACTAC-3'
and subcloned in HindIII and XbaI sites of pBLCAT8+ replacing the thymidine kinase promoter. The construct
73Col-Luc, which contains the collagenase promoter fused to
luciferase, has been described previously (41). Expression vectors for
human VDR, RXR
, RAR
, and SRC-1 cloned in pSG5 (35, 42, 43) and RSV-Jun (44) have been also described. The expression vectors for the
VDR point mutants K264A, L417S, and E420Q were obtained by PCR using
established protocols (45). In the first PCR reaction the sense
oligonucleotides 5'-CGCCCTCTGTGCTCGAA-3' (for L417S),
5'-CCCCTTGTGCTCCAAGTG-3' (for E420Q), and
5'-GCTTTGCTGCGATGATACC-3' (for K246A) containing the desired
nucleotide changes, as well as the antisense C-terminal oligonucleotide
5'-CGGGATCCTCAGGAGATCTCATTGCC-3' with a BamHI site were used
to generate mutated fragments. After an elongation phase, a second PCR
reaction was performed. In the case of L417S and E420Q the sense
oligonucleotide for this reaction was 5'-CCTTCACCATGGACGACAT-3'
containing the unique NcoI site of the VDR cDNA. For the
K246A mutant the sense oligonucleotide, containing a BstXI
site was 5' -GGAGCAGCAGCGCATCATT-3'. The resulting products were cloned
into the pSG5 expression vector, and the mutations were confirmed by
sequencing. pGST-VDR, which expresses a fusion protein between
glutathione S-transferase (GST) and VDR, was obtained by PCR
using the pSG5-VDR plasmid as a template and the oligonucleotides
5'-CGGGATCCATGGAGGCAATGGCGG-3' and 5'-GGAATTCTCAGGAGATCTCATTGC-3' to generate the complete VDR cDNA. This fragment was then
subcloned into BamHI/EcoRI sites of the pGEX-2T
plasmid. Similarly, pGST-VDR(K246A) was generated by subcloning a
BstXI-NcoI restriction fragment from VDR K246A
into the pGST-VDR previously digested with the same enzymes. The
plasmids GST-ACTR and GST-CBP, which express the cDNAs coding for
the amino acids 621-821 and 1-1099 of ACTR and CBP, respectively,
have been previously described (27, 31). These fragments contain the
nuclear receptor-interacting sequences of both proteins.
Cell Culture and Transfections--
HeLa and COS-7 cells were
cultured in Dulbecco's modified Eagle's medium containing 10% fetal
calf serum and were plated 24 h prior to transfection by calcium
phosphate with the reporter constructs. GH4C1 cells were cultured in
the same medium and were transfected by electroporation as described
previously (46). The cells from each electroporation were split into
different culture plates in Dulbecco's modified Eagle's medium
containing 10% AG1 × 8 resin-charcoal stripped newborn calf
serum. Treatments were administered in serum-free medium. In
cotransfection experiments the reporter plasmids were transfected with
receptor expression vectors or with a vector encoding SRC-1 as
indicated in the corresponding figures. In all cases the total amount
of DNA among different transfections was kept constant by the addition
of empty noncoding expression vectors. Each transfection also received
0.5 µg of a luciferase or a CAT vector as a control for transfection
efficiency. CAT activity was determined by incubation of the cell
extracts with [14C]chloramphenicol. The unreacted and
acetylated [14C]chloramphenicol were separated by thin
layer chromatography and quantified with an InstantImager. The data
are expressed as the percentages of acetylated forms after each
treatment. Each treatment with the ligands was performed at least in
duplicate cultures that normally exhibited less than 10% variation in
CAT activity, and the experiments were repeated at least three times. The results are expressed as the means ± S.D. of the CAT or
luciferase values obtained.
Protein Preparations--
VDR, VDR mutants, RXR, SRC-1, and
luciferase cloned in pSG5 were used for in vitro
transcription and translation following the manufacturer
recommendations of the TNT7 Quick coupled transcription/translation System (Promega). Reactions were performed in the presence of 40 µCi
of [35S]methionine (Amersham) (for the pull-down assays)
or with the same amount of unlabeled amino acid (for gel retardation
assays). 5 µl of the reaction product were resolved in 10% SDS-PAGE.
The gel was dried and autoradiographed overnight. The GST fusion
proteins VDR, VDR(K246A), ACTR, and CBP were expressed in the bacterial strain BL21 (DE3). They were grown at 37 °C in 2× YT (tryptone 16 g/liter, yeast extract, 5 g NaCl/liter, pH 7) until the absorbance reached 0.6. Then the induction was performed at 30 °C for 3 h with 0.4 mM
isopropyl-
-D-thiogalactopyranoside. GST and GST fusion proteins were expressed and purified by standard techniques following the recommendations of Pharmacia Biotech Inc. The expression of correctly sized proteins was monitored by SDS-PAGE.
Western Blot Analysis--
Extracts from cells transfected with
wild type or mutant VDRs or with the noncoding vector pSG5-0
containing equal amounts of proteins were run in 10% polyacrylamide
gels. The proteins were transferred to nitrocellulose membranes. The
membranes were blocked with a buffer containing 5% nonfat milk,
washed, and incubated with a 1:3000 dilution of a monoclonal antibody
(Chemicon, MAB1360) raised against the DNA-binding domain of VDR. The
proteins were visualized by chemiluminiscence using the ECL detection system.
Limited Proteolytic Digestion--
In vitro
translated 35S-labeled wild type or K246A VDR were
incubated in glass tubes with 100 nM vitamin D3 or ethanol
for 20 min at room temperature. Aliquots of receptors were then
incubated with increasing amounts of trypsin (between 0 and 50 µg/ml)
for 10 min. Proteolyis was stopped by adding SDS sample buffer and boiling for 10 min. The proteolytic fragments were separated by SDS-PAGE in a 12% polyacrylamide gel and identified by autoradiography.
Mobility Shift Assays--
Gel retardation assays were performed
with the in vitro translated receptors and the
oligonucleotide corresponding to the VDRE of the osteopontin-1 (Spp-1)
promoter: 5'-agctcAGGTCAAGGAGGTCAg-3'. For the binding reaction, the
proteins were incubated on ice for 15 min in a buffer (20 mM Tris-HCl, pH 7.5, 75 mM KCl, 1 mM dithiothreitol, 5 µg/ml bovine serum albumin, 13%
glycerol) containing 3 µg of poly(dI-dC) and then for 15-20 min at
room temperature with approximately 50,000 cpm of labeled
double-stranded oligonucleotide end-labeled with
[32P]dCTP, using Klenow fragment as kinase. DNA-protein
complexes were resolved on 6% polyacrylamide gels in 0.5 × TBE
buffer. The gels were then dried and autoradiographed at
70 °C.
Protein-Protein Interactions--
GST pull-down assays were
performed with 5 µl of the in vitro translated
L-[35S]methionine-labeled proteins. These
proteins were incubated with 1 µg of the GST fusion protein or with
the same amount of GST as a control and immobilized in
glutathione-Sepharose beads. The proteins were first incubated in the
presence of 100 nM vitamin D or ethanol for 20 min at room
temperature in glass tubes. The reaction with the beads was performed
for 1 h at 4 °C in a binding buffer containing 25 mM Hepes KOH, pH 7.9, 1% glycerol, 5 mM
MgCl2, 1 mM dithiothreitol, 0.05% Triton
X-100, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride. Free proteins were washed from the beads with a buffer containing increasing concentrations (50, 100, and 200 mM)
of KCl, and the bound proteins were analyzed by SDS-PAGE and autoradiography.
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RESULTS |
Transcriptional Activation by Mutant Vitamin D Receptors--
As
shown in Fig. 1, the amphipathic
-helix that contains the core AF-2 domain as well as sequences
contained in helix 3 and helix 4 are well conserved among different
nuclear receptors. A glutamic acid residue at position 420 in the
predicted helix 12 of VDR is also present in thyroid, steroid, and
retinoid receptors. We investigated the importance of the glutamic acid
residue in the transcriptional actions of VDR by replacing it with a
glutamine to generate E420Q. Additionally, the leucine residue 417 conserved in most receptors was changed by a serine (L417S). These
mutations in VDR have been previously shown to impair transactivation
and to be essential for interaction with coactivators (14). On the other hand, lysine residue 246 at the C terminus of the predicted helix
3 in VDR was mutated to an alanine to render K246A. This lysine residue
is also extremely well conserved in the different nuclear receptors and
also appears to play an important role in transactivation (35-39).

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Fig. 1.
Alignment of nuclear receptor sequences
corresponding to helices 3 and 12 in the ligand-binding domain of
nuclear receptors. The structure of VDR is schematically
presented, showing the location of the predicted helix 3 and helix 12 of the ligand-binding domain with black boxes. Also
illustrated is an amino acid sequence comparison of these regions among
several nuclear receptors. The lysine residue at the C terminus of
helix 3 is indicated by a shaded box on the left.
This lysine was mutated to an alanine to give VDR K246A. The central
conserved glutamic acid residue in helix 12 is also shown in a
shaded box on the right. The glutamic acid was
substituted by a glutamine in the E420Q VDR mutant. The leucine residue
at position 417 of VDR, which is also conserved in most nuclear
receptors, was mutated to a serine to generate VDR L417S.
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We compared K246A, L417S, and E420Q with the native VDR for the ability
to stimulate transcription from a CAT reporter gene containing the VDRE
of the osteopontin (Spp-1) gene fused to the thymidine kinase promoter
in transiently transfected COS-7 cells. These cells contain low levels
of endogenous VDR. Fig. 2A
shows that upon transfection with wild type VDR, treatment with vitamin D activated the Spp1-tk-CAT construct in COS-7 cells, whereas the helix
12 and helix 3 mutants were transcriptionally inactive. Fig.
2B shows that, as assessed by Western blot analysis, K246A, L417S, E420Q, and the wild type receptor were expressed in COS-7 cells
at similar levels. The DNA binding properties of the mutant receptors
were determined in gel retardation assays with the Spp-1 VDRE. As shown
in Fig. 2C, in the presence of the heterodimeric partner
RXR, the mutant VDRs bound DNA as strongly as the native receptor.

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Fig. 2.
A, transcriptional activation by VDR
mutants in COS-7 cells. The cells were transiently transfected with 5 µg of the reporter plasmid Spp1-tk-CAT (which contains the VDRE of
the osteopontin promoter) and 250 ng of a noncoding vector ( ), an
expression vector encoding wild type VDR (wt) or the same
amount of vectors for the VDR mutants indicated. The cells were treated
for 24 h in the presence (black bars) or absence
(shaded bars) of 100 nM vitamin D, and CAT
activity was determined. B, expression of VDR mutants in
COS-7 cells. Cell lysates from two independent cultures were used to
analyze the levels of expression of the transfected receptors by
Western blot with the monoclonal MAB1360 anti-VDR antibody.
C, the mutant receptors bind to the Spp-1 VDRE as
heterodimers with RXR. Gel retardation assays were performed using 1 µl of each in vitro translated VDR mutants indicated in
the presence (+) and absence ( ) of the same amount of in
vitro translated VDR. Translation efficiency was identical for the
different VDRs. Lane 1 shows the mobility of the unretarded
Spp-1 oligonucleotide, lane 2 shows that the unprogrammed
reticulocyte lysate did not cause retardation, and lane 3 shows that RXR by itself does not bind to the VDRE.
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The transcriptional activity of the helix 3 and helix 12 mutants was
also tested in HeLa cells in which vitamin D causes a 6-7-fold
activation of the Spp-1-tk-CAT construct. Whereas overexpression of
wild type VDR increased further the response of the reporter plasmid to
vitamin D, the mutant receptors had a dominant negative activity and
blocked the response to the vitamin mediated by endogenous VDR (data
not shown).
VDR and VDR K264A Present Identical Ligand-dependent
Protease Sensitivity--
Ligand-induced conformational changes within
the receptors appear to result in an increased resistance to limited
proteolytic digestion. To analyze whether the inability of the helix 3 mutant to activate transcription could result from an inadequate
conformation upon ligand binding, we used a trypsin digestion assay.
Fig. 3 shows that both VDR and the K246A
mutant are digested to small peptides at a trypsin concentration of 50 µg/ml within 10 min at room temperature. At lower trypsin
concentrations several proteolytic fragments with molecular masses
between 38 and 25 kDa were also observed. Incubation with vitamin D
strongly inhibited the proteolysis of these fragments. The size of the
main resistant fragment was found to be approximately 33 kDa. Trypsin
digestion of the helix 3 mutant in the presence of vitamin D generated
a proteolytic pattern indistinguishable from that obtained with the
native VDR.

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Fig. 3.
Ligand-dependent proteolytic
pattern of VDR and the K246A VDR mutant.
[35S]Methionine-labeled in vitro translated
wild type (wt) VDR (left panel) or the mutant
VDR(K246A) (right panel) were preincubated with vehicle
alone (lanes 1-6) or with 100 nM vitamin D
(lanes 7-12) for 20 min. 1-µl aliquots of trypsin were
added to 9-µl aliquots of receptors to give the indicated final
concentrations, and the mixtures were incubated for 10 min. Samples
were analyzed by SDS-PAGE and autoradiography. The sizes of the
molecular mass markers in kilodaltons (kDa) are indicated at the
right. The arrows at the left show the
size of the undigested VDRs (white arrow) and the size of
the resistant protein fragments obtained after incubation with vitamin
D (black arrows). The main resistant fragment had a
molecular mass of approximately 33 kDa in both VDRs.
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The Core AF-2 Domain as Well as Lys-246 Are Required for
Transrepression of the RAR
2 Promoter--
We have observed that
incubation with vitamin D reduces retinoic acid-mediated
transactivation of the RAR
2 promoter in pituitary GH4C1 cells
(47). VDR interferes with the activation
of this promoter, which contains a strong retinoic acid response
element (40). We then examined whether mutations in helix 3 and helix 12 could also affect transrepression mediated by VDR. Fig.
4 compares the influence of VDR and the
mutants K246A, L417S, and E420Q on the response of the RAR
2 promoter
in GH4C1 cells (panel A) and in HeLa cells (panel
B). In GH4C1 cells incubation with 1 µM retinoic acid increased by about 7-fold the activity of the RAR
2 promoter, and this response was reduced to approximately 5-fold in cells incubated in the presence of 1 nM vitamin D. Transfection
with native VDR enhanced this inhibitory response, whereas the helix 12 and helix 3 mutants were unable to elicit a further reduction in the
response to retinoic acid. Similar results were observed in HeLa cells.
In this cell type vitamin D repressed the retinoic acid response in
cells transfected with VDR, and the different mutants were again
inactive.

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Fig. 4.
The helix 3 and helix 12 VDR mutants do not
repress the response of the RAR 2 promoter to
retinoic acid. The effect of vitamin D (vit.D) on the
response of the construct R-140 CAT that contains the RAR 2 promoter
was analyzed in pituitary GH4C1 cells (panel A) and in HeLa
cells (panel B). GH4C1 cells were transfected by
electroporation with 25 µg of R-140 CAT and a noncoding vector, an
expression vector for wild type VDR (wt), or vectors
expressing the VDR mutants L417S, E420Q, and K246A. HeLa cells were
transfected by calcium phosphate with 5 µg of R-140 CAT and 2.5 µg
of expression vectors for the wild type or mutants VDRs and also
received 0.5 µg of a RAR expression vector. CAT activity was
determined after 24 h of incubation with 1 µM
retinoic acid and/or 1 nM vitamin D in GH4C1 cells and 100 nM vitamin D in HeLa cells as indicated.
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The AF-2 Mutants, but Not Wild Type VDR, Affect Collagenase
Promoter
Activity--
12-O-Tetradecanoyl-phorbol-13-acetate (TPA)
leads to the induction of genes through activation of the AP-1 complex,
and different nuclear receptors have been shown to functionally
interact with TPA-inducible gene expression (8). The collagenase
promoter has been widely used as a model for the study of
AP-1-dependent gene activation. In Fig.
5A we examined the effect of
wild type VDR and the mutants K246A, L417S, and E420Q on the activity
of the collagenase promoter in HeLa cells. Activation by TPA was not
modified by the different VDRs in the absence of ligand. In the
presence of vitamin D the collagenase response to TPA was minimally
increased in cells transfected with the wild type receptor. However,
both the helix 12 mutants and the helix 3 mutant significantly enhanced
the response to TPA. The data shown in Fig.
6B confirm that these mutants
are able to mediate a ligand-dependent increase in
AP-1-dependent collagenase activation. In this case, the
effect of the receptors was assessed in cells transfected with
c-jun, a component of the AP-1 complex. Whereas vitamin D
did not activate the collagenase promoter in HeLa cells expressing the
native VDR, the K246A, L417S, and E420Q mutants mediated a significant
activation.

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Fig. 5.
Influence of mutant VDRs on collagenase
promoter activity. A, HeLa cells were transfected with 5 µg of the reporter plasmid 73Col-Luc. In this construct the
collagenase promoter that contains an AP-1 site is fused to the
luciferase gene. The reporter plasmid was cotransfected with 2.5 µg
of an empty expression vector ( ), an expression vector for wild type
(wt) VDR, or expression vectors for the VDR mutants L417S,
E420Q, and K246A. CAT activity was determined in cells incubated in the
presence or absence of vitamin D (vit.D) for 24 h
and/or with 100 nM TPA during the last 6 h. The data
are expressed as the percentages of the response obtained in the cells
treated with 100 nM TPA in each case. B, the
cells were transfected with 73Col-Luc and the VDRs indicated in the
presence of 1 µg of an expression vector for c-jun. CAT
activity was determined in untreated cells and in cells treated with
100 nM vitamin D for 24 h. CAT activity is expressed
as a percentage of the values obtained in the corresponding untreated
cells.
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Fig. 6.
Mutation of lysine 246 disrupts the vitamin
D-dependent interaction between VDR and coactivators.
A, GST alone and the GST fusion proteins wild type VDR
(wt) and VDR(K246A) were immobilized in
glutathione-Sepharose beads. In vitro translated
35S-labeled SRC-1, RXR, and Luciferase were incubated with
the beads in the absence ( ) or presence (+) of 100 nM
vitamin D (Vit.D). B, in vitro
translated 35S-labeled VDR wild type (wt),
VDR(L417S), VDR(E420Q), VDR(K246A), and luciferase were incubated with
immobilized GST or the fusion proteins GST-ACTR or GST-CBP in the
presence and absence of vitamin D. In both panels the bound proteins
were analyzed by SDS-PAGE and visualized by autoradiography. The
first lane in each case shows a 20% of the input of the
corresponding labeled protein.
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Lys-246 Is Required for Vitamin D-dependent Binding of
Coactivators--
Transcriptional stimulation by nuclear receptors
correlates with their ability to interact with coactivator proteins in
a ligand-dependent manner. Therefore, we tested the effect
of K246A mutation on in vitro interaction between VDR and
SRC-1 in GST pull-down assays. As shown in Fig. 6A in the
presence of vitamin D a significant portion of the input of
35S-labeled SRC-1 was specifically retained by wild type
GST-VDR fusion protein immobilized in glutathione-agarose beads,
whereas no significant binding was observed either in the absence of
vitamin D or by GST alone. The helix 3 mutation dramatically reduced
binding to the coactivator, and the 35S-labeled SRC-1 was
not retained by GST-VDR(K246A) in the presence of vitamin D. That this
reduction is specific for the coactivator is shown by the finding that
association with 35S-labeled RXR was unaffected by mutation
K246A. In addition, no interaction of the receptors with
35S-labeled luciferase used as a negative control was detected.
The ability of wild type and mutant K246A, L417S, and E420Q receptors
to interact with the coactivators ACTR and CBP was also analyzed. In
these assays the coactivators fused to GST, and in vitro
translated 35S-labeled receptors were used. As illustrated
in Fig. 6B, wild type VDR was significantly retained by
GST-ACTR in the presence of vitamin D, whereas mutation K246A abolished
this association. In addition, the helix 12 mutants were unable to
interact with GST-ACTR in a vitamin D-dependent manner.
Similar results were obtained with a fragment of GST-CBP (amino acids
1-1099) that contains the receptor interaction domain. In this case,
whereas 35S-labeled luciferase was not retained by the CBP
fragment, some interaction with the different VDR mutants in the
absence of vitamin D was observed. However, incubation with vitamin D
increased binding of GST-CBP to the wild type receptor, whereas binding
to the helix 3 or helix 12 mutants was unaffected.
SRC-1 Does Not Enhance Transactivation by the K246A Mutant
VDR--
In view of the differences in the ability of wild type and
mutant K246A VDR to interact with coactivators in vitro, we
analyzed the effect of overexpression of SRC-1 on the activity of these receptors in COS-7 cells transfected with Spp1-tk-CAT. As shown in Fig.
7, the vitamin D-stimulated activity of
the wild type receptor was enhanced with increasing amounts of SRC-1.
In contrast, the K246A mutant was a very poor transcriptional
activator, and in agreement with the lack of binding to SRC-1 in
vitro, its activity was unaffected by expression of SRC-1.

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Fig. 7.
Influence of SRC-1 on the transcriptional
activity of the wild type and K246A VDR. COS-7 cells were
transfected with 5 µg of Spp1-tk-CAT and expression vectors for VDR
or VDR(K246A) (20 ng) in the presence of the amounts indicated of SRC-1
expression vector. After transfection the cells were treated with
medium containing no additions or 100 nM vitamin D
(vit.D) for 24 h, and CAT activity was
determined.
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DISCUSSION |
Previous work has shown the importance of the C-terminal helix 12 for AF-2 activity of nuclear receptors. Helix 12 contains residues with
significant negative charges and forms an amphipathic
-helix that
functions as an interaction surface for coactivators (17, 18). In this
study we confirmed the requirement of an intact AF-2 core domain for
vitamin D-activated transcription. Point mutants E420Q and L417S were
transcriptionally silent, although they have been described to bind
vitamin D with equilibrium constants similar to wild type VDR (14).
These amino acids in helix 12 are required for the interaction with
SRC-1, SUG-1, or RIP140 in the yeast two-hybrid system (14), and our
in vitro interaction assays indicate that in the presence of
vitamin D the coactivators ACTR (the human homologue of mouse p/CIP)
and CBP also fail to associate in a ligand-dependent manner
with VDRs containing these inactivating mutations in helix 12. This
defect can explain the lack of transcriptional activation.
Although the minimal AF-2 domain of VDR (residues 408-427) appears to
be sufficient to mediate ligand-dependent transactivation as well as interaction with coactivators, the activity of this region
is only partial (14). This suggested that additional elements of the
LBD outside the core AF-2 domain were important for generating an
efficient AF-2. These elements could include helix 3 because this helix
is in close proximity to helix 12 in the holo-receptors (16-21). Our
studies demonstrate that indeed the conserved lysine 246 at the
predicted C terminus of helix 3 plays an important role in the
transcriptional actions of VDR. The replacement of this lysine with
alanine dramatically reduced AF-2 activity without affecting
heterodimerization with RXR or DNA binding activity.
It was possible that the inactivity of the lysine 246 mutant could be a
consequence of an altered receptor conformation upon ligand binding. It
has been postulated that the ligand-induced conformational changes
within the receptor result in a more compact folding of the LBD and
increased resistance to proteolysis (48). Our results show that vitamin
D treatment of wild type and K246A receptors generates
indistinguishable peptide maps with a 33-kDa fragment becoming
resistant to tryptic digestion. Given that the helix 3 mutation does
not affect this property, it can be concluded that the overall
structure of the mutant VDR resembles that of the wild type receptor
and that both undergo similar conformational changes after binding of
vitamin D. On the other hand, the changes elicited by vitamin D
demonstrate that the loss of transcriptional activation by the K246A
mutant was not due to a defect in ligand binding. It had been
previously shown that replacing this lysine with a glycine also
impaired transcriptional activity of VDR and that this mutant receptor
was normal with respect to ligand binding (39).
Our studies also reveal that not only the C-terminal domain of the
receptor but also the conserved lysine 246 residue are required for
interaction with SRC-1. The transcriptionally inactive mutant receptor
K246A was found to be defective for in vitro binding to the
coactivators SRC-1, ACTR, and CBP. Functional studies also show that
overexpression of SRC-1 in COS-7 cells enhanced
ligand-dependent transcriptional activation by VDR and that
lysine 246 is essential for this function. These results suggest that
the transcriptional defect of this mutant results from its inability to
recruit coactivators upon vitamin D binding. The fact that this mutant
does not interact with the coactivators suggests subtle differences in
conformation between the native and mutant VDR that were not detected
at the level of resolution afforded by the protease digestion assay. The transcriptional activities of the thyroid hormone and the estrogen
receptors were reported to be also strongly reduced when the conserved
lysine was replaced with another amino acid (35-38). Furthermore, the
mutant estrogen receptor also fails to interact with SRC-1 upon
estrogen binding (35). These results as well as our results with the
VDR indicate that residues in helix 12 as well as the lysine residue in
helix 3 are required to form the surface by which the nuclear receptors
interact with different coactivators. Mutation K246A could destabilize
the helix 3-helix 12 interaction that appears to be important for
generating an efficient AF-2. Because both the charged residues in
helix 12 and the lysine residue in helix 3 are exposed on the surface
of the ligand-binding domain (9), they may generate the hydrophilic surface of interaction with the coactivators required to mediate AF-2
activity (35).
In addition to their inability to stimulate transcription of a reporter
gene containing a positive VDRE, our results also reveal that the VDR
mutants are unable to inhibit retinoic acid-mediated transactivation of
the RAR
2 promoter. We have previously observed that the ability of a
mutant VDR lacking the last 12 C-terminal amino acids to mediate
transrepression by vitamin D was strongly decreased (47). Our present
results indicate that the integrity of not only helix 12 but also helix
3 is essential for the dominant negative activity of VDR. The
requirement of the residues responsible for AF-2 activity suggests that
titration of coactivators or common associated proteins may be involved
in the inhibitory effect of vitamin D, and in agreement with this
hypothesis over-expression of E1A, which can act as a RAR
2
promoter-specific coactivator, significantly reversed repression by
vitamin D (47). These results also indicate that vitamin
D-dependent transactivation and transrepression might
depend on similar or the same interaction surfaces, although we cannot
dismiss the possibility that the proteins interacting might be different.
Our results have also demonstrated an unanticipated property of the VDR
mutants. They can enhance the response of the AP-1 containing
collagenase promoter to TPA, a characteristic not shared by the native
receptor. Although several receptors have been shown to antagonize the
effect of TPA or c-Jun on the collagenase promoter, the functional
interaction of nuclear receptors with AP-1 is complex, and Fos and Jun
can have cell-specific inhibitory and stimulatory effects on
transcription activation by nuclear receptors (8). Interestingly, some
unliganded mutants of the thyroid hormone receptors have an effect
similar to that of the liganded VDR mutants shown here (49). The fact
that the VDR mutants have the common characteristic of being
AF-2-defective suggests the involvement of coactivators in this
property. A good candidate for being implicated in this function is
CBP/p300, which also plays an essential coactivator role for several
classes of transcription factors, including the AP-1 complex (26-29,
33, 34). Overexpression of CBP has been shown to relieve AP-1
antagonism by the retinoic acid and glucocorticoid receptors,
consistent with the hypothesis that competition between AP-1 and
nuclear receptors for limiting amounts of CBP/p300 accounts for
transrepression (26). We have shown that vitamin D does not enhance
binding of CBP to the helix 3 and helix 12 VDR mutants, which could
allow a greater availability of this common coactivator for the
TPA-inducible signal transduction pathway. On the other hand, we cannot
dismiss the possibility that the mutations create a new surface for
interaction with other coactivator proteins or that they abolished
interaction with putative inhibitory factors. Therefore, the mechanism
by which the VDR mutants enhance c-Jun-mediated transactivation is
unclear, and further work will be required to explain this effect.
In summary, our results have established that AF-2 activity of the VDR
depends on the conserved lysine in helix 3 and support the view that
recruitment of coactivators is essential in vitamin D-dependent transactivation as well as in vitamin
D-dependent transrepression. Moreover, the facts that
mutation of this lysine also abolishes ligand-independent
transactivation by the thyroid hormone receptor (37, 38) and that this
residue is conserved in most orphan receptors, which are believed to be
constitutively active, suggest the existence of common mechanisms for
ligand-dependent and ligand-independent transactivation.
Future studies will hopefully clarify the role of different coactivator
complexes in the diverse transcriptional actions of the nuclear receptors.