©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Affinity Labeling of the 1,25-Dihydroxyvitamin D Receptor (*)

(Received for publication, September 6, 1995; and in revised form, November 14, 1995)

Rahul Ray (1)(§) Narasimha Swamy (1) Paul N. MacDonald (2)(¶) Swapna Ray (1) Mark R. Haussler (2) Michael F. Holick (1)

From the  (1)Bioorganic and Protein Chemistry, Vitamin D Laboratory, Boston University School of Medicine, Boston, Massachusetts 02118 and the (2)Department of Biochemistry, The University of Arizona College of Medicine, Tucson, Arizona 85724

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Genomic actions of the calciotropic hormone 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) involves a multistep process that is triggered by the highly specific binding of 1,25(OH)(2)D(3) to 1alpha,25-dihydroxyvitamin D(3) receptor, VDR. In order to study this key step in the cascade, we synthesized 1alpha,25-dihydroxy[26(27)-^3H]vitamin D(3)-3-deoxy-3beta-bromoacetate (1,25(OH)(2)[^3H]D(3)-BE) and 1alpha,25-dihydroxyvitamin D(3)-3beta-[1-^14C]bromoacetate (1,25(OH)(2)D(3)-[^14C]BE), binding-site directed analogs of 1,25(OH)(2)D(3), and affinity-labeled baculovirus-expressed recombinant human VDR (with 1,25(OH)(2)[^3H]D(3)-BE), and naturally occurring VDRs in cytosols from calf thymus homogenate and rat osteosarcoma (ROS 17/2.8) cells (with 1, 25(OH)(2)D(3)-[^14C]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 1alpha,25(OH)(2)D(3). These results strongly suggested that 1,25(OH)(2)[^3H]D(3)-BE and 1,25(OH)(2)D(3)-[^14C]BE covalently modified the 1,25(OH)(2)D(3)-binding sites in baculovirus-expressed recombinant human VDR and naturally occurring calf thymus VDR and rat osteosarcoma VDR, respectively.


INTRODUCTION

Multiple and diverse physiological actions of the calciotropic hormone 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) (^1)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)(2)D(3) 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)(2)D(3), is currently available as a drug for treating psoriasis(6, 7) , and several synthetic analogs of 1,25(OH)(2)D(3) are currently under investigation as drugs against breast cancer(8) . These diverse biologic properties of 1,25(OH)(2)D(3) are manifested by its high affinity binding to 1,25-dihydroxyvitamin D(3) 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)(2)D(3).

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)(2)D(3), 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)(2)D(3), 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)(2)D(3) or with a radiolabeled photoaffinity analogs of 1,25(OH)(2)D(3) have met with very limited success (21, 22, 23, 24, 25, 26) . This led us to synthesize 1alpha,25-dihydroxyvitamin D(3)-3beta-bromoacetate (1,25(OH)(2)D(3)-BE), a potential affinity labeling reagent for VDR(27) . In a recent publication, we have demonstrated that 1,25(OH)(2)D(3)-BE functions as a substrate-analog for chick intestinal VDR(27) . In the present investigation, we synthesized 1alpha,25-dihydroxy[26(27)-^3H]vitamin D(3)-3-deoxy-3beta-bromoacetate (1,25(OH)(2)[^3H]D(3)-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 ^3H in 1,25(OH)(2)[^3H] D(3)-BE. To circumvent this problem we synthesized 1,25(OH)(2)D(3)-[^14C]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.


EXPERIMENTAL PROCEDURES

1,25(OH)(2)D(3) 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)(2)[^3H]D(3)] (specific activity, 175 Ci/mmol) was from Amersham Corp., and [^14C]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).

Receptor Preparations

A partially enriched recombinant human VDR (hVDR) sample was obtained by using the baculovirus expression system according to the procedure by MacDonald et al.(28) . Briefly, two cytosol samples were prepared by infecting S. frugiperda ovarian cell cultures (Sf9 cultures) with wild-type virus AcMNPV, and mutant-type virus pVDR1392. Both cytosol samples, prepared in KTED buffer containing 0.1 mmol of zinc sulfate and 20% glycerol, pH 7.4, were enriched with hVDR by blue dextran-chromatography (28) however, without the addition of any 1,25(OH)(2)[^3H]D(3). Competitive 1,25(OH)(2)[^3H]D(3) binding assays were carried out according to the published procedure(29) .

Calf thymus cytosol was prepared according to the procedure described by Reinhardt et al.(30) .

Cell Culture

Rat osteoblast-like osteosarcoma cells (ROS 17/2.8) were grown in roller bottles at 37 °C in Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 10% fetal calf serum, streptomycin (0.1 mg/ml), and penicillin (100 units/ml)(31) . The cells were grown to approximately 80% confluence and harvested by trypsinization. The nuclear extract of the cells was prepared by a method by Shapiro et al.(32) .

Synthetic Procedures

A standard sample of 1,25(OH)(2)D(3)-BE was prepared according the method developed by us(27) .

Synthesis of ^3H-1,25(OH)(2)D(3)-BE

Toluene solutions of vitamin D(3) (100 µg), bromoacetic acid (59 µg), 4,4`-dimethylaminopyridine (3.47 µg), and 1,25(OH)(2)[^3H]D(3) (1 µCi; specific activity, 175 Ci/mmol) were mixed and dried under argon. To this mixture was added a solution of dicyclohexylcarbodiimide (117 µg) dissolved in 200 µl of anhydrous dichloromethane. The reaction mixture was stored in a stoppered vial under argon for 24 h followed by the removal of solvent under argon. The reaction mixture was redissolved in 5% isopropyl alcohol in hexane and chromatographed (HPLC), where fractions were collected at 0.25-min intervals. The fractions corresponding to an authentic sample of 1,25(OH)(2)D(3)-BE were pooled. Radioactive counting of an aliquot of the pooled sample indicated that the total yield of 1,25(OH)(2)[^3H]D(3)-BE was 0.27 µCi (27%). Radiochemical purity of 1,25(OH)(2)[^3H]D(3)-BE was determined by HPLC-analysis of a sample containing 1,25(OH)(2)[^3H]D(3)-BE and 1,25(OH)(2)D(3)-BE-standard.

Synthesis of 1,25(OH)(2)D(3)-[^14C]BE

(a) A toleune solution of 1alpha,25-dihydroxyvitamin D(3)-1,25-di-t-butyldimethylsilyl ether (1.3 mg) (24) and ^14C-bromoacetic acid] (specific activity, 18.65 mCi/mmol, 25 µCi) and 4,4`-dimethylaminopyridine (0.5 µg) was dried under argon, and a solution of dicyclohexylcarbodiimide (0.4 mg) in 200 µl of anhydrous dichloromethane was added to it. The solution was stored in a stoppered vial under argon atmosphere for 24 h. Then, the reaction mixture was applied to a 1000-µm silica plate, which was eluted with 1% ethyl acetate in hexane. The UV active band, corresponding to a standard sample of 1alpha,25-dihydroxyvitamin D(3)-1,25-di-t-butyldimethylsilyloxy-3-bromoacetate (24) was isolated by extraction with ethyl acetate (9.9 µCi, 39.6%).

(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)(2)D(3)-BE was isolated. The yield of the desired product (1,25(OH)(2)D(3)-[^14C]BE) was 4.48 µCi (18% overall). HPLC analysis (as described in the case of 1,25(OH)(2)[^3H]D(3)-BE) of 1,25(OH)(2)D(3)-[^14C]BE, mixed with an authentic sample of 1,25(OH)(2)D(3)-BE, indicated that this material was radiochemically homogeneous.

Affinity Labeling of Baculovirus-expressed hVDR with 1,25(OH)(2)[^3H]D(3)-BE

Samples of 1,25(OH)(2)[^3H]D(3)-BE (22,500 cpm, 0.13 pmol), dissolved in 7.5 µl of ethanol, were added to each of 90-µl aliquots (9 µg of protein) of the wild-type and mutant-type previously frozen extracts. The solutions were incubated on ice for 3 h followed by the addition of electrophoresis sample buffer containing SDS and dithiothreitol (90 µl) to each sample. The samples were boiled for 5 min, and 100-µl aliquots were loaded on to a 10% SDS-polyacrylamide gel. Also loaded on the gel were 100-µl aliquots each of wild-type and mutant-type cytosol samples diluted 1:2000, 1:200, and 1:20 with KTED buffer. A sample containing a mixture of ^14C-labeled protein molecular weight marker (Amersham Corp.) was also loaded onto the gel. After the electrophoretic run, one-half of the gel was fixed, enhanced (sodium salicylate solution), dried, and exposed to a Kodak X-Omat film at -70 °C for 115 h. The other half of the gel was transferred to Immobilon membrane and subjected to Western blot analysis using 9A7 monoclonal antibody for hVDR(28) .

In another experiment, 30-µl samples of mutant-type cell extract were incubated with either 1,25(OH)(2)[^3H]D(3)-BE (100,000 cpm, 0.57 pmol) or 1,25(OH)(2)[^3H]D(3)-BE (100,000 cpm, 0.57 pmol) and 1,25(OH)(2)D(3) (1 µg, 2.4 nmol) at 0 °C for 3 h. After the incubation, the samples were electrophoresed and autoradiographed as described earlier.

Affinity Labeling of Calf Thymus VDR (cVDR) and Rat VDR (rVDR) in ROS 17/2.8 Cells with 1,25(OH)(2)D(3)-[^14C]BE

Two samples (50 µl) each of calf thymus cytosol and nuclear extract from ROS 17/2.8, diluted with phosphate buffer, pH 7.4, were incubated for 3 h at 0 °C with 10 µl of ethanolic solutions of either 1,25(OH)(2)D(3)-[^14C]BE (specific activity, 18.65 Ci/mmol; 10,000 cpm; 0.33 nM) alone or 1,25(OH)(2)D(3)-[^14C]BE (10,000 cpm, 0.33 nM, and 20 µg of 1,25(OH)(2)D(3), 48 nM). Following the incubation, the samples were boiled briefly with electrophoresis sample buffer containing SDS and dithiothrieotol, and electrophoressed on an SDS gel along with molecular weight markers. After the elctrophoretic run, the gel was dried and subjected to radioactivity scanning in a PhosphorImager.


RESULTS AND DISCUSSION

alpha-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)(2)D(3)-BE, an alpha-bromo ester derivative of 1,25(OH)(2)D(3)(27) . This synthetic procedure, however, was useless in the case of 1,25(OH)(2)[^3H]D(3)-BE due to practical infeasibility of starting with nanogram quantity of 1,25(OH)(2)[^3H]D(3) of very high specific activity (175 Ci/mmol), and carry out several synthetic steps. Alternatively, we coupled commercially available 1,25(OH)(2)[^3H]D(3) (specific activity, 175 Ci/mmol) with bromoacetic acid in the presence of a large excess of vitamin D(3) 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-3beta-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). (^2)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 1alpha,25-dihydroxy[26(27)-^3H]vitamin D(3)-3-3beta- bromoacetate [^3H-1,25(OH)(2)D(3)-BE]. Vitamin D(3)-3beta-bromoacetate (I); 1,25(OH)(2)[^3H]D(3)-BE (II); 1alpha,25-dihydroxy[26(27)-^3H]vitamin D(3)-1alpha-bromoacetate (III); 1alpha,25-dihydroxy[26(27)-^3H]vitamin D(3)-1alpha,3beta-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)(2)D(3)-BE and a sample of 1,25(OH)(2)[^3H]D(3)-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)(2)[^3H]D(3)-BE produced a single radioactive band (M(r) 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 ^3H-1,25(OH)(2)D(3)-BE. Lane 1, VDR-negative cytosol + 1,25(OH)(2)[^3H]D(3)-BE; lane 2, VDR-positive cytosol + 1,25(OH)(2)[^3H]D(3)-BE; Lane 3, ^14C-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)(2)D(3), the labeling of 50-kDa hVDR band was drastically reduced (Fig. 5, lane 2), compared with the sample without 1,25(OH)(2)D(3) (Fig. 5, lane 1). The results of all the experiments described above strongly suggested that 1,25(OH)(2)[^3H]D(3)-BE specifically and covalently labeled the 1,25(OH)(2)D(3)-binding site in hVDR.


Figure 5: Affinity labeling of baculovirus-expressed hVDR with 1,25(OH)(2)[^3H]D(3)-BE. Lane 1, VDR-positive cytosol + 1,25(OH)(2)[^3H]D(3)-BE. Lane 2, VDR-positive cytosol + 1,25(OH)(2)[^3H]D(3)-BE + 1,25(OH)(2)D(3) (large excess). Positions of standard molecular weight marker proteins are indicated on the left.



If we consider a simple competition between 1,25(OH)(2)D(3) and 1,25(OH)(2)D(3)-BE for the binding site on VDR, 4000-fold molar excess of 1,25(OH)(2)D(3) 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)(2)D(3)-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)(2)D(3) and 1,25(OH)(2)D(3)-BE. Thus, certain amount of covalently labeled VDR will always contaminate the mixture. Alternatively, binding characteristics of 1,25(OH)(2)D(3) and 1,25(OH)(2)D(3)-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)(2)D(3)-BE is a potent agonist of 1,25(OH)(2)D(3) in inhibiting the nuclear uptake of [^3H]thymidine in cultured human keratinocytes(34) . Further research is required to determine the exact mechanism of action of 1,25(OH)(2)D(3)-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)(2)[^3H]D(3)-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)(2)[^3H]D(3)-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)(2)D(3)-[^14C]BE (Fig. 6). Although specific activity of 1,25(OH)(2)D(3)-[^14C]BE (18.65 mCi/mmol) was much lower than that of 1,25(OH)(2)[^3H]D(3)-BE (175 Ci/mmol), we reasoned that the higher energy beta-particles in [^14C]radionucleide may be enough to detect any labeled band in the gel.


Figure 6: Scheme for the synthesis of 1alpha,25-dihydroxyvitamin D(3)-3beta[1-^14C]bromoacetate (1,25(OH)(2)D(3)-[^14C]BE).



Incubation of nuclear extracts from calf thymus and ROS 17/2.8 cells with 1,25(OH)(2)D(3)-[^14C]BE produced a labeled protein band (M(r) 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)(2)D(3), 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(r) 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)(2)D(3)-binding sites in cVDR and rVDR were covalently modified by 1,25(OH)(2)D(3)-[^14C]BE.


Figure 7: Affinity-labeling of nuclear extracts from calf thymus (cVDR) and ROS 17/2.8 cells (cVDR) with 1,25(OH)(2)D(3)-[^14C]BE. Lane 1, calf thymus nuclear extract + 1,25(OH)(2)D(3)-[^14C]BE; lane 2, calf thymus nuclear extract + 1,25(OH)(2)D(3)-[^14C]BE + 1,25(OH)(2)D(3) (excess); lane 3, ROS 17/2.8 nuclear extract + 1,25(OH)(2)D(3)-[^14C]BE; lane 4, ROS 17/2.8 nuclear extract + 1,25(OH)(2)D(3)-[^14C]BE + 1,25(OH)(2)D(3) (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)(2)D(3)-binding domain of VDR using the ^14C-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)(2)D(3) (as in the case of 1,25(OH)(2)D(3)-BE). These analogs will allow us identify several contact points within the 1,25(OH)(2)D(3)-binding cavity in VDR. Identification of these important ``recognition markers'' in the 1,25(OH)(2)D(3)-binding domain of VDR will be crucial for developing next generation 1,25(OH)(2)D(3)-based drugs with broad spectrum anticancer activities. In general, these studies will provide important structural information about VDR and 1,25(OH)(2)D(3) in relation to their functions.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants RO1 DK 47418 and 44337 (to R. R.), AR 15781 and DK 33351 (to M. R. H.), and DK 43690 and AR 36963 (to M. F. H.). Part of this work was presented at the 14th annual meeting of the American Society for Bone and Mineral Research (Ray, R., Swamy, N., MacDonald, P. N., Ray, S., Haussler, M. R., and Holick, M. F.(1992) J. Bone Miner. Res. 7, Suppl. 1, 169). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118. Tel.: 617-638-8199; Fax: 617-638-8882.

Present address: Dept. of Pharmacological and Physiological Science, St. Louis University, School of Medicine, St. Louis, MO 63104.

(^1)
The abbreviations used are: 1,25(OH)(2)D(3), 1alpha,25-dihydroxyvitamin D(3); VDR, 1,25-dihydroxyvitamin D(3) receptor; 1,25(OH)(2)D(3)-BE, 1alpha,25-dihydroxyvitamin D(3)-3beta-bromoacetate; HPLC, high performance liquid chromatography; hVDR, human VDR; cVDR, calf thymus VDR; rVDR, rat VDR.

(^2)
1alpha,25-Dihydroxyvitamin D(3)-1-bromoacetate (Fig. 1, III) was synthesized by treating 1alpha,25-dihydroxyvitamin D(3)-3-t-butyldimethylsilyl ether (25) with bromoacetic acid, dicyclohexylcarbodiimide, and 4,4`-dimethylaminopyridine in anhydrous dichloromethane followed by the removal of the silylether protecting group with aqueous hydrofluoric acid/acetonitrile. 1alpha,25-Dihydroxyvitamin D(3)-1,3-dibromoacetate (Fig. 1, IV) was synthesized by treating 1alpha,25(OH)(2)D(3) with an excess of bromoacetic acid in the presence of dicyclohexylcarbodiimide and 4,4`-dimethylaminopyridine in anhydrous dichloromethane. All the unknown compounds were characterized by NMR. Prior to the HPLC-purification of the radioactive mixture, compounds II, III, and IV were analyzed by HPLC (under the same conditions as described under ``Experimental Procedures'') to determine their relative retention times.


ACKNOWLEDGEMENTS

We thank Dr. Tai C. Chen for assistance in the preparation of the cytosol from calf thymus and Dr. Daniel T. Baran for kindly providing the ROS 17/2.8 cells.


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