(Received for publication, April 25, 1996, and in revised form, October 25, 1996)
From the Department of Medical Specialties,
University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 and the Department of Biochemistry, University of California,
Riverside, California 92521
An important focus of structure-function studies
of synthetic ligands for the vitamin D receptor (VDR) concerns the
chiral center at carbon 20 of the steroid side chain;
20-epi analogues are 100-10,000 times more potent
transcriptionally than the natural hormone 1,25-dihydroxyvitamin
D3 (1
,25-(OH)2D3). We have
compared the binding properties of three pairs of analogues either with a natural (N) or 20-epi (E) orientation. In intact cells,
45-60% of VDR·N-analogue complexes, but only 5-20% of
VDR·E-analogue complexes, dissociated over a 3-h interval. The two
groups of ligands induced distinct changes in VDR conformation as
revealed by protease clipping assays. Mapping of ligand-VDR binding
activity by deletions indicated that amino acids 420-427 were
important for high affinity of VDR·N-analogue complexes, but not for
VDR·E-analogue complexes. Site-directed mutagenesis revealed that
residues 421 and 422 were essential for
1
,25-(OH)2D3-induced conformational changes,
high affinity of 1
,25-(OH)2D3 for VDR, and
transcriptional activity, but not for binding of its 20-epi
analogue. In contrast, deletion of residues 396-427 abolished binding
of 1
,25-(OH)2D3, but binding of its
20-epi analogue was still detectable. The results suggest
that the ligand-binding domain of VDR has multiple and different
contact sites for the two families of side chain-modified ligands,
resulting in VDR·ligand complexes with different half-lives and
transcriptional activities.
The action of the vitamin D receptor
(VDR),1 like that of other nuclear
receptors, is dependent primarily on interaction with its biologically
active ligand, 1,25-dihydroxyvitamin D3 (1-3). The
binding of ligand to its nuclear receptor leads to conformational changes in the receptor (4-6), promotes self-dimerization and heterodimerization with the retinoid X receptor (RXR) (7-11), and
enhances binding to DNA and transcriptional activities (12-15). It is
generally accepted that the transcriptional activities of nuclear
receptors are directly correlated with their affinity for their
respective ligands (16-18). There are, however, several exceptions to
this rule. For example, the effective dose required to reach 50%
saturation (ED50) of the estrogen receptor by its ligand is
several orders of magnitude greater than the ED50 for maximal transcriptional activity (19). A possible explanation for this
is that ligand-activated estrogen receptor acts in a cooperative manner
with unoccupied estrogen receptor molecules during interaction with its
DNA response element or with the transcriptional apparatus, whereas
other steroid hormone receptors do not. Another exception is the
progesterone antagonist RU486; this synthetic ligand binds tightly to
the progesterone receptor, but does not induce transcriptional activity
(20). The explanation for this discrepancy is that the
analogue/antagonist interacts with the progesterone receptor at a
different site from progesterone, thus creating a unique conformational
change in the receptor and preventing its normal action (21).
Recently, we began to analyze the mechanism of action of analogues of
1,25-(OH)2D3 that regulate receptor-mediated
transcription more effectively than the natural hormone, although their
affinity for VDR is not greater (6). For example, a concentration of 10
11 M is required to induce 50% of maximal
DNA binding and transcriptional activities of VDR by the
20-epi analogue MC 1288 (1-E; see Fig. 1), but this
concentration of 1-E is 200-fold lower than the ED50 of
2 × 10
9 M for saturation of VDR binding
sites in equilibrium (6, 22). These results suggest that enhanced
activation of VDR occurs after binding of the analogue to VDR, but
before induction of transcription. Because analogue 1-E induces a
unique conformational change in VDR in vitro and enhances
dimerization of VDR with RXR in vivo (6), we speculated that
the conformation of 20-epi analogue-activated VDR promotes
better binding to DNA by stabilizing the VDR heterodimer. The
conformational differences between
1
,25-(OH)2D3·VDR and 20-epi analogue·VDR complexes are probably due to differences in the sites
where ligands contact the receptor, as are the differences in the
interaction of progesterone and RU486 with the progesterone receptor
(21). To test this hypothesis, it is necessary to map the
ligand-binding domain of VDR, to identify the amino acids that are
required for the binding of 1
,25-(OH)2D3 to
it, and to determine whether the same amino acids are also required for binding of the 20-epi analogues (E-analogues). In the study
presented here, we identified the amino acids required for interaction
of 1
,25-(OH)2D3 at the C-terminal region of
VDR. We also showed that the same amino acids were required for binding
of 1
,25-(OH)2D3 and analogues with a natural
orientation of the side chain (N-analogues). However, all of the
E-analogues examined had different binding requirements at this region,
and these differences in binding requirements were associated with
increased stability of the ligand-receptor complexes.
Synthetic oligonucleotides were prepared by the
Macromolecular Synthesis and Analysis Facility of the M. D. Anderson
Cancer Center. [35S]Methionine and
1,25-(OH)2[26,27-3H]D3 were
obtained from Amersham Corp. A coupled transcription/translation kit
and a site-directed mutagenesis kit were obtained from Promega. The
analogues used in this study were a generous gift from Dr. L. Binderup
(Leo Pharmaceuticals, Ballerup, Denmark). The structural formulas for
and abbreviations of these ligands are shown in Fig. 1.
Single-stranded DNA templates were prepared from Escherichia coli JM109 cells bearing the plasmid pAlter (Altered Sites in vitro mutagenesis system, Promega) with an insert encoding the wild-type human VDR cDNA, a tetracycline resistance gene, and a mutated ampicillin resistance gene. A VDR primer with the desired mutation and an oligonucleotide containing repair sequences for the mutation in the ampicillin resistance gene were annealed to the single-stranded DNA, and then synthesis of complementary strand was catalyzed by T4 DNA polymerase. Synthetic plasmids were transformed into E. coli strain BMH71-18 (mutS) and isolated from ampicillin-resistant colonies. Plasmid DNA was transformed into JM109 for a second screening, and then DNA was isolated from individual colonies and screened for mutations by DNA sequencing through the mutated site. Subsequently, the DNA coding for a mutant VDR was subcloned into the plasmid pGEM-4 for in vitro studies with synthetic receptor or into a eukaryotic expression vector for functional studies in intact cells.
Cell Culture and TransfectionsRat osteosarcoma ROS 17/2.8 cells and monkey kidney CV-1 cells were plated in 35-mm dishes at a density of 3 × 105/dish. ROS 17/2.8 cells were transfected with 2 µg of plasmid ocVDRE containing the vitamin D-responsive element (VDRE) from the human osteocalcin gene (GGTGACTCACCGGGTGAACGGGGGCATT) (23). This response element was attached to the thymidine kinase promoter/growth hormone fusion gene. Monkey kidney CV-1 cells were transfected with the osteocalcin VDRE/reporter fusion gene (4 µg/dish) and the recombinant human VDR expression vector (2 µg/dish). Monkey kidney COS-1 cells were plated in 150-mm dishes at a density of 6 × 105/dish and transfected with recombinant human VDR plasmids (20 µg/dish).
All transfections were performed by the DEAE-dextran method (6). Medium samples for measurements of growth hormone were collected 2 days after transfection. Growth hormone production from the reporter gene was measured by a radioimmunoassay as described by the manufacturer (Nichols Institute, San Juan Capistrano, CA).
Ligand Binding AssaysTo assess the relative affinity of
1,25-(OH)2D3 and the E-analogues for
wild-type and mutant VDRs in vitro, whole-cell homogenates from COS-1 cells transfected with VDR expression plasmids were prepared
in KTED buffer (0.3 M KCl, 10 mM Tris-HCl, pH
7.4, 1.5 mM EDTA, and 1 mM dithiothreitol) as
described previously (6). The homogenates were then aliquoted into
tubes containing 0.2 pmol of
1
,25-(OH)2[3H]D3 and
increasing concentrations of nonradioactive ligand. For Scatchard
analysis of 1
,25-(OH)2D3 binding to
wild-type and mutant VDRs, the homogenates were incubated with
increasing concentrations of
1
,25-(OH)2[3H]D3 (0.05-5
nM). The mixtures were incubated on ice for 3-4 h, and
then the free ligand was separated from bound by hydroxylapatite (24).
The bound ligand was released from the hydroxylapatite by ethanol
extraction, and the radioactivity was measured by scintillation counting. For the competition assays, the results were plotted as the
inverse value of percent maximal binding against competitor concentration by the method of Wecksler and Norman (24).
To assess receptor occupancy by the ligand as well as the dissociation
rate of the ligand in vivo, monolayers of VDR-transfected COS-1 cells were washed three times in PBS and incubated for 1 h
with ligand in serum-free medium. Then the medium was discarded, the
cells were washed three times in PBS, and fresh medium was added. At
various times, the medium was discarded again, and the cells were
washed three times in cold PBS, scraped into 10 ml of PBS, centrifuged,
resuspended in KTED buffer , and homogenized. Aliquots (0.2 ml) of the
homogenates were incubated on ice for 3-4 h with 0.2 pmol of
1,25-(OH)2[3H]D3 with or
without a 100-fold excess of unlabeled ligand. To assess the number of
unoccupied VDR sites, the free ligand was separated from the bound by
hydroxylapatite as described above.
To assess exchange of unlabeled 1,25-(OH)2D3
or its analogues with
1
,25-(OH)2[3H]D3, monolayers
of VDR-transfected COS-1 cells were washed three times in PBS and
incubated for 1 h with ligand in serum-free medium. Then the
medium was discarded, and the cells were washed three times in cold
PBS, scraped into 10 ml of PBS, centrifuged, resuspended in KTED
buffer, and homogenized. Aliquots of the homogenates (0.2 ml) were
incubated at 30 °C with 0.2 pmol of
1
,25-(OH)2[3H]D3 for various
times and then transferred to ice for an additional 3 h. The free
radioactive ligand was separated from the bound by hydroxylapatite as
described above. Exchange was assessed by comparing the amount of
1
,25-(OH)2[3H]D3 bound to
unoccupied VDR and the amount of
1
,25-(OH)2[3H]D3 bound to
in vivo bound VDR at each time point.
Synthetic wild-type
and mutant human VDRs labeled with [35S]methionine (1000 Ci/mmol) were prepared by in vitro coupled
transcription/translation in reticulocyte lysates (Promega) with the
human VDR cDNA inserted into the pGEM-4 plasmid. The receptor
preparations were incubated with the indicated concentrations of
1,25-(OH)2D3 or analogues for 10 min at room
temperature. Then 0-25 µg/ml trypsin (Calbiochem) was added, and the
mixtures were incubated for another 10 min. The digestion products were
analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis, and the gels were dried and autoradiographed.
A recent study from our
laboratories showed that the E-analogue 1-E had a 200-fold greater
transcriptional activity through a VDRE than did
1,25-(OH)2D3 (6). Another E-analogue with a
modified chemistry of the side chain, 3-E, also had enhanced transcriptional activity. These results suggested that side chains with
20-epi configuration facilitated receptor action. To further study the role of the chemistry and stereochemistry of the side chain
in VDR actions, we examined the transcriptional activity of three
E-analogues and their N-analogue homologues (Table I). The transcriptional activity of these ligands was examined in ROS
17/2.8 cells transfected with a fusion gene containing the osteocalcin
VDRE fused to the thymidine kinase promoter/growth hormone reporter
gene. The cells were treated with the ligand for 1 h in serum-free
medium to allow ligand uptake and saturation of cellular VDR
ligand-binding sites in the absence of serum-binding proteins. The
results of this assay (Table I) showed that extension of the
1
,25-(OH)2D3 side chain (as in 2-N) enhanced
transcriptional activity by 50-fold, but the addition of oxygen at
position 22 (as in 3-N) to this chemically modified side chain
decreased transcriptional activity by 2-fold relative to 2-N. The
20-epi stereochemistry of each of these side chains further
enhanced their transcriptional activities. The E-analogue 2-E had the
greatest transcriptional activity, up to 5000-fold greater than that of
1
,25-(OH)2D3. Therefore, we concluded that
the 20-epi configuration enhanced the transcriptional
activity of these ligands whether or not their side chains were
chemically modified.
|
In our previous study (6), we confirmed the earlier report
(Binderup et al. (22)) that the increased growth inhibitory and differentiating activities of the E-analogues were not associated with a greater affinity for VDR. However, another aspect of ligand binding was not tested in these studies: the stability of the ligand-receptor complexes in nonequilibrium. This is an important aspect of analogue action because, in our experiments, cellular VDR
binding sites were first saturated with the ligand under equilibrium conditions, but then the ligand was removed. Therefore, we tested the
transcriptional activity of the VDR·ligand complexes under nonequilibrium conditions. Similar conditions may develop naturally during prolonged incubation of cell cultures with the ligand or in vivo when the ligand is removed by catabolism. Therefore,
we tested the possibility that the enhanced transcriptional activity of
the analogues may in part be due to a change in their rate of
dissociation from the VDR complexes. VDR-transfected COS-1 cells were
incubated with 108 M
1
,25-(OH)2D3 or its analogues for 1 h;
the excess ligand was then removed; and the rate of appearance of
unoccupied binding sites was measured over the next 3 h (Fig.
2A). We found that unoccupied binding sites
appeared at a rapid rate when cellular VDR was first saturated with
1
,25-(OH)2D3 or with the N-analogues: in
3 h, only 35% of the VDR binding sites remained occupied with 1
,25-(OH)2D3, and 50% of the VDR binding
sites remained occupied with the analogues 2-N and 3-N. We also
concluded that the chemistry of these analogues' side chains did not
have a significant effect on the stability of the ligand-receptor
complexes. On the other hand, 20-epi configuration of these
side chains significantly slowed down the replenishment of unoccupied
binding sites. After 3 h of incubation, 80% of the VDR binding
sites remained occupied by the E-analogues 1-E and 3-E, and all of the
binding sites remained occupied by the most active 20-epi
analogue, 2-E. Therefore, we conclude that ligands with a natural
orientation of the side chain dissociate rapidly from VDR, whereas
ligands with the 20-epi side chain either dissociate very
slowly from VDR or induce irreversible inactivation of VDR
ligand-binding sites by an unknown mechanism.
To determine whether the increase in the number of unoccupied binding
sites in intact cells was due to dissociation of ligand from VDR or due
to reactivation of inactive VDR, we performed an in vitro
exchange assay. Again, VDR-transfected COS-1 cells were incubated with
a 108 M concentration of each ligand for
1 h in serum-free medium; the ligands were removed; and the cells
were washed three times and homogenized. The homogenates were incubated
at 30 °C with 1
,25-(OH)2[3H]D3, and exchange
of unlabeled and radioactive ligands was measured at various times. The
results (Fig. 2B) show that the exchange rate of
receptor-bound unlabeled 1
,25-(OH)2D3 with
1
,25-(OH)2[3H]D3 in
vitro was very rapid in the first 15 min of incubation: 55% of
the receptor-bound unlabeled ligand was exchanged for the radioactive
ligand. On the other hand, 45% of the receptor binding sites did not
bind the radioactive ligand even after 1 h of incubation. A
similar pattern of rapid exchange of ligand from a high percentage of
the occupied binding sites was seen with the two N-analogues. However,
rapid exchange of 1-E occurred with only 20% of the occupied VDR
binding sites, and no detectable rapid exchange occurred with VDR
binding sites occupied with the analogue 2-E. Only 3-E had an exchange
rate similar to that of its N-analogue homologue (3-N). So, although
the dissociation rate of this analogue in the cells was very slow, its
exchange rate in vitro was not affected by the orientation
of the side chain. It is possible that the presence of the oxygen atom
at position 22 controls the interaction with the ligand-binding domain
so that the orientation of the side chain does not affect the complex
process of exchange that requires the simultaneous movement of 3-E out
of the ligand-binding domain and
1
,25-(OH)2[3H]D3 into it.
The results presented above, together with our earlier
observations of differences in the conformation of VDR complexed with 1,25-(OH)2D3 and the E-analogues (6),
support the hypothesis that the mode of interaction of the
20-epi analogues with VDR is different from that of
1
,25-(OH)2D3 and the N-analogues. One possible explanation for these differences is that the contact points
of the 20-epi analogues with the ligand-binding domain are
different from the contact points of ligands with natural side chain
stereochemistry. To test this hypothesis, we mapped the ligand-binding
domain of VDR by deletions, testing the binding activities of the
mutants to 1
,25-(OH)2D3 and the analogues. The mutation strategy was based on earlier studies by McDonnell et al. (25) and Nakajima et al. (7), who
identified a C-terminal sequence between amino acids 390 and 427 as
essential for ligand binding activity. To further map this region, we
prepared deletion mutants by inserting stop codons at the positions
shown in Fig. 3A. These deletion mutants were
first examined for protein expression in COS-1 cells and then for
binding and transcriptional activities. Analysis by Western blotting
showed that all the mutants in Fig. 3 expressed similar levels of
immunoreactive VDR in COS-1 cells (data not shown). Fig. 3C
shows that the transcriptional activity of VDR was abolished by
deleting the C-terminal residues 420-427. This deletion did not
abolish the binding activity of 1
,25-(OH)2D3 to VDR (Fig. 3B), but did decrease it by 50-70%. Further
deletion of residues 410-420 had no marked effect on binding of
1
,25-(OH)2D3 to VDR, but deleting residues
403-410 induced an additional dramatic decrease, leaving only residual
binding activity. This residual binding activity was completely
abolished by deleting residues 396-403. From these mapping
experiments, we concluded that at the C-terminal region, the amino
acids important for binding of 1
,25-(OH)2D3
were located between residues 420 and 427 and between residues 396 and
410. Therefore, some of the amino acids required for binding of
1
,25-(OH)2D3 overlap with amino acids that
are essential for transcriptional activity of VDR.
Comparison of the Binding Requirements of the E- and N-analogues
To determine whether the binding requirements of the
E-analogues were different from those of the N-analogues, we performed binding assays in which wild-type or mutant VDRs were incubated with
1,25-(OH)2[3H]D3 and
increasing concentrations of unlabeled competitors. The data were
plotted as the inverse value of percent maximal binding against
competitor concentration. The slopes of the linear plots reflect the
affinity of each ligand for the
1
,25-(OH)2D3-binding site in wild-type and
mutant VDRs. Fig. 3D shows that the slope of the competition
plot of 1
,25-(OH)2D3 was significantly
reduced by deleting residues 420-427, suggesting that the affinity of 1
,25-(OH)2D3 for this mutant was reduced.
There was no significant reduction in the slope of the competition plot
of 1
,25-(OH)2D3 using VDR that had an
additional deletion of residues 410-420, suggesting that this deletion
did not introduce another change in the affinity of
1
,25-(OH)2D3 for the receptor. To confirm the change in affinity of these deletion mutants for
1
,25-(OH)2D3, we performed saturation
assays. Scatchard analysis of these assays in one experiment revealed
that the equilibrium dissociation constants (Kd) for
1
,25-(OH)2D3 binding to wild-type VDR, to
deletion mutant 420/TGA, and to deletion mutant 410/TAA were 0.9, 4.5, and 3.75 nM, respectively. In another experiment, the
Kd values were 1.2 nM (wild-type VDR),
7.8 nM (mutant 420/TGA), and 7.5 nM (mutant
410/TAA).
The competition assays with the E-analogue 1-E showed that deleting
residues 420-427 had no effect on the ability of 1-E to compete for
the binding of
1,25-(OH)2[3H]D3 to VDR, but
deleting residues 410-420 induced a small but consistent reduction in
the ability of 1-E to compete for the binding of
1
,25-(OH)2[3H]D3 to VDR.
Therefore, we concluded that the last seven amino acids of VDR were
important for the binding of 1
,25-(OH)2D3, but not for 1-E, and that residues 410-420 seem to be more important for the binding of the E-analogue than for the binding of
1
,25-(OH)2D3.
Because the most significant difference in the binding requirements of
1,25-(OH)2D3 and the E-analogue was within
residues 420-427, we wished to determine if this difference was indeed due to the stereochemistry of the side chain and not due to its chemistry. Therefore, we repeated the competition assays with the six
ligands shown in Fig. 1, using wild-type VDR and the VDR deletion
mutant 420/TGA. Fig. 4 shows that the ability of
1
,25-(OH)2D3 and the N-analogues to compete
for the binding of
1
,25-(OH)2[3H]D3 to VDR was
significantly reduced by removal of residues 420-427. However, this
deletion had either little or no effect on the ability of the
E-analogues to compete for the binding of
1
,25-(OH)2[3H]D3 to VDR. The
results of these experiments strongly suggest that the binding
requirements of the E-analogues are upstream from the eight C-terminal
amino acids of VDR, although we do not know which other residues are
required for binding of these compounds.
To determine which residues between positions 420 and 427 were
important for binding of 1,25-(OH)2D3, we
mapped this region by point mutations (Fig.
5A). The glutamic acid residue at position 420 was replaced with alanine (E420A). The two hydrophobic residues (valine at position 421 and phenylalanine at position 422) were mutated
simultaneously to methionine and alanine, respectively (V421M/F422A),
and the glutamic acid residue at position 425 was replaced with
glutamine (E425Q). Each of these mutants was tested for its
transcriptional activity in response to
1
,25-(OH)2D3 and its binding to
1
,25-(OH)2[3H]D3 by one-point
binding analysis and by competition assays. Fig. 5A shows
that amino acids 420-422 are located within a cluster of hydrophobic
residues. A comparison with other steroid hormone receptors showed that
this cluster overlaps a conserved transcriptional activation function 2 domain (TAF-2) (12, 13). As expected, these residues are also important
for transcriptional activity of VDR (Fig. 5B). Mutating the
glutamic acid residue at position 420 reduced the transcriptional
activity of VDR, and the double mutation of amino acids 421 and 422 completely abolished it. The latter mutant did not have detectable
transcriptional activity in response to the E-analogues (data not
shown). In contrast, mutating the glutamic acid residue at position
425, which is located outside this cluster, had no effect on
transcriptional activity.
These mutants were also tested by one-point binding assay for binding
of 1,25-(OH)2[3H]D3 (Fig.
5B) and for changes in affinity for
1
,25-(OH)2D3 and its E-analogue by
competition assays (Fig. 5C). In the one-point saturation
analysis, there was some decrease in the binding of 1
,25-(OH)2D3 to the VDR mutants E420A and
V421M/F422A, but no change in the binding activity of the VDR mutant
E425Q. When we tested the effect of these mutations on the affinity of
1
,25-(OH)2D3 and the E-analogue 1-E, we
found that only the double mutation at amino acids 421 and 422 significantly decreased the affinity of
1
,25-(OH)2D3 for VDR; the other point
mutations had no such effect. However, the ability of the E-analogue
1-E to compete for binding of
1
,25-(OH)2[3H]D3 to these
three mutants was the same as for wild-type VDR, suggesting that these
mutations had no effect on the affinity of 1-E for VDR. Therefore, we
concluded that amino acids downstream from residue 420 were completely
unnecessary for binding of the E-analogues, although residues 421 and
422 were required for transcriptional activity of these ligands.
Our recent studies have shown that binding of
1,25-(OH)2D3 changes the sensitivity of VDR
to protease digestion, producing a 34-kDa protease-resistant fragment
(probably encompassing the entire ligand-binding domain (25)) and a
28-kDa fragment. The binding of the E-analogues also induced resistance
to protease digestion, but with a different pattern: in addition to the
34- and 28-kDa protease-resistant fragments, there was a 32-kDa
fragment. Therefore, the conformation of VDR
·1
,25-(OH)2D3 complexes was clearly
different from the conformation of the VDR·E-analogue complexes. To
determine the relationship between ligand-induced receptor conformation
and its action, we performed a protease sensitivity assay with receptor
mutants (Fig. 6). Deleting residues 420-427 was
sufficient to diminish 1
,25-(OH)2D3-induced
resistance to protease digestion of synthetic VDR (Fig. 6A),
although this mutant receptor still had significant binding activity
(Fig. 3). Incubation of the same receptor mutant with the E-analogue
1-E did induce a significant resistance to protease digestion. However, no 34-kDa protease-resistant fragment was produced; only the 32- and
28-kDa fragments were seen, and their intensity was significantly greater than with wild-type VDR. Therefore, residues 420-427 were clearly required for proper receptor folding whether or not ligand binding activity was detectable.
Because deletion of these amino acids diminished transcriptional
activity, we presumed that the loss of amino acids that were necessary
for proper folding of VDR in response to the ligand also caused a loss
of transcriptional activity. To further test this hypothesis, we
examined the ligand-induced conformational changes in the three VDRs
with the point mutations described above (Fig. 6, C and
D) and in another mutant (E396Q/H397N) that had a wild-type
phenotype with respect to ligand binding and transcriptional activities
(data not shown). We found that 1,25-(OH)2D3
induced the wild-type pattern of resistance to protease digestion in
the VDR mutants E420A and E425Q and in E396Q/H397N, but this activity was completely lost with the double mutant V421M/F422A. When the effect
of the E-analogue 1-E on the conformation of these mutants was
examined, we found that it was identical to the wild type in mutants
E420A, E425Q, and E396Q/H397N, but had changed in the transcriptionally
inactive double mutant V421M/F422A. In this double mutant, the pattern
of resistance to protease digestion was similar to that induced by the
E-analogue 1-E in VDR by deleting residues 420-427: the 34-kDa
fragment was less intense, and the 32- and 28-kDa fragments were more
intense. From these experiments, we concluded that residues 421 and 422 were essential for both transcriptional activity of VDR and the
induction of correct conformational changes by both E-analogues and
1
,25-(OH)2D3. Furthermore, ligand binding
alone was not sufficient to induce either the proper conformational change in VDR or transcriptional activity.
The experiments described above strongly suggested that ligand-induced
resistance to protease digestion does not necessarily reflect the
affinity of VDR mutants for the ligand. However, it is reasonable to
assume that as long as ligand-dependent resistance to
protease digestion is detectable, VDR will still display ligand binding
activity. Therefore, the protease sensitivity assay could be used to
map the binding sites for the E-analogue 1-E in the deletion mutants
that could not be examined by the competition assays because they had
lost binding for
1,25-(OH)2[3H]D3. Fig. 6
(A and B) shows that the analogue 1-E continued
to induce resistance to protease digestion even after deletion of amino
acids 390-427 (390/TGA). The intensity of the protease-resistant fragment induced by deleting residues 390-427 was weaker than that of
the fragment induced by deleting residues 396-427 (396/TAA), but so
was the translation efficiency of the 390/TGA deletion mutant. To
determine whether there was a real difference in ligand-induced resistance to protease digestion between these two mutants, we assessed
the dose response to 1-E with the two mutants (Fig.
7) and found that they induced resistance to protease
digestion in the same concentration range, which confirmed that the
binding activity of 1-E depended not on residues between positions 390 and 396, but on residues farther upstream.
The results of the binding and competition assays are combined with the
results of the protease sensitivity assays and summarized schematically
in Fig. 8. From this illustration, we concluded that
critical amino acids required for binding of
1,25-(OH)2D3 were located between positions
420 and 427 and between positions 396 and 410. On the other hand, the
critical amino acids required for binding of 1-E were between positions
410 and 420 and between positions 396 and 403, and the residual binding
activity of this ligand depended on amino acids upstream from position
390. Therefore, the binding requirements of these two ligands, which
differ only in the orientation of their side chains, were clearly
different.
This study is a direct extension of our previous work on the
mechanism of action of analogues of
1,25-(OH)2D3 (6). In the previous study, we
demonstrated for the first time that the E-analogues can activate VDR
differently from the natural ligand and hypothesized that the
E-analogues interacted with VDR at contact points not used by the
natural hormone. We also speculated that the association at these
alternative contact points facilitated dimerization with RXR, binding
to DNA, and transcriptional activation of VDR. Here we confirmed a part
of this hypothesis by comparing the binding properties and amino acid
requirements for interaction of two groups of ligands with VDR: ligands
with natural side chain orientation and 20-epi analogues.
However, it is still necessary to investigate the relationships between
specific binding requirements of the analogues and facilitated
dimerization of VDR with RXR.
The ligand-binding domain of human VDR is a 37-kDa polypeptide
encompassing two-thirds of the receptor molecule. Earlier studies have
identified an upstream region between amino acids 114 and 166 (25) and
a downstream region between amino acids 390 and 427 that are essential
for binding of 1,25-(OH)2D3 to VDR (7, 25).
But these experiments did not define the exact residues that are
required for ligand interaction with VDR or their role in other
receptor functions. In our mapping experiments, we focused only on the
C-terminal region for technical convenience, but also because this
region has a significant degree of functional similarities and amino
acid homology to the ligand-binding sites of other steroid hormone
receptors (7, 13). Furthermore, we could use the information obtained
recently from the crystal structure of unoccupied RXR (26) and the
ligand-occupied retinoic acid receptor and thyroid hormone receptor
(27, 28) to interpret our results and to predict the location of
structures that are potential building blocks of the ligand-binding
pocket of VDR and the location of amino acids that probably contact the
ligand. The alignment of the C-terminal region of the ligand-binding
domain of VDR and RXR (using the Genetics Computer Group program to
predict secondary structures) in Fig. 9 clearly shows
that helices 7, 10, and 11, which form the putative ligand-binding
pocket B in RXR, aligned very well with similar secondary structures in
VDR. Interestingly, our mutational analysis showed that the C-terminal
structures homologous to helices 10 and 11 in RXR were also essential
for binding of 1
,25-(OH)2D3 and its
analogues to VDR. Furthermore, amino acids 396-410, which were
important for binding of 1
,25-(OH)2D3, overlapped with a region essential for contact of estradiol,
glucocorticoids, and androgens with their respective receptors
(29-33). These striking similarities in the mapping of ligand binding
activity strongly suggest that there is a certain rigidity of the
ligand-binding pocket in different steroid hormone receptors and that
the amino acids mapped in this study are probably contact sites for the ligands.
Does changing the stereochemistry of the side chain affect the
requirement of ligands for the amino acids between positions 396 and
410? Our binding analysis showed that residues 403-410 were important
for binding of 1,25-(OH)2D3 and that
residual binding activity was lost by deleting residues 396-403.
Analysis of the binding requirement of the E-analogue 1-E by
competition assays and by the protease sensitivity assay strongly
suggested that the residues required for binding of this analogue were
between positions 396 and 403 and upstream from position 390. Therefore, from these data, it appears that binding of the E-analogue
is shifted within the putative ligand-binding pocket into a region that
overlaps with the ninth heptad repeat of the dimerization domain (7).
We hypothesize that this shift in binding site also reflects a shift in
the physical contact of the analogue in the ligand-binding pocket and
away from the C terminus.
Another piece of evidence supporting a general shift in binding
requirements of the E-analogues was seen in a region closer to the C
terminus of VDR. The N-analogues and
1,25-(OH)2D3 required residues 420-427 for
interaction with VDR, whereas the E-analogues did not. The C-terminal
region contains a cluster of hydrophobic amino acids termed TAF-2. This
region is conserved in ligand-binding nuclear receptors (13), and its
function is to modulate transcriptional activity. In VDR, these
residues are also essential for maintenance of the appropriate
ligand-induced receptor conformational change. Because TAF-2 forms a
contact with the ligand in the thyroid hormone receptor and retinoic
acid receptor (27, 28), it is tempting to speculate that in VDR, these
TAF-2 residues actually make contact with side chains that have the
natural orientation, but not with 20-epi side chains.
A crystallography study of the unoccupied ligand-binding domain of RXR suggested that in the absence of ligand, the hydrophobic residues in TAF-2 are masked and therefore not available for protein-protein interaction with transcription coactivators or for interaction between various domains of the receptor (26). However, when the structure of the ligand-occupied retinoic acid receptor and the structure of unoccupied RXR were superposed, the most distinct conformational difference between the two was in TAF-2 (27). It has been suggested that when the ligand enters in the binding pocket, it induces a conformational change that modifies the steric position of TAF-2 residues so that they become available for protein-protein interactions. We hypothesize that in VDR, ligands with natural side chain orientation actually make contact with these hydrophobic residues, thus preventing optimal folding and exposure of these residues for interaction with coactivators. In contrast, the E-analogues, which do not use these residues for binding, change the conformation of VDR in a manner that allows an optimal folding of the hydrophobic residues and thus promotes a more efficient interaction with coactivators.
The general shift in the binding requirements of the E-analogues may
also explain their slow dissociation and exchange rates from VDR. We
speculate that when the E-analogues enter the binding pocket, the VDR
conformation changes so that the ligands become "locked" into the
binding pocket. On the other hand, the ligand-binding pocket remains
partially open after binding of 1,25-(OH)2D3
or the N-analogues, and this allows rapid dissociation of these ligands from VDR. The dissociation rate of these two groups of ligands may be
regulated by the hydrophobic residues at the C-terminal region.
However, we cannot exclude the possibility that amino acids inside the
ligand-binding pocket may also be important. To test this hypothesis,
it will be necessary to examine the effect of individual point
mutations on the dissociation rate of ligands with natural side chain
orientation and 20-epi analogues.
We thank Dr. L. Binderup for the generous gift of the analogues CB 966, KH 1139, MC 1288, MC 1301, and KH 1060. We thank Dr. J. W. Pike for the human vitamin D receptor expression plasmid. We also thank J. Zhu for assistance with the statistical analysis.