WO1998056908A1

WO1998056908A1 - Vitamin d receptor isoform proteins

Info

Publication number: WO1998056908A1
Application number: PCT/JP1997/002052
Authority: WO
Inventors: Shigeaki Kato; Kenju Ueno
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 1997-06-13
Filing date: 1997-06-13
Publication date: 1998-12-17
Also published as: AU3107097A

Abstract

Genes encoding vitamin D receptor isoform proteins which are DNAs containing a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1, nucleotide sequences encoding proteins having an amino acid sequence represented by SEQ ID NO: 1 having a partial substitution, deletion or addition and showing a vitamin D receptor isoform protein activity or nucleotide sequences hybridizable therewith; recombinant vectors containing these genes; prokaryotic or eucaryotic host cells transformed by these recombinant vectors; vitamin D receptor isoform proteins obtained by culturing these host cells; antibodies recognizing these proteins; a method for determining bone density by using these proteins, and a method for screening vitamin D-like substances by using these proteins.

Description

Specification

Vitamin D receptor isoform protein

Branch art

The present invention relates to a gene encoding a novel vitamin D receptor isoform protein, a recombinant vector containing the gene, a host cell transformed with the recombinant vector, and a vitamin obtained by culturing the host cell. The present invention relates to a D receptor isoform protein, an antibody recognizing the protein, a method for diagnosing bone density using the protein, and a method for screening a vitamin D-like substance using the protein.

Background art

1,25-Dihydroxyvitamin D ₃ [1,25 (OH) ₂ D ₃ ] has biological activities such as controlling calcium homeostasis and cell differentiation, but most of its biological activities are in the nucleus. It also acts by the expression of genes mediated by the vitamin D receptor (VDR) (Darish and DeLuca, Crit. Rev. tukaryotic Gene Express., 3: 89-116, 1993). VDR is known to be a member of the nuclear receptor-superfamily that functions as a ligand-inducible transcription factor (Green and Chambon, Trends Genet., 4: 309-314, 1988; Parker, Curr. Opin. Cell Biol., 5: 499-504, 1993). This family includes nuclear receptors for steroid hormones, thyroid hormone and retinoic acid, and an unknown ligand called the orphan 'receptor. Based on similarities in structure and function, VDRs are associated with retinoic acid receptors (RARs), 9-cis retinoic acid receptors (RXRs), and thyroid hormone receptors (TRs) along with subfamilies within nuclear receptors. Forming a ribbon.

Like RAR and TR, VDR is known to form a heterodimer with RXR. These heterodimers bind to different but similar target enhancer elements. This target enhancer element consists of two repetitive A AGGTCA motif (or a related 6-base motif). The spacer existing between the two core motifs is 3 bp (DR 3) in the case of the RX R / VDR heterodimer, and the length is 7! In ¾, it is 413 (DR4), and in RXR / RAR, it is 2 bp (DR2) and 5 bp (DR5). Distinguish (Umesono et al., Cell 6δ: 1255-1266, 1991; astineja d et al., Ature 375: 203-211, 1995)

In addition to the heterodimer formation between RXR and VDR described above, VDR forms a homodimer at several vitamin D response elements (VDREs), indicating that vitamin D has two signaling pathways. Suggest (Carber et al., Nature 361: 657-660, 1993; Towers et al., Proc. Natl. Acad. Sci. USA 90: 6310-6314, 1993).

Despite the functional and structural similarities of these receptors, only one type of VDR protein has been found to date. On the other hand, in RAR, RXR and TR, multiple subtypes and isoforms with altered proteins encoded by them have been found (Kastner et al., Proc. Natl. Acad. Sci. USA 87: 2700-2704, 1990; Leroy et al., EMBO J. 10: 59-69, 1991; Zelent et al., EMBO J. 10: 71-81, 1991). Eight chopcho! The ^ isoform is thought to result from alternative splicing and the control of Z or a different promoter. Further, according to the functional analysis using transient expression Atsusi, than between receptors one isoform has been shown to transfer promoting activity is different (Nagpal, et al, Cell 70 :. 1007-1019, 1992) 0 In addition, genetic experiments using mouse strains lacking the RAR and RXR receptor isoform subtypes have shown that tissue- and development-specific effects of each subtype and isoform on retinoic acid signaling pathways. Important roles have been elucidated (Kastner et al., Cell 78: 987-1003, 1994). Thus, nuclear receptor subtypes and isoforms appear to differentially regulate the biological effects of the ligand.

In vitro functional analysis of VDR (Nakajima et al., Mol. Endocrinol. 8: 159-172, 1994) and hereditary _{1, 25 (OH) 2 D} 3 resistance rickets (HVDRR) genetic mutations seen in patients (Kristjansson et al, J. Clin Invest 92:... 1 2-16, 1993) has shown the importance of VDR for the vitamin D signaling pathway. More recently, a variant of the allele that maps to intron 8 of the human VDR gene (hVDR) is closely linked to osteocalcin (a protein involved in bone formation and maintenance) and bone density. (Morrison et al., Ature 367: 284-287, 1994) ₀ Based on these findings, they used an allelic variant of the hVDR gene to predict bone density, It is proposed that this be used to predict the risk of fracture in adults. They suggest that alterations in the 3 ′ untranslated region (UTR) sequence of mRNA due to splicing alterations due to allelic differences in the human VDR gene may result in different transcriptional half-lives. However, there has been no molecular biological study of how allelic variation affects the vitamin D signaling pathway.

Therefore, considering the various metabolic derivative conductor broad effect and _{1, 25 (OH) 2 D} 3 vitamin D, there may be subtypes or isoforms of VDR protein that affects the activity of vitamin D .

Exons containing a stop codon in many nuclear receptors are known to be longer than other exons. Various genetic polymorphisms are known in the nucleotide sequence of this final exon and the introns before and after it. These genetic polymorphisms are those that occur in non-coding regions, or that the encoded amino acids remain unchanged. It is known that the stability and expression level of mRNA are changed by mutation of bases due to polymorphism. Abnormal splicing associated with disease due to base changes has been described as 1) exon 'skipping, 2) activation of hidden splice sites, 3) generation of pseudo-exons within introns, 4) exonization of introns. Are known and are associated with many diseases: Abnormal splicing is particularly likely around long exons.

As described above, regarding mRNA polymorphisms in nuclear receptors, there are various subtypes with different genes in TR, RAR, RXR, etc. It is known that mRNA polymorphism due to selective slicing occurs under physiological conditions from these genes. In addition, it is thought that the encoded protein changes to produce isoforms and that the stability of mRNA changes, contributing to more precise gene expression regulation.

However, such a phenomenon is not yet known for the vitamin D receptor (VDR); therefore, the object of the present invention was to examine the presence or absence of isoforms resulting from the alternative splicing of VDR, and To study function-Disclosure of invention

In order to achieve the above object, the present inventors have designed DNA fragments encoding various regions of canonical rat VDR (hereinafter referred to as rVDR0) and oligonucleotides of P box in the DNA binding domain (hereinafter, referred to as rVDR0). Parker, Curr. Op in. Cell Biol. 5: 499-504, 1993) to encode novel VDR isoforms by screening cDNA libraries from various murine and avian tissues. The gene could be isolated and its structure determined.

After inserting the gene into an appropriate vector, the transformant transformed with the expression vector is cultured, and the produced target protein is separated and purified to obtain a novel VDR isoform protein. I was able to. This is the first example of such an isoform identified at the vitamin D receptor.

In addition, the function of the novel VDR isoform protein was examined, and it was confirmed that this function negatively regulates the vitamin D signaling pathway.

Therefore, the present invention provides a gene encoding a VDR isoform protein. A preferred gene of the present invention is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1. A nucleotide sequence that encodes a protein having a partially substituted, deleted or added amino acid sequence and having vitamin D receptor isoform protein activity, or These are DNAs containing nucleotide sequences that hybridize to them, and this gene is derived from rat. A preferred gene derived from such a rat is as shown in SEQ ID NO: 2. A preferred gene of the present invention is also a nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide obtained by partially substituting, deleting or adding the nucleotide sequence. A DNA comprising a sequence or a nucleotide sequence that hybridizes to them, the gene being derived from humans.

The present invention also provides a recombinant vector comprising a gene encoding a VDR isoform protein.

The present invention further provides a prokaryotic or eukaryotic host cell transformed by a recombinant vector comprising a gene encoding a VDR isoform protein. The present invention further provides a set comprising a gene encoding a VDR isoform protein It is intended to provide a VDR isoform protein obtained by culturing a transformant obtained by transforming with a recombinant vector.

The present invention further provides an antibody that recognizes a VDR isoform protein _{; the} present invention further provides a method for diagnosing bone density using a VDR isoform protein.

The present invention further provides a method for screening a vitamin D-like substance using a VDR isoform protein. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide and amino acid sequences of rVDR0 and rVDR1 cDNA isolated from one of the rat kidney cDNA libraries.

FIG. 2 shows the structure of the rat VDR genomic region near exons 7-9 and the protein of the two rVDR isoforms (rVDRO and rVDR1) generated by alternative splicing. r Schematic representation of the A to E regions of VDR and their amino acid residues. In the restriction enzyme sites of the rat VDR genomic region, S represents SmaI, H represents Hindm, K represents Kpnl, and P represents Psttl.

Figure 3 shows the expression of rVDR0 and rVDR1 transcripts in rat intestine and kidney. FIG. 4 is a diagram (photograph of electrophoresis) analyzed by Northern blot using these poly A (+) mRNAs.

FIG. 4 shows the results of a dominant negative activity test of rVDR0 against rVDR1 using CAT technology.

FIG. 5 shows the results of a dose-dependent activity test of rVDR1 using CAT Atsey.

FIG. 6 shows the results of a study using CAT Atsey to study the effect of rVDR1 on thyroid hormone and retinoic acid signaling pathways.

FIG. 7 is a graph showing that the dominant negative activity of I ″ VDR1 is sequence-specific.

Figure 8 shows samples obtained by expressing rVDRO and rVDR1 as GST fusion proteins in Escherichia coli (GST-rVDR0 and GST-rVDR1), and digested and purified with thrombin. _The molecular weight measured on a polyacrylamide SDS gel using rVDR0 and rVDR1 samples is shown (photograph of electrophoresis). FIG. 9 shows the results of gel shift assay performed on various amounts of purified mouse RXR (RXR), purified rVDRO and rVDR1 using DR3T as a probe (electrophoresis pictures).

Figure 10 shows the gel shift assay performed on various amounts of purified rVDRI, chicken TRH (TR) and mouse RARa (RAR) using 0 shaku 4 and 0! ¾5 as probes. The results are shown (photograph of electrophoresis).

Figure 1 1 is a synthetic recombinant r VDR protein (r VDR O or r VDR 1) at E. coli, and _{1, 2 5 (OH) 2} D 3 l nM labeled with ^[3 H], labels and Tei no 1, 2 5 (OH) ₂ D ₃ was added at various concentrations, 4 ° C, 1 to 6 hours ink Yupeto, vitamin D not bound except by centrifugation, the recombinant r VDR protein The result of measuring the radioactivity of the bound ligand is shown. BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, the gene encoding the VDR isoform protein Was able to get by. However, the method for obtaining the gene of the present invention is not limited to this.It is possible to prepare cDNA from tissues, cells, etc., which express the gene of the present invention, using methods known to those skilled in the art, as described below. Can ₃

In this way, the gene encoding the cloned VDR isoform protein can be transformed into other prokaryotic or eukaryotic host cells by incorporating it into the appropriate vector DNA.

Furthermore, by introducing an appropriate promoter and a sequence relating to expression into these vectors, the gene can be expressed in each host cell. In addition to the gene of interest, a gene encoding a polypeptide can be expressed. By ligating and expressing as a fusion protein, purification can be facilitated, or the expression level can be increased, and the target protein can be cut out by subjecting it to an appropriate treatment in the purification process. In the examples described below, after expressing and purifying as a GST fusion protein, GST was removed and the VDR isoform protein was isolated.

In general, eukaryotic genes are considered to exhibit polymorphism, as is known at the human interferon gene (eg, Ni shi et al .: [. Biochem. 97 153 198 5]). In some cases, one or more amino acids may be replaced by a shape phenomenon, and in other cases, the amino acid may not change at all even though the nucleotide sequence changes.

Also, a polypeptide lacking or adding one or more amino acids in the amino acid sequence of SEQ ID NO: 1 or a polypeptide in which an amino acid is substituted with one or more amino acids. However, it may have the activity of VDR isoform protein. For example, it is already known that a polypeptide obtained by converting a nucleotide sequence corresponding to cysteine of the human interleukin 2 ((L-2) gene to a sequence corresponding to serine retains IL-12 activity. (Wang et al., Science, 224 1431 1984) _c

In addition, when expressed in eukaryotic cells, sugar chains are often added, but the addition of sugar chains can be regulated by converting one or more amino acids. May have the activity of

Further, the obtained polypeptide has VDR isoform protein activity, The present invention also includes a gene encoding a polypeptide containing the amino acid sequence shown in SEQ ID NO: 1 or a gene which hybridizes with the nucleotide sequence shown in SEQ ID NO: 2 or 3. The conditions may be the same as those used in probe hybridization which is usually performed (for example, Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold spring Habor Laboratory Press, 1989).

The expression vector of the present invention contains an origin of replication, a selection marker, a promoter located in front of the gene to be expressed, an RNA splice site, a polyadenylated sidanal, and the like.

Prokaryotic host cells among the hosts used in the expression system of the present invention include, for example, Escherichia coli, Bacillus subtilis, Bacillus thermophilus, and the like. No. Among eukaryotes, host cells for eukaryotic microorganisms include, for example, Saccharomyces cerevisiae, and host cells derived from mammals include, for example, COS cells and Chinese hamster ovary. (CHO) cells, C127 cells, 3T3 cells, Hela cells, BHK cells, Nabarba cells and the like. The transformant of the present invention may be cultured by appropriately selecting culture conditions suitable for the host cell.

As described above, the transformant transformed with the gene encoding the desired VDR isoform protein is cultured, and the produced VDR isoform protein is isolated from inside or outside the cell. It can be purified to homogeneity.

Separation and purification of the VDR isoform protein, which is the target protein of the present invention, may be performed by a separation and purification method used for ordinary proteins, and is not limited in any way. For example, various types of chromatography, ultrafiltration, salting, dialysis and the like can be appropriately selected and used in combination.

To measure the activity of a VDR isoform protein, for example, a transient expression assay, which is a method for measuring gene transcription activity using a CAT (chloramphenicylacetyltransferase) gene as a reporter gene, described below. (C AT Atsushi).

Maintain the HeLa cells in Dulbecco's modified Eagle's medium (without phenol red, supplemented with 5% dextran coated charcoal-treated fetal serum). Transform 40-50% confluent cells in a 9-cm petri dish using a total of 20 μg of DNA by the calcium phosphate method: DNA used is 2 μg of CAT reporter plasmid and receptor expression Add 500 ng of vector and pCH100 (Pharmacia), a 3-galactosidase expression vector, which is used as an internal control to normalize the variation due to transformation efficiency, and adjust the total amount of DNA. Use Bluescribe M1 3 + (Stra tagene) as a carrier for

One hour after transformation and at the time of each medium change, add a ligand (1 μM of all-trans-retinoic acid or 0.1 μM of thyroid hormone or vitamin D O. 1 μM) to the medium. After a 20 hour incubation with the calcium phosphate precipitated DNA, wash the cells with fresh medium and incubate for an additional 20 to 24 hours. A cell extract is prepared by lyophilization, and the CAT is assayed after normalizing the j3-galactosidase activity by the method described in the literature (Sasaki et al., Biochemistry 34: 370-377, 1995).

The antibody recognizing the VDR isoform protein of the present invention may be a polyclonal antibody or a monoclonal antibody. In preparing a polyclonal antibody, a conventional method (for example, Shinsei Chemistry Laboratory Course 1, Protein I, According to p389-139, 1992), the antigen (VDR isoform protein) is immunized to animals such as egrets, rats, goats, sheep, and mice, and is produced in vivo. The antibody can be obtained by collecting the antibody. The titer of the obtained antibody can be measured by a method known in the art. Monoclonal antibodies can also be prepared according to a conventional method (for example, Kohler et al., Nature 256: 496, 1975; Kohler et al., Eur. J. Immunol. 6: 511, 1976). That is, as described above, an animal is immunized to obtain antibody secreting somatic cells, which are fused with a myeloma cell line, and a hybrid that produces antibodies is selected. Also, binding fragments of such monoclonal and polyclonal antibodies, eg, Fab, F (ab,), Fv fragments, can be used as the antibodies of the present invention: Antibody fragments can be intact antibodies such as papain or pepsin. It can be obtained by conventional methods after digestion.

The present invention will be specifically described below.

Isolation of genes encoding VDR isoform proteins

Using the ligand-binding domain of canonical rat VDR (rVDRO) (Sasaki et al., Biochemistry 34: 370-377, 1995), 10 positive clones closely related to rVDRO cDNA Was isolated. These are then performed by volimerase chain reaction (PCR) using primer pairs corresponding to exons encoding the rVDR0 protein (Burmester et al., Pro Natl. Acad. Sci. USA 85: 1005-1009, 1988). Were analyzed and two clones encoding the same isoform (hereinafter referred to as rVDR1) were found.] RVDR0 and rVDRl were sequenced and 285 nuclei specific for rVDR1 were sequenced. Leotide was identified (Figure 1). In rVDR1, 1134 bp was inserted into the ligand binding domain of rVDR0, but the other sequences of rVDR1 were identical to the open reading frame of rVDR0 (FIG. 1).

r By analyzing the genomic structure and sequence of the VDR gene, it was found that the inserted sequence was derived from the intron between exons 8 and 9 (Fig. 2), and that the sequence between exons and intro was C This is consistent with Hambon's general rule (5, sides on both sides of the intron is GT, 3 on the side is AG), thus suggesting that this isoform was generated by the primary transcript by alternative splicing: Retaining intron 8 as an exon of rVDR1 will result in a relatively large exon located in the most downstream region of the locus. This gene structure of the rat VDR is very similar to the structure of other members of the receptor superfamily with large final exons (Gronemeyer, Annu. Rev. Gen et. 25: 89-123, 1991).

Therefore, the VDR isoform protein (rVDRl) of the present invention is The gene to be loaded has the nucleotide sequence shown in SEQ ID NO: 2, and the amino acid sequence shown in SEQ ID NO: 1. The deduced amino acid sequence of rVDR1 lacks 86 amino acids at the C-terminus and has an extra 19 amino acids compared to rVDR0.) This is due to the 1 1 of the intron existing between exon 8 and exon 9. Probably because translation was stopped by the stobcodon located at 34 bp (see FIGS. 1 and 2) .: Then, this exon of rVDR1 was used as a specific probe for Northern blots. The presence of the transcript (Sasaki et al., Biochemistry 34: 370-377, 1995) was detected. As a result, the transcript r VD R 0 is detected in the kidney and intestine expressing (FIG. 3) ₃ - Non comprising 1 04 2 bp of how, intron 6 located between Ekison 6 and 7 Specific transcripts could not be detected using the specific probe: Analysis of the specific band with a densitometer showed that the amount of rVDR1 transcript was 1/15 to 1 Z20 of the amount of rVDR0. Met. In addition, poly (A) ⁺ mRNA from various tissues was converted into cDNA using reverse transcriptase, and then amplified with PCR to detect rVDR1 transcript in the cytosolic mRNA fraction. The presence of the VDR1 transcript was confirmed: thus, it was suggested that the rVDR1 transcript was localized in the cytosol as mature mRNA for transcription.

r Functional role of VDR 1

A recent report that the exact location of the ligand-binding domain at the C-terminus of human VDR maps to 403-427 amino acid residues (399-423 amino acids in rVDR0) (Nakajima et al., ol. Endocrinol., 8: 159-172, 1994), the rVDR1 protein synthesized in vitro showed no ligand binding (Fig. 11).

One of the two domains required for heterodimer formation with RX R (heptad 9: 399-407 amino acids in rVDRO) was absent from rVDRl but involved in specific DNA binding The other region (heptad 4: 321 to 328 amino acids in rVDRO) was present in rVDRl.

Therefore, the transcription promoting activity of rVDR1 was evaluated using transiently expressed atsate (CAT atsee) (Sasaki et al., Biochemis try 34: 370-377, 1995). r V I 2

Vectors expressing DR0, rVDR1 and rat RXRc, and a CAT reporter plasmid containing a consensus vitamin D response element (VDRE) were prepared. This consensus vitamin D response element is one in which two AGTTCA motifs are directly linked via the 3 bp spacer (DR3T) described above. This motif is said to be a stronger VDR binding motif than AG GTCA (Freedman et al., Mol. Endcrinol., 8: 265-273, 1994): As a result, rVDR1 itself promotes transcription. Did not have any activity: When co-transfection of rVDRO (0.5 μg) and rVDRl (2 μg), all cells tested (see below) In the examples, HeLa cells are shown as a representative example), and the ligand-induced transcription promoting activity by rVDR0 was inhibited (see FIG. 4). In addition, when the amount of the rVDR1 expression vector added was increased in the presence of rVDRO (expression vector-0.5 Mg), the inhibitory activity of rVDR1 became more remarkable (see FIG. 5). The addition of 5 μg of the rVDR0 expression vector did not suppress ligand-induced transcription-promoting activity (Figure 5), indicating that this inhibitory effect was limited by the interaction between the two isoforms with limited nuclear factors. Just antagonism

(Tasset et al., Cell 62: 1177-1187, 1990). Further, although no data is described herein, no clear difference was observed between the three subtypes of RXR (α, β, γ).

The dominant negative activity of rVDR1 is sequence-specific

The degree of inhibition of rVDR1 on rVDR0 was sequence-specific, and was more pronounced for mouse 'osteobontin than for human' osteocalcin VDRE. This is to be because the VDR homodimer prefers binding with murine Osuteoponchin target VDRE. (Cheskis et al, Molec Cell Biol 14:.... 3329-3338, 1994) found the following ₀ AGTTCA motif The notion of superiority as a binding core for VDRs over the AGGTCA motif is supported by the fact that DR3T is more active as a VDRE than DR3G (see Figure 7). On the other hand, rVDR1 is a consensus response element for retinoic acid (DR5) and a consensus for thyroid hormone when the same type of receptor is present. It did not suppress the ligand-induced transcription-promoting activity of the sponge element (DR4) (see Fig. 6). When the same type of response element was present, r VDRl was replaced by another retinoic acid response element (0 2) and the estrogen response element (consensus ERE) had no apparent effect.c Therefore, these results indicate that the rVDR1 isoform acts as a dominant negative receptor for rVDR0. Suggest that.

rVPR1 binds to VDRE as a homodimer but does not form a heterodimer complex with RXR

To investigate the molecular mechanism of dominant negative activity of rVDR1, binding activity to consensus VDRE (DR3T) was tested and compared to that of rVDRO.

For this reason, rVDRO protein and rVDR1 protein were produced by genetic recombination. The open reading frames of the rDNAs of rVDR0 and rVDR1 were predicted to be 48 KDa and 40 KDa proteins, respectively: The purified rVDR0 and rVDR1 proteins were SDS- It moved to the position of the predicted molecular weight on the PAGE gel (Figure 8).

As a result of the binding test with DR3T, the purified recombinant rVDRO protein was homodimer-free without RXR, as described in the literature (Freedman et al., Ol. Endocrinol. 8: 265-273, 1994). As DR3T (Fig. 9). r VDR l homodimer also bound DR 3 T to the same extent. Then, when mouse RX Rct was added to rVD R0, DNA binding was significantly increased by heterodimer formation, and a specific monoclonal antibody against RXR α recognized and bound this DNΑ-heterodimer, resulting in electrical The migration band shifted. This confirmed that RXR was present in the complex. However, rVDRl cannot form a heterodimer with rRXRa, which is a mutant without the C-terminus of human VDR (hVDR) (Nakajima et al., Mol.Endocrinol. 8: As in 159-172, 199 4), it seems to be because it does not have the C-terminal domain necessary for heterodimer formation. rVDR1 does not specifically bind to the consensus thyroid response element (DR4) and retinoic acid response element (DR5) (Figure 10), indicating that rVDR1 has specificity for the target enhancer element. Suggests that it is the same as VDR 0.

In VDR, similar to RAR, RXR and TR, conventional observations show that the dimer interface formed between DNA binding domains specifies receptor-dimer binding recognition with its cognate response element (eg, Rastinejad et al. , ature 375: 203-211, 1995) and this result agree well.

To summarize the results of the transcriptional activation activity, rVDR1 acts as a dominant negative receptor in the vitamin D signaling pathway by competitively binding to the target VDRE (vitamin D response element) as a homodimer. Shows

The novel rat VDR isoform (rVDRl) obtained in the present invention is a primary rVDR transcript generated by alternative splicing, whereas rVDR1 is an extra gene present in intron 8 in rVDR0. With exon. This new exon

The stop codon at (intron 8 in rVDR0) loses part of the C-terminal ligand binding domain (86 amino acids) but adds 19 amino acids.

Isolation of human VDR isoforms

Primers were designed to specifically amplify intron 8 from the known human VDR intron 8 sequence (WO 94-036333), and intron 8 was amplified from human genomic DNA. Using this intron 8 as a probe incorporating ³² P-d CTP with random primers, a human leukocyte cDNA library (C1 ontech) was screened, and human VDR intron 7, exon 8, intron 8 A cDNA fragment containing 8 was obtained: in humans, unlike in rats, intron 7 is also different from rat, because intron 7 is in full-length frame and ligated to exon 8 during amino acid translation. The fragment of the retained isoform cDNA could be cloned.

The nucleotide sequence of the obtained cDNA fragment was determined, and the nucleotide sequence shown in SEQ ID NO: 3 was determined. It was confirmed to have the peptide sequence.

In humans, clones that also retained intron 7 were obtained, so other isoforms (eg, isofs with only intron 7; arms, isoforms with introns 7 and 8, only intron 8) The nucleotide sequence of intron 7 in the human VDR isoform is shown in SEQ ID NO: 4 in the Sequence Listing, and the nucleotide sequence of intron 8 is shown in SEQ ID NO: 5. .

Functions of VDR isoforms and their use

Due to the similarity of structure and function, it forms a subfamily in the nuclear receptor Hoover family with VDI ^ tRAR, RXR and TR. However,

VDR is unique within this subfamily because the RAR, RXR and TR isoform proteins consist of various exons combined by alternative splicing and the use of Z or different promoters It can be said that. For some genes, it is already known that retaining introns as exons results in functionally distinct isoform proteins (Nakaraura et al., Science 257: 1138—1142, 1992). This is the first example of a superfamily receptor isoform: this is why, unlike RAR, RXR and TR, the VDR isoform has been cloned despite its cDNA cloning. It appears that the VDR isoform protein of the present invention acts as a dominant negative receptor in the vitamin D signaling pathway: a recent report (Morrison et al., Pro atl. Acad. Sci. USA 89: 6665-6669, 1994), allelic changes in the human VDR gene indicate changes in blood osteocalcin concentration and bone mineral density. Closely related. Bone density can be a predictor of osteoporotic fracture risk: The report states that allelic changes in the human VDR gene that predict bone density are located in intron 8. Thus, VDR isoform protein that r VD R 1 obtained in the present invention is caused by holding the Intoron 8 rats VDR gene is extremely interesting _Q present invention It can be expected to elucidate the regulation mechanism of the vitamin D signaling pathway and to use it to improve bone density. In addition, it is possible to diagnose bone density using the VDR isoform proteins of the invention:

Further, it is considered that the expression level of the VDR isoform of the present invention is related to various diseases. Diseases such as osteoporosis, fractures, secondary hyperthyroidism, immune diseases, and skin diseases are related to the onset of the expression of the isoform of the present invention in all diseases considered to be associated with VDR. Therefore, the isolation and characterization of the VDR isoforms of the present invention has great significance in elucidating and treating these diseases.

Furthermore, the VDR isoform protein of the present invention can be used to screen for a vitamin D-like substance .: For example, a gene encoding the VDR protein of the present invention is transfected into an appropriate cell, A substance having at least one of vitamin D-like actions (eg, calcium-bone metabolism, differentiation-inducing action, immunosuppression, antitumor action, fat-lipid metabolism suppression) using the cells (eg, steroids) , Retinoic acid).

By using the gene for the VDR isoform protein of the present invention in this screening system, a system closer to physiological conditions can be constructed. Therefore, the accuracy is expected to be higher than that of the screening system using the existing VDR gene.

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto. Example

Example 1: Cloning of cDNA and Genomic DNA

c Cloning of DNA

r An XhoI-PstI restriction enzyme fragment containing the ligand binding domain of VDR0 was radiolabeled and used as a probe to screen a rat kidney cDNA library (Clontech). 50% Holm for the hybridization mixture I7 amide, lx Denhards solution, 5 x SSPE, 100 μg / m 1 denatured salmon sperm DNA and 10 ^e cpm of ³² P-labeled probe DNA: 42 C of dinitrocellulose membrane Hybridized for 16 hours, washed twice in 2 × SSC, 0.1% SDS, 55 ^C C, 30 minutes. The film was exposed to 180 using a sensitive screen for 16 hours.

rVD RO Polymer (Burmester et al., Proc. Natl. Acad. Sci. USA 85: 1005-1009, 1988). Polymers using primers corresponding to exons—zechase reaction (PCR) analyzing the emissions from 1 0 ⁶ plaques were selected 1 0 positive clones: was further select two clones encoding the r VDR 1 is the same Aisofu ohms from this. That is, when the nucleotide sequence was determined, it was found that out of the 10 positive clones, 8 were rVDR0 and 2 were rVDR1.

Cloning of genome DNA

Using the SmaI-KpnI fragment of rVDRlcDNA as a probe, a genomic fragment of about 20 kb having a BamHI site derived from a rat genomic library (cio ech) was cloned. A 3.5 kb DNA fragment with a PstI site containing intron 8 was subcloned and sequenced.

The nucleotide sequence of the obtained rVDRl is shown in SEQ ID NO: 2, and the deduced amino acid sequence is shown in SEQ ID NO: 1. The sequence compared to rVDR0 is shown in FIG. 1, and the relevant region of the VDR genome and the mapping of rVDR1 generated by alternative splicing are shown in FIG. 2 in comparison with rVDR0. Example 2: Northern plot analysis

Expression of rVDRO and rVDR1 transcripts was tested by Northern plot analysis using 3 g of Bori A) mRNA from rat intestine and kidney. Exon 8 of rVDR1 was used as a probe. Fig. 3 shows the obtained results. The rVDR1 transcript was expressed in the gut and kidney where rVDR0 was expressed. The relative amount of rVDR transcript was calculated by scanning the specific band with a densitometer: When the average of three or more samples was determined, the amount of rVDR1 transcript was 1/15 to 1/20 of the amount of rVDR0. Example 3: Construction of plasmid

Synthetic oligonucleotides with Hind II and Xba I restriction sites were inserted into the corresponding restriction sites of the plasmid pGCAT (Kato et al., Cell 68: 731-742, 1992) to insert the CAT reporter gene. It was constructed. The oligonucleotide sequences used as probes in the CAT reporter gene and gel shift assay are as follows:

DR3G: 5'-AGCTTCAGGTCAGGAAGGTCAGT-3 ';

DR3T: 5'-AGCTTCAGTTCGGAAGTTCAGT-3 ';

DR4: 5'-AGCTTCAGGTCACGGAAGGTCAGT-3 ';

DR5: δ'-AGCTTCAGGTCACCGGAAGGTCAGT-3 ';

Human osteocalcin VD RE (O C): Mouse osteopontin VD RE (Ο ΡΝ):

δ'-AGCTTGCTCGGGTAGGGTTCACGAGGTTCACTCGACTCGT-3 '

In addition, by replacing the SacI—BamHI fragment of rVDR0 with the rVDR1 fragment (911 to L071 bp in FIG. 1) amplified by PCR, rVDRl An expression vector was constructed. Example 4: Expression and purification of recombinant VDR protein

The cDNAs encoding rVDR0 and rVDRl were amplified by PCR using BamHI and EcoRI restriction sites and inserted into the corresponding sites of plasmid pGEX-2T (Pharmacia). did. Escherichia coli (DH5c) was transformed with these vectors and induced with IPTG (0.1 mM).

The resulting GST fusion protein was purified with Daltathione Sepharose 4B. After digesting 500 g of each GST fusion protein with thrombin (5 U), Thrombin and GST were removed through Thion Sepharose. The flow-through fraction was further supplemented with 50 mM Tris-HCI Knuffer-1 (pH 8.0) containing 1 M NaCI, 0.1 mM DTT and 10% glycerol, Sephadex G20. It was applied to column 0. The purity of these proteins was over 95% on SDS-PAGE. Example 5: Effect of r VDR 1 by CAT technology

The dominant negative activity of rVDR1 against rVDR0 was tested.

CAT reporter plasmid containing two 5'-AG TTCA motifs via a 3 bp (DR 3 T) spacer, as well as mouse RXR a (0.5 μg), r VDR 0 (0.5 μg) and rVDR1 (2 μg) were transformed into HeLa cells. The transformed cells 1, 2 5 - (OH) and maintained 4 4 hours in the presence of _{_{2 D 3 (1 0 n)}} (+) or absence (1), expressed by p CH ll O internal control vector CAT activity was normalized by β-galactosidase activity and is expressed as the mean soil standard deviation from at least three independent tests: The results obtained are shown in FIG.

As is clear from the figure, rVDR1 itself did not have a transcription promoting activity. However, when the r VDR 0 and r VDR 1 co to transflector Ekushi Yon, H e L in a cell, r VDR 0 _c embodiments inhibit transcription promoting activity of ligand-induced by 6: dose of r VDR 1 Dependent activity

Cells r VDR 0 expression vector 0. 5 / ig, as well as 1, 2 5 - (OH) in the presence of _{_{2 D 3 (1 0 n M}} ), the amount of VDR expression base Kuta one shown in FIG. 5 (r VDRO Or rVDR1). CAT activity was calculated in the same manner as in Example 5. As is clear from FIG. 5, the inhibitory activity of rVDR1 became more prominent when the amount of the rVDR1 expression vector added was increased in the presence of rVDR0. r When 5 μg of the VDRO expression vector was added, the ligand-induced transcription promoting activity was not suppressed. Example 7: Effect of rVDR1 on thyroid hormone and retinoic acid signaling pathway

Cat reporter plasmid containing two 5'-AGTT CA motifs via a 4 bp (DR4: consensus thyroid hormone response element) or 5 bp (DR5: consensus retinoic acid response element) promoter , And a vector that expresses mouse RXR and chicken TR ct (for DR4) or RXR α and mouse RARct (for DR 5), and the same type of ligand [Ι ΜηΜ thyroid hormone (Τ ₃ ), 1 μΜ all-trans retinoin The cells were transformed in the presence or absence of [acid]. The effect of rVDR1 was tested by co-transfection with the rVDR1 expression vector (2 μ). CAT activity was calculated in the same manner as in Example 5.

The results obtained are shown in FIG. r VDR1 was found not to suppress ligand-induced transcriptional activation of retinoic acid consensus response element (DR5) or thyroid hormone consensus response element (DR4) in the presence of the same type of receptor. Example 8: Sequence specificity of rVDR1 inhibitory activity against rVDR0

Using CAT reporter plasmids, including DR3 G, DR 3T, hyosteocalcin VDRE (OC) and mouse steobontin VDRE (OPN), as well as RXR and VDR (rVDR0 or rVDR1) expression vectors HeLa cells were transformed in the presence or absence of 1,25- (OH) ₂ D ₃ (Ι ΜηΜ). CAT activity was calculated in the same manner as in Example 5.

Fig. 7 shows the obtained results. The degree of inhibition of rVDR1 against rVDR0 was sequence-specific, and was more pronounced for mouse osteobontin than for human osteocalcin VDRE. It was also observed that DR 3 T was more active as VDR E than DR 3 G. Example 9: Formation of homodimers and heterodimers of rVDR1 and their DNA binding properties

(A) r Molecular weight of VDR l

According to the method of Example 4, rVDR0 and rVDR1 were expressed as GST fusion proteins in Escherichia coli, purified with daltathione.sepharose 4Β, and then digested with thrombin. The digested sample was applied to a Sephadex G-100 column to further purify rVDRO and rVDR1 proteins. The GST fusion protein (shown as GST-rVDR0 or GST-rVDR1 in Figure 8) and the purified rVDR protein (shown as rVDR0 or rVDR1 in Figure 8) are 5% polyacrylamide. Electrophoresis was performed on a do SDS gel, and the molecular weight was determined from the molecular weight marker: the respective molecular weights expected from the open reading frames of rVDR0 and rVDR1 (48 KDa for rVDR0;! ") At ¥ 01¾1, a band was observed at 401: 0 a).

(B) Gersif Totssey

Electrophoretic migration shift assay (EMSA) and antibody supershift were performed using the method described in the literature (Sasaki et a] .., Biochemistry 34: 370-377, 1995). The following purified receptors were also used in this assay:

RAR: partially purified mouse RARct lacking AB region generated in E. coli

(mRAR αΔΑΒ);

RXR: partially purified mouse lacking AB region generated in E. coli

(mRX R αΔΑΒ);

TR: Partially purified chicken produced in E. coli TR

The antibody super one shift using a monoclonal antibody 4 RX (for RXR): receptor and ^[32 Ρ] - 5- end-labeled synthetic oligonucleotide (DR3 T, DR 3G, DR4 and DR5) coupled reaction mixture containing, And poly (did C) (Pharmacia, 2 μg) in a binding buffer [10 mM Tris-HC1 (pH 7.5), 1 mM dithiothreitol, 1 mM EDTA, 100 mM KC 10% [Glycerol] for 15 minutes at 25 ° C. ink Yubeshiyo emission start documents antibody was added during _{(s asa ki et al, Biochemistry} 34:. 370-377, 1995) by the method described, it was dissolved the product obtained in 5% polyacrylamide Riruami de gel.

Using DR3T as a probe, gel shift assays were performed with various amounts of purified mouse RXRα (RXR), purified rVDR0 and rVDR1 shown in FIG. The results obtained are shown in Figure 9: The presence of DR 3 T one RXR-VDR complex by super shift trials with monoclonal antibodies (4 RX for mouse RX R _alpha) was confirmed. In FIG. 9, the band in lane 6 shows the position of the complex supershifted with the anti-RXR antibody: This shows that the rVDR1 homodimer binds to the consensus VDRE (DR3T).

Next, gel shift assays were performed with DR 4 and DR 5 as probes using various amounts of purified rVDR1, chicken TRc (TR) and mouse RAR ct (R AR) as shown in FIG. The results obtained are shown in Figure 10: rVDRl homodimer did not bind to the consensus thyroid hormone response element or the retinoic acid response element (0.40 and 0! ^ 5). Example 10: Isolation of human VDR isoform

VDR intron 8 was specifically amplified from human genomic DNA by PCR, radiolabeled by random prime method, and used as a probe to screen a human leukocyte cDNA library (Clontech). Hybridization mixture 50% formamide in, 5 x D enhardt, s solution, 5 x SSC, 0. 1% SDS, 200 μ g / m 1 denatured salmon sperm DNA and 1 0 ⁶ cpm ³² P- labeled probes DNA was included. The nitrocellulose membrane was hybridized at 42 ° C for 16 hours, once in 4 x SSC, 0.1% SDS, once in 2 x SSC, 0.1% SDS, lx SSC, 0.1% Each was washed once in SDS at room temperature for 10 minutes. The film was exposed for 16 hours at 180 ° C using a sensitizing screen. 4 xl 0 ⁶ claw N'yori about 1. 1 was obtained positive clones with Insato of 4 kb.

The nucleotide sequence of the obtained clone was determined, and the nucleotide sequence of SEQ ID NO: 3 was determined. Sequence obtained: This nucleotide sequence contains human VDR intron 7, exon 8, intron 8. The nucleotide sequence of intron 7 in the human VDR isoform is shown in SEQ ID NO: 4 in the sequence listing, and the nucleotide sequence of intron 8 is shown in SEQ ID NO: 5.

[Sequence list]

SEQ ID NO: 1

Array length: 3 5 6

Sequence type: amino acid

Topo mouth: linear

Sequence type: Peptide

Array

SEQ ID NO: 2

Array length: 1 0 7 1

Sequence type: nucleic acid

Number of chains: two

Topology: linear

Sequence type: c D N A

Array

ATGGAGGCAA CAGCGGCCAG CACCTCCCTG CCCGACCCTG GTGACTTTGA CCGGAACGTG 60 CCCCGGATCT GTGGAGTGTG TGGAGACCGA GCCACAGGCT TCCACTTCAA TGCTATGACC 120 TGTGAAGGCT GCAAAGGTTT CTTCAGGCGG AGCATGAAGC GGAAGGCCCT GTTCACCTGT 180 CCCTTCAATG GAGATTGCCG CATCACCAAG GACAACCGGC GACACTGCCA GGCCTGCCGG 240 CTCAAACGCT GTGTGGACAT CGGCATGATG AAGGAGTTCA TCCTGACAGA TGAGGAGGTA 300 CAGCGTAAGA GGGAGATGAT AATGAAGAGA AAAGAGGAAG AGGCCTTGAA GGACAGTCTG 360 AGGCCCAAGC TATCTGAAGA ACAACAGCAC ATCATAGCCA TCCTGCTGGA CGCCCACCAC 420 AAGACCTATG ACCCCACCTA CGCTGACTTC AGGGACTTCC GGCCTCCAGT TCGTATGGAC 480

GGAAGTACAG GGAGCTATTC TCCAAGGCCC ACACTCAGCT TCTCCGGGAA CTCCTCCTCC 540

TCCAGCTCTG ACCTGTACAC CACCTCACTA GACATGATGG AACCATCCGG CTTTTCCAAC 600

CTGGATCTGA ACGGAGAGGA TTCTGATGAC CCGTCTGTGA CTCTGGACCT GTCTCCTCTC 660

TCCATGCTGC CCCACCTGGC TGACCTTGTC AGTTACAGCA TCCAAAAGGT CATCGGCTTT 720

GCCAAGATGA TCCCAGGATT CAGGGATCTC ACCTCCGATG ACCAGATTGT CCTGCTTAAG 780

TCAAGCGCCA TTGAGGTGAT CATGTTACGC TCCAACCAGT CTTTCACCAT GGATGATATG 840

TCCTGGGACT GTGGCAGCCA GGACTACAAG TACGACGTCA CCGATGTCTC CAAAGCTGGG 900

CACACCCTGG AGCTGATCGA GCCCCTCATA AAGTTCCAGG TGGGGCTGAA GAAGCTGAAC 960

TTACATGAGG AAGAGCATGT CCTTCTCATG GCCATCTGCA TTGTCTCCCC GGGTACGGAG 1020

CCTGGCAGGG AGGAGCTGAG GGACCTGGGA CACGTTGGGG ACTGTGAATG A 1071 SEQ ID NO: 3

Array length: 1 4 0 4

Sequence type: nucleic acid

Number of chains: two

Topology: linear

Sequence type: c D N A

Array

ATGGAGGCAA TGGCGGCCAG CACTTCCCTG CCTGACCCTG GAGACTTTGA CCGGAACGTG 60 CCCCGGATCT GTGGGGTGTG TGGAGACCGA GCCACTGGCT TTCACTTCAA TGCTATGACC 120 TGTGAAGGCT GCAAAGGCTT CTTCAGGCGA AGCATGAAGC GGAAGGCACT ATTCACCTGC 180

CCCTTCAACG GGGACTGCCG CATCACCAAG GACAACCGAC GCCACTGCCA GGCCTGCCGG 240

CTCAAACGCT GTGTGGACAT CGGCATGATG AAGGAGTTCA TTCTGACAGA TGAGGAAGTG 300

CAGAGGAAGC GGGAGATGAT CCTGAAGCGG AAGGAGGAGG AGGCCTTGAA GGACAGTCTG 360

CGGCCCAAGC TGTCTGAGGA GCAGCAGCGC ATCATTGCCA TACTGCTGGA CGCCCACCAT 420

AAGACCTACG ACCCCACCTA CTCCGACTTC TGCCAGTTCC GGCCTCCAGT TCGTGTGAAT 480

GATGGTGGAG GGAGCCATCC TTCCAGGCCC AACTCCAGAC ACACTCCCAG CTTCTCTGGG 540 GACTCCTCCT CCTCCTGCTC AGATCACTGT ATCACCTCTT CAGACATGAT GGACTCGTCC 600

AGCTTCTCCA ATCTGGATCT GAGTGAAGAA GATTCAGATG ACCCTTCTGT GACCCTAGAG 660

CTGTCCCAGC TCTCCATGCT GCCCCACCTG GCTGACCTGG TCAGTTACAG CATCCAAAAG 720

GTCATTGGCT TTGCTAAGAT GATACCAGGA TTCAGAGACC TCACCTCTGA GGACCAGATC 780

GTACTGCTGA AGTCAAGTGC CATTGAGGTC ATCATGTTGC GCTCCAATGA GTCCTTCACC 840

ATGGACGACA TGTCCTGGAC CTGTGGCAAC CAAGACTACA AGTACCGCGT CAGTGACGTG 900

ACCAAAGGTA TGCCTAGNNN NCACCTCCTG GGGAGTCTTT TTCAGCTCCC AGATTCTGGC 960

TCCACCCGTC CTGGGGTTTG GCTCCAATCA GATACATGGG AGGGAGTTAG GCACCAACAG 1020

GCAGAGAAGG GCGAGGGTCA GACCCATGGG GTTGGAGGTG GGTGGGCGGC TCCTCAGCTC 1080

TTGCCCGCAG TACCTCCCCA TTGTCTCTCA CAGGCCGGAC ACAGCCTGGA GCTGATTGAG 1140

CCCCTCATCA AGTTCCAGGT GGGACTGAAG AAGCTGAACT TGCATGAGGA GGAGCATGTC 1200

CTGCTCATGG CCATCTGCAT CGTCTCCCCA GGTATGGGGC CAGGCAGGGA GGAGCTCAGG 1260

GACCTGGGGA GCGGGGAGTA TGAAGGACAA AGACCTGCTG AGGGCCAGCT GGGCAACCTG 1320

AAGGGAGACG TAGCAAAAGG AGACACAGAT AAGGAAATAC CTACTTTGCT GGTTTGCAGA 1380

GCCCCTGTGG TGTGTGGACG CTGA 1404 SEQ ID NO: 4

Array length: 2 1 0

Sequence type: nucleic acid

Number of chains: two

Topology: linear

Sequence type: c D N A

Array

GGTATGCCTA GNNNNCACCT CCTGGGGAGT CTTTTTCAGC TCCCAGATTC TGGCTCCACC 60 CGTCCTGGGG TTTGGCTCCA ATCAGATACA TGGGAGGGAG TTAGGCACCA ACAGGCAGAG 120 AAGGGCGAGG GTCAGACCCA TGGGGTTGGA GGGCGCTCTC GC

GCAGTACCTC CCCATTGTCT CTCACAGGCC 210 SEQ ID NO: 5

Array length: 1 7 3

Sequence type: nucleic acid

Number of chains: two

Toboguchi: straight chain

Sequence type: c D N A

Array

GTATGGGGCC AGGCAGGGAG GAGCTCAGGG ACCTGGGGAG CGGGGAGTAT GAAGGACAAA 60 GACCTGCTGA GGGCCAGCTG GGCAACCTGA AGGGAGACGT AGCAAAAGGA GACACAGATA 120 AGGAAATACC TACTTTGCTG GTTTGCAGAG CCCCTGTGGT GTGTGGACGC TGA 173

Claims

The scope of the claims

1. The gene encoding the vitamin D receptor isoform protein:

2. The gene according to claim 1, wherein the gene encoding the vitamin D receptor isoform protein is derived from rat.

3. A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which a part of the amino acid sequence shown in SEQ ID NO: 1 is substituted, deleted or added, and is a vitamin D receptor isoform protein 3. The gene according to claim 1, which is a nucleotide sequence encoding a protein having an activity, or a DNA containing a nucleotide sequence hybridizing thereto.

4. The gene according to claim 3, which has the nucleotide sequence shown in SEQ ID NO: 2.

5. The gene according to claim 1, wherein the gene encoding the vitamin D receptor isoform protein is derived from human.

6. The gene according to claim 5, comprising the nucleotide sequence shown in SEQ ID NO: 4 and / or SEQ ID NO: 5.

7. The gene according to claim 5 or 6, which is a nucleotide sequence represented by SEQ ID NO: 3, a nucleotide sequence obtained by partially substituting, deleting or adding the nucleotide sequence, or a DNA comprising a nucleotide sequence hybridizing thereto. .

8. A recombinant vector containing the gene according to claim 1.

9. A prokaryotic or eukaryotic host cell transformed by the recombinant vector according to claim 8.

10. A vitamin D receptor isoform protein obtained by culturing the host cell according to claim 9.

11. An antibody that recognizes the vitamin D receptor isoform protein according to claim 10.

12. A method for diagnosing bone density using the vitamin D receptor isoform protein according to claim 10.

13. The vitamin D receptor isoform protein according to claim 10 is used. Vitamin D-like substance screening method ,: