JP2001519441A - Novel vitamin D receptor-related polypeptides, nucleic acid sequences encoding such polypeptides and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates to a novel vitamin D receptor-related (VDRR) polypeptide and a preparation containing the same. Also disclosed are nucleic acid sequences encoding VDRR polypeptides, expression vectors containing such sequences, and host cells transformed with such expression vectors, as well as methods for expressing the novel VDRR polypeptides of the present invention. The present invention further relates to the use of a VDRR polypeptide as a medicament and the use of a substance that affects VDRR signal transduction to produce a medicament for treating a metabolic, proliferative or inflammatory condition. The present invention also relates to a method for identifying a clone encoding a VDRR polypeptide, a method for identifying a ligand for VDRR, and a method for identifying a substance for treating a condition affected by a VDRR polypeptide. More specifically, the novel VDRR polypeptide can be a polypeptide called VDRRRγ, which can be regulated by small chemical molecules that are structurally similar to known ligands for nuclear receptors.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] The present invention relates to novel vitamin D receptor-related (VDRR) polypeptides. Also disclosed are nucleic acid sequences encoding such polypeptides, expression vectors containing such sequences, and host cells transformed with such expression vectors, as well as methods for expression of the novel VDRR polypeptides of the invention and their use.

BACKGROUND OF THE INVENTION Nuclear hormone receptors are a large group of conditionally regulated transcription factors. These receptors are activated in response to binding to various small chemical molecules (ligands), including steroids, vitamin D3, retinoids, eicosanoids (prostanoids), thyroid hormones and cholesterol derivatives, to regulate target gene expression. Adjust.

[0003] While an increasing number of structurally related receptors are being identified, their ligands have not yet been identified. This group of receptors are orphan nuclear receptors (ONR).
). A discussion of ONR is provided by Enmark et al., Mol. Endo. ,
10, 11 (1966) p. 1293-1307, which is incorporated herein by reference. Many ONRs have been shown by biochemical and genetic approaches to be of central importance for processes such as metabolic homeostasis, cell differentiation and development. In addition, some ONRs have also been implicated as key factors in various common diseases and disorders such as diabetes, obesity, inflammatory conditions and proliferative diseases.

[0004] Based on these findings, it is generally believed that the new ONRs will be potential drug targets for therapeutic inventions for common diseases. Therefore, it is very important to identify such receptors.

SUMMARY OF THE INVENTION [0005] The present invention relates to novel vitamin D receptor-related (VDRR) polypeptides and formulations containing the same. Also disclosed are nucleic acid sequences encoding VDRR polypeptides, expression vectors containing such sequences, and host cells transformed with such expression vectors, as well as methods for expressing the novel VDRR polypeptides of the present invention. The present invention further relates to the use of a VDRR polypeptide as a medicament and the use of a substance that affects VDRR signal transduction for the manufacture of a medicament for treating a metabolic, proliferative or inflammatory condition. The present invention also relates to a method for identifying a clone encoding a VDRR polypeptide, a method for identifying a ligand for VDRR, and a method for identifying a substance for treating a condition affected by a VDRR polypeptide. More specifically, novel VDRR polypeptides can be regulated by small chemical molecules that are structurally similar to known ligands for nuclear receptors.
It can be a polypeptide called VDRRRγ.

Detailed Description of the Invention The above objects are achieved by the present invention with respect to mammalian, preferably human, isolated or recombinant nucleic acids comprising flanking nucleic acid sequences encoding a vitamin D receptor-related (VDRR) polypeptide. . VDRR polypeptides are of suitable origin.

[0007] In a preferred embodiment of the invention, the nucleic acid encoding the VDRR polypeptide is
A DNA binding domain containing approximately 77 amino acids and 9 cysteine residues (DBD
). DBD further comprises human vitamin D receptor (hVDR) and orphan nuclear receptor 1 (x) isolated from Xenopus laevis.
Each of the ONDs is characterized by the following amino acid sequence similarity to the DBD of ONR1 = XOR-6): (i) at least about 60% amino acid sequence similarity to the hVDR DBD; and (ii) to the xONR1 DBD. At least about 65% amino acid sequence similarity.

More specifically, the amino acid sequence similarities of hVDR and xONR1 to the DBD are as follows: (i) about 65% amino acid sequence similarity to the hVDR DBD; and (ii) about 2.5% to the xONR1 DBD. 71% amino acid sequence similarity.

[0009] In a preferred embodiment of the invention, the nucleic acid encoding the VDRR polypeptide is
(i) at least about 30% amino acid sequence similarity to the hVDR LBD, suitably at least 35% amino acid sequence similarity to the hVDR LBD to hVDR and xONR1, respectively; It contains a ligand binding domain (LBD) characterized by amino acid sequence similarity of 40% amino acid sequence similarity, suitably LBD of xONR1 and at least 45% amino acid sequence similarity.

More specifically, the amino acid sequence similarities of hVDR and xONR1 to the LBD are as follows, respectively: (i) about 42% amino acid sequence similarity to the hVDR LBD; and (ii) about the same as xONR1 to the LBD. 54% amino acid sequence similarity.

“Amino acid sequence similarity” is defined as consensus length セ ン サ (consensus length +
(Mismatch + gap) × 100.

[0012] The term amino acid sequence identity may also be used. Amino acid sequence identity is calculated by comparing absolute amino acid residue identity. In FIG. 13, the new gene VDRRg-
1 shows amino acid sequence identity between 1 and VDRRg-2 and known genes.

In a particularly preferred embodiment, the nucleic acid sequences of the invention are substantially the same as those shown in FIG. 1 or FIG. 7, or are the same or alleles thereof.

[0014] The present invention also relates to probes for detecting a nucleic acid sequence encoding a VDRR polypeptide in a sample. Suitably, the probe comprises at least 14 contiguous nucleotides, preferably at least 28 contiguous nucleotides of the nucleic acid sequence shown in FIG. 1 or FIG. Nucleic acid probes can be used in a method to identify clones encoding a VDRR polypeptide, wherein a genomic or cDNA library is screened with the probe under low stringency hybridization conditions, and a high degree of Identifying clones that exhibit hybridization.

[0015] The invention further relates to an isolated or recombinant VDRR polypeptide. Polypeptides can be of full length, wherein the sequence of amino acids is identical to the corresponding sequence found in common mammals, and especially in humans. In the present invention, the polypeptide may be a truncated, extended or mutated form of the full-length polypeptide. Truncated and extended forms refer to a VDRR polypeptide in which one or more amino acids are missing or added, respectively, at the N-terminus of the polypeptide chain. Variants relate to VDRR polypeptides in which one or more amino acids have been replaced by another amino acid. Suitably, an isolated or recombinant VDR
The R polypeptide has the amino acid sequence shown in FIG. 4 or FIG.

[0016] The N-terminal sequence of the nucleic acid encoding a VDRR polypeptide, and the VDRR polypeptide
The amino acid sequence of a polypeptide can vary. Accordingly, various N-terminal isoforms are envisioned, for example, Transcription Factor 3: Nuclear receptor, Protein Profile, Vol. 2, No. 11, (1995), p. 11
Α1, α2, β1, β2, β3, β disclosed in FIG.
4, either γ1 or γ2 is assumed. More specifically, vitamin D receptors and related orphans, such as ONR1, are described in p. 1191-1992.

[0017] The invention further relates to a pharmaceutical formulation comprising the isolated or recombinant VDRR polypeptide and one or more therapeutically acceptable excipients. Examples of excipients that can be used are carbohydrates, for example monosaccharides, disaccharides, and sugar alcohols such as saccharose and sorbitol. Further examples include amino acids such as histidine and arginine, surfactants such as polyoxyethylene sorbitan fatty acid esters, inorganic salts such as sodium chloride and calcium chloride, and complexing agents such as EDTA and citric acid. Agent included.

The preparation may be in the form of a ready-to-use aqueous solution or in a dry form, in particular a lyophilized form. In the latter case, the preparation is reconstituted with a liquid, for example, sterile water or saline, before use.

The present invention further relates to isolated or recombinant nucleic acids, ie vitamin D receptor related (
(VDRR) polypeptides comprising nucleic acids carrying adjacent nucleic acid sequences. The invention also relates to cells containing such an expression vector.

The invention further relates to the claimed nucleic acids, namely the vitamin D receptor related (VD
RR) a cell comprising a nucleic acid carrying a flanking nucleic acid sequence encoding a polypeptide.

[0021] The present invention further relates to the preparation of vitamin D in suitable host cells, preferably eukaryotic cells.
The present invention relates to a process for recombinantly producing a VDRR polypeptide by expressing a claimed or isolated flanking nucleic acid sequence encoding a receptor-associated (VDRR) polypeptide.

[0022] The present invention further provides for the use of V-cells, eg, cell-based reporter assays, transgenic animal reporter assays, or in vitro binding assays.
Methods for identifying ligands for DRR. The invention also relates to metabolism,
A method for identifying a substance for the treatment of a condition affected by a VDRR polypeptide, comprising screening for an agent or antagonist of a VDRR polypeptide signal transduction used to treat a proliferative or inflammatory condition. .

The invention further provides for the use of a VDRR polypeptide as a medicament, as well as for metabolism,
It relates to the use of substances that affect VDRR signal transduction for the manufacture of a medicament for treating a proliferative or inflammatory condition. More specifically, the present invention can be used to manufacture a medicament for treating obesity, diabetes, anorexia, lipoprotein deficiency, hyperlipidemia, hypercholesterolemia or hyperlipoproteinemia. . The invention also relates to osteoporosis, rheumatoid arthritis, benign and malignant tumors,
It can also be used to manufacture a medicament for treating hyperproliferative skin disease or hyperparathyroidism.

The present invention further relates to a method for treating a metabolic, proliferative or inflammatory condition by introducing a nucleic acid vector encoding expression of a VDRR polypeptide into a mammal. The nucleic acid vector is capable of transforming a cell in vivo and expressing the polypeptide in such a transformed cell.

The present invention further relates to substances that affect VDRR signal transduction, in particular VDR
A method for treating a metabolic, proliferative or inflammatory condition by administering a therapeutically effective amount of an R polypeptide.

As used herein, the term “isolated” in reference to a VDRR polypeptide or a nucleic acid encoding the same relates to a nucleic acid or polypeptide isolated from a natural source, for example, human liver, small intestine or colon. The isolated VDRR polypeptides or nucleic acids of the present invention are unique in the sense that they are not actually found in pure or isolated form. Use of the term "isolated" indicates that a naturally occurring sequence has been transferred from its normal cellular environment. That is, the sequence is considered to be in a cell free environment or a different cell environment. Such terms do not imply that the sequence is the only nucleic acid or amino acid sequence present, but that the sequence is the predominant nucleic acid or amino acid sequence present. In addition, the nucleic acid or polypeptide should be essentially free of non-amino acid or non-nucleic acid material naturally associated with the respective product. In this context, essentially free includes a purity of 80% or more, suitably 90% or more, preferably 95% or more.

When referring to the nucleic acid sequence of FIG. 1 or FIG. 7 and the amino acid sequence of FIG. 4 or FIG. 8, the term “substantially the same” refers to the sequence in which they are depicted in the figure. Derived and have the same function as the arrangement in the figure.

The present inventors have surprisingly isolated novel nucleic acid sequences and polypeptides encoded by such nucleic acid sequences. That is, a novel cDNA encoding a polypeptide designated VDRRRγ was cloned and characterized. This polypeptide is
Based on amino acid sequence similarity, it is a new member of the nuclear (hormone) receptor supergene family. The Hidden Markov Model (HMM), which combines phylogenetic analysis such as the Neighbor-Joining phylogenetic method with other statistical algorithms, has a VDR
Rγ is associated with vitamin D receptor (VDR) and xONR1 (Smith et al., Nucl.
. Acids Res. , 22 (1994), no. 1, p. 66-71)
Alternatively, it belongs to the subfamily of VDR-like receptors from Xenopus laevis called XOR-6, as in WO 96/22390. Therefore, VDRRRγ is a member of a family of vitamin D receptor-related (VDRR) polypeptides.

The most closely related receptors, XOR-6, hVDR and CAR (WO 93/1
The degree of amino acid similarity in the DBD and LBD of VDRRg is similar to that between other different but related nuclear receptors (see FIG. 12). Thyroid hormone (TRb) and retinoic acid receptor (RARb) are about 60% and 40% identical at the amino acid level in DBD and LBD, respectively. By comparison, tightly related but unique genes encoding human RARa and RARb nuclear receptors are 97% and 8% in DBD and LBD, respectively.
2% identical.

As will be appreciated by those skilled in the art of nuclear receptors, DBDs have the highest conservation (amino acid identity) both between different nuclear receptors (paralogs) and between identical receptors from different species (orthologs). Is shown. The two "zinc fingers" in the DBD are two evolutionarily conserved amino acid motifs, Cys-X2-Cys-X13-Cys-X2-Cys, in which two pairs of cysteines are chelated on zinc ions. Amino terminal or primary zinc finger) and Cys-Xn-Cys-X9-Cys
Generated by -X2-Cys (carboxy terminus or secondary zinc finger). Very many nuclear receptors are secondary zinc fingers (Cys-X5-Cys-
X9-Cys-X2-Cys) has five amino acid residues between the first two Cys residues (for details see Gronemeyer and Laudet (Pr
protein profile 1995, 2, 11)). The only exception currently known for this role is that it has three amino acid residues (Cys-X3-
(Cys-X9-Cys-X2-Cys) PPAR and 7 amino acid residues (
(Cys-X7-Cys-X9-Cys-X2-Cys) TLL group receptors. Another feature characterizing the novel VDRRg polypeptides described herein is that
The number of amino acid residues in this part of the DBD is six (Cys-X6-Cys-X9-Cys-X2-Cys) as shown in FIGS. Current,
The only other nuclear receptor-like sequence found in the TREMBLE database that has the same number of amino acid residues between two cys residues is the worm C. elegans. elegans
Are two sequences from (Q20097 and Q18155). However, these putative C.I. The entire DBD of the elegans nuclear receptor has only a distant affinity to the DBD of VDRRg. Taken together, the comparison of the DBD and LBD of the nuclear receptor VDRRg described here (see FIG. 12) shows that this receptor is ONR-1 (Smit
h et al., 1994, Nucleic Acids Res. , 22, p. 66-7
1) or XOR-6 (WO96 / 22390), hVDR and CAR
(WO 93/17041), clearly showing a new member of the nuclear receptor supergene family, distinct from other known nuclear receptors most closely related to VDRRg.

This finding, combined with the highly restricted expression pattern observed for human VDRRRγ (liver, small intestine, and colonic mucosa), and also for tissue-specific such as peroxisome proliferator-activated receptor (PPAR) By analogy with other nuclear receptors showing expression patterns, it has been suggested that VDRRRγ plays important physiological functions in the liver, small intestine and colon. Therefore, VDRRγ is considered to be an important sensor of key metabolic pathways affecting metabolism / homeostasis of lipids, carbohydrates or amino acids. In addition, a highly selective tissue-specific expression pattern is
Suggests that γ may be involved in cell differentiation and development of these tissues.

Additional Human VDRRRγ cDNA with an Alternative Spliced 5 ′ End
Was identified (see FIG. 7). Therefore, the VDRRRγ cDNA is the VDRRRγ shown in FIG.
At least one alternative N-terminal variant (FIG. 8) can be encoded in addition to the polypeptide. By analogy with other members of the nuclear receptor supergene family such as RORα and RARα, these N-terminal isoforms of VDRRRγ are:
It is thought to define different functions, including DNA binding specificity and / or promoter specific activation (Gronemeyer and Laudet, 1995).

As used herein, the term VDRRRγ relates to various polypeptides corresponding to the differentially spliced VDRRRγ cDNA, including VDRRRγ-1 and VDRRRγ-2. However, when referring to FIGS. 1 and 4, the VDRRγcD
NA and VDRRRγ are, in particular, VDRRRγ-1 cDNA and VDRRRγ-1 respectively.
About. Similarly, when referring to FIGS. 7 and 8, VDRRRγ cDNA and VDRRRγ specifically relate to VDRRRγ-2 cDNA and VDRRRγ-2, respectively.

In contrast to the VDRRRγ-2 cDNA, VDRRγ-1 cDNA is a classic AU
It does not include the G start codon, but will instead start with an alternative CUG codon. This putative non-AUG start site is located in a sequence environment that favors efficient initiation from the alternative start site and is located within the frame containing the entire open reading frame, after the stop codon.

Taken together, VDRRs, and in particular VDRRRγ, are generally 1) metabolic diseases such as obesity, diabetes (types I and II), lipoprotein disorders, 2) small intestine and colon tumors (benign and Malignant), 3) small intestine and colon ulcers-inflammatory diseases such as Crohn's disease and ulcerative colitis, and 4) congenital abnormalities of the small intestine and colon.

The fact that VDRRRγ and VDR show high amino acid sequence identity in both the DNA binding domain (DBD) and the ligand binding domain (LBD) indicates that these two receptors have overlapping but different functional properties It suggests. By analogy, retinoic acid receptor (RAR) and retinoid X receptor (RXR)
And VDRRRγ have similar amino acid sequence identity in the DBD and LBD regions.
RAR and RXR have obvious functional similarities, both receptors bind 9-cis retinoic acid, and have overlapping DNA binding specificities,
Thus, it has been shown to regulate overlapping gene networks.
Based on these findings, it is believed that VDRRRγ is regulated by small chemical molecules that are structurally similar to known ligands for nuclear receptors but are not necessarily identical to ligands for 1α, 25-dihydroxyvitamin D3 receptor. Can be
In addition, VDRRγ is thought to regulate the vitamin D3-responsive gene network by binding to a vitamin D-responsive element (VDRE) -like DNA sequence. In the present application, the 1α, 25-dihydroxyvitamin D3 receptor is abbreviated as vitamin D receptor (VDR).

In the present invention, the substance that affects VDRR signal transduction may be a small chemical molecule of natural or synthetic origin, for example a carbohydrate such as an aromatic compound. Small molecules can have a molecular weight ranging from about 100 to about 500 Da. Suitably, the small chemical molecule has a molecular weight in the range of about 200 to 400 Da. Preferably, the small chemical molecule has a molecular weight of about 300 Da.

[0038] Human VDRRRγ polypeptides, including VDRRRγ-1 and VDRRRγ-2,
For example, activated by pregnenolone and estradiol (weakly)
It has been shown not to be activated by some other steroid hormones, such as cortisol, aldosterone, progesterone and estrogen, and probably not by progestin and glucocorticoids. Therefore,
Human VDRRγ is not activated by the glucocorticoid antagonist pregnenolone 16α-carbonitrile (PCN). For this reason, human VDRRRγ
May also be referred to as human pregnenolone-activated (nuclear) receptor (hPAR). Information on pregnenolone can be found, for example, in Merck Index, Edition 11,
Merck & Co. , Inc. Rahway, N .; J. , USA, p. 77
35, 1989.

Activators (hPAR-1 and hPAR-2, respectively) for human VDRRRγ polypeptides, including VDRRRγ-1 and VDRRRγ-2, are like pregnane-one, pregnane-dione, pregnane-trione and pregnane-diol. And androstanes such as, but not limited to, pregnenolone, androstan-ol and androstan-diol. Suitably, the pregnenolone has a non-planar structure, especially 5β-pregnane.

[0040] Activators of human VDRRRγ polypeptides, including VDRRRγ-1 and VDRRRγ-2, and possibly specific examples of ligands, are available from Sigma, Sweden.
a-the following compounds marketed by Aldrich: i) 5β-pregnane-3,20-dione ii) 3α-hydroxy-5β-pregnane-11,20-dione methanesulfonate iii) 5β-pregnane-3α, 20β-diol iv) pregnenolone v) pregnu-4-eno [16,17-δ] [2] isoxazoline-3,20-
Dione, 6α-methyl-3′-phenyl-, ethyl ether solvate vi) pregna-1,4,9 (11) -triene-3,20-dione, 21- [
4- [6-Methoxy-2- (4-morpholinyl) -4-pyrimidinyl] -1-piperazinyl] -16-methyl-, (16α) -vii) estran-3-ol, 17-[[[3- ( Trifluoromethyl) phenyl] methyl] amino]-, (E) -2 butenedioate (1: 1) (salt) viii) 9α-fluoro-5α-androstan-11β, 17β-diol ix) spiro [5α- Androstane-3,2'-benzothiazoline] -11
On, 17β-hydroxy-17-methyl-x) spiro [pregnane-3,2′-thiazolidine] -4′-carboxylic acid, 11
α-hydroxy-20-oxo-, sodium salt xi) 17β-dimethylamino-17-ethynyl-5α-androstane-11
β-ol xii) 6β-hydroxy-3,5-cyclo-5α-pregnan-20-one,
Nitrite xiii) 3α-hydroxy-5β-pregnane-11,20-dione, acetate, 20-O- (methylsulfonyl) -oxime xiv) 17α-methyl-5α-androstan-11β, 17-diol xv) 5β-pregnane -3,11,20-trione, trioxime xvi) 3 with hydrazide of 1- (carboxymethyl) pyridinium chloride
α-hydroxy-5β-pregnane-11,20-dione, 20-hydrazone.

One possible use of VDRRg antagonists is in HIV protease inhibitors and cyclosporins to suppress the expression of CYP3A4, thereby increasing the bioavailability of drugs with low pharmacokinetic levels for CYP3A4 metabolism. The co-administration of a VDRRg antagonist may be co-administered with other agents, such as, but not limited to. Genes encoding polypeptides such as human vitamin D receptor-related gamma (hVDRRRg) can be obtained by incorporating a DNA fragment encoding the polypeptide into a recombinant DNA vehicle, such as a vector, to obtain a suitable prokaryotic or eukaryotic host cell. It can be cloned by transformation. Such recombinant DNA techniques are well known and include, for example, Metho.
ds in Enzymology, Academic Press, San
Diego, CA, USA (1994), 65 and 68 (1979), 100
And 101 (1983).

[0042] Host cells for use in the present invention are prokaryotic or eukaryotic cells, preferably eukaryotic cells. Suitable eukaryotic host cells are yeast, such as Saccharo
cells from C. myces, insect cells, and Chinese hamster ovary (CHO)
And mammalian cells such as, but not limited to, baby hamster kidney (BHK), COS, and the like. Suitable prokaryotic host cells are Enterobacteriaceae.
riaea), such as E. coli, Bacillus and Streptomyce
s, including but not limited to cells from S.

[0043]

【Example】

The following examples are given for illustrative purposes only, and are not to be construed as limiting the scope of the invention in any way, which is defined by the claims.

Example 1 Expression and Isolation of Human VDRRg cDNA The Expressed Sequence Tag (EST) database was
Screened for nuclear receptor-related sequences with a nuclear receptor DNA binding domain (DBD) profile. This search profile is based on a multiple alignment of a selected set of nuclear receptor subdomains, followed by a so-called hidden Markov model (HM) of various subfamily members of the nuclear receptor supergene family.
M) by statistical calculation to obtain One cDNA of the identified nuclear receptor-related EST sequence (Incyte clone No. 2211526) was analyzed in detail by sequencing. After DNA sequencing of the entire Incyte cDNA clone (about 2200 base pairs), such clones were identified as xONR-1 and vitamin D.
It was found to encode a putative ligand binding domain (LBD) with 54% and 44% similarity to the receptor (VDR), respectively. The cDNA of the Incyte clone was not full length and did not encode the sequence corresponding to the full DBD.

[0045] Randomly primed cDNA from human liver RNA (Invitroge
n) 5′-RACE (rapid amplification of cDNA ends), followed by cloning and DNA sequencing, showed that the 5 ′ portion of the cDNA corresponding to the Incyte clone encodes a DBD characteristic of nuclear receptors and that xONR- 1 and VDR and 71 each
% And 65% sequence similarity. Multiple alignments combined with neighbor-joining evolutionary phylogenetic analysis classify the polypeptides encoded by the cDNA (specified in FIG. 1) into groups of VDRs (FIGS. 2 and 3) and associate them with human vitamin D receptor-related It was named gamma (VDRg). The deduced amino acid sequence of VDRRg is shown in FIG.

Example 2 Expression of VDRRg mRNA in Human Tissues VD in Adult Tissues Using Multiple Tissue Northern Blots (Clontech)
The expression pattern of RRg was examined. As shown in FIG. 5, VDRRg is abundantly expressed in the small intestine, mucosal lining of the colon and liver, but spleen, thymus, prostate, testis, ovary, peripheral vascular leukocytes, heart, brain, placenta, lung, skeletal muscle, It is not expressed in some other tissues, including kidney and pancreas. To determine if VDRRRγ was expressed at lower levels in any of the other tissues examined, filters were used for a long time (
(One week for one night). After this prolonged contact (data not shown), expression was again only detected in the same tissue and not in any other test tissue. The limited expression pattern of VDRRg suggests that this receptor may have important regulatory functions in liver and intestine.

Example 3 Transient Transfection of GAL4-DBD / VDRRRγ-LBD Fusion Protein Using Vitamin D3 Transient transfection was performed to analyze whether vitamin D3 activates VDRRRγ polypeptide. did. To this end, an expression plasmid encoding a fusion protein of GAL4-DBD fused to VDR or LBD of VDRR,
Transient co-transfection of CV-1 cells was performed using a reporter plasmid containing five GAL4 response elements upstream of the luciferase gene. After transfection, cells were treated with vehicle (DMSO) alone or with vitamin D3 for 48 hours, after which cells were harvested and luciferase activity in cell extracts was measured. As shown in FIG. 6, vitamin D3 (1 μM) transactivated GAL4-DBD / VDR-LBD under these conditions, while the corresponding GAL
The 4-DBD / VDRRRγ-LBD polypeptide did not activate. This is 2
This suggests that the two receptors have different ligand binding specificities.

Example 4 Identification and Isolation of Human VDRRRγ cDNA Encoding Multiple N-Terminal Isoforms Replaced by 5′-RACE of cDNA from Human Liver RNA (see Example 1) and Subsequent Cloning and DNA Sequencing An additional human VRDRγ cDNA with an alternatively spliced 5 ′ end (see FIG. 7) was identified. Therefore VDRR
The γ cDNA can encode at least one alternative N-terminal variant (FIG. 8) in addition to the VDRRγ polypeptide shown in FIG. The polypeptides disclosed in FIGS. 4 and 8 that correspond to the differentially spliced VDRRRγ cDNA are referred to as VDRRRγ-1 and VDRRRγ-2, respectively.

Example 5 VDRRγ Heterodimerizes with RXR and Binds Direct Repeats (DRs), Three or Four Nucleotide Spacing.

The expression plasmid containing the VDRRRγ or RXRβ cDNA is transcribed using T7 polymerase, and the TNT reticulocyte lysate (Prome
ga, Madison, WI, USA). To determine the DNA binding specificity of VDRRRγ, a native gel mobility assay was used essentially as described (Berkenstam et al., Cell, 69, 401-412, 1992). That is, in vitro translated VDRRRγ was incubated with various 32P-labeled direct repeats (DR-1 to DR-5) as shown in FIG. 9 in the presence or absence of in vitro translated RXRβ. The direct iteration is RA
R-β2 promoter (de The et al., Nature, 343, 177-18).
0, 1990) and modified to be separated by 1 to 5 nucleotides (Pettersson et al., Mechanisms).
of Dev. , 54, 1-13, 1995). Protein-DNA complexes were separated by native 5% polyacrylamide / 0.25 × TBE gel followed by autoradiography. As shown in FIG. 9, out of the five DRs tested, efficient VDRRRγ binding was relative to DRs separated by 3 or 4 nucleotides.
In addition, detection was possible only in the presence of RXR. However, DR-2 and DR-
Weak RXR-dependent binding was also observed for one element. These results are
Reveals that Rγ requires RXR heterodimerization for efficient DNA binding to a specific subset of DRs. However, these results indicate that VDR
This does not exclude the possibility that Rγ binds to different but related DNA sequences as monomers, dimers or heterodimers. Importantly, our results are:
VDRRγ and VDR (eg, Markose, ER, et al., Proc. Nat.
l. Acad. Sci. USA, 87, 1701-1705, 1990), TH
R (eg, Groneymeyer, H. and Moras, D., Nature, 3).
75, 190-191, 1995), LXR (Willy, PJ et al., Gen.).
es. Dev. , 9, 1033-1045, 1995) have shown that different nuclear overlapping DNA sequences can be present and thus regulate overlapping gene networks.

Interestingly, ONR-1 (Smith et al., 1994, Nucleic A
cids Re. , 22, p. 66-71) or XOR-6 (WO96 / 2)
The most closely related nuclear receptor, designated No. 2390), has been reported to "bind well with the retinoic acid responsive element, bRARE" (p. 11, line 30 in WO 96/22390). However, the novel nuclear receptor VDRRg reported here is
Like XOR-6, it has 71% amino acid similarity in DBD (FIG. 12).
, VDRRg do not appear to bind to the same bRARE sequence (DR-5 in FIG. 9).

Example 6 Pregnenolone Derivatives as Activators of VDRRRγ In order to identify activators or ligands for VDRRRγ, different classes of activators and ligands for nuclear receptors have been structurally biased towards ligands. Searched library. Transient Caco-2 (TC7) cells (Carr
The activation of VDRRRγ was analyzed in a reporter gene assay (iee et al., 1994). In this initial screen, a synthetic material capable of activating DRDRγ was found to be structurally similar to pregnenolone (data not shown). Based on these results, naturally occurring pregnenolone derivatives were examined for VDRRRγ activation. The result is shown in FIG. As can be seen from FIG. 10, VDRRRγ is pregnenolone, 5β-pregnane-3,20.
-Dione, 5β-pregnane-3α, 20β-diol and 3α-hydroxy-
About 5 to 1 depending on 5β-pregnane-11,20-dionemethanesulfonate
It was activated twice. In contrast to the efficient activation observed by 5β-pregnane-3,20-dione, the corresponding planar steroid derivative, 5α-pregnane-3,20-dione, did not activate the receptor. As is clear from FIG.
Unlike all planar pregnenolone derivatives studied, the other 5β-pregnane also activated VDRRRγ efficiently.

Example 7 Pregnenolone 16α-carbonitrile (PC
N), dexamethasone and antiprogestin (RU486).

The glucocorticoid antagonist pregnenolone 16α-carbonitrile (P
Further experiments were performed to determine whether CN) or dexamethasone was an activator of VDRRRγ. For this, Caco-2 cells were transfected with VDRRRγ as described above, and the cells were treated with 10 μM PCN or dexamethasone and analyzed for activation. The result is shown in FIG. FIG.
As can be seen, VDRRRγ is not activated by these
It suggested that Rγ was not a human PCN receptor. This suggestion is supported by the finding that only antiprogestin RU486 slightly increased (2-fold) the VDRRRγ-mediated reporter gene activity, as evident from FIG.

Activators of XOR-6 such as butyl 4-NH2 benzoate (WO96 /
FIG. 3 in 22390 did not activate VDRRRγ in a similar reporter assay used in WO 96/22390 (data not shown).
.

[Sequence list]

[Brief description of the drawings]

FIG. 1A shows the cDNA sequence encoding a novel nuclear receptor polypeptide, vitamin D receptor related gamma (VDRg).

FIG. 1B shows the cDNA sequence encoding a novel nuclear receptor polypeptide, vitamin D receptor associated gamma (VDRg).

FIG. 2. Evolutionary neighbor-joining (neighbor-joining) phylogenetic tree for VDRRg obtained by DBD-HMM alignment.

FIG. 3. Evolutionary neighbor-joining phylogenetic tree for VDRRg obtained by LBD-HMM alignment.

FIG. 4 shows the deduced amino acid sequence of VDRRg.

FIG. 5. VDRRg expression in adult human tissues. Numbers on the right indicate kilobase pairs of mRNA.

FIG. 6. Vitamin D3 is GAL in transient transfection of CV-1 cells.
Transactivates the 4-DBD / VDR-LBD fusion protein, but not GAL4-DB
The D / VDRRRγ-LBD fusion protein does not activate. The numbers on the left indicate the relative luciferase activity of the GAL-4 luciferase reporter gene.

FIG. 7A. VDRR with alternatively spliced 5 ′ end compared to VDRRg
cDNA sequence encoding g-2.

FIG. 7B. VDRR with an alternatively spliced 5 ′ end compared to VDRRg
cDNA sequence encoding g-2.

FIG. 8 shows the deduced amino acid sequence of VDRRg-2.

FIG. 9 shows heterodimerization of VDRRg with retinoid X receptor (RXR).

FIG. 10 shows the effect of a pregnenolone derivative as a VDRRg activator.

FIG. 11. Pregnenolone 16α-carbonitrile (P
CN), dexamethasone and antiprogestin (RU486).

FIG. 12. Novel genes VDRRg-1 and VDRRg-2, and known genes XOR-6,
Percent similarity between HVDR, CAR-1 and CAR-2.

FIG. 13 shows novel genes VDRRg-1 and VDRRg-2 and known gene XOR-6.
, HVDR, percent identity between CAR-1 and CAR-2.

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Claims (31)

[Claims] 1. A mammalian, preferably human, isolated or recombinant nucleic acid comprising a flanking nucleic acid sequence encoding a vitamin D receptor-related (VDRR) polypeptide. 2. The isolated or recombinant DNA / nucleic acid or allele thereof according to FIG. 1 or 7, which encodes a novel VDRR polypeptide. 3. Includes a DNA binding domain (DBD) comprising about 77 amino acids and 9 cysteine residues, the DBD comprising: (i) at least 60% amino acid sequence similarity to the hVDR DBD; and ii) The nucleic acid of claim 1 or 2, which encodes a VDRR polypeptide characterized by an amino acid sequence similarity of at least 65% amino acid sequence similarity with the DBD of xONR1. 4. The DBD is characterized by amino acid sequence similarity of (i) about 65% amino acid sequence similarity with hVDR DBD; and (ii) about 71% amino acid sequence similarity with xONR1 DBD. The nucleic acid according to claim 3. 5. The polypeptide according to claim 5, wherein the ligand binding domain (LBD) of the polypeptide is hV.
Each of which is characterized by (i) at least about 30% amino acid sequence similarity to the hVDR LBD; and (ii) at least about 40% amino acid sequence similarity to the xONR1 LBD, respectively. The nucleic acid of any one of claims 1 to 4, which encodes a VDRR polypeptide. 6. The LBD is characterized by amino acid sequence similarity of (i) at least about 35% amino acid sequence similarity to hVDR LBD; and (ii) at least about 45% amino acid sequence similarity to xONR1 LBD. The nucleic acid according to claim 5, wherein 7. The LBD is characterized by amino acid sequence similarity of (i) about 42% amino acid sequence similarity to hVDR LBD; and (ii) about 54% amino acid sequence similarity to xONR1 LBD. The nucleic acid according to claim 6. 8. The nucleic acid according to claim 1, wherein the nucleic acid sequence is one shown in FIG. 1 or FIG. 7 or an allele thereof. 9. The nucleic acid of claim 8, wherein said nucleic acid sequence is the same or substantially the same as that shown in FIG. 1 or FIG. 10. A nucleic acid probe for detecting a nucleic acid sequence encoding a VDRR polypeptide in a sample. 11. The nucleic acid probe of claim 10, wherein said probe comprises at least 14 contiguous nucleotides of the nucleic acid sequence shown in FIG. 1 or FIG. 12. A method for screening a genomic or cDNA library with the nucleic acid probe according to claim 10 under low stringency hybridization conditions, and identifying a clone showing a high degree of hybridization with the probe. A method for identifying a clone encoding a VDRR polypeptide, comprising: An expression vector comprising the nucleic acid according to any one of claims 1 to 9. A cell comprising the nucleic acid according to any one of claims 1 to 9. 15. A cell comprising the expression vector according to claim 14. 16. A process for recombinant production of a VDRR polypeptide, comprising expressing the nucleic acid of claim 1 in a suitable host cell. 17. The process according to claim 16, wherein the host cell is a eukaryotic cell. 18. An isolated or recombinant mammalian, preferably human, VDRR polypeptide. 19. The isolated or recombinant VDRR polypeptide of claim 18, which comprises an amino acid sequence substantially or identical to that shown in FIG. 4 or FIG. 20. The method of claim 18, comprising injecting the protein into a mammal.
A method for producing a specific monoclonal or polyclonal antibody against the polypeptide according to any one of the above. 21. The isolated or recombinant VDRR according to claim 18 or 19.
A pharmaceutical formulation comprising a polypeptide and one or more therapeutically acceptable excipients. 22. VDRR by cell-based reporter assay, transgenic animal reporter assay or in vitro binding assay.
For identifying ligands to the same. 23. For the treatment of a condition affected by a VDRR polypeptide, including screening for an agent or antagonist of VDRR polypeptide signal transduction used to treat a metabolic, proliferative or inflammatory condition. Method of identifying a substance. 24. A mammalian, preferably human VDRR polypeptide according to any of claims 18 and 19 for use as a medicament. 25. Use of a substance that affects VDRR signal transduction, such as an agent or antagonist of VDRR polypeptide signal transduction, for the manufacture of a medicament for treating a metabolic, proliferative or inflammatory condition. 26. Influence of VDRR signal transduction for the manufacture of a medicament for treating obesity, diabetes, anorexia, lipoprotein deficiency, hyperlipidemia, hypercholesterolemia or hyperlipoproteinemia. 26. Use according to claim 25 of the influencing substance. 27. Influence VDRR signal transduction for the manufacture of a medicament for the treatment of osteoporosis, rheumatoid arthritis, benign and malignant tumors, hyperproliferative skin diseases or hyperparathyroidism. Use of substance. 28. A substance that affects the transduction of a VDRR signal,
28. The use according to any of claims 25 to 27, which is a chemical molecule of natural or synthetic origin having a molecular weight in the range of 0 to about 500 Da, preferably of about 300 Da. 29. A method comprising introducing a nucleic acid vector according to claim 13 encoding the expression of a VDRR polypeptide into a mammal, wherein the nucleic acid vector is in vivo.
A method for treating a metabolic, proliferative or inflammatory condition, which comprises transforming a cell with vo and expressing the polypeptide in the transformed cell. 30. A method for treating a metabolic, proliferative or inflammatory condition by administering a therapeutically effective amount of a substance that affects VDRR signal transduction. 31. A substance which affects VDRR signal transduction,
31. The method according to claim 30, which is a chemical molecule of natural or synthetic origin having a molecular weight in the range of 0 to about 500 Da, preferably of about 300 Da.