Full text of DOI:10.1359/jbmr.2001.16.1.39

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 16, Number 1, 2001
©
2001 American Society for Bone and Mineral Research
The Unique Tryptophan Residue of the Vitamin D
Receptor Is Critical for Ligand Binding
and Transcriptional Activation
1
,2
1,2
3
1,4
CYNTHIA SOLOMON, MICHAEL MACORITTO, XIAO-LING GAO, JOHN H. WHITE, and
1
RICHARD KREMER
ABSTRACT
The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily of transcriptional
regulators. Here we show that tryptophan 286 of the hVDR is critical for ligand binding and transactivation
of 1,25-dihydroxyvitamin D [1,25(OH) D ] target genes. Two mutants of the hVDR were produced, W286A
3
2 3
and W286F, in which the tryptophan was replaced with an alanine or a phenylalanine, respectively. The
W286A mutant did not bind 1,25(OH) D , interact with steroid receptor coactivator 1 (SRC-1) in vitro, or
2
3
activate transcription. Moreover, the W286A receptor did not heterodimerize in a ligand-dependent manner
with the human retinoid X receptor ␣ (hRXR␣). Although the W286F receptor heterodimerized with hRXR␣,
interacted with SRC-1, and bound 1,25(OH) D , its capacity to transactivate was attenuated severely. Thus,
2
3
tryptophan 286 of hVDR plays an important role in specific 1,25(OH) D ligand interaction and subsequently
2
3
in hVDR/RXR interaction, SRC-1 binding, and ligand-dependent transactivation of 1,25(OH) D target genes.
2
3
These results identify the first amino acid that is absolutely required for ligand binding in the VDR and further
define the structure-function relationship of 1,25(OH) D interaction with its receptor. (J Bone Miner Res
2
3
2
001;16:39–45)
Key words: vitamin D receptor, 1,25-dihydroxyvitamin D , tryptophan, ligand binding, coactivators
3
INTRODUCTION
The crystal structures of the ligand binding domains
(3)
(
LBDs) of the retinoid X receptor ␣ (RXR␣), the retinoic
(4)
HE BIOLOGICALLY active form of vitamin D , 1,25- acid receptor ␥ (RAR␥), and the thyroid hormone recep-
3
(5)
T
dihydroxyvitamin D3 [1,25(OH) D ], directly modu- tor ␣ (TR␣) have been determined. All were found to
2 3
lates the transcription of several target genes by binding to share a similar secondary structure of 12 ␣-helices and a
(6)
the vitamin D receptor (VDR), a member of the nuclear very low content of ␤-pleated sheet in which helix 12 is
receptor family of transcriptional regulators. Binding of the highly conserved AF-2 domain. No structural data has
ligand activates nuclear receptors by inducing a change in yet been published for the VDR; however, other nuclear
the position of the C-terminal activation function 2 (AF-2) receptors may serve as models on which to base information
(1)
domain. The AF-2 domain mediates transactivation by about the VDR.
creating an interface for ligand-dependent interaction with
The amino acid sequence of human RAR␥ revealed a
tryptophan residue at position 227 in the LBD. Tryptophan
(2)
coactivators, a bridge to the transcriptional machinery.
1
Department of Medicine, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada.
These authors contributed equally to this work.
Department of Chemistry, McGill University, Montreal, Quebec, Canada.
Department of Physiology, McGill University, Montreal, Quebec, Canada.
2
3
4
3
9

4
0
SOLOMON ET AL.
fluorescence studies showed that fluorescence was Western blot analysis
quenched in a dose-dependent manner on addition of all-
Western blotting was used to determine the cellular dis-
tribution of hVDR. Nuclear and cellular fractions from
transiently transfected COS-7 cells (15 ␮g) were boiled for
trans retinoic acid indicating that tryptophan 227 of RAR␥
(6)
is in close contact with bound ligand. Saturation of the rat
VDR with 1,25(OH) D quenches the fluorescence of tryp-
2
3
5
minutes, resolved on 10% sodium dodecyl sulfate (SDS)-
tophan 282, suggesting that the ligand and tryptophan also
(7)
polyacrylamide gels and transferred to polyvinylidene di-
fluoride (PVDF) membranes (BIO-RAD, Hercules, CA,
USA). Protein content was determined with a protein assay
kit (BIO-RAD). Blots were probed with a polyclonal anti-
VDR antibody (diluted to 1:200; Affinity Bioreagents,
Neshanic Station, NJ, USA). After incubation with a horse-
radish peroxidase–conjugated secondary antibody (BIO-
9) RAD), the bands were visualized by enhanced chemilumi-
nescence (ECL; Amersham).
are in close proximity in the rat VDR. Similar results also
have been observed for the human RXR␣ LBD.
(8)
The exact amino acids in the human VDR (hVDR) that
contact 1,25(OH) D remain unknown. Extensive work
2
3
with deletion mutants of the LBD has contributed to the
elucidation of regions important for ligand binding. Dele-
tion mapping of the hVDR LBD has revealed that amino
(
acids 232–382 are critical for interaction with ligand.
Here, we show that tryptophan 286 of the hVDR is critical
for ligand binding, coactivator interaction, and transactiva-
tion.
Gel mobility shift analysis
Two micrograms of transiently transfected COS-7 cellu-
lar extracts were incubated for 20 minutes on ice in the
Ϫ7
MATERIALS AND METHODS
presence or absence of 10 M 1,25(OH) D , and 1 ␮g
2
3
polydeoxyinosine-deoxycytidilic acid (poly dI.dC) in a
Preparation of the mutant VDRs
binding buffer (25 mM Tris-HCl, pH 8.0, 5% glycerol, and
3
0
.5 mM DTT). Five femtomoles of the [ P]-labeled mouse
The hVDR expression vector hVDRpSG5 was con-
structed by inserting an EcoRI fragment containing the
entire coding region of the hVDR into the EcoRI site of
osteopontin VDR element (mOP VDRE; 5Ј-GTACAA-
GGTTCACGAGGTTCACGTCTTA-3Ј) were added and in-
cubated for 20 minutes at room temperature. The anti-RXR
antibody 4X1D12 (a kind gift of Dr. P. Chambon, College
de France, Illkirch, France) was then added where appro-
priate and further incubated for 20 minutes at room temper-
ature. The samples were electrophoresed on 5% nondena-
turing polyacrylamide gels, dried, and exposed to KODAK
XAR-5 film (Eastman Kodak Company, Rochester, NY,
USA).
(10)
pSG5.
The hVDRpSG5 was used as a template to intro-
duce point mutations into hVDR through a double polymer-
(11)
ase chain reaction.
The tryptophan at position 286 of
hVDR was replaced by either an alanine or a phenylalanine
to produce W286A and W286F, respectively.
Cell culture and transfections
COS-7 cells were grown in Dulbecco’s modified Eagle’s
Medium (DMEM) (Gibco-BRL, Burlington, Ontario, Can-
ada) supplemented with 10% fetal bovine serum (FBS).
Transfections were performed by incubating 5 ␮g of each
Expression of glutathione-S-transferase fusion proteins
in Escherichia coli
Glutathione-S-transferase (GST)-hRXR␣, GST-hVDR,
plasmid DNA with 15 ␮g lipofectAMINE (Gibco-BRL) for GST-hVDR W286A, and GST-hVDR W286F expression
2
0 h in serum-free OPTI-MEM (Gibco-BRL) and then vectors were constructed by inserting a fragment encoding
replacing the medium with fresh DMEM containing 10% the LBDs of hRXR␣, wild-type hVDR, hVDR W286A, or
FBS.
hVDR W286F into the EcoRI site of pGEX-2TK (Pharma-
cia, Baie d’Urf e´ , Quebec, Canada). The GST fusion proteins
were expressed in E. coli. Bacteria transformed with the
GST fusion protein expression plasmids were grown to an
optical density at a wavelength of 595 nm (A595) of 0.5 at
Cellular extracts of COS-7 cells
Transiently transfected cells were harvested for gel retar-
dation assays 48 h after transfection by washing cells twice
with phosphate-buffered saline (PBS) followed by scraping
the cells in 1 ml of PBS. Cells were centrifuged at 2000 rpm
for 10 minutes at 4°C and the pellets were resuspended in 1
ml of buffer A (25 mM Tris, pH 7.5, 0.3 mM dithiothreitol
3
7°C and expression was subsequently induced with 0.5
mM isopropyl ␤-D-thiogalactopyranoside. GST alone also
was expressed in E. coli for control purposes. Cells were
centrifuged and resuspended in 12 ml lysis buffer (50 mM
Tris-HCl, pH 7.5, 100 mM KCl, 10 ␮M ZnCl , 0.1 mM
2
DTT, 10% glycerol, 100 mg/ml lysozyme, and protease
inhibitors). The lysate was sonicated and pelleted by ultra-
centrifugation for 1 h at 30,000 rpm. Aliquots of the super-
natants were stored at Ϫ80°C.
[DTT], 0.1 M KCl, and 20% glycerol). Cells were lysed by
three freeze-thaw cycles and then centrifuged at 11,500 rpm
for 15 minutes at 4°C. Supernatants were stored at Ϫ80°C.
Nuclear extracts of COS-7 cells
In vitro transcription and translation
Nuclear extracts were prepared from transiently trans-
fected COS-7 cells as previously described.
Plasmids encoding wild-type VDR or the mutant hVDRs
were transcribed with T7 polymerase and translated in vitro
(12)

TRYPTOPHAN 286 OF THE hVDR IS CRITICAL FOR LIGAND BINDING
41
with a T T T7-coupled reticulocyte lysate system (Pro-
N
3
5
mega, Madison, WI, USA) in the presence of [ S]methi-
onine according to the manufacturer’s instructions. Plas-
mids encoding steroid receptor coactivator 1 (SRC-1) were
transcribed with T3 polymerase (Pharmacia) and translated
in vitro as described previously. Translation efficiency was
monitored by running 0.5 ␮l of the reaction on an sodium
dodecyl sulfate (SDS)–polyacrylamide gel.
GST pull-down assays
FIG. 1. Cellular distribution and expression of wild-type
and mutant VDRs. Nuclear and cytosolic fractions were
prepared from COS-7 cells transfected with the wild-type
VDR (lanes 1 and 2), VDR W286A (lanes 3 and 4), or VDR
W286F (lanes 5 and 6). Fifteen micrograms of each fraction
were used for SDS-polyacrylamide gel electrophoresis
GST fusion proteins were purified from 500 ␮l of bacte-
rial lysate by incubation with 100 ␮l of glutathione-
Sepharose 4B beads (Pharmacia) overnight at 4°C. The
beads were washed three times with GST buffer (50 mM
Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl , 0.3 mM
2
DTT, 5% glycerol, 0.1% NP-40, and protease inhibitors)
and the protein content was determined by the Bradford
method. Beads bound to GST fusion proteins or GST alone
were incubated with the in vitro translated proteins in the
(PAGE) and Western blotting as described in the Materials
and Methods section. N, nuclear fraction; C, cytosolic frac-
tion.
Ϫ7
presence or absence of 10 M 1,25(OH) D at room tem-
2
3
perature for 1 h. The beads were then washed three times
with 400 ␮l GST buffer, boiled for 10 minutes, and elec-
trophoresed on 10% SDS-polyacrylamide gels. Gels were
then treated with Enlightening, an autoradiography en-
hancer (Mandel Scientific-NEN, St. Laurent, Quebec, Can-
ada), for 20 minutes, dried, and exposed to KODAK XAR-5
film overnight.
NEN) in 25 ␮l and 5 ␮l of vehicle for total binding or 5 ␮l
of nonlabeled 1,25(OH) D for a final concentration of 250
2
3
nM for nonspecific binding for 16 h at 4°C. Proteins and
bound hormone were pelleted and washed twice rapidly
with KTEDG. The pellet was resuspended in 300 ␮l of
KTEDG and 100 ␮l was assessed by liquid scintillation
counting.
Transcriptional activation assay
4
COS-7 cells were plated at a density of 4 ϫ 10 cells/well
into 6-well plates and transfected with 4 ␮g of the
RESULTS
1
,25(OH) D -sensitive chloramphenicol acetyltransferase
2 3
(13)
Cellular distribution of wild-type and mutant hVDRs
expressed in COS-7 cells
(CAT) reporter plasmid, mOP3,
4 ␮g of either the wild-
type or one of the mutant hVDR constructs, and 0.1 ␮g of
(14)
a human growth hormone (hGH) reporter plasmid
as an
Western blot analysis was performed using equal
amounts of protein from both nuclear and cytosolic extracts
prepared from COS-7 cells transfected with expression plas-
mids encoding either wild-type VDR, the W286A mutant,
or the W286F mutant (Fig. 1). An antibody specific for the
hVDR revealed a protein complex in the nuclear fraction of
the COS-7 cell extracts from cells transfected with wild-
type VDR and from cells transfected with the mutant VDRs
internal control for transfection efficiency. Cells were incu-
bated overnight in serum-free OPTI-MEM and then treated
with varying concentrations of 1,25(OH) D for 24 h. The
2
3
medium was retained for the hGH assay and the cells were
trypsinized washed in PBS, resuspended in 0.25 M Tris-
HCl, pH 8.0, and lysed by five freeze-thaw cycles. The cell
lysate was centrifuged and aliquots of cell extracts were
used for the CAT assays. Assays were performed using a
CAT ELISA kit (5 Prime-3 Prime, Boulder, CO, USA). The
hGH assays were performed using an hGH ELISA kit
(
Fig. 1, lanes 1, 3, and 5). No protein complex correspond-
ing to the VDR was detected in the cytosolic fractions
extracted from the transfected COS-7 cells (Fig. 1, lanes 2,
(Boehringer Mannheim, Laval, Quebec, Canada).
4
, and 6), indicating that mutation of W286 does not affect
nuclear localization of the receptor.
Ligand binding assays
GST-VDR fusion proteins were expressed in bacteria and
the fusion proteins were then purified and quantified as
described previously. One microgram of purified protein
1
,25(OH) D ligand binding in vitro
2 3
Saturation binding revealed a single class of binding sites
was suspended in KTEDG buffer (10 mM Tris-HCl, pH 7.6, (Fig. 2A). Wild-type hVDR had a K of 1.536 Ϯ 0.087 nM.
d
1
mM EDTA, 300 mM KCl, 1 mM DTT, 0.1 mM phenyl- Mutation of tryptophan 286 of the hVDR to an alanine
methylsulfonyl fluoride [PMSF], and 1 Complete Mini pro- completely abolished the capacity of the receptor to bind to
tease inhibitor cocktail tablet [Boehringer Mannheim]) to a 1,25(OH) D while mutation of tryptophan 286 to phenyl-
2
3
volume of 170 ␮l. The proteins were incubated with varying alanine did not considerably interfere with ligand binding,
3
concentrations of [H ]-1,25(OH) D (Mandel Scientific- giving a K of 1.216 Ϯ 0.110 nM (Fig. 2B).
2
3
d

4
2
SOLOMON ET AL.
FIG. 3. Ability of in vitro translated wild-type VDR,
VDR W286A, and VDR W286F to interact with GST-
RXR␣ LBD fusion proteins. Experiments were performed
with GST alone (lanes 4,7, and 10) or GST fusion proteins
containing the LBD from the human RXR␣ (lanes 5, 6, 8, 9,
1
1, and 12) incubated with either in vitro translated wild-
type VDR (lanes 4–6), VDR W286A (lanes 7–9), or VDR
Ϫ7
W286F (lanes 10–12), in the presence or absence of 10
M
1
,25(OH) D as indicated. Aliquots (0.5 ␮l) of the in vitro
2
3
FIG. 2. Saturation ligand binding and Scatchard analysis
of wild-type and mutant GST-hVDR LBDs. GST proteins
were prepared as described and 1 ␮g of each was incubated
translation products are shown in lanes 1–3 as indicated.
3
with increasing concentrations of [ H]-1,25(OH) D in the
2
3
absence or presence of an excess of unlabeled 1,25(OH) D
was monitored on an mOP VDRE. The wild-type VDR
2
3
for 16 h at 4°C. (A) Saturation binding curve showing the (Fig. 4, lanes 1–3) and the W286F mutant (Fig. 4, lanes 7–9)
specific binding of the wild-type receptor (Ⅺ), the GST formed complexes with hRXR␣ on the mOP VDRE, which
protein alone (f), W286A (E), and W286F (ࡗ). Each point could be enhanced to similar degrees by addition of 10Ϫ7
M
represents the mean Ϯ SEM of three determinations. (B) 1,25(OH) D and supershifted with an anti-RXR antibody
2
3
Saturation binding data were transformed to yield the indi- (Fig. 4, lanes 3 and 9). However, the extracts from COS-7
cated dissociation constants (K ) and SEM.
cells transfected with hRXR␣ and the W286A mutant did
d
not respond to 1,25(OH) D as indicated by the absence of
2
3
enhanced complex formation in the presence of ligand (Fig.
, lane 5), thus indicating that the W286A mutant is com-
pletely defective for heterodimerization with hRXR␣.
4
Interaction of wild-type and mutant VDRs with hRXR␣
in vitro
Heterodimerization of wild type and mutant VDR LBDs
was examined by in vitro translation (Fig. 3, lanes 1–3) and
Interaction of the mutant VDRs with coactivators
in vitro
incubation with GST-hRXR␣ fusion proteins in the pres-
Ϫ7
ence or absence of 10
M 1,25(OH) D . The W286F
GST fusion proteins of the wild-type and mutant VDR
2
3
mutant displayed reduced levels of ligand-dependent inter- LBDs were incubated with in vitro translated SRC-1 in the
Ϫ7
action with the GST-hRXR␣ fusion protein (Fig. 3, lane 12) presence or absence of 10 M 1,25(OH) D (Fig. 5). The
2
3
as compared with the wild-type VDR (Fig. 3, lane 6). In wild-type VDR and W286F mutant interacted with SRC-1
contrast, the W286A mutant did not interact with hRXR␣ in in a ligand-dependent manner (Fig. 5, lanes 5 and 9, respec-
the presence of 1,25(OH) D above basal levels (Fig. 3, tively). In contrast, the W286A mutant was unable to bind
2
3
lanes 8 and 9). Each of the in vitro translated VDR proteins SRC-1 (Fig. 5, lane 7).
that were incubated with GST alone as a control did not
show any significant interaction (Fig. 3, lanes 4, 7, and 10).
Ligand-dependent transactivation in COS-7 cells
transfected with wild-type or mutant VDRs
Heterodimerization was analyzed further in gel mobility
shift assays with full-length VDR. Under these conditions,
DNA binding domain dimerization on a VDRE would be
Transcriptional activation in response to increasing con-
Ϫ10
Ϫ7
expected to stabilize any weak interaction between hRXR␣ centrations of 1,25(OH) D (10 M–10 M) was exam-
2
3
and hVDR. Cellular extracts were prepared from COS-7 ined by cotransfecting into COS-7 cell plasmids expressing
cells transfected with hRXR␣ and wild-type or mutant wild-type hVDR or the mutants W286A or W286F and the
VDRs, and formation of specific VDR/hRXR␣ complexes mOP3 reporter construct, which contains three repeats of

TRYPTOPHAN 286 OF THE hVDR IS CRITICAL FOR LIGAND BINDING
43
FIG. 5. Ability of the in vitro translated coactivator
SRC-1 to interact with GST-VDR, GST-VDR W286A, and
GST-VDR W286F fusion proteins. Experiments were per-
formed with GST alone (control; lanes 2 and 3), GST-VDR
wild-type (lanes 4 and 5), GST-VDR W286A (lanes 6 and
7
), or GST-VDR W286F fusion proteins (lanes 8 and 9) in
Ϫ7
the presence or absence of 10 M 1,25(OH) D as indi-
2
3
cated. An aliquot (0.5 ␮l) of the in vitro translation product
FIG. 4. Heterodimerization of the wild-type and mutant is shown in lane 1.
VDRs with hRXR␣. Cellular extracts from COS-7 cells
transfected with either wild-type VDR (lanes 1–3), VDR
W286A (lanes 4–6), or VDR W286F (lanes 7–9), and
hRXR␣ expression plasmids were treated as described in
the Materials and Methods section and incubated in the pres-
Ϫ7
ence or absence of 10 M 1,25(OH) D and an anti-RXR
2
3
antibody as indicated. Diamonds indicate the presence of the
VDR/RXR heterodimerization complex while asterisks indi-
cate the presence of the supershifted VDR/RXR complex.
(14)
the mOP VDRE upstream of a CAT reporter gene.
A
reporter plasmid encoding hGH was used as an internal
(15)
control for transfection efficiency.
Results were ex-
pressed as a percent of vehicle-treated control values. The FIG. 6. Effect of 1,25(OH) D on transcriptional activa-
2
3
ligand-dependent transcriptional activation in cells trans- tion via wild-type and mutant hVDRs. COS-7 cells were
(13)
fected with the W286F receptor was attenuated severely transfected with the mOP3-CAT reporter plasmid,
the
whereas no transactivation was observed in cells transfected GH expression plasmid long terminal repeat driven GH
(14)
with the W286A mutant (Fig. 6).
expression plasmid (pLTR-GH),
and either wild-type
VDR, the W286A mutant VDR, or the W286F mutant
VDR. The cells were transfected as described in the Mate-
rials and Methods section and subsequently treated with 5%
charcoal stripped FBS and increasing concentrations of
DISCUSSION
Ϫ10
Ϫ7
This study shows that tryptophan 286 of the hVDR is 1,25(OH) D (10 –10 M). CAT activity was assayed
2
3
critical for both high affinity binding of 1,25(OH) D to the after 24 h and normalized for transfection efficiency by the
2
3
receptor and for subsequent heterodimerization and gene corresponding hGH activity. Each value represents the
transactivation. W286 mutants heterodimerized less effi- mean Ϯ SD of three determinations and is representative of
ciently with RXR␣ in GST pull-down assays (Fig. 3). In three different experiments. Asterisks indicate a significant
addition, mutation of W286 abolished transactivation in difference from vehicle-treated control values (100%),
gene transfer experiments and in the case of the alanine while open circles indicate a significant difference between
mutation eliminated the ligand-dependent interaction of the cells transfected with wild-type VDR and W286F or
receptor with the coactivator SRC-1 in GST pull-down W286A at the 1,25(OH) D concentration indicated.
2
3
assays (Figs. 5 and 6). Nuclear fractions of COS-7 cells
transfected with wild-type, W286A, or W286F mutants
contained similar amounts of receptor (Fig. 1). Thus, mu-
The substitution of W286A was increasingly more dis-
tation of W286 did not affect hVDR expression levels or ruptive than the more conservative W286F mutation. The
nuclear localization of the hVDR. W286A mutant was completely defective for ligand bind-

4
4
SOLOMON ET AL.
TABLE 1. EFFECT OF NATURAL AND SYNTHETIC MUTATIONS IN THE HVDR ON LIGAND BINDING, TRANSACTIVATION, AND
HETERODIMERIZATION WITH HRXR〈
1
,25(OH) D
Heterodimerization
2
3
Mutation in hVDR
Type
interaction
Transactivation
with hRXR␣
a
H305Q
R271L
Natural
Natural
Reduced 8-fold
Reduced 100-fold
Reduced 5- to 10-fold
Normal in the
presence of 10
ϩϩϩ
ND
b
Ϫ6
M 1,25(OH) D
Reduced
2
3
c
I314S
Natural
Natural
Synthetic
Comparable to
wild type
Comparable to
wild type
ϩϩ
ϩ
c
R391C
Reduced
d
C288G
Reduced 3-fold at
Normal in the
presence of 10
ϩϩϩ
Ϫ6
4
°C
M 1,25(OH) D
2
3
W286A
W286F
Synthetic
Synthetic
Completely
abolished at 4°C
Attenuated at 4°C
Completely abolished
0
Completely abolished
ϩϩ
Tryptophan 286 of hVDR is absolutely required for 1,25(OH) D binding and transactivation.
2
3
ND, not determined.
a
b
c
d
18
Kristjansson et al.
19
Malloy et al.
Whitfield et al.
20
16
Nakajima et al.
ing, heterodimerization, interaction with SRC-1 in vitro, SRC-1 was observed (Fig. 5). This, along with the obser-
and transactivation. The W286F receptor retained its ability vation of ligand-dependent heterodimerization of the
to interact with 1,25(OH) D in ligand binding assays (Fig. W286F mutant, suggests that it can form activated DNA-
2
3
2
). However, we did detect slightly attenuated heterodimer- bound heterodimers at high concentrations in vitro. Despite
ization of W286F with RXR␣ in GST pull-down assays and the ligand-inducible interaction of W286F with SRC-1, it is
unaffected heterodimerization with RXR␣ in gel mobility possible that this mutant is unable to bind other important
shift assays (Figs. 3 and 4). Taken together, mutation of the coactivators for hVDR. This may explain the lack of ligand-
large, aromatic tryptophan residue to alanine eliminates dependent transactivation observed with this receptor mu-
ligand binding while mutation to the more conservative tant and implies that W286 may be essential for interaction
large, aromatic residue phenylalanine eliminates transacti- of the VDR with components of the transcription machinery
vation. Therefore, tryptophan 286 is critical for both ligand in addition to its role in the stabilization of ligand binding.
binding and transactivation.
The vitamin D receptor mediates the bone mineral ho-
The crystal structure of the hormone binding domain of meostatic actions of 1,25(OH) D and therefore natural
2
3
(
3)
RXR␣ showed that ligand interacts with a hydrophobic mutations in the VDR often result in hereditary hypocalce-
(
17)
pocket formed by helices 5 and 7, the C terminus of helix 10 mic vitamin D–resistant rickets (HVDRR).
Several mu-
W286 is tations have been characterized that are located within the
positioned in an area corresponding to the ␤-strand region LBD of the VDR and result in the HVDRR phenotype
(16)
and the N terminus of helix 11, and ␤-strands.
(18)
(19)
of the VDR LBD. Indeed, it recently has been found that including R271L
and H305Q.
In the VDR, R274L is
W286 directly contacts bound 1,25(OH) D via packing located in helix 5, H305Q is in the loop between helix 6 and
2
3
interactions between the aromatic ring of tryptophan and the helix 7, and W286 is positioned in the ␤-turn between helix
A-ring of 1,25(OH) D (Dino Moras, personal communica- 5 and helix 6, all at important interfaces for ligand binding.
2
3
tion, 1999). This is consistent with our observation that Mutagenesis studies with the VDR indicate that the LBD is
(20)
replacement of tryptophan 286 with a nonaromatic amino well aligned with those in the hRXR␣ and hRAR␥.
acid such as alanine completely abolishes ligand binding. Tryptophan is critical for ligand binding and transactivation
(6)
(8)
Both the conservative phenylalanine and the more dra- in the hRAR␥, hRXR␣, and hVDR, and conservation of
matic alanine substitution completely eliminated transacti- the unique tryptophan in the LBD of other members of the
vation by the VDR, even at high ligand concentrations. nuclear receptor superfamily provides insight concerning
Interaction of the W286A mutant with SRC-1 was elimi- the putative structure of the VDR with respect to the
nated, consistent with effects of this mutation on ligand hRXR␣ and hRAR␥. Furthermore, mutation of cysteine 288
binding and thus ligand-dependent movement of the AF-2 (C288G) was shown to reduce the affinity of the receptor for
(16)
domain from an inactive to an active conformation. How- 1,25(OH) D .
Table 1 summarizes the effect of natural
2
3
ever, significant interaction of the W286F receptor with and synthetic mutations characterized within the LBD of the

TRYPTOPHAN 286 OF THE hVDR IS CRITICAL FOR LIGAND BINDING
45
VDR thus far on ligand binding and transactivation. Tryp-
tophan 286 is the only amino acid shown absolutely to be
required for 1,25(OH) D interaction with hVDR and trans-
activation.
In summary, our experiments have shown that tryptophan
9. Jin CH, Kerner SA, Hong MH, Pike JW 1996 Transcriptional
activation and dimerization functions in the human vitamin D
receptor. Mol Endocrinol 10:945–957.
2
3
1
0. Green S, Isseman I, Scheer E 1988 A versatile in vivo and in
vitro eukaryotic expression vector for protein engineering.
Nucleic Acids Res 16:369.
2
86 is essential for 1,25(OH) D interaction with hVDR. 11. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR 1989 Site
2 3
Mutation of tryptophan 286 to alanine completely abolishes
the ability of the receptor to interact with 1,25(OH) D .
This, in turn, affects the ligand-activated functions of the
receptor including heterodimerization, interaction with co-
regulatory proteins, and transactivation.
directed mutagenesis by overlap extension using the polymer-
ase chain reaction. Gene 77:51–59.
2. Solomon C, Sebag M, White JH, Rhim J, Kremer R 1998
Disruption of vitamin D receptor-retinoid X receptor het-
erodimer formation following ras transformation of human
keratinocytes. J Biol Chem 273:17573–17578.
2
3
1
1
3. Sebag M, Henderson J, Rhim J, Kremer R 1992 Relative
resistance to 1,25-dihydroxyvitamin D3 in a keratinocyte
model of tumor progression. J Biol Chem 267:12162–12167.
4. Ferrara J, McCuaig KA, Hendy GN, Uskokovic M, White JH
ACKNOWLEDGMENTS
1
We are grateful to M. Gratton, I. Bolivar, and S.S. Sol-
omon for technical assistance and to Dino Moras (Illkirch,
France) for communicating crystallographic data before
publication. R.K. is a recipient of Medical Research Council
of Canada grant MT-10839. J.H.W. is a recipient of Medical
Research Council of Canada grant MT-11704 and is a
chercheur-boursier of the Fonds de Recherche en Sant e´ du
Qu e´ bec.
1
994 Highly potent transcriptional activation by 16-ene deriv-
atives of 1,25-dihydroxyvitamin D . Lack of modulation by
9-cis-retinoic acid of response to 1,25-dihydroxyvitamin D3 or
its derivatives. J Biol Chem 269:2971–2981.
3
1
5. Banville D, Boie Y 1990 Retroviral long terminal repeat is the
promoter of the gene encoding the tumor associated calcium-
binding protein oncomodulin in the rat. J Mol Biol 207:481–
4
90.
16. Nakajima S, Hsieh J, Jurutka P, Galligan M, Haussler C,
Whitfield G, Haussler M 1996 Examination of the potential
functional role of conserved cysteine residues in the hormone
binding domain of the human 1,25-dihydroxyvitamin D re-
ceptor. J Biol Chem 271:5143–5149.
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Address reprint requests to:
Richard Kremer
687 Pine Avenue West
Room H4–67
Montreal, Quebec, H3A 1A1, Canada
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. Cheng L, Norris AW, Tate BF, Rosenberger M, Grippo JF, Li
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