Mapping the Domains of the Interaction of the Vitamin D Receptor and Steroid Receptor Coactivator-1
Rajbir K. Gill,
Loretta M. Atkins,
Bruce W. Hollis and
Norman H. Bell
Departments of Medicine and Pediatrics Medical University of
South Carolina Department of Veterans Affairs Medical Center
Charleston, South Carolina 29401-5799
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ABSTRACT
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The vitamin D receptor (VDR) binds to the vitamin
D response element (VDRE) and mediates the effects of the biologically
active form of vitamin D, 1,25-dihydroxyvitamin
D3
[1,25-(OH)2D3], on
gene expression. The VDR binds to the VDRE as a heterodimeric complex
with retinoid X receptor. In the present study, we have used a yeast
two-hybrid system to clone complementary DNA that codes for
VDR-interacting protein(s). We found that the human steroid receptor
coactivator-1 (SRC-1) interacts with the VDR in a ligand-dependent
manner, as demonstrated by ß-galactosidase production. The
interaction of the VDR and the SRC-1 takes place at physiological
concentrations of 1,25(OH)2D3. A
48.2-fold stimulation of ß-galactosidase activity was observed in the
presence of 10-10 M
1,25-(OH)2D3. In
addition, a direct interaction between the ligand-activated
glutathione-S-transferase-VDR and
35S-labeled SRC-1 was observed in
vitro. Deletion-mutation analysis of the VDR established that the
ligand-dependent activation domain (AF-2) of the VDR is required for
the interaction with SRC-1. One deletion mutant, pGVDR-(1418), bound
the ligand but failed to interact with the SRC-1, whereas another
deletion mutant, pGVDR-(1423), bound the ligand and interacted with
the SRC-1. We demonstrated that all the deletion mutants were expressed
as analyzed by a Gal4 DNA-binding domain
antibody. Deletion mutation analysis of the SRC-1 demonstrated that 27
amino acids (DPCNTNPTPMTKATPEEIKLEAQS-QFT) of the SRC-1 are essential
for interaction with the AF-2 motif of the VDR.
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INTRODUCTION
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The vitamin D receptor (VDR) is a member of the nuclear hormone
receptor family, which also includes retinoid, thyroid hormone, and
steroid hormone receptors. These receptors function as ligand-inducible
transcription factors by binding to specific DNA sequences known as
hormone response elements in the promoters of the target genes (1, 2, 3, 4).
The VDR mediates the actions of the biologically active form of vitamin
D, 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3], by binding to the vitamin D
response element (VDRE) and modulates transcription of the target
genes, presumably by interacting with the basal transcriptional
machinery (5). The VDRE sequences described to date, including those of
rat (6, 7, 8) and human (9) osteocalcin, mouse osteopontin (10), rat
25-hydroxyvitamin D3-24-hydroxylase (11), avian integrin
ß3 (12), and mouse calbindin-D28k (13),
consist of two hexamers arranged in direct tandem repeats and a
composite response element as described in c-fos (14).
Studies with VDR obtained from an overexpression system indicate that
the VDR alone binds to VDRE with low affinity and requires an
additional nuclear protein(s), termed receptor or nuclear auxiliary
factor, for high affinity binding to the VDREs (15, 16, 17, 18). Several groups
have demonstrated that isoforms of the retinoid X receptor (RXR)
function as auxiliary factors, as RXRs mimic receptor auxiliary factor
activity (19, 20, 21). In addition, involvement of RXR in VDR-mediated
transcription is supported by the observation that vitamin D-dependent
transcription is augmented by exogenous RXR in the transient expression
system (19, 21), and VDR mutants that fail to interact with RXR also
fail to activate transcription (22). The role of RXR in VDR-mediated
transcription has been confirmed in yeast that does not express any
endogenous RXR (23, 24). Thus, transcriptional activation by the VDR
appears to require heterodimeric interaction with another nuclear
receptor, such as RXR.
Recently a steroid receptor coactivator has been cloned and shown to
bind and regulate transcription mediated by progesterone receptor (PR),
estrogen receptor (ER), thyroid hormone receptor (TR), and RXR (25, 26). In the current study, we demonstrate that the VDR forms a direct
protein-protein interaction with the newly described steroid receptor
coactivator-1 (SRC-1) and that the interaction is dependent on the
presence of the ligand. In addition, we mapped the regions of the VDR
and the SRC-1 that are required for this interaction.
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RESULTS
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Isolation of SRC-1 from Human Kidney cDNA Library with
Gal4-VDR-(1427)
In the preliminary experiment, the VDR (1427 amino acids)
expressed as a fusion protein with Gal4
DNA-binding domain (Gal4DB), was assayed in the
two-hybrid system to test for background transcriptional activity. When
Gal4DB-VDR alone (Fig. 1
, A and B) or coexpressed with the
pGAD10 [Gal4 activation domain
(Gal4AD)] plasmid (Fig. 1
, C and D),
ß-galactosidase activity was not observed in the absence or presence
of 1,25-(OH)2D3 (10-7
M). Similarly, transcriptional activity was not detected in
the presence or absence of 1,25-(OH)2D3 when
the VDR was coexpressed with an unrelated plasmid (data not shown).
Thus, we established that the VDR has little if any transcriptional
activity in yeast in the presence or absence of ligand. To identify the
cDNA coding for the VDR-interacting proteins, we transformed YPB6 cells
expressing Gal4-VDR with a cDNA library
constructed in pGAD10 vector [Gal4 activation
domain plasmid (Gal4AD)] and tested the double
transformants for ß-galactosidase activity and histidine prototrophy
in the presence of 10-7 M
1,25-(OH)2D3. Of 2.5 x 107
individual clones examined in the presence of
1,25-(OH)2D3, 73 of them demonstrated histidine
prototrophy and expressed ß-galactosidase, as determined by filter
assay. The specificity of interaction with
Gal4-VDR of these 73 clones was tested by curing
the yeast cells of the bait plasmid and testing them for
ß-galactosidase activity. In 63 clones, loss of
Gal4-VDR resulted in concordant loss of
ß-galactosidase activity, demonstrating that ß-galactosidase
production was due to interaction of the protein and the VDR.

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Figure 1. Demonstration of Lack of Intrinsic Transcriptional
Activity of the VDR in the Yeast Two-Hybrid System
Gal4DB-VDR fusion protein does not contain transcriptional
activation functions in yeast. YPB6 cells were transformed with
pGVDR-(1427) (Gal4-VDR) alone (A and B) or cotransformed
with pGAD10 (Gal4AD; C and D). Transformed cells were
plated on selection medium containing vehicle (A and C) or
10-7 M 1,25-(OH)2D3 (B
and D). Colonies were tested for ß-galactosidase activity by filter
assay.
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Among the 63 positive clones obtained by screen, 13 of them were
identified to be SRC-1 (25, 26) by sequencing and comparison with the
GenBank database. Amino acids of SRC-1 coded by different cDNA inserts
are as follows: nine clones coded amino acids 11260, two clones coded
amino acids 342-1440, one clone coded amino acids 711-1160, and one
clone coded amino acids 745-1180.
Requirement of
1,25-(OH)2D3 for
Interaction of VDR and SRC-1
To determine whether the ligand is required for interaction, yeast
cells containing both plasmids were plated on
Leu-,Trp- synthetic MEM (SD medium) in the
absence or presence of 10-7 M
1,25-(OH)2D3 and assayed for ß-galactosidase
activity (Fig. 2
). Transcriptional
activation of ß-galactosidase was observed in the presence of ligand,
as demonstrated by the development of blue color (Fig. 2
, A1 and B). In
the absence of ligand, VDR and SRC-1 failed to interact with each other
(Fig. 2A
2). In addition, we examined the interaction between VDR and
SRC-1 in vitro. As shown in Fig. 2C
, very little binding of
35S-labeled SRC-1 was observed with unactivated
glutathione-S-transferase (GST)-VDR (in the absence of
ligand), and binding was significantly enhanced when GST-VDR was
activated with 1,25-(OH)2D3. These results
demonstrated that the VDR undergoes direct interaction with the SRC-1
in a ligand-dependent manner. To determine whether these interactions
occur at physiological concentrations of
1,25-(OH)2D3, the cells containing both
plasmids were cultured at concentrations ranging from
10-12-10-7 M
1,25-(OH)2D3, and ß-galactosidase
activity was determined by liquid assay. Transcriptional activation was
dose dependent and reached a maximum level at 10-8
M 1,25-(OH)2D3 (Fig. 2B
). A
significant stimulation (48.2-fold) of ß-galactosidase activity was
observed at 10-10 M
1,25-(OH)2D3. These results demonstrate that
SRC-1 binds to VDR in a dose-dependent manner.

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Figure 2. Interaction of the VDR with the SRC-1 in the
Presence of Ligand
A, Stimulation of ß-galactosidase activity due to interaction of
Gal4DB-VDR (bait plasmid) and Gal4AD-SRC-1 in
the absence (A2) and presence of 10-7 M
1,25-(OH)2D3 (A1). B, Dose response of
ß-galactosidase activity stimulation. The pGVDR-(1427) was
cotransformed with Gal4AD-SRC-1. Transformants were
cultured in selection medium, and ß-galactosidase activity was
quantitated by liquid assay. C, In vitro interaction of
the SRC-1 with the VDR. SRC-1 (3.4 kb) radiolabeled with
[35S]methionine was incubated in batch with
yeast-expressed GST (lanes 1 and 2) or GST-VDR fusion protein (lanes 3
and 4) in the absence (lanes 1 and 3) or presence of ligand (lanes 2
and 4). Bound SRC-1 was eluted and analyzed on SDS-PAGE.
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Requirement for the Ligand-Dependent Activation Domain (AF-2)
Region of the VDR for Interaction with the SRC-1
To determine the domains of the VDR required for interaction with
the SRC-1, deletion mutant fragments of the VDR were PCR amplified as
shown in Fig. 3A
. Deletion fragments were
cloned to express Gal4 fusion proteins. These
deletion mutant constructs were cotransformed with SRC-1 into
YBP6 and cultured onto Leu-,Trp- SD medium
containing 1,25-(OH)2D3. As shown in Fig. 3A
, loss of amino acids 1116 of the VDR had no effect on the interaction
or on ligand binding. These results demonstrate that the conserved
DNA-binding domain is not essential for ligand binding and interaction
between these proteins. Loss of amino acids 117382 of the VDR
resulted in the failure of ligand to bind the mutant VDR (Fig. 3A
), and
as interaction of VDR and SRC-1 takes place in the presence of ligand,
these VDR deletion mutants fail to interact with SRC-1. A deletion of
amino acids 419427 of the VDR resulted in failure to interact with
the SRC-1. However, this mutant could bind ligand (Fig. 3A
). Deletion
of amino acids 423427 of the VDR had no effect on ligand binding and
interaction with the SRC-1 (Fig. 3A
).

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Figure 3. Identification of the Region of the VDR Required
for Interaction with SRC-1
A, Diagrammatic representation of deletion fragments of the VDR that
are fused in-frame with Gal4DB to express them as fusion
protein. The VDR deletion mutant construct was cotransformed with SRC-1
into YBP6. Transformants were plated on selection medium containing
10-7 M 1,25-(OH)2D3
and tested for ß-galactosidase activity by filter assay.
Transformants were cultured in selection medium in the absence of
ligand, and whole cell extracts were analyzed for ligand binding. +,
The presence of ß-galactosidase activity or ligand binding; -, the
absence of ß-galactosidase activity or ligand binding. B, Expression
of VDR deletion mutants. Twenty and 5 µg of total protein were
analyzed by immunological assay with Gal-DB monoclonal antibodies.
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The failure of interaction between SRC-1 and the VDR deletion mutants
could be due to the loss of a binding site in the VDR deletion mutant.
Alternatively, loss of interaction could also result from impaired
protein synthesis in the deletion mutant or degradation of the
synthesized protein. To distinguish between the loss of a binding site
in deletion mutants vs. the failure of fusion protein
synthesis, we analyzed the Gal4-VDR deletion
mutants by immunological assay. As demonstrated in Fig. 3B
, analysis
with anti-Gal4DB monoclonal antibody revealed
that all fusion proteins are expressed, and loss of interaction is due
to the loss either of the binding site for SRC-1 in the deletion mutant
or of the sequence that is essential for ligand binding. Thus, the lack
of ß-galactosidase activity is due to the failure of the two proteins
to interact, which results in formation of a functional Gal-4
transcription factor, and functional Gal-4 is required for
lacZ transcription.
These results demonstrate that the AF-2 region of the VDR is required
for ligand-dependent interaction with the SRC-1. Amino acids 417422
of the VDR represent consensus for AF-2, as shown by bold
letters in Fig. 4B
, and are
conserved in the nuclear receptor. The main features of this motif are
a central acidic amino acid (E) flanked by two hydrophobic amino acids
(Fig. 4B
).

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Figure 4. Demonstration That the AF-2 Domain of the VDR
Interacts with the SRC-1
A, Diagrammatic representation of the amino acids essential for
interaction in the two regions. B, Alignment of the AF-2 region of
various nuclear receptors. The consensus sequence is  XE and
is represented as bold letters. represents conserved
hydrophobic amino acids, X represents nonconserved amino acids, and E
is conserved glutamic acid residue. Nuclear receptors with their
accession numbers are human VDR (J03258), human T3R
(M24899), human T3Rß (m26747), human RAR (X06538),
RARß (X07282 or Y00291), RAR (M57707), human RXR (X52773),
RXRß (X63522), mouse RXR (M84819), human glucocorticoid receptor
(X03225), human ER (M11457), and human PR (M15716). C, Helical wheel
representation of amphipathic helix of the AF-2 region of the VDR
showing charged and polar amino acids on one side of the helix.
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Identification of the SRC-1 Region That Interacts with the VDR
Deletion mutation analysis was performed to identify the region of
the SRC-1 that binds the VDR. We isolated 13 clones in the library
screening that coded for 4 SRC-1 peptides (C1C4; Fig. 5A
). Two additional deletion mutants (C5
and C6) were constructed in which amino acids 745809 and 745781,
respectively, were used as Gal4 fusion proteins.
These deletion mutants were cotransformed with
Gal4DB-VDR-(1427) into YPB6 and analyzed for
stimulation of lacZ transcription. Proteins coded by C1C5
bound the VDR, as shown by stimulation of ß-galactosidase assay (Fig. 5A
), whereas protein coded by C6 failed to bind the VDR, as
demonstrated by lack of ß-galactosidase activity (Fig. 5A
). These
results show that amino acids 782809 of SRC-1 (Fig. 5B
) are involved
in binding the VDR. These residues of SRC-1 form an amphipathic helix
with charged and polar residues on one side of the helix, as shown in
Fig. 5C
.

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Figure 5. Identification of the Region of the SRC-1 That
Interacts with the VDR
A, Deletion mutant analysis of the SRC-1. SRC-1 deletion mutation
constructs were cotransformed with Gal4DB-VDR, and
transformants were analyzed for ß-galactosidase activity. B,
Diagrammatic representation of SRC-1 required for interaction with the
VDR. C, Helical wheel representation of amphipathic helix of SRC-1
amino acids 781809.
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In summary, six amino acids in the ligand-binding domain of the VDR,
which represent AF-2, are involved in the interaction (Fig. 4
).
Twenty-seven amino acids of the SRC-1 are required to interact with the
VDR in the presence of the ligand (Fig. 5
).
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DISCUSSION
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The VDR, like other steroid receptors, functions as a
ligand-dependent transcription factor. Enhancement of transcription
initiation by sequence-specific DNA binding of these factors and their
interaction with specific components of the basal transcriptional
machinery are principal mechanisms for regulating transcription
(27, 28, 29). Steroid receptors may interact with the basal machinery or
interact through an adapter. A direct protein-protein interaction of
transcription factor IIB (TFIIB) and the VDR both in vivo
and in vitro as well as the role of TFIIB in
ligand-dependent transcription regulation have been demonstrated (30, 31).
The present report demonstrates that human SRC-1 interacts with the VDR
in a ligand-dependent manner (Fig. 2A
). Similar to the VDR, SRC-1 has
been shown to interact directly with the PR, ER, retinoic acid receptor
(RAR), RXR, and TFIIB in the presence of ligand (25, 26). The
interaction between the SRC-1 and the VDR occurs at physiological
concentrations of 1,25-(OH)2D3, as
demonstrated by stimulation of lacZ transcription (Fig. 2B
).
A significant stimulation of ß-galactosidase activity was observed at
10-10 M 1,25-(OH)2D3,
with maximum stimulation at 10-8 M. In
addition, SRC-1 has been shown to stimulate transcription mediated
by PR, ER, TR, RXR, and glucocorticoid receptor by their respective
ligands (25).
In addition to SRC-1, several other potential factors have been
demonstrated to interact with the steroid hormone receptors in the
presence of ligand. These include mouse bromodomain-containing protein,
known as TIF-1, Trip-1 and other TR-associated proteins, and the
ER-associated proteins, p160, RIP160 (receptor interacting
protein-160), and RIP80 (32, 33, 34, 35). Even though these proteins interact
with steroid receptors, their roles as potential coactivator need to be
demonstrated. RIP140 (36) and GRIP (glucocorticoid receptor interacting
protein) (37) have recently been identified as coactivators, and N-CoR
(nuclear receptor corepressor) (38, 39) as corepressor.
Our studies reveal that an AF-2 core domain plays a central role in
SRC-1-mediated signaling. Deletion mutation analysis of the VDR (Fig. 3
) demonstrated that the C-terminal domain of the VDR is required for
interaction with the SRC-1. As SRC-1 binds to the
1,25-(OH)2D3-VDR complex, we analyzed deletion
mutants for ligand binding. We found that in a yeast system, deletion
of 34 amino acids at the C-terminus of the VDR (
393427) resulted
in failure to bind ligand, and deletion of amino acids 1116, the
DNA-binding domain, had no effect on ligand binding or on the
interaction with SRC-1 (Fig. 3A
). These results support previous
studies in mammalian cells in which deletion of the DNA-binding domain
(amino acids 1116) had no effect on ligand binding, and removal of
amino acids 383427 resulted in failure to bind ligand (40, 41). In
addition, a deletion mutant lacking amino acids 418427 of the VDR can
bind ligand. These results are in agreement with those of previous
studies in which
VDR-(1409) was shown to bind ligand (40, 41). The
C-terminus amino acids 419423 of the VDR are required for the
interaction with SRC-1 (Figs. 3A
and 4A
). This region contains
consensus AF-2 motif (amino acids 416422) that is conserved among all
nuclear receptors (Fig. 4B
). It has been established that the integrity
of AF-2 is required for ligand-dependent receptor-mediated activity
(42, 43, 44). Mutagenesis of this conserved region abrogates AF2 activity
without significantly altering DNA binding, heterodimerization, and
ligand binding (42, 43, 44). Interestingly, the corresponding motif is
absent in v-erbA, which has no AF2 activity (45, 46), and a
point mutation in this region has little or no effect on steroid or DNA
binding (40, 41, 42, 43, 44). In the present study, we established that deletion of
this AF-2 region abrogates the binding to SRC-1 without affecting
ligand binding (Fig. 3A
). The conserved AF-2 region contains amino
acids with significant negative charges and forms an amphipathic
-helix. Amphipathic helixes have been implicated in the function of
a variety of transcription factors (for review, see Ref.47). Thus,
deletion of amino acids encompassing this putative amphipathic helix in
the VDR resulted in failure to interact with the SRC-1. These results
demonstrate that the SRC-1 activates the transcription by interacting
with the AF-2 activation domain of the steroid hormone receptor.
Interestingly, these amino acids are found adjacent to a heptad repeat
9 that has been shown to function as a dimerization interface (37).
Recent crystal structural analysis of the ligand-binding domains of TR,
RXR, and RAR suggests that the AF-2 core domain, which forms an
amphipathic helix also known as helix 12, undergoes striking
conformational changes upon hormone binding (48, 49, 50). In the unoccupied
receptor, helix 12 projects into the solvent (48). In the
hormone-occupied receptor, the helix folds back toward the receptor to
form part of the ligand binding cavity (49, 50). The helix is packed
loosely with hydrophobic residues facing inward toward the
ligand-binding pocket, and charged residues extend into the solvent
(49, 50). It is possible that in this conformation, the helix presents
itself for interaction with SRC-1. Recently, Henttu et al.
(51) established that AF-2 activity and lysine 366 of the ER are
essential for interaction with the SRC-1.
Both the RXR, as demonstrated previously (25, 26), and the VDR, as
shown in the present study, were shown to interact with the SRC-1. As
the VDR-RXR heterodimer mediates
1,25-(OH)2D3-regulated gene transcription, it
is not clear whether one SRC-1 molecule binds both heterodimer partners
or each partner binds to a different SRC-1 molecule. However, in
studies using RXR mutants in which the AF-2 domain of RXR was deleted,
a significantly reduced trans-activation was observed by
RXR-RAR, -TR, and -VDR heterodimers in the presence of hormones
specific for the RXR heterodimeric partner (52), demonstrating that the
AF-2 domain of the RXR is necessary for hormone-dependent
trans-activation. It is possible that the SRC-1 integrates
the AF-2 functions of both partners of the VDR-RXR heterodimer in the
same manner, as previously demonstrated for AF-1 and AF-2 functions of
the ER (53).
The 27 amino acids of SRC-1 that are required for interaction with the
VDR also form an amphipathic
-helix. As shown in Fig. 5C
, helical
wheel analysis of these amino acids of SRC-1 demonstrates that there
are clusters of charged and polar amino acids on one side of the
amphipathic helix of this region of SRC-1 that may be involved in
interaction. These charged amino acids probably extend into the solvent
from amphipathic
-helix formed by the AF-2 region of the VDR.
In summary, the present study demonstrate that the AF-2 domain of the
VDR interacts with 27 amino acids of the SRC-1. Six amino acids in the
ligand-binding domain of the VDR, which represent AF-2, are involved in
this interaction (Fig. 4
). The results provide further insight into the
molecular events associated with ligand-dependent gene activation by
1,25-(OH)2D3.
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MATERIALS AND METHODS
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Bacterial and Yeast Strains
Yeast strains SFY526 [genotype MATa, Ura352,
His3200, Ade2101, Lys2801,
Trp1901, Leu23112, Canr,
Gal4542, Gal80538,
Ura3::Gal1-lacZ] and DY150 [genotype
MATa, Ura352, Leu23112,
Trp11, Ade21, His311, and
Can1100) were purchased from Clontech (Palo Alto,
CA). Yeast strain YPB6 [genotype MATa, Ura352,
His3200, Ade2101, Lys2801,
Trp190, Leu23112, CanR,
Gal4542, Gal80538,
Gal-lacZ::Ura3,Gal1-His3(pBM1499)::Lys2]
was a gift from Dr. Paul Bartel (Department of Microbiology, State
University of New York, Stony Brook, NY). The Escherichia
coli strain of DH5
(Life Technologies, Grand Island, NY) was
used for rescue of plasmids from yeast cells.
Construction of Plasmids
Gal4DB-VDR Fusion Protein and Gal4DB-VDR
Deletion Mutant Fusion Protein Plasmids
Yeast shuttle vector pGBT9 (Gal4-DB) was a gift
from Dr. Paul Bartel (Department of Microbiology, State University of
New York, Stony Brook, NY). The pGBT9 was described in detail
previously (54). The pGBT9 contains the DNA-binding domain of the yeast
transcription factor Gal4. A cDNA clone coding
for VDR (55) was purchased from American Type Culture Collection
(Rockville, MD). To express VDR as a Gal4 fusion
protein, a full-length coding region (1427 amino acids) of the VDR
was PCR amplified with primer containing EcoRI and
BamHI linkers at 5' and 3', respectively. After digestion
with EcoRI and BamHI, the PCR-amplified VDR cDNA
was subcloned into EcoRI and BamHI of pGBT9, and
this plasmid was designated pGVDR-(1427). The pGVDR-(1427) was
sequenced to confirm in-frame fusion of the VDR with the
Gal4-DB. Similarly, the deletion mutants of VDR
were PCR amplified and cloned into pGBT9, and these plasmids were
designated pGVDR followed by the amino acid position in the VDR
protein.
GST-VDR Fusion Protein Plasmid
To express VDR as a GST fusion protein, the cDNA insert was removed
from pGVDR-(1427) by digestion with EcoRI and
SalI restriction enzymes and ligated into EcoRI-
and SalI-digested pYEX 4T1 yeast vector. This recombinant
vector was pGST-VDR. The resulting plasmid produced an in-frame fusion
of GST and VDR from Met1-Ser427.
In vitro Transcription and Translation Vector
A clone containing 3.5 kb cDNA corresponding to the SRC-1 was subcloned
into pBluescript, and the recombinant clone was digested with
XbaI before in vitro transcription and
translation.
Two-Hybrid Library Screening
We used the two-hybrid system developed by Field and Song (56).
Briefly, one hybrid is a fusion protein between the
Gal4 DNA-binding domain and the VDR, whereas the
other hybrid is a fusion protein between the activation domain of
Gal4 and a second protein.
Trans-activation of His3 and the
lacZ reporter gene occurs only if both proteins of interest
interact with each other when coexpressed in an appropriate yeast
strain. The interaction can, therefore, be monitored by
ß-galactosidase activity and/or prototrophy for histidine. The YPB6
yeast reporter strain was used for the library screening. Yeast was
grown in SD containing 2% sucrose (wt/vol) and 0.67% nitrogen base
without amino acids (wt/vol; Difco, Detroit, MI) supplemented with the
appropriate amino acids. To identify proteins that interact with the
VDR in the presence of 1,25-(OH)2D3, the
Saccaromyces cerevisiae YPB6 reporter strain containing
pGVDR-(1427) was transformed with a human kidney cDNA library
constructed in pGAD10 vector. Approximately 2.5 x 107
transformants were plated onto
Trp-,Leu-,His- SD containing 30
mM 3-aminotrizole and 10-7 M
1,25-(OH)2D3. His+ colonies that
appeared from 48 days after plating were tested for ß-galactosidase
by the filter assay with the substrate
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside as
previously described (57). The library plasmids
(Gal4 activation domain plasmid) were rescued by
growing the yeast clone in Leu- SD medium for 40 h
and then selecting for growth on Leu- SD medium and not
Leu-,Trp- SD medium. This plasmid was
transformed into either the YPB6 or SFY526 strain alone or
cotransformed with either pGVDR or unrelated plasmid
(Gal4DB-lamin) to confirm interaction and to test
for false positive clones.
Subcloning and Sequencing of the cDNA
The partial sequence of each clone was determined by double-stranded
sequence with the Sequenase 2.0 kit (U.S. Biochemical Corp., Cleveland
OH) according to the manufacturers instructions. The partial sequence
of each clone was compared to determine the number of times each clone
was independently isolated and to determine the reading frame of the
fusion protein. The cDNA insert from the rescued plasmid was excised
with restriction enzymes and subcloned into pBluescript for sequencing.
A search for homology with known sequences from GenBank was carried out
with the Fasta A software of Wisconsin package (Genetic Computer Group,
Madison, WI).
Quantitation of ß-Galactosidase Activity by Liquid Assay
The liquid ß-galactosidase assay was performed by following the
published protocol (57). Briefly, the YPB6 cells transformed with the
indicated plasmid pair were cultured into
Leu-,Trp- SD in the absence or presence of
1,25-(OH)2D3 to midlog phase (OD600
= 0.30.7), and a 100-µl aliquot of culture was used for the liquid
ß-galactosidase assay. After the reaction, absorbance was measured at
OD420. The specific ß-galactosidase activity was
calculated with the following formula and expressed as Miller units
(mean of at least four determinations ± SEM):
ß-galactosidase assay = 1000 x [OD420/(t
x V x OD600)], where t is time of incubation in
minutes, V is the volume of culture added in milliliters,
OD600 is optical density of yeast culture at
600,
OD420 is absorbance at
420.
Protein-Protein Interactions
The yeast DY150 strain was transformed with pGST-VDR. The GST-VDR
fusion protein was expressed according to the published protocol (58).
Yeast whole cell extract was prepared in KTEDM buffer [300
mM KCl, 10 mM Tris-HCl (pH 8.0), 1.5
mM EDTA, and 10 mM sodium molybdate]
containing soybean trypsin inhibitor (10 µg/ml), leupeptin (2
µg/ml), pepstatin (2 µg/ml), and aprotinin (2 µg/ml). The GST-VDR
was activated in vivo by the addition of 10-8
M 1,25-(OH)2D3 during induction of
the protein. Yeast cell extracts were treated for an additional 15 min
at room temperature with 10-6 M hormone before
purification. Approximately 400 µg total protein extract were
incubated with 20 µl GST-Sepharose beads in suspension for 2 h
at 4 C. Resins were then washed twice with NENT buffer [20
mM Tris-HCl (pH 8.0), 100 mM NaCl, 1
mM EDTA, 0.5% Nonidet P-40, and 0.5% milk powder] and
washed twice more with transcription buffer [20 mM HEPES
(pH 7.9), 60 mM NaCl, 1 mM dithiothreitol, 6
mM MgCl2, 0.1 mM EDTA, and 10%
glycerol]. Subsequently, the beads were mixed with 20 µl in
vitro transcribed and translated
[35S]methionine-labeled SRC-1, and the interaction was
allowed to occur for 4 C for 1 h. The bound protein was eluted
with SDS sample buffer, fractionated on SDS-PAGE, and analyzed by
fluorography for 35S.
Ligand Binding Assay
Whole cell extract of YPB6 cells expressing
Gal4DB-VDR-(1427) and various deletion
mutations was prepared in KTEDM buffer as described above. The cell
suspension was centrifuged at 210,000 x g for 35 min
at 4 C. The protein concentration of the supernatant was determined by
the method of Bradford (59). The ligand binding assay was performed
according to a published protocol (60).
Analysis of Gal4-VDR Fusion Protein
Expression
Whole cell yeast extracts of YPB6 cells and YPB6 cells
transformed with the Gal4DB-VDR and
Gal4DB-VDR deletion mutants were prepared in
KTEDM. The proteins were applied to Immobilon-P
(polyvinyldifluoridine) with a slot blotter, and then the blot was
dried for 20 min at 37 C as described previously (61). The membranes
were blocked in 1% BSA in Tris-buffered saline and then incubated with
anti-Gal4DB monoclonal antibody (Clontech, Palo
Alto, CA). The membrane was incubated with secondary antimouse
antibodies conjugated to peroxidase for 1 h at room temperature,
followed by development of color.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Paul Bartel and Stanley Fields for yeast
matchmaker plasmids and yeast strain YPB6.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Rajbir K. Gill, Ph.D., Research Service/Veterans Administration Medical Center, 109 Bee Street, Charleston, South Carolina 29401-5799.
This work was supported in part by an institutional grant from the
Medical University of South Carolina (to R.G.).
Received for publication July 25, 1997.
Accepted for publication October 16, 1997.
 |
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