(Received for publication, September 6, 1995; and in revised form, November 14, 1995)
From the
Genomic actions of the calciotropic hormone
1,25-dihydroxyvitamin D
(1,25(OH)
D
) involves a multistep process
that is triggered by the highly specific binding of
1,25(OH)
D
to 1
,25-dihydroxyvitamin D
receptor, VDR. In order to study this key step in the cascade, we
synthesized 1
,25-dihydroxy[26(27)-
H]vitamin
D
-3-deoxy-3
-bromoacetate
(1,25(OH)
[
H]D
-BE) and
1
,25-dihydroxyvitamin
D
-3
-[1-
C]bromoacetate
(1,25(OH)
D
-[
C]BE),
binding-site directed analogs of 1,25(OH)
D
, and
affinity-labeled baculovirus-expressed recombinant human VDR (with
1,25(OH)
[
H]D
-BE), and
naturally occurring VDRs in cytosols from calf thymus homogenate and
rat osteosarcoma (ROS 17/2.8) cells (with 1,
25(OH)
D
-[
C]BE). In each
case, specificity of labeling was demonstrated by the drastic reduction
in labeling when the incubation was carried out in the presence of an
excess of nonradioactive 1
,25(OH)
D
. These
results strongly suggested that
1,25(OH)
[
H]D
-BE and
1,25(OH)
D
-[
C]BE
covalently modified the 1,25(OH)
D
-binding sites
in baculovirus-expressed recombinant human VDR and naturally occurring
calf thymus VDR and rat osteosarcoma VDR, respectively.
Multiple and diverse physiological actions of the calciotropic
hormone 1,25-dihydroxyvitamin D
(1,25(OH)
D
) (
)include
absorption of calcium and phosphorus in the intestine, mobilization of
calcium from bone, mediation of bone remodelling, conservation of
minerals in the kidney, and modification of
T-lymphocytes(1, 2, 3, 4) . In
addition, 1,25(OH)
D
has recently been found to
be a potent inhibitor of proliferation of cancer cells(5) , for
example, Calcipotriol, a synthetic analog of
1,25(OH)
D
, is currently available as a drug for
treating psoriasis(6, 7) , and several synthetic
analogs of 1,25(OH)
D
are currently under
investigation as drugs against breast cancer(8) . These diverse
biologic properties of 1,25(OH)
D
are manifested
by its high affinity binding to 1,25-dihydroxyvitamin D
receptor (VDR) in the nucleus of the target cell(9) .
After the initial binding, the ligand-receptor complex, in association
with a nuclear factor, interacts with the vitamin D-controlled genes
with a resulting change in the transcription/gene regulation leading
toward protein synthesis and changes in cellular
functions(10, 11) . In essence, VDR, a
ligand-dependent nuclear transcription factor, is responsible for
various physiological properties of 1,25(OH)
D
.
VDR, in terms of its mechanism of action, closely resembles receptors for all of the members of the steroid/thyroid receptor superfaminly (11) . In general, these proteins consist of a highly conserved N-terminal DNA-binding region and a relatively large C-terminal hormone-binding area. During the past few years, structure-functional studies of VDR (12, 13, 14) have shown that the N-terminal boundary of the ligand-binding domain lies between 114 and 166(15) , while C-terminal boundary is considered to be between 403 and 427(16) . A sequence comparison among various members of the steroid receptor superfamily has revealed that the C-terminal sequence is unique for each receptor (17) .
Since the
transcription-regulatory role of VDR is triggered by the highly
specific-binding of its ligand, i.e. 1,25(OH)D
, determination of the topography
of ligand-binding domain of VDR, which includes identification of
``contact points,'' is crucial for the proper understanding
of the diverse physiological properties of
1,25(OH)
D
, particularly in relation to other
steroid hormones. This information will also aid in the development of
agonists and antagonists of the ligand with potential pharmacological
importance.
Affinity/photoaffinity labeling studies have been used
to covalently label and identify an enzyme or a receptor in a
heterogenous sample (18) . These methods have also been
successfully applied to map the binding sites of several enzymes and
receptors including those of estrogen and glucocorticoid
receptors(19, 20) . During the past several years,
efforts from our laboratory and others to covalently label VDR in chick
and pig intestinal cytosol by photoaffinity labeling with either
radiolabeled 1,25(OH)D
or with a radiolabeled
photoaffinity analogs of 1,25(OH)
D
have met
with very limited success (21, 22, 23, 24, 25, 26) .
This led us to synthesize 1
,25-dihydroxyvitamin
D
-3
-bromoacetate
(1,25(OH)
D
-BE), a potential affinity labeling
reagent for VDR(27) . In a recent publication, we have
demonstrated that 1,25(OH)
D
-BE functions as a
substrate-analog for chick intestinal VDR(27) . In the present
investigation, we synthesized
1
,25-dihydroxy[26(27)-
H]vitamin
D
-3-deoxy-3
-bromoacetate
(1,25(OH)
[
H]D
-BE) of high
specific activity (175 Ci/mmol) and carried out affinity labeling
studies of baculovirus-expressed recombinant human VDR. We, however,
were unsuccessful in affinity labeling naturally occurring VDRs from
calf thymus and rat osteosarcoma ROS 17/2.8 cells, possibly due to the
low detection limit of
H in
1,25(OH)
[
H] D
-BE. To
circumvent this problem we synthesized
1,25(OH)
D
-[
C]BE and
affinity labeled VDRs in cytosols from calf thymus and ROS 17/2.8
cells. Results of these studies are presented in this communication.
1,25(OH)D
was a generous gift from
Dr. Milan Uskokovic (Hoffmann-La Roche Inc., Nutley, NJ). All other
chemicals were purchased from Aldrich, E. Merck Science, Gibbstown, NJ
(HPLC solvents).
1,25(OH)
[
H]D
]
(specific activity, 175 Ci/mmol) was from Amersham Corp., and
[
C]bromoacetic acid (specific activity, 18.65
mCi/mmol) was from Sigma. HPLC analysis of the samples (Econosil silica
columns (Altech Associates, State College, PA), 5% isopropyl alcohol in
hexane; flow rate, 2 ml/min) were carried out in a Waters HPLC system
consisting of a M660A solvent delivery pump, a U6K injector, and a 440
UV (254 nm) detector. In some cases effluents from HPLC were directly
introduced into a Radiomatic FloOne Radioactivity detector (Radiomatic
Corp., Tampa, FL).
Calf thymus cytosol was prepared according to the procedure described by Reinhardt et al.(30) .
(b) The product from the above step was
dissolved in 0.4 ml of acetonitrile, and 40 µl of aqueous
hydrofluoric acid (48%) was added to it. The solution was stirred at 25
°C for 20 h followed by careful neutralization with saturated
sodium bicarbonate solution to a pH of approximately 8. The aqueous
solution was extracted with ethyl acetate. The organic extract was
dried over anhydrous magnesium sulfate, and solvent was removed under
argon. The resulting reaction mixture was purified by preparative
thin-layer chromatography on a 1000-µm silica plate (33.3% ethyl
acetate in hexane), and the UV active band corresponding to an
authentic sample of 1,25(OH)D
-BE was isolated.
The yield of the desired product
(1,25(OH)
D
-[
C]BE) was
4.48 µCi (18% overall). HPLC analysis (as described in the case of
1,25(OH)
[
H]D
-BE) of
1,25(OH)
D
-[
C]BE, mixed
with an authentic sample of 1,25(OH)
D
-BE,
indicated that this material was radiochemically homogeneous.
In another
experiment, 30-µl samples of mutant-type cell extract were
incubated with either
1,25(OH)[
H]D
-BE (100,000
cpm, 0.57 pmol) or
1,25(OH)
[
H]D
-BE (100,000
cpm, 0.57 pmol) and 1,25(OH)
D
(1 µg, 2.4
nmol) at 0 °C for 3 h. After the incubation, the samples were
electrophoresed and autoradiographed as described earlier.
-Halo ester derivatives of biomolecules, particularly
steroids, have been popular in designing affinity analogs due to their
relative ease of synthesis and their high reactivity toward
nucleophilic amino acid residues in the ligand-binding
pocket(18) . We have recently described a multistep procedure
to synthesize 1,25(OH)
D
-BE, an
-bromo
ester derivative of 1,25(OH)
D
(27) .
This synthetic procedure, however, was useless in the case of
1,25(OH)
[
H]D
-BE due to
practical infeasibility of starting with nanogram quantity of
1,25(OH)
[
H]D
of very high
specific activity (175 Ci/mmol), and carry out several synthetic steps.
Alternatively, we coupled commercially available
1,25(OH)
[
H]D
(specific
activity, 175 Ci/mmol) with bromoacetic acid in the presence of a large
excess of vitamin D
as a carrier (Fig. 1). HPLC
analysis of the reaction mixture (Fig. 2, left panel)
demonstrated that, although it consisted of several UV-absorbing peaks
including that of vitamin D
-3
-bromoacetate (I) (Fig. 2, left panel, top), there
were only three radioactive peaks (Fig. 2, left panel, bottom). These radioactive peaks represented the 1,3-diester
derivative (IV) and 3- and 1-monoester derivatives (II and III, respectively) of
1,25(OH)
[
H]D
(retention
times, 4.6, 10.2, and 11.3 min, respectively). (
)By careful
fractionation, as described under ``Experimental
Procedures,'' our desired compound, i.e. 1,25(OH)
[
H]D
-BE (II) was separated to the base line from other isomers and
products of the reaction. HPLC analysis of a mixture containing
1,25(OH)
[
H]D
-BE (II) and a standard sample of
1,25(OH)
D
-BE (27) confirmed that
1,25(OH)
[
H]D
-BE was
radiochemically homogeneous (Fig. 2, right panel).
Figure 1:
Scheme for the
synthesis of 1,25-dihydroxy[26(27)-
H]vitamin
D
-3-3
- bromoacetate
[
H-1,25(OH)
D
-BE]. Vitamin
D
-3
-bromoacetate (I);
1,25(OH)
[
H]D
-BE (II);
1
,25-dihydroxy[26(27)-
H]vitamin
D
-1
-bromoacetate (III);
1
,25-dihydroxy[26(27)-
H]vitamin
D
-1
,3
-dibromoacetate (IV)
Figure 2:
Left panel, HPLC-analysis of the reaction
mixture depicted in Fig. 1(Silica column, 5% isopropyl alcohol
in hexane, 1.3 ml/minute). Top, UV absorption at 254 nm; bottom, radioactivity in cpm. Right panel, HPLC
analysis of a mixture containing a standard sample of
1,25(OH)D
-BE and a sample of
1,25(OH)
[
H]D
-BE isolated
from the reaction mixture. Top, UV absorption at 254 nm; bottom, radioactivity in cpm.
Incubation of recombinant-type (VDR positive) or the wild-type (VDR
negative) cytosols with
1,25(OH)[
H]D
-BE produced
a single radioactive band (M
50,000) in the case
of VDR positive sample (Fig. 3, lane 2), which was
completely absent in the case of wild-type (VDR negative) sample (Fig. 3, lane 1). In the latter sample, however, a low
molecular weight protein band was labeled to a minor extent, possibly
due to nonspecific labeling.
Figure 3:
Affinity labeling of baculovirus-expressed
hVDR with H-1,25(OH)
D
-BE. Lane
1, VDR-negative cytosol +
1,25(OH)
[
H]D
-BE; lane
2, VDR-positive cytosol +
1,25(OH)
[
H]D
-BE; Lane
3,
C-labeled protein molecular weight
markers.
In a Western blot analysis, the 50-kDa protein band strongly cross-reacted with the monoclonal antibody for hVDR in VDR-positive sample (Fig. 4, lane 2), while immunoreactivity was completely absent in VDR-negative sample (Fig. 4, lane 1). When the protein content of the samples was increased 10-fold, several immunoreactive bands were observed for the mutant-type (Fig. 4, lane 4), indicating some degradation of the VDR. Intact VDR, however, represented greater than 90% of the immunoreactive products in the extract. For the wild-type, there was no observable cross-reactivity even with 500 ng of protein (Fig. 4, lane 3).
Figure 4:
Western blot analysis: the gel containing
cytosolic samples was probed with various amounts of monoclonal
antibody (9A7) for hVDR. Lanes 1 and 3,
VDR-negative cytosolic samples + 9A7
(50 and 500 ng,
respectively); lanes 2 and 4, VDR-positive cytosolic
samples + 9A7
(50 and 500 ng respectively). Positions of
standard molecular weight marker proteins are indicated on the
right.
Finally, when the incubation was carried out in the presence of a
large excess of 1,25(OH)D
, the labeling of
50-kDa hVDR band was drastically reduced (Fig. 5, lane
2), compared with the sample without 1,25(OH)
D
(Fig. 5, lane 1). The results of all the
experiments described above strongly suggested that
1,25(OH)
[
H]D
-BE
specifically and covalently labeled the
1,25(OH)
D
-binding site in hVDR.
Figure 5:
Affinity labeling of baculovirus-expressed
hVDR with
1,25(OH)[
H]D
-BE. Lane
1, VDR-positive cytosol +
1,25(OH)
[
H]D
-BE. Lane
2, VDR-positive cytosol +
1,25(OH)
[
H]D
-BE +
1,25(OH)
D
(large excess). Positions of standard
molecular weight marker proteins are indicated on the left.
If we
consider a simple competition between 1,25(OH)D
and 1,25(OH)
D
-BE for the binding site on
VDR, 4000-fold molar excess of 1,25(OH)
D
is
expected to eliminate the labeling completely. This was, however, not
the case in reality (Fig. 5, lane 2). A possible
explanation would be that if the interaction between
1,25(OH)
D
-BE and VDR is rapid and irreversible,
covalently labeled VDR would accumulate with time, even in the presence
of a reversible competition between 1,25(OH)
D
and 1,25(OH)
D
-BE. Thus, certain amount of
covalently labeled VDR will always contaminate the mixture.
Alternatively, binding characteristics of 1,25(OH)
D
and 1,25(OH)
D
-BE with VDR could be
different, so that the latter could be a pure agonist, a pure
antagonist, or anything in between. For example, a whole spectrum of
estradiol-agonists and antagonists are known. They share a common
binding site in the estrogen receptor but bring about different
conformational changes in the apoprotein(33) . In a recent
preliminary study, we observed that
1,25(OH)
D
-BE is a potent agonist of
1,25(OH)
D
in inhibiting the nuclear uptake of
[
H]thymidine in cultured human
keratinocytes(34) . Further research is required to determine
the exact mechanism of action of 1,25(OH)
D
-BE.
Once successful labeling of the partially enriched recombinant hVDR
was achieved, we began a study to label naturally occurring VDR.
Incubation of cVDR with
1,25(OH)[
H]D
-BE produced
no labeled protein band, and almost all the radioactivity appeared at
the bottom of the SDS-polyacrylamide gel (results not shown). We were,
however, encouraged by the fact that there was very little
``random labeling'' indicating the specificity of the
reagent. We also realized that low-energy tritium nucleide (as in
1,25(OH)
[
H]D
-BE) is not
suitable for the very low level detection of radioactivity (due to
extremely low natural abundance of VDR in mammalian tissues). This
observation prompted us to synthesize
1,25(OH)
D
-[
C]BE (Fig. 6). Although specific activity of
1,25(OH)
D
-[
C]BE (18.65
mCi/mmol) was much lower than that of
1,25(OH)
[
H]D
-BE (175
Ci/mmol), we reasoned that the higher energy
-particles in
[
C]radionucleide may be enough to detect any
labeled band in the gel.
Figure 6:
Scheme for the synthesis of
1,25-dihydroxyvitamin
D
-3
[1-
C]bromoacetate
(1,25(OH)
D
-[
C]BE).
Incubation of nuclear extracts from calf
thymus and ROS 17/2.8 cells with
1,25(OH)D
-[
C]BE produced
a labeled protein band (M
50,000) (Fig. 7, lanes 1 and 3, respectively). When the incubation was
carried out in the presence of 145-times molar excess of
1,25(OH)
D
, the intensity of labeling was
significantly reduced (Fig. 7, lanes 2 and 4,
respectively). In calf thymus cytosol, cVDR represented the only
distinctly labeled protein band (Fig. 7, lanes 1 and 2); but in, ROS 17/2.8 nuclear extract, there were several
minor labeled protein bands including a band (M
25,000) possibly representing a proteolytic digestion product of
rVDR. (Fig. 7, lanes 3 and 4). In both cases,
most of the radioactivity appeared at the dye-front of the gel,
possibly representing the free label. These results strongly indicated
that 1,25(OH)
D
-binding sites in cVDR and rVDR
were covalently modified by
1,25(OH)
D
-[
C]BE.
Figure 7:
Affinity-labeling of nuclear extracts
from calf thymus (cVDR) and ROS 17/2.8 cells (cVDR) with
1,25(OH)D
-[
C]BE. Lane 1, calf thymus nuclear extract +
1,25(OH)
D
-[
C]BE; lane 2, calf thymus nuclear extract +
1,25(OH)
D
-[
C]BE +
1,25(OH)
D
(excess); lane 3, ROS 17/2.8
nuclear extract +
1,25(OH)
D
-[
C]BE; lane 4, ROS 17/2.8 nuclear extract +
1,25(OH)
D
-[
C]BE +
1,25(OH)
D
(excess). Positions of standard
molecular weight marker proteins are indicated on the right.
We
are currently in the process of developing a bacterial overexpression
system to obtain substantial quantity of hVDR (35) for
``mapping'' the 1,25(OH)D
-binding
domain of VDR using the
C-labeled affinity labeling
process described in this communication. We are also in the process of
synthesizing next generation affinity analogs containing affinity
probes at sites other than the 3-hydroxyl group of
1,25(OH)
D
(as in the case of
1,25(OH)
D
-BE). These analogs will allow us
identify several contact points within the
1,25(OH)
D
-binding cavity in VDR. Identification
of these important ``recognition markers'' in the
1,25(OH)
D
-binding domain of VDR will be crucial
for developing next generation 1,25(OH)
D
-based
drugs with broad spectrum anticancer activities. In general, these
studies will provide important structural information about VDR and
1,25(OH)
D
in relation to their functions.