A Unique Point Mutation in the Human Vitamin D Receptor Chromosomal Gene Confers Hereditary Resistance to 1,25Dihydroxyvitamin D3

Teruki Sone, Stephen J. Marx, Uri A. Liberman, and J. Wesley Pike* Departments of Pediatrics (T.S., J.W.P.) and Cell Biology (J.W.P. Baylor College of Medicine Houston, Texas 77030 Mineral Metabolism Section National Institutes of Health (S.J.M.) Bethesda, Maryland 20892 Metabolic Diseases Beilinson Medical Center Peta Tiqva Sackler School of Medicine Tel-Aviv University (U.A.L.) Tel-Aviv, Israel

The syndrome of hereditary resistance to 1,25-dihydroxyvitamin D3 is due to defective function of the vitamin D receptor (VDR). The recent cloning and nucleotide sequence determination of the human VDR chromosomal gene have enabled a direct evaluation of the genetic basis for this disease in affected patients. In this report we employed polymerase chain reaction techniques to amplify the gene exons that encode the DNA-binding domain of the VDR from two 1,25-dihydroxyvitamin D3-resistant patients whose receptors displayed defective binding to nonspecific DNA. Although their families were apparently unrelated, each patient displayed an identical homozygous point mutation within the third exon, a mutation that causes substitution of a glutamine for an arginine residue highly conserved within the entire steroid receptor superfamily. We introduced this base change into the normal VDR cDNA via site-directed mutagenesis, transfected an expression vector containing this cDNA into cells, and examined the functional properties of the resultant VDR expression product. The produced mutant receptor bound 1,25-dihydroxy vitamin D3 with normal affinity, but displayed weak affinity for the nuclear fraction and for heterologous DNA. More importantly, the protein was inactive in promoting transcription in a cotransfection assay employing a chloramphenicol acetyltransferase gene reporter

fused down-stream of the VDR-inducible osteocalcin gene promoter-enhancer. These results provide the genetic and functional basis for the phenotype of rickets in this inherited disease. (Molecular Endocrinology 4: 623-631, 1990)

INTRODUCTION The hereditary human syndrome of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] resistance is characterized by rickets, hypocalcemia, secondary hyperparathyroidism, and moderately to extremely elevated levels of 1,25(OH)2D3(1-5). Investigations of fibroblasts derived from patients with this disease have revealed these cells to be biologically resistant to the action of this steroid hormone and have allowed preliminary characterization of abnormalities within the vitamin D effector pathway (6-12). These include the demonstration of several different functional defects within the intracellular receptor protein that both binds to and mediates the action of 1,25-(OH)2D3 (6-12). The apparently defective vitamin D receptors (VDR) associated with this resistance syndrome have been assigned to three classes of functional defects: hormone binding defects, DNA recognition abnormalities, and nuclear translocation or retention defects (6-12). The normal mechanism of vitamin D action involves association of the 1,25-(OH)2D3-receptor complex with specific DNA sequence elements located up-stream of hormone-responsive gene pro-

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moters and the subsequent modulation of transcription analogous to the mechanism for other steroid hormones (13-16). Indeed, comparisons of the deduced amino acid sequence of the VDR with that of other proteins clearly show that the VDR belongs to the steroid receptor protein superfamily of genes (17-20). Using a strategy based upon prior structural and functional analysis of the VDR (15) as well as insights derived from determination of the organization and nucleotide sequence of the VDR chromosomal gene (Kesterson, R. A., and J. W. Pike, submitted), the genetic basis for 1,25-(OH)2D3 resistance in two kindreds with VDR DNA recognition defects has recently been defined (21, 22). The point mutations that cause single amino acid substitutions for highly conserved residues within the DNA-binding domain of the VDR were identified (22). Recreation of the point mutations within the normal VDR cDNA followed by expression in host cultured cells resulted in receptors whose DNA-binding phenotypes were identical to those of the patients and whose abilities to direct transcription in vitro were completely compromised (23). In addition, a point mutation has been defined also in several related families that results in premature termination, producing a protein that does not bind hormone and is equally incapable of activating transcription (24). In the current report we examined the VDR gene from two additional apparently unrelated patients with resistance to 1,25(OH)2D3. We focused exclusively upon amplification and sequence analysis of the two exons that encode the DNA-binding region of the VDR, as the receptors from these patients also belong within the DNA recognition class of defects (10). We identify a unique point mutation common to the genes of both patients that is located within the third exon encoding the carboxyl portion of the DNA-binding domain. This mutation is distinct from the two DNA-binding mutations identified previously (22). Nevertheless, we find that the mutant receptor is likewise unable to bind DNA or activate transcription after transfection into cultured cells.

RESULTS Identification of the Molecular Defect in the VDR Gene in P3 and P7 Cells Table 1 summarizes the properties of the VDR in cultured skin fibroblasts from patients P3 and P7 (6, 10). While the 1,25-(OH)2D3-binding affinity and capacity of the receptor were normal, the ability of the VDR to remain associated with the nuclear fraction and interact with heterologous DNA was altered. Thus, these receptor defects belong to the DNA recognition class of abnormalities. As the DNA-binding domain of the VDR is encoded by exons 2 and 3 of the VDR gene (Fig. 1A), we amplified these regions from genomic DNA isolated from P3 and P7 cells using oligonucleotides 2a and 2b and 3a and 3b, and then sequenced the amplified products. As illustrated for P3 DNA by direct sequence analysis (Fig. 1B) and similarly identified in P7 DNA (data not shown), we identified at nucleotide 327

Table 1. Summary of the Properties of VDR in Cultured Skin Fibroblasts from Two Patients with Resistance to 1,25(OH)2D3 (from Refs. 6 and 10) Saturation Analysis Patient

Wt P3 P7

Soluble extract

Nuclear uptake

DNA Binding (Molar KCI cone, at Peak Elution)

N 50% D 70% D

0.22 0.10-0.13 0.09-0.11

N, Normal binding capacity and binding affinity for 1,25(OH)2D3; D, decreased binding capacity for 1,25-(OH)2D3; Wt, wild type.

of exon 3 a single point mutation converting a G to an A (partial sequence of normal exon 3 is also shown in parallel). This base change causes the substitution of a glutamine residue for arginine residue 77 that is highly conserved within the steroid, thyroid, and retinoic acid receptor gene family. The sequence of exon 2 from the DNA of both P3 and P7 cells was normal (data not shown). Functional Properties of the VDR Harboring Mutant Amino Acid 77 Site-directed mutagenesis was used to introduce the mutant nucleotide at base 327 into the normal or wildtype human (h) VDR cDNA. The mutant cDNA was cloned into the p91023b expression vector (Fig. 2A), and expressed in COS-1 cells through DNA transfection. Western blot analysis of cellular extracts revealed the expression of a 50-kDa mutant receptor at steady state concentrations equivalent to those of the wildtype receptor (Fig. 2B). 1,25-(OH)2D3saturation analysis demonstrated quantitatively that both the mutant and the normal receptor were expressed equivalent^ and displayed an identical affinity for 1,25-(OH)2D3 (Table 2). Thus, the stability and turnover rate of the mutant VDR are probably similar to those of the wild-type receptor. In contrast, however, the ability of the mutant 1,25(OH)2D3-VDR complex to remain bound to the nuclear fraction in 0.05 M KCI was significantly lower than that of the wild-type receptor and analogous to that of the receptors in P3 or P7 cells (see Table 1). DNA Recognition Properties of the Mutant Receptor To examine the ability of the receptor to interact with DNA, we prepared soluble extracts from COS-1 cells transfected with either normal or mutant receptor cDNAs, labeled the receptors in vitro with 1,25-(OH)2[3H]D3, and chromatographed the complexes on DNA cellulose. Wild-type receptor bound well to DNA cellulose and eluted at a KCI concentration of 0.2 M. In contrast, a high percentage of the mutant receptor was not retained by the column resin, and the fraction that was retained eluted at approximately 0.09-0.11 M KCI (Fig. 3). Thus, this mutant VDR retained the same

Vitamin D Receptor Gene Mutation

625

A. 10

Kilobases

5B6B

1B 2B

3B

11

1 1

8B 9B

Primer Pairs

Exon 1A

2A

1,25(OH) 2 D 3

DNA

B. 5' G C C *A G C T C A A A C G 3'

C G G C G A G T T T G C

P3

=

G A T CG AT C Exon

C G G C C G A G T T T G C

5' G C C G** G C T C A A A C G 3'

3

Fig. 1. A, Strategy for detection of genetic mutation in the hVDR gene. The diagram illustrates the organization of the hVDR gene comprised of nine exons which span 43.2 kb of genomic DNA. Seven pairs of 24- to 33-mer oligonucleotide primers (each containing 5' £coRI restriction ends) which hybridize to intron sequence either 5' or 3' of the intron/exon boundary of each exon were available for DNA amplification. The oligonucleotide nomenclature is as follows. Numbers indicate the VDR gene exon that is amplified, and the letters a and b reflect either the oligonucleotide hybridizing to the 5' or 3' side of the exon, respectively. The VDR DNA-binding domain is encoded by exons 2 and 3; the steroid-binding domain is encoded by exons 4-9. The kilobase scale is shown. B, Direct sequence analysis of exon 3 amplified from a normal subject and patient 3 DNA. Patient 3 DNA contains a point mutation altering codon 77 from normal CGG to CAC. The relevant sequence from patient (P3) and normal (N) exon 3 DNA is depicted where the mutant and normal bases are indicated by a single and double asterisk, respectively. G, A, T, and C sequencing lanes are indicated.

functional phenotype as the receptors previously characterized in both P3 and P7 fibroblasts (Table 1). Transactivation Properties of the Mutant VDR The transactivation capabilities of the mutant VDR were evaluated in a transcriptional activation assay that used a VDR-inducible osteocalcin gene reporter plasmid (see Fig. 4). Cotransfection of increasing concentrations of wild-type receptor expression vector together with the osteocalcin reporter plasmid resulted in a hormonedependent induction of transcription, as measured by increased chloramphenicol acetyltransferase (CAT) activity (depicted as fold induction; Fig. 5A). This induction was likewise dependent upon the presence and concentration of hormone (Fig. 5B). In contrast, the mutant receptor exhibited no capacity to induce transcription from the osteocalcin gene promoter, as a function of either expression plasmid concentration (Fig. 5A) or hormone concentrations as high as 10~7 M (Fig. 5B). These experiments suggest that the mutation found in exon 3 of the VDR gene in P3 and P7 cells produces a

receptor that is severely defective or inactive in vitro and is probably the cause of 1,25-(OH)2D3 resistance in the patients' cells in culture. It suggests further that this mutation also produces a defective VDR in vivo and, thus, represents the precise genetic cause of the disease phenotype of these two patients with hereditary 1,25-(OH)2D3-resistant rickets.

DISCUSSION The VDR belongs to the steroid, thyroid, and retinoic acid hormone receptor gene family of proteins that mediate the action of their respective cognate ligands through the regulation of gene transcription (25). The proteins are modular in nature and are essentially comprised of a DNA-binding domain and a hormone-binding domain (26-29). The latter regulates in unknown fashion the ability of the DNA-binding domain of the protein to recognize DNA sequence elements of responsive genes and, in turn, to modulate the activity of those

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A.

B.

SV40 Origin

Ori—

Tripartite Leader Intron coRI pAd-hVDR

VDR

PBR322 ori

SV40 Poly(A

3 4 5 6 Fig. 2. A, Schematic of the plasmid vector p91023b used for expression of the mutant VDR. The point mutation identified in P3/ P7 DNA was introduced into the normal VDR cDNA by site-directed mutagenesis in M13, as indicated in Materials and Methods, and then transferred in positive orientation to p91023b to create pAd-hVDR. B, Western blot analysis of mutant hVDR after transfection into COS-1 cells. Expression plasmids (20 Mg/100-mm plate) containing wild-type receptor in positive (lane 1) or negative (lane 2) orientation relative to the promoter and the mutant receptor in positive orientation (lane 4) were transfected into COS-1 cells, as indicated in Materials and Methods. Additional plasmid preparations expressing hVDR with unrelated point mutations in positive orientation (lanes 3, 5, and 6) were transfected similarly for comparison of expression. After a 48-h culture, cells were harvested, cytosols were prepared, and 30 ng protein was analyzed by immmunoblot (15). Standard protein migrations are indicated to the left of the autoradiogram where ori is origin, and dye is dye front. The hVDR is indicated by the arrow.

Table 2. Expression and Biochemical Properties of WildType and Mutant VDRs in COS-1 Cells Expression

Wt (0.7-2.8)

2.12 1.53

76 28

DNA Binding

Mol

0.22 0.10

50 50

Wt, Wild-type VDR; R-77, P3 and P7 mutant VDR.

genes. Extensive dissection of the DNA recognition domain of this protein family has shown two highly conserved DNA-binding loops or finger structures, each with four positionally conserved cysteine residues that serve to coordinate a zinc atom (30). This structure within the VDR is illustrated schematically in Fig. 6. We have shown through deletion analysis that this domain structure is likewise a feature of the VDR, that these intact domains are important for transactivation of the human osteocalcin gene promoter, and that a transactivation domain responsible for at least a portion of the ability of this protein to activate osteocalcin gene transcription resides in a region located within the N-terminal half of the hormone-binding domain (15). We have also identified a short DNA sequence element within the osteocalcin gene promoter that acts in cis together with the VDR to enhance osteocalcin gene transcription (16). This element displays characteristics typical of steroid hormone-inducible enhancers, including its ability to act in an orientation-independent fashion and to transfer 1,25-(OH)2D3-inducibility to gene promoters not otherwise responsive to vitamin D. These observations, with regard to both receptor structure and its interaction with a specific DNA element, confirm the hypothesis advanced many years ago that 1,25-(OH)2D3 functions as a steroid hormone (31).

In this report we have identified a point mutation in the VDR genes of two patients with hereditary 1,25(OH)2D3-resistant rickets. Because the receptors from these patients displayed abnormal DNA-binding behavior in vitro and because of previous structural insights about the VDR, we amplified from each patient only the two exons encoding this function (15). We identified within exon 3 a single homozygous mutation that resulted in the substitution of a glutamine for an arginine residue (Fig. 6). Most importantly, this mutation is quite different from two mutations identified previously at the tips of each zinc finger (22) (see Fig. 6) and is located on the carboxyl-terminal side of the second finger structure (32-34). As the patients' abnormal receptor phenotype of weak nuclear and DNA-binding activity was confirmed after recreation of the mutant receptor by mutagenesis and subsequent expression, and because of the striking defect of the recreated VDR for transactivation in vitro, we did not amplify and sequence the remaining six structural exons. If another point mutations) exists within any of these regions, it is unlikely to contribute importantly to the defective VDR phenotype. Finally, while there is no known kinship information to suggest that these patients are related (except that the two kindreds are from North Africa and of the same ethnic origin), the nature of this rare mutation suggests a distant ancestral relationship. The parents of both patients were unavailable for evaluation. Mutations within the DNA-binding domain have been shown to compromise steroid receptor function (3234). At least three potentially overlapping types of mutations are possible in this domain and include 1) mutations that alter the structure of the domain, 2) mutations that alter the charge, and 3) mutations that alter contacts with nucleotides within an enhancer element located in a gene promoter. The point mutation we have

Vitamin D Receptor Gene Mutation

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3.5r

10

20

30

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Fraction Number 25r

60

B

identify. The development of assays capable of evaluating the receptor's interaction with specific osteocalcin DNA sequences is in progress, but it is possible that important distinctions about how mutations disrupt receptor-DNA interactions will come about only after insight into the three-dimensional structure of the receptor protein itself emerges. The existence of a human disease such as hereditary 1,25-(OH)2D3-resistant rickets was proposed many years ago (35). While the direct action of 1,25-(OH)2D3 on transcription involves multiple nuclear proteins, it is probable, although not proven, that the specificity of transcriptional activation by vitamin D lies in the VDR itself. Thus, it is not surprising that generalized resistance to 1,25-(OH)2D3arises from mutations in the VDR, analogous to those receptor mutations suspected to exist in androgen (36), cortisol (37), T3 (38), and mineralocorticoid (39) resistance states. It is important to note, however, that while complete resistance to the vitamin D hormone is readily apparent in such patients as those evaluated here for genetic abnormalities, it is possible that other mutations within the VDR might lead to partial resistance and unknown phenotypes. Moreover, these possibilities do not preclude also the occurrence of vitamin D resistance that might arise as a result of changes in other elements of the vitamin D-inducible transcription pathway, either in specific transcriptional factors or in the important target genes themselves. Presumably, each might have a unique clinical presentation that is currently unrecognized.

MATERIALS AND METHODS

10

20 30 40 Fraction Number

50

Fig. 3. Interaction of Expressed Mutant Receptor with Immobilized Heterologous DNA Cytosols derived from COS-1 cells transfected with plasmid expressing either wild-type VDR or P3/P7 mutant VDR were labeled with 1,25-(OH)2-[3H]D3 (2 riM) for 2 h and then chromatographed on a column (5 ml) of DNA cellulose. The column was eluted with a linear gradient of KCI. A, Normal VDR eluted at 0.2 M KCI; B, mutant VDR either did not bind to the column or eluted at 0.1 M KCI.

identified at amino acid 77 clearly alters the charge of that residue (basic to neutral) similarly to the altered charge effects that arise from the previously identified mutant VDRs (22) (Fig. 6). Thus, it is possible that this change in charge is responsible for the receptor's inability to properly interact with DNA and, as a result, its transcriptional inactivity. It is difficult, however, to discern the difference between mutations that alter structure and those that alter charge using an assay based upon the receptor's interaction with nonspecific DNA, as employed here. It is equally likely that mutations that affect contact sites with specific DNA will be difficult to

All molecular biological reagents, including modifying and restriction enzymes, were obtained from Boehringer Mannheim (Indianapolis, IN). 1,25-(OH)2D3 was provided by Dr. M. Uskokovic of Hoffman LaRoche (Nutley, NJ). 1,25-(OH)2-[23,24-N3 H]D3, [14C]chloramphenicol, and [«-35S]dATP were obtained from Amersham Corp. (Arlington Heights, IL). [a-32P]dCTP and [125l]protein-A were obtained from ICN Biochemicals (Irvine, CA). Sequenase was purchased from U.S. Biochemical Corp. (Cleveland, OH). Taq\ polymerase was obtained from Perkin Elmer Cetus Corp. (Norwalk, CT). Synthetic oligonucleotides were purchased from Genetic Designs, Inc. (Houston, TX). Cell Culture Skin fibroblasts obtained from previously described patients with hereditary 1,25-(OH)2D3-resistant rickets were maintained in culture in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, as previously reported (10). Monkey kidney fibroblasts (COS-1, CRL 1650, and CV1, CCL 70) were obtained from American Type Culture Collection (Rockville, MD) and grown in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum, as described previously (15). Detection of VDR Mutations Genomic DNA from fibroblasts of patient 3 (P3) and patient 7 (P7) was prepared by the methods described by Maniatis et al. (40). DNA amplifications were performed as described by

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TATAAA S

A

N |P

J-509-489J

I

I

P

-1339

+ 10

Fig. 4. Diagram of the Human Osteocalcin Gene Promoter-CAT Reporter Plasmid An osteocalcin gene fragment with coordinates -1339/+10 containing the vitamin D response element at - 5 0 9 / - 4 8 9 (derived from a human genomic clone seen in the upper portion of the figure) was cloned into the CAT gene expression plasmid pBL CAT3, as previously described (16), and designated phOC-1339. The four exons in the gene are cross-hatched. B, eamHI; P, Pst\; S, Sacl, A, Apa\; N, A/col; H, H/ndlll; X, Xho\; Bg, BglW. TATAAA represents the TATA homolog.

25,-

2 Or

.2 15

10

0.01

0.1 1.0 log hVDR Expression Vector (pg)

10

°4r

10 9 8 -log 1,25(OH)2D3 (M)

Fig. 5. A, assessment of the transactivation function of mutant VDR. CV-1 cells were transfected with increasing concentrations of either wild-type (O) or mutant (•) receptor expression plasmid together with 10 ^g reporter plasmid phOC-1339 and then treated with either ethanol (0.1% final concentration) or 10~8 M 1,25-(OH)2D3 (final concentration) in ethanol. Cell extracts containing 100 Mg protein prepared 72 h after transfection were used for overnight CAT assays at 37 C. Fold hormone induction represents the ratio of hormone-induced to hormone-uninduced CAT activity. B, Effect of graded concentrations of 1,25-(OH)2D3on transactivation activity. CV-1 cells were transfected as described above with 0.5 ^g of either wild-type (O) or mutant (•) VDR expression plasmid and then treated with ethanol or increasing concentrations of hormone as indicated.

Vitamin D Receptor Gene Mutation

629

(Hughes, et.al.)

(Hughes, et.al.)

Fig. 6. Location in the VDR Gene of Genetic Mutations That Compromise the DNA-Binding Function of the VDR The amino acid sequence of the N-terminus of the VDR encoded by exons 2 and 3 that contains the two zinc fingers is schematically shown. Conserved amino acids within the steroid receptor gene family are cross-hatched, and the positions of the two introns of the VDR gene are indicated by the arrows. The mutation identified in P3/P7 is indicated at residue 77. Two previously identifed mutations within the DNA-binding domain are indicated as Hughes et al. (22).

the manufacturer in a reaction using 1 fig DNA and 5 U Taq\ polymerase. Thirty reaction cycles were carried out using oligonucleotide primers 2a and 2b or 3a and 3b (see Fig. 1 for nomenclature; 100 pmol/reaction) annealed at 55 C, extended at 72 C, and denatured at 94 C. The oligonucleotide sequences of the primers are as follows: 2a, 5'-AGGAATTCAGCTGGCCCTGGCACTGACTCTGCTCT-3'; 2b,5' CTGCCTTCATGGAAACACCTTGCTTCTTCTCCCTC-3'; 3a,5'-GTG AATTC AGGGTGAGGAGCCGGAAGTTCAGTGAC-3'; and 3b,5' GCGAATTCCTTTCCCTGACTCCACTTCAGGCCCAA-3'. Amplified products were treated with EcoRI, isolated from low melting point agarose gels, and cloned into an EcoRI-restricted alkaline phosphatase-treated pSP73 vector obtained from Promega Corp. (Madison, Wl). Denatured plasmid DNA from transformed bacterial HB101 cells was sequenced on both strands by the Sanger method using the Sequenase polymerase enzyme (41). Amplified DNA products were also sequenced directly by methods described previously using the Taq\ polymerase enzyme (42). Mutagenesis of VDR cDNA The 2.1-kilobase (kb) hVDR cDNA was cloned into M13 mp18. It was then subjected to oligonucleotide-directed mutagenesis, as outlined by Kunkel (43), using a 21-base synthetic DNA sequence which contained at nucleotide 327 the mutant nucleotide base identified within the VDR genes of the two patients. Mutagenized M13 clones were identified via DNA sequencing of single stranded templates. Double stranded DNA preparations were then prepared, and the mutant 2.1-kb VDR cDNA insert was isolated with EcoRI and cloned into the expression vector p91023b (15). Both wild-type and mutant VDR expression plasmids were isolated and purified via banding through two cesium chloride gradients.

cellulose chromatography were carried out as previously documented (15). VDR intranuclear retention assays were performed as follows. COS-1 cells were washed twice with serumfree medium 48 h after transfection with VDR expression plasmid, as described above, and then incubated for 2 h at 37 C in medium containing 1 % serum and 10 DM 1,25-(OH)2-[3H] D3(17.6 Ci/mmol) in the absence or presence of 1 HM radioinert 1,25-(OH)2D3. Cells were harvested, washed in PBS, and lysed by three freeze-thaw cycles in 0.1 ml TKD-0.05. Lysates were centrifuged at 12,000 x g for 15 min at 4 C, and the supernatants were removed. All supernatants were subjected to triplicate evaluation of hormone-bound receptor by hydroxylapatite assay methods (15). Transcriptional Activation Assay This cellular cotransfection assay has been described previously (15). Briefly, the hVDR cDNA expression plasmids were transfected using polybrene (45) into VDR-negative CV-1 cells together with a CAT gene reporter plasmid controlled by the human osteocalcin gene promoter enhancer (phOC-1339). This reporter plasmid contains a vitamin D-responsive element at -509/-489 and is dependent upon both 1,25-(OH)2D3 and the VDR (16). Transfected cells were treated immediately with ethanol or 1,25-(OH)2D3(10"8 M) in ethanol (final concentration of ethanol, 0.1%) and harvested 48-72 h later. Cell lysates containing 100 ^g protein were used in an overnight CAT assay (46). Conversion of [14C]chloramphenicol to acetylated products was evaluated by TLC, and after autoradiography, the radioactive products were excised from the TLC plates and quantitated by liquid scintillation techniques.

Acknowledgments

Expression and Functional Properties of the VDR

The authors thank L. Chen, S. Kerner, and R. Scott for their expert technical assistance in this work.

COS-1 cells, plated 24 h previously were transfected with VDR expression plasmid (20 /ig/100-mm plate) using the diethylaminoethyl-dextran (DEAE-dextran) method (44). Forty-eight to 72 h after transfection, cells were harvested, washed with PBS, and sonicated in 10 mM Tris-HCI (pH 7.6), 0.3 M HCI, and 5 HIM dithiothreitol (TKD-0.3). Homogenates were subjected to ultracentrifugation at 220,000 x g to prepare the TKD-0.3-soluble cell fraction. Western blot analysis, saturation analysis with 1,25-(OH)2D3, hydroxylapatite assays, and DNA

Received December 29, 1989. Revision received January 26,1990. Accepted January 26,1990. Address requests for reprints to: J. Wesley Pike, Ph.D., Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030. This work was supported by grants from the NIH (AR38130 and DK-38170) and the March of Dimes Birth Defects Foundation (1-1009).

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* Established Investigator of the American Heart Association. 18.

REFERENCES 19. 1. Rosen JF, Fleischman AR, Finberg L, Hamstra A, DeLuca HF 1979 Rickets with alopecia: an inborn error of vitamin D metabolism. J Pediatr 94:729-735 2. Marx SJ, Speigel AM, Brown EM, Gardner DG, Downs RW, Attie M, Hamstra AJ, DeLuca HF 1978 A familial syndrome of decrease in sensitivity to 1,25-dihydroxycholecalciferol. J Clin Endocrinol Metab 47:1303-1310 3. Liberman UA, Halabe A, Samuel R, Kauli R, Edelstein S, Weisman Y, Papapoulos SE, Fraher LJ, Clemens TL, O'Riordan JLH 1980 End-organ resistance to 1,25-dihydroxycholecalciferol. Lancet 1:504-507 4. Beer S, Tieder M, Kohelet D, Liberman UA, Vure E, BarJoseph G, Gabizon D, Borochowitz ZU, Varon M, Modai D 1981 Vitamin D resistant rickets with alopecia: a form of end organ resistance to 1,25-dihydroxyvitamin D. Clin Endocrinol (Oxf) 14:395-402 5. Takeda E, Kuroda Y, Saijo T, Naito E, Kobashi H, Yokota I, Miyao M 1987 1-hydroxyvitamin D3 treatment of three patients with 1,25-dihydroxyvitamin D-receptor-defect rickets and alopecia. Pediatrics 80:97-101 6. Liberman UA, Eil C, Marx SJ 1983 Resistance to 1,25dihydroxyvitamin D. J Clin Invest 71:192-200 7. Gamblin GT, Liberman UA, Eil C, Downs RW, DeGrange DA, Marx SJ 1985 Vitamin D-dependent rickets type II. J Clin Invest 75:954-960 8. Balsan S, Garabedian M, Liberman UA, Eil C, Bourdeau A, Guillozo H, Grimberg R, Le Deunff MJ, Lieberherr M, Guimbaud P, Broyer M, Marx SJ 1983 Rickets and alopecia with resistance to 1,25-dihydroxyvitamin D: two different clinical courses with two different cellular defects. J Clin Endocrinol Metab 57:803-811 9. Pike JW, Dokoh S, Haussler MR, Liberman UA, Marx SJ, Eil C 1984 Vitamin D3-resistant fibroblasts have immunoassayable 1,25-dihydroxyvitamin D3 receptors. Science 224:879-881 10. Liberman UA, Eil C, Marx SJ 1986 Receptor-positive hereditary resistance to 1,25-dihydroxyvitamin D: chromatography of hormone-receptor complexes on deoxyribonucleic acid-cellulose shows two classes of mutation. J Clin Endocrinol Metab 62:122-126 11. Chen TL, Hirst MA, Cone CM, Hochberg Z, Tietze H, Feldman D 1984 1,25-Dihydroxyvitamin D resistance, rickets, and alopecia: analysis of receptors and bioresponses in cultured fibroblasts from patients and parents. J Clin Endocrinol Metab 59:383-388 12. Hirst MA, Hochman HI, Feldman D 1985 Vitamin D resistance and alopecia: a kindred with normal 1,25-dihydroxyvitamin D binding, but decreased receptor affinity for deoxyribonucleic acid. J Clin Endocrinol Metab 60:490495 13. Pike JW 1987 Emerging concepts on the biologic role and mechanism of action of 1,25-dihydroxyvitamin D3. Steroids 49:3-27 14. Minghetti PP, Norman AW 1988 1,25-(OH)2-vitamin D3 receptors: gene regulation and genetic circuitry. FASEB J 2:3043-3053 15. McDonnell DP, Scott RA, Kerner SA, O'Malley BW, Pike JW 1989 Functional domains of the human vitamin D3 receptor regulate osteocalcin gene expression. Mol Endocrinol 3:635-644 16. Kerner SA, Scott RA, Pike JW 1989 Sequence elements in the human osteocalcin gene confer basal activation and inducible response to hormonal vitamin D3. Proc Natl Acad Sci USA 86:4455-4459 17. McDonnell DP, Mangelsdorf DJ, Pike JW, Haussler MR, O'Malley BW 1987 Molecular cloning of complementary

20.

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25. 26.

27.

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30.

31.

32.

33.

34.

35. 36.

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