WO1998056908A1 - Proteines isoformes du recepteur de la vitamine d - Google Patents

Proteines isoformes du recepteur de la vitamine d Download PDF

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WO1998056908A1
WO1998056908A1 PCT/JP1997/002052 JP9702052W WO9856908A1 WO 1998056908 A1 WO1998056908 A1 WO 1998056908A1 JP 9702052 W JP9702052 W JP 9702052W WO 9856908 A1 WO9856908 A1 WO 9856908A1
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vitamin
vdr
gene
rvdr1
nucleotide sequence
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PCT/JP1997/002052
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Shigeaki Kato
Kenju Ueno
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Chugai Seiyaku Kabushiki Kaisha
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Publication of WO1998056908A1 publication Critical patent/WO1998056908A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • 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.
  • 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).
  • VDR vitamin D receptor
  • 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.
  • RARs retinoic acid receptors
  • RXRs 9-cis retinoic acid receptors
  • TRs thyroid hormone receptors
  • 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 3 ⁇ 4, it is 413 (DR4), and in RXR / RAR, it is 2 bp (DR2) and 5 bp (DR5). Distinguish (Umesono et al., Cell 6 ⁇ : 1255-1266, 1991; astineja d et al., Ature 375: 203-211, 1995)
  • 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).
  • VDR hereditary 1, 25 (OH) 2 D 3 resistance rickets
  • HVDRR hereditary 1, 25 (OH) 2 D 3 resistance rickets
  • 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.
  • Abnormal splicing is particularly likely around long exons.
  • 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.
  • VDR vitamin D receptor
  • 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.
  • 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.
  • 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.
  • 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.
  • rVDRO and rVDR1 the protein of the two rVDR isoforms generated by alternative splicing.
  • rVDRO and rVDR1 the protein of the two rVDR isoforms (rVDRO and rVDR1) generated by alternative splicing.
  • S represents SmaI
  • H represents Hindm
  • K represents Kpnl
  • P Psttl.
  • 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.
  • FIG. 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! 3 ⁇ 45 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.
  • the gene encoding the VDR isoform protein was able to get by.
  • 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
  • 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.
  • the gene can be expressed in each host cell.
  • a gene encoding a polypeptide can be expressed.
  • 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.
  • GST was removed and the VDR isoform protein was isolated.
  • 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]).
  • 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.
  • 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 may have the activity of VDR isoform protein.
  • 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
  • sugar chains 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
  • 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.
  • 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.
  • VDR isoform protein which is the target protein of the present invention
  • Separation and purification of the VDR isoform protein may be performed by a separation and purification method used for ordinary proteins, and is not limited in any way.
  • a separation and purification method used for ordinary proteins for example, various types of chromatography, ultrafiltration, salting, dialysis and the like can be appropriately selected and used in combination.
  • 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).
  • DNA used is 2 ⁇ g of CAT reporter plasmid and receptor expression
  • 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.
  • a ligand (1 ⁇ M of all-trans-retinoic acid or 0.1 ⁇ M of thyroid hormone or vitamin D O. 1 ⁇ M) to the medium.
  • a ligand (1 ⁇ M of all-trans-retinoic acid or 0.1 ⁇ M of thyroid hormone or vitamin D O. 1 ⁇ M
  • 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.
  • a conventional method for example, Shinsei Chemistry Laboratory Course 1, Protein I, According to p389-139, 1992
  • the antigen VDR isoform protein
  • 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.
  • RVDR0 and rVDRl were sequenced and 285 nuclei specific for rVDR1 were sequenced.
  • 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 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.
  • 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.
  • VDRE consensus vitamin D response element
  • rVDR1 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).
  • 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
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • the VDR isoform protein of the present invention can be used to screen for a vitamin D-like substance .
  • 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).
  • vitamin D-like actions eg, calcium-bone metabolism, differentiation-inducing action, immunosuppression, antitumor action, fat-lipid metabolism suppression
  • the cells eg, steroids
  • Retinoic acid e.g, Retinoic acid
  • 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.
  • PCR zechase reaction
  • 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
  • rVDRO and rVDR1 transcripts were 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
  • 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
  • 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 ⁇ g), r VDR 0 (0.5 ⁇ g) and rVDR1 (2 ⁇ g) were transformed into HeLa cells.
  • rVDR1 itself did not have a transcription promoting activity.
  • 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
  • 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 ⁇ ).
  • CAT activity was calculated in the same manner as in Example 5.
  • 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.
  • DR5 retinoic acid consensus response element
  • DR4 thyroid hormone consensus response element
  • 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 ( ⁇ ⁇ ). 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
  • rVDR0 and rVDR1 were expressed as GST fusion proteins in Escherichia coli, purified with daltathione.sepharose 4 ⁇ , 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
  • 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 ⁇ 013 ⁇ 41, a band was observed at 401: 0 a).
  • 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
  • RXR partially purified mouse lacking AB region generated in E. coli
  • 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 ⁇ ] - 5- end-labeled synthetic oligonucleotide (DR3 T, DR 3G, DR4 and DR5) coupled reaction mixture containing, And poly (did C) (Pharmacia, 2 ⁇ g) 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.
  • 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 ⁇ 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 type nucleic acid
  • TCCATGCTGC CCCACCTGGC TGACCTTGTC AGTTACAGCA TCCAAAAGGT CATCGGCTTT 720
  • Sequence type nucleic acid
  • CTGTCCCAGC TCTCCATGCT GCCCCACCTG GCTGACCTGG TCAGTTACAG CATCCAAAAG 720
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

L'invention concerne: des gènes codant des protéines isoformes du récepteur de la vitamine D qui sont des ADN contenant une séquence nucléotidique codant la séquence d'acides aminés représentée par SEQ ID NO: 1, des séquences nucléotidiques codant les protéines présentant une séquence d'acides aminés représentée par SEQ ID NO: 1, présentant une substitution, une délétion ou une addition partielle ainsi qu'une activité de protéine isoforme du récepteur de vitamine D, ou des séquences nucléotidiques pouvant être hybridées avec lesdites séquences; des vecteurs de recombinaison contenant ces gènes; des cellules hôtes procaryotes ou eucaryotes transformées par ces vecteurs de recombinaison, des protéines isoformes de récepteur de la vitamine D obtenues par mise en culture de ces cellules hôtes; des anticorps reconnaissant ces protéines; un procédé de détermination de la densité osseuse au moyen de ces protéines; et un procédé de criblage de substances analogues à la vitamine D au moyen de ces protéines.
PCT/JP1997/002052 1997-06-13 1997-06-13 Proteines isoformes du recepteur de la vitamine d WO1998056908A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU31070/97A AU3107097A (en) 1997-06-13 1997-06-13 Vitamin d receptor isoform proteins
PCT/JP1997/002052 WO1998056908A1 (fr) 1997-06-13 1997-06-13 Proteines isoformes du recepteur de la vitamine d

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP1997/002052 WO1998056908A1 (fr) 1997-06-13 1997-06-13 Proteines isoformes du recepteur de la vitamine d

Publications (1)

Publication Number Publication Date
WO1998056908A1 true WO1998056908A1 (fr) 1998-12-17

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AU (1) AU3107097A (fr)
WO (1) WO1998056908A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2801311A1 (fr) * 1999-11-22 2001-05-25 Centre Nat Rech Scient Polypeptides derives du recepteur nucleaire de la vitamine d, et leurs utilisations notamment dans le cadre du criblage d'analogues de la vitamine d

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1994003633A1 (fr) * 1992-07-31 1994-02-17 Garvan Institute Of Medical Research Evaluation de variation allelique de facteurs agissant en trans

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1994003633A1 (fr) * 1992-07-31 1994-02-17 Garvan Institute Of Medical Research Evaluation de variation allelique de facteurs agissant en trans

Non-Patent Citations (4)

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Title
ANDREW R.B. et al., "Cloning and Expression of Full-Length cDNA Encoding Human Vitamin D Receptor", PROC. NATL. ACAD. SCI. USA, (1988), Vol. 85, p. 3294-3298. *
EBIHARA K. et al., "Intron Retention Generates a Novel Isoform of the Murine Vitamin D Receptor that Acts in a Dominant Negative Way on the Vitamin D Signaling Pathway", MOL. CEL. BIOL., (Jul. 1996), Vol. 16, No. 7, p. 3393-3400. *
HIDEAKI KATO, "Analysis of Vitamin D Intranuclear Receptor Mechanisms (in Japanese)", TEAM FOR RESEARCH AND STUDY OF HORMONE RECEPTOR MECHANISM ANOMALY, ONE OF THE SPECIFIED DESEASES AS STIMULATED BY THE MINISTRY OF HEALTH AND WELFARE 1995 COMPREHENSIVE RESEARCH PROJECT REPORT, (Dec. 1996), p. 38-40. *
NIGEL A.M. et al., "Prediction of Bone Density from Vitamin D Receptor Alleles", NATURE, (1994), Vol. 367, p. 284-287. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2801311A1 (fr) * 1999-11-22 2001-05-25 Centre Nat Rech Scient Polypeptides derives du recepteur nucleaire de la vitamine d, et leurs utilisations notamment dans le cadre du criblage d'analogues de la vitamine d
WO2001038393A1 (fr) * 1999-11-22 2001-05-31 Centre National De La Recherche Scientifique Polypeptides derives du recepteur nucleaire de la vitamine d, et leurs utilisations notamment dans le cadre du criblage d'analogues de la vitamine d

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