AU732079B2 - Novel vitamin D receptor related polypeptides, nucleic acid sequence encoding the same and uses thereof - Google Patents

Description

WO 99/19354 PCT/SE98/01548 NOVEL VITAMIN D RECEPTOR RELATED POLYPEPTIDES. NUCLEIC ACID SEQUENCE ENCODING THE SAME AND USES THEREOF FIELD OF THE INVENTION The present invention relates to novel vitamin D receptor related (VDRR) polypeptides. Nucleic acid sequences encoding the same, expression vectors containing such sequences and host cells transformed with such expression vectors are also disclosed, as are methods for the expression of the novel VDRR polypeptides of the invention, and uses thereof.

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

A growing number of structurally related receptors have been identified for which no ligands yet have been identified. This group of receptors is referred to as orphan nuclear receptors (ONRs). A review of the ONRs can be found in Enmark et al, Mol. Endo., vol. No. 11 (1996) pp. 1293-1307, which is hereby incorporated by reference. The pivotal importance of a number of ONRs for processes such as metabolic homeostasis, cell differentiation and development have been demonstrated both by biochemical and genetic techniques. In addition, several ONRs have also been implicated as key factors in a variety of common diseases and disorders such as diabetes, obesity, inflammatory conditions and proliferative diseases.

Based on these findings it is generally believed that novel ONRs are going to become potential drug targets for therapeutic invention of common diseases. Thus, it is of great importance to identify such receptors.

WO 99/19354 PCT/SE98/01548 SUMMARY OF THE INVENTION The present invention relates to novel vitamin D receptor related (VDRR) polypeptides, and formulations containing the same. Nucleic acid sequences encoding the VDRR polypeptides, expression vectors containing such sequences and host cells transformed with such expression vectors are also disclosed, as are methods for the expression of the novel VDRR polypeptides of the invention. The invention further relates to VDRR polypeptides for use as medicaments, and use of substances affecting VDRR signal transduction for the manufacture of medicaments for treating metabolic, proliferative or inflammatory conditions. The present invention also relates to methods for identifying clones encoding a VDRR polypeptide, methods for identifying ligands to a VDRR and methods for identifying substances for treatment of conditions affected by a VDRR polypeptide. More specifically, the novel VDRR polypeptide can be the polypeptide designated VDRRy, which may be regulated by any small chemical molecule similar in structure to known ligands for nuclear receptors.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The cDNA sequence encoding the novel nuclear receptor polypeptide vitamin D receptor related gamma (VDRRg) is shown.

Figure 2 Evolutionary neighbor-joining tree for VDRRg as given by DBD-HMM alignment.

Figure 3 Evolutionary neighbor-joining tree for VDRRg as given by LBD-HMM alignment.

Figure 4 The deduced amino acid sequence of VDRRg is shown.

Figure 5 Expression of VDRRg in adult human tissues. The numbers on the right hand side, refer to kilobasepairs of the mRNA.

Figure 6 Vitamin D3 transactivate a GAL4-DBD/VDR-LBD fusion protein but not a GAL4-DBD/VDRRy-LBD fusion protein in transient transfections of CV-1 cells. The number on the left hand side refer to relative luciferase activity of the GAL4-luciferase reporter gene.

WO 99/19354 PCT/SE98/01548 Figure 7 The cDNA sequence encoding VDRRg-2 with an alternatively spliced end compared to VDRRg is shown.

Figure 8 The deduced amino acid sequence of VDRRg-2 is shown.

Figure 9 Heterodimerization of VDRRg with a retinoid X receptor (RXR) is shown.

Figure 10 The effect of pregnenolone derivatives as activators of VDRRg are shown.

Figure 11 The effect of pregnenolone 16a-carbonitrile (PCN), dexamethasone and an antiprogestin (RU486) as activators of VDRRg are shown.

Figure 12 Percent similarity between the new genes VDRRg-1 and VDRRg-2 and the known genes XOR-6. HVDR, CAR-1 and CAR-2.

Figure 13 Percent identity between the new genes VDRRg-I and VDRRg-2 and the known genes XOR-6. HVDR, CAR-1 and CAR-2.

DETAILED DESCRIPTION OF THE INVENTION The objects above are met by the present invention, which relates to a mammalian, preferably human, isolated or recombinant nucleic acid comprising a contiguous nucleic acid sequence encoding a vitamin D receptor related (VDRR) polypeptide. The VDRR polypeptide is suitably origin.

In preferred embodiments of the present invention, the nucleic acid encoding the VDRR polypeptide contains a DNA-binding domain (DBD) comprising about 77 amino acids with 9 cysteine residues. The DBD is further characterized by the following amino acid sequence similarity relative to the DBDs of human Vitamin D Receptor (hVDR) and Orphan Nuclear Receptor 1 isolated from Xenopus laevis (xONR1 XOR-6), respectively: at least about 60% amino acid sequence similarity with the DBD of hVDR; and (ii) at least about 65% amino acid sequence similarity with the DBD of xONRl.

More particularly, the amino acid sequence similarity relative to the DBDs of hVDR and xONRI, respectively is about 65% amino acid sequence similarity with the DBD of hVDR; and WO 99/19354 4 PCT/SE98/01548 (ii) about 71% amino acid sequence similarity with the DBD of xONR1.

In preferred embodiments of the present invention, the nucleic acid encoding the VDRR polypeptide contains a ligand-binding domain (LBD) characterized by the following amino acid sequence similarity, relative to the LBDs of hVDR and xONR1, respectively: at least about 30% amino acid sequence similarity with the LBD of hVDR, suitably at least 35% amino acid sequence similarity with the LBD of hVDR; and (ii) at least about 40% amino acid sequence similarity with the LBD of xONR1, suitably at least 45% amino acid sequence similarity with the LBD of xONR1.

More particularly, the amino acid sequence similarity relative to the LBDs of hVDR and xONRl, respectively is about 42% amino acid sequence similarity with the LBD of hVDR; and (ii) about 54% amino acid sequence similarity with the LBD of xONRl.

amino acid sequence similarity" refers to: 100x Consensus Lenght divided by Consencus Length Mismatsches Gaps.

The term amino acid sequence identity can also be used. Amino acid sequence identity is calculated by comparing the absolute amino acid residue identity. In Figure 13 the amino acid sequence identity between the new genes VDRRg-1 and VDRRg-2 and the known genes are shown.

In particularly preferred embodiments, the nucleic acid sequences of the present invention are substatially the same as those given in Fig. 1 or Fig. 7, the same or alleles thereof.

The present invention also relates to a nucleic acid probe for the detection of a nucleic acid sequence encoding a VDRR polypeptide in a sample. Suitably, the probe comprises at least 14 contiguous nucleotides, and preferably at least 28 contiguous nucleotides, of the nucleic acid sequences given in Fig. 1 or Fig. 7. The nucleic acid probe can be used in a method for identifying clones encoding a VDRR polypeptide, wherein the method comprises screening a genomic or cDNA library with the probe under low stringency hybridization conditions, and identifying those clones which display a substantial degree of hybridization to said probe.

The present invention further relates to an isolated or recombinant VDRR polypeptide. The polypeptide can be full-length, at which the sequence of amino acids is identical to WO 99/19354 PCT/SE98/01548 the corresponding sequence found in mammals in general, and in human beings in particular. In the present invention, the polypeptide can also be a truncated, extended or mutated form of the full-length polypeptide. Truncated and extended forms relate to VDRR polypeptides where one or more amino acids are missing or have been added, respectively, at the N terminal end of the polypeptide chain. Mutated forms relate to VDRR polypep-tides where one or more amino acid has been substituted by another amino acid. Suitably, the isolated or recombinant VDRR polypeptide exhibits the amino acid sequences given in Fig.

4 or Fig. 8.

The N-terminal sequence of the present nucleic acids encoding VDRR polypeptides, as well as the amino acid sequence of the present VDRR polypeptides, may vary. Thus, various N-terminal isoforms are envisaged, e.g. any of al a2, P 1, p2, P3, P4, yl or y2 as disclosed in Fig. 7B of Transcription Factors 3: nuclear receptors, Protein Profile, vol. 2, issue 11 (1995), pp. 1173-1235. This review of nuclear receptors generally is hereby incorporated by reference. More specifically, Vitamin D receptors and related orphans, e.g.

ONR1, are discussed at p. 1191-1992.

The present invention further relates to pharmaceutical formulations comprising an isolated or recombinant VDRR polypeptide, and one or more therapeutically acceptable excipients. Examples of excipients that can be used are carbohydrates, e.g. monosaccharides, disaccharides and sugar alcohols, such as saccharose and sorbitol. Further examples include amino acids, e.g. histidine and arginine, surfactants, e.g. polyoxyethylene sorbitan fatty acid esters, inorganic salts, e.g. sodium chloride and calcium chloride, and complexing agents, e.g. EDTA and citric acid.

The present formulation can be in the form of an aqueous solution ready-for-use, or dried, particularly lyophilized. In the latter case, the formulation is reconstituted with a liquid, e.g. sterile water or saline, before use.

The present invention further relates to an expression vector comprising an isolated or recombinant nucleic acid, the nucleic acid comprising a contiguous nucleic acid sequence encoding a Vitamin D receptor related (VDRR) polypeptide. The invention also relates to a cell containing such an expression vector.

WO 99/19354 PCT/SE98/01548 The present invention further relates to a cell containing the claimed nucleic acid, the nucleic acid comprising a contiguous nucleic acid sequence encoding a Vitamin D receptor related (VDRR) polypeptide.

The present invention further relates to a process for recombinant production of a VDRR polypeptide, by expressing the claimed isolated or recombinant contiguous nucleic acid sequence encoding a Vitamin D receptor related (VDRR) polypeptide in a suitable host cell, preferably an eukaryotic cell.

The present invention further relates to method for identifying a ligand to a VDRR, e.g. by a cell-based reporter assay, transgenic-animal reporter assay or in vitro-binding assay. It also relates to a method for identifying a substance for treatment of a condition affected by a VDRR polypeptide, comprising screening for an agonist or an antagonist of VDRR polypeptide signal transduction to be used for treating metabolic, proliferative or inflammatory conditions.

The present invention further relates to a VDRR polypeptide for use as a medicament, as well as use of a substance affecting VDRR signal transduction for the manufac-ture of a medicament for treating metabolic, proliferative or inflammatory conditions.

More particularly, the present invention can be used for the manufacture of medicaments for treating obesity, diabetes, anorexia, lipoprotein defects, hyperlipidemia, hypercholeste-remia or hyperlipoproteinemia. The present invention can be used also for the manufacture of medicaments for treating osteoporosis, rheumatoid artritis, benign and malign tumors, hyperproliferative skin disorders or hyperparathyroidism.

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

The present invention further relates to a method for treatment of a metabolic, proliferative or inflammatory condition by administration of a therapeutically effective amount of a substance affecting VDRR signal transduction, specifically a VDRR polypeptide.

In the present invention, the term "isolated" in connection with VDRR polypeptides or nucleic acids encoding the same, relates to nucleic acids or polypeptides that have been isolated from a natural source, e.g. the liver, small intestine or colon of a human being. The WO 99/19354 PCT/SE98/01548 isolated VDRR polypeptides or nucleic acids of the present invention are unique in the sense that they are not found in a pure or separated form in nature. Use of the term "isolated" indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free environment or in a different cellular environment. The term does not imply that the sequence is the only nucleic acid or amino acid sequence present, but that it is the predominant nucleic acid or amino acid sequence present. Furthermore, 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 relates to more than 80%, suitably more than 90%, and preferably more than 95% purity.

The term "sustantially the same "when referring to the nucleic acid sequences in Fig 1 or Fig 7 and when referring to the amino acid sequences in Fig. 4 or Fig.8 means that they are derived from the sequences given in the figures and have the same function as those.

The inventors of the present invention, have surprisingly isolated a novel nucleic acid sequence, and a polypeptide encoded by said nucleic acid sequence. Thus, a novel cDNA encoding a polypeptide designated VDRRy has been cloned and characterized. This polypeptide is, based on amino acid sequence similarity, a novel member of the nuclear (hormone) receptor supergene family. Hidden Markov Models (HMMs) in combination with phylogenetic analysis such as neighbor-joining tree methods and other statistical algorithms shows that VDRRy belong to a sub-family of vitamin D receptors (VDRs) and a VDR-like receptor from Xenopus laevis designated xONRI (see Smith et al., Nucl. Acids Res., 22 (1994), No. 1, pp. 66-71) or XOR-6 as in W096/22390. The VDRRy, therefore, is one member of a family of Vitamin D receptor related (VDRR) polypeptides.

The degree of amino acid similarity in the DBD and LBD of VDRRg as compared to the most closely related receptors XOR-6, hVDR and CAR (see WO 93/17041) is similar to the relationship between other distinct, but related nuclear receptors. (See Fig. 12). The thyroid hormone (TRb) and retinoic acid receptor (RARb) are approximately 60% and 40% identical at the amino acid level in the DBD and LBD, respectively. By comparison, the closely related but unique genes encoding human RARa and RARb nuclear receptors are 97% and 82% identical in the DBD and LBD, respectively.

WO 99/19354 PCT/SE98/01548 As recognized by those skilled in the art of nuclear receptors, the DBD displays the highest degree of conservation (amino acid identity) both between different nuclear receptors (paralogous) and between identical receptors from different species (orthologues). The two "zink-fingers" in the DBD are generated by two evolutionary conserved amino acid motifs Cys-X2-Cys-X13-Cys-X2-Cys (amino-terminal or first zink-finger) and Cys-Xn-Cys-X9- Cys-X2-Cys (carboxy-terminal or second zink-finger) in which two pairs of cysteins chelate on zink ion. The vast majority of nuclear receptors have five amino acid residues between the firs two Cys residues in the second zink-finger (Cys-X5-Cys-X9-Cys-X2-Cys) see Gronemeyer and Laudet (Protein Profile 1995, 2, issue 11) for details. The today only known exception to this role are the PPARs which have three amino acid (Cys-X3-Cys-X9- Cys-X2-Cys) residues and the TLL group of receptors which have seven (Cys-X7-Cys-X9- Cys-X2-Cys). Thus another feature which is characteristic of the novel VDRRg polypeptide 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.4 and 8. Today, the only other nuclear receptor like sequences found in the TREMBLE data base with the same number of amino acid residues between the two cys residues are two sequences (Q20097 and Q18155) from the worm C. elegans (Q20097 and Q18155). However, the entire DBD of these putative C.

elegans nuclear receptors are only distantly related to the DBD of VDRRg. Taken together, the comparison of the DBD and LBD of the nuclear receptor VDRRg described herein (See Fig. 12), clearly demonstrate that this receptor is a novel member of the nuclear receptor super-gene family which is distinct from other known nuclear receptors that are most closely related to the VDRRg including ONR-I (in Smith et al., 1994, Nucleic Acids Res., 22, pp66- 71) or XOR-6 (in WO 96/22390), hVDR and CAR (WO 93/17041).

This finding, in combination with the highly restricted expression pattern we observe for human VDRRy (liver, small intestine and mucosa of colon) and in analogy to other nuclear receptors exhibiting a tissue specific expression pattern such as the peroxisome pro-liferatoractivated receptors (PPARs) suggest that VDRRy performs important physiolo-gical functions in liver, small intestine and colon. Accordingly, VDRRy is likely to be an important sensor of key metabolic pathways affecting lipid, carbohydrate or amino acid metabolism/homeostasis. In addition, the highly selective tissue specific expression pattern WO 99/19354 PCT/SE98/01548 suggest that VDRRy may participate in cellular differentiation and development of these tissues.

An additional human VDRRy cDNA with an alternatively spliced 5'-end has been identified (see Fig. The VDRRy cDNAs are thus able to encode at least one alternative N-terminal variant (Fig. 8) in addition to the VDRRy polypeptide shown in Fig. 4. In analogy to other members of the nuclear receptor supergene family such as RORca and RARax these N-terminal isoforms of VDRRy may specify different functions including DNA-binding specificity and/or promoter specific activation (Gronemeyer and Laudet, 1995).

In the present specification, the term VDRRy relates to the various polypeptides corresponding to the differentially spliced VDRRy cDNAs including VDRRy-1 and VDRRy-2. However, when reference is made to Fig. I and Fig. 4, VDRRy cDNA and VDRRy relates specifically to VDRRy-1 cDNA and VDRRy-1, respectively. In the same way, when reference is made to Fig. 7 and Fig. 8, VDRRy cDNA and VDRRy relates specifically to VDRRy-2 cDNA and VDRRy-2, respectively.

In contrast to the VDRRy-2 cDNA, the VDRRy-1 cDNA does not contain a classical AUG initiation codon but instead may initiate at an alternative CUG codon. This putative non-AUG start site is located in a favorable sequence context for efficient initiation from alternative start sites and is in frame with the entire open reading frame and preceded by a stop codon.

Taken together, the VDRRs in general, and more specifically the VDRRy, may be important in 1) metabolic diseases such as obesity, diabetes (type I and II), lipoprotein disorders, 2) proliferative conditions such as tumors (benign and malignant) of the small intestine and colon, 3) ulcero-inflammatory diseases of small intestine and colon such as Crohn's disease and ulcerative colitis, and 4) congenital anomalies of small intestine and colon.

The high amino acid sequence identity of VDRRy with the VDR both in the DNAbinding domain (DBD) and ligand-binding domain (LBD) indicate that these two receptors WO 99/19354 PCT/SE98/01548 may also have overlapping yet distinct functional characteristics. In analogy, retinoic acid receptors (RARs) and retinoid X receptors (RXRs) have similar amino acid sequence identities in the DBD and LBD region as the VDR and VDRRy. RARs and RXRs have been shown to have distinct functional similarities such that both receptors bind 9-cis retinoic acid and have overlapping DNA-binding specificities and accordingly regulate overlapping gene networks. Based on these findings, VDRRy may be regulated by small chemical molecules similar in structure to known ligands for nuclear receptors but not necessarily identical to ligands for the la, 25-dihydroxy vitamin D3 receptor. Furthermore, VDRRy may regulate vitamin D3 responsive gene networks by binding to a Vitamin D responsive element (VDRE)-like DNA sequence. In the present application, the la, 25-dihydroxy vitamin D3 receptor is abbreviated as the Vitamin D receptor (VDR).

In the present invention, the substance affecting VDRR signal transduction can be any small chemical molecule of natural or synthetic origin, e.g. a carbohydrate such as an aromatic compound. The small molecule may have a molecular weight in the range of from about 100 up to about 500 Da. Suitably, the small chemical molecule has a molecular weight in the range of from 200 up to 400 Da. Preferably, the small chemical molecule has a molecular weight of about 300 Da.

The human VDRRy polypeptides, including VDRRy-1 and VDRRy-2, have been shown to be activated e.g. by pregnenolones and estradiol (weakly), but not by certain other steroid hormones such as cortisol, aldosterone, progesterone and estrogen, and most likely not by progestines and glucocorticoids. Thus, human VDRRy is not activated by pregnenolone 16a-carbonitrile (PCN), a glucocorticoid antagonist. For this reason, human VDRRy can also be designated human pregnenolone activated (nuclear) receptors (hPAR).

Information about pregnenolone can be found e.g. in the Merck Index, 11th ed., Merck Co., Inc. Rahway, USA, p. 7735, 1989.

Activators for human VDRRy polypeptides, including VDRRy-1 and VDRRy-2, (hPAR-l and hPAR-2, respectively), include but are not limited to pregnenolones, such as pregnane-ones, pregnane-diones, pregnane-triones, and pregnane-diols, and androstanes, such as androstane-ols, and androstane-diols. Suitably, the pregnenolones are non-planar, particularly 51-pregnanes.

WO 99/1 9354 PCT/SE98/O1 548 Specific examples of activators and possibly ligands for human VDRRy polypeptides, including VDRRy- 1 and VDRRy-2, are the following compounds. which are marketed by Sigma-Aldrich of Sweden: i) 5 p-pregnane-3 ii) 3 ct-hydroxy-5p~-pregnane-I 1,20-dione methanesulphonate iii) 5f3-pregnane-3c,2O03-diol iv) pregnenolone v) Pregn-4-eno[ 16,1 7-6] isoxazolline-3,20-dione, 6ax-methyl-3'-phenyl-, ethyl ether solvate vi) Pregna- 1,4,9(11 )-triene-3 ,20-dione, 21 [6-methoxy-2-(4-morpholinyl)-4-pyrimidinyl]- 1 -piperazinyl]- 1 6-methyl-, (1 6ax)vii) Estran-3-ol, 1 7- -(trifluoromethyl)phenyI] methy1] amino]-, (E)-2-butenedioate 1) (salt) *viii) 9c-Fluoro-5cc-androstane- I1~ I 7P-diol ix) Spiro [5 a-androstane-3 ,2'-benzothiazolin]- 11I-one, 1 7p-hydroxy- I17-methylx) Spiro [pregnane-3 ,2'-thiazolidine] -4 t -carboxylic acid, 1 I sodium salt xi) I 7f-Dimethylamino- I 7-ethynyl-5Sa-androstane- 11 3-ol xii) 6P-Hydroxy-3 ,5-cyclo-5ac-pregnan-20-one, nitrite xiii) 3cc-Hydroxy-50f-pregnane- 1,20-dione, acetate, xiv) 1 7cx-Methyl-5ca-androstane- 11I P3,I 7-diol xv) 5f3-Pregnane-3,1 1,20-trione, trioxime xvi) 3ct-Hydroxy-5f3-pregnane-1 1,20-dione, 20-hydazone with hydrazide of 1 -(carboxymethyl) pyridiniumn chloride.

A possible use of a VDRRg antagonist, could be a synergistic co-administration of the VDRRg antagonist together with other drugs such as, but not limited to, HIV protease inhibitors and cyclosporin to inhibit the expression of CYP3A4 and thus increase the bioavailability of drugs with poor pharinacokinetics due to CYP3A4 metabolism.

Genes coding for polypeptides, such as human vitamin D receptor related gamma (hVDRRg), may be cloned by incorporating a DNA fragment coding for the polypeptide into a recombinant DNA vehicle, e.g. a vector, and transforming suitable prokaryotic or WO 99/19354 PCT/SE98/01548 eukaryo-tic host cells. Such recombinant DNA techniques are well known and e.g. described in Methods in Enzymology, Academic Press, San Diego, CA, USA (1994), vols. 65 and 68 (1979), and vols. 100 and 101 (1983).

The host cells for use in the present invention can be prokaryotic or eukaryotic, preferably eukaryotic cells. Suitable eukaryotic host cells include but are not limited to cells from yeast, e.g. Saccharomyces, insect cells and mammalian cells such as Chinese Hamster Ovary (CHO), Baby Hamster Kidney (BHK), COS and the like. Suitable prokaryotic host cells include but are not limited to cells from Enterobacteriacea, e.g. E. coli, Bacillus and Streptomyces.

EXAMPLES

The following Examples are provided for purposes of illustration only and are not to be construed as in any way limiting the scope of the present invention, which is defined by the appended claims.

EXAMPLE 1 Identification and isolation of human VDRRg cDNA Expressed Sequence Tag (EST) databases were screened for nuclear receptor related sequences with a DNA-binding domain (DBD) profile of nuclear receptors. This search profile was created by multiple alignment of a selected set of nuclear receptor sub-domains followed by a statistical calculation to obtain a so called Hidden Markov Model (HMM) of different subfamily members of the nuclear receptor supergene family. The cDNA of one of the nuclear receptor related EST sequences identified (Incyte clone no 2211526) was analyzed in detail by sequencing. After DNA sequencing of the entire Incyte cDNA clone (approximately 2200 basepairs) the clone was found to encode a putative ligand-binding domain (LBD) with 54% and 44% similarity to xONR-1 and to the vitamin D receptor (VDR), respectively. The cDNA of the Incyte clone was not full-length and did not encode a sequence corresponding to a complete DBD.

WO 99/19354 PCT/SE98/01548 (rapid amplification of cDNA ends) of random primed cDNA from human liver RNA (InVitrogen) followed by cloning and DNA sequencing showed that the 5'-part of the cDNA corresponding to the Incyte clone encoded a DBD characteristic for nuclear receptors and with 71% and 65% sequence similarity to xONR-I and VDR, respectively.

Multiple alignments in combination with evolutionary neighbor-joining tree analysis placed the polypeptide encoded by the cDNA (specified in Fig. 1) in the group of VDRs (Figs. 2 and 3) and was named human vitamin D receptor related gamma (VDRRg). The deduced amino acid sequence of VDRRg is given in Fig. 4.

EXAMPLE 2 Expression of VDRRg mRNA in human tissues Multiple tissue northern blots (Clontech) was used to determine the expression pattern of VDRRg in adult human tissues. As shown in Fig. 5, VDRRg is abundantly expressed in small intestine, mucosal lining of colon and liver but not in several other tissues including spleen, thymus, prostate, testis, ovary, peripheral blood leukocytes, heart, brain, placenta, lung, skeletal muscle, kidney and pancreas. To investigate ifVDRRy was expressed at lower levels in any of the other tissues examined, the filter was exposed for an extended time (one week as compared to overnight). Even after this prolonged exposure (data not shown), expression could still only be detected in the same tissues and not in any of the other tissues examined. The restricted expression pattern of VDRRg suggest that this receptor is likely to have an important regulatory function in liver and intestine.

EXAMPLE 3 Transient transfections of GAL4-DBD/VDRR-LBD fusion protein using Vitamin D3 Transient transfections were performed to analyze if vitamin D3 activate the VDRRy polypeptide. To this end, transient co-transfections of CV-1 cells were performed with expression plasmids encoding fusion proteins of the GAL4-DBD fused to the LBD of either the VDR or the VDRR together with a reporter-plasmid containing five GAL4 responsive elements upstream of the luciferase gene. After transfection, cells were treated with vehicle (DMSO) alone or with vitamin D3 for 48 hours followed by harvesting of the cells and measurement of the luciferase activity in cell extracts. As shown in Fig. 6, vitamin D3 (1 WO 99/19354 PCT/SE98/01548 p.M) transactivate the GAL4-DBD/VDR-LBD but not the corresponding GAL4-DBD/- VDRRy-LBD polypeptide under these conditions. This indicates that the two receptors may have distinct ligand-binding specificities.

EXAMPLE 4 Identification and isolation of human VDRRy cDNAs encoding multiple N-terminal isoforms (see Example 1) of cDNA from human liver RNA followed by cloning and DNA sequencing identified an additional human VDRRy cDNA with alternatively spliced 5'-end (see Fig. The VDRRy cDNAs are thus able to encode at least one alternative Nterminal variant (Fig. 8) in addition to the VDRRy polypeptide shown in Fig. 4. The polypeptides disclosed in Fig. 4 and Fig. 8 which correspond to the differentially spliced VDRRy cDNAs are designated as VDRRy-1 and VDRRy-2, respectively.

EXAMPLE VDRRy heterodimerise with RXR and bind to direct repeats (DRs) spaced by three or four nucleotides Expression plasmids containing VDRRy or RXRp cDNAs were transcribed using T7 polymerase and translated in vitro in TNT reticulocyte lysates (Promega, Madison, WI, USA). To investigate the DNA-binding specificity of VDRRy a native gel mobility assay was employed essentially as described (Berkenstam et al., Cell, 69, 401-412, 1992) in which in vitro translated VDRRy was incubated in the presence or absence of in vitro translated RXRp with different 32P-labelled direct repeats (DR-1 to DR-5) as indicated in Fig. 9. The direct repeats were derived from the DR-5 element in the RAR-P2 promoter (de The et al., Nature, 343, 177-180, 1990) and modified to be separated by one to five nucleotides (Pettersson et al., Mechanisms of Dev., 54, 1-13, 1995). Protein-DNA complexes were separated on native 5% polyacryl-amide/0.25xTBE gels followed by autoradiography. As shown in Fig. 9, of the five DRs tested efficient VDRRy binding could only be detected with DRs separated by three or four nucleotides and only in the presence of RXR. However, weaker RXR-dependent binding could also be observed to DR-2 and DR-1 elements. These WO 99/19354 l, PCT/SE98/01548 results demonstrate that VDRRy require RXR heterodimerisation for efficient DNA-binding to a specific subset of DRs. These results, however, do not exclude the possibility that VDRRy may bind as a monomer, dimer or heterodimer to distinct but related DNAsequences. Importantly, our results demonstrate that VDRRy and other nuclear receptors including the VDR Markose, E. R. et al., Proc. Natl. Acad. Sci. USA, 87, 1701-1705, 1990), THRs Gronemeyer, H. and Moras, Nature, 375, 190-191, 1995), LXRs (e.g.

Willy, P. J. et al., Genes. Dev., 9, 1033-1045, 1995), have distinct but overlapping DNAsequence and thus may regulate overlapping gene networks.

Interestingly, the most closely related nuclear receptor called ONR-1 (in Smith et al., 1994, Nucleic Acids Res., 22, pp66-71) or XOR-6 (in WO 96/22390) have been reported to "bind well to a retinoic acid response element, bRARE" 11, line 30 in WO 96/22390).

However, although the novel nuclear receptor VDRRg reported herein has 71% amino acid similarity in the DBD as compared to XOR-6 (fig 12), VDRRg does not appear to bind to the same bRARE sequence (DR-5 in Fig. 9).

EXAMPLE 6 Pregnenolone derivatives as activators of VDRRy For identifying activators or ligands for VDRRy, a library of substances structurally biased towards different classes of activators and ligands for nuclear receptors were tested.

The activation of VDRRy was analyzed in a reporter gene assay in transiently Caco-2 (TC7) cells (Carriere et al, 1994). In this initial screen, the synthetic substances with ability to activate VDRRy were found to be structurally similar to pregnenolones (data not shown).

Based on these results, naturally occuring pregnenolone derivatives were examined for activation of VDRRy. The results are shown in Fig. 10. As is evident from Fig. 10, VDRRy was activated about 5 to 12 fold by pregnenolone, 5p-pregnane-3,20-dione, 3a,20p-diol and 3a-hydroxy-53-pregnane- 1,20-dione methanesulphonate. In contrast to the efficient activation observed by the 5 -pregnane-3,20-dione, the corresponding planar steroid derivative 5a-pregnane-3,20-dione did not activate the receptor. Other also activated VDRRy efficiently as opposed to all planar pregnenolone derivatives tested, as is also evident from Fig.

I

P:\OPERUMS90131-98-p 16.do,-207/00 -16- EXAMPLE7 Pregnenolone 16 a-carbonitrile (PCN), dexamethasone and an antiprogestin (RU486) as activators if VDRRy.

Further experiments were performed to find out if pregnenolone 16 a-carbonitrile (PCN), a glucocorticoid antagonist or dexamethasone are activators of VDRRy. To this effect, Caco-2 cells were transfected as before with VDRRy and the activation was analyzed after treatment of the cells with 10 ptM PCN or dexamethasone. The results are shown in Fig.11. As is evident from Fig. 11, VDRRy was not activated by these substances, indicating that VDRRy is not the human PCN receptor. This suggestion is corroborated by the observation that also the antiprogestin RU486 only caused a slight increase (two fold) in VDRRy mediated reporter gene activity as is evident from Fig. 11.

Activators of XOR-6 (Fig.3 in WO 96/22390) such as butyl 4-NH2 Benzoate did not activate VDRRy (data not shown) in similar reporter assays as used in WO 96/22390.

15 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the g common general knowledge in Australia.

0 *500 S 0 )00 17 SEQUENCE LISTING <110> Pharmacia Upjohn AB <120> Novel vitamin D receptor related polypeptides, nucleic acid sequence encodinq the same and uses thereof <130> 1788 <140> PCT/SE98/01548 <141> 1998-08-31 <160> 4 <170> Patentln Ver. 2.1 <210> <211> <212> <213> 1 2905

DNA

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<400> 1 cctctgaagg tacttcagtg gagaataagc tgatcgatcc aacttaccac aqctqgaacc agtgtcaacq gccactggct gccatgaaac cggaaqaccc aagaagqaqa aagaaaagtg cggatgatga catttcaaqa ctqcagqccc tctttqaagg ccagccgaca acctacatgt cccatcgagg ttcaacacag ttggaagaca tacatgctga ctcttctccc caattcqcca ttcttgttcc acccaqcggc ttqttcggca aqacccagag cgacaatgcc tcctcaggaa agactcttac ccctttcctt tcaagqtgtg cccacgtttg ttctagaatc ggaatctcgg taatactcct tttgcaccgg caagcagtcc atgctgactt cagatgagga atcacttcaa gcaacgcccg ggcgacagtg tgatcatgtc aacggacagg tcagggagct atttccggct catcgaggga tctctctgca gtggcgggaa tcaaaggcat accagatctc tgttcaacgc ctgcaggtgg agaagctgca cagaccgccc ttactctqaa tgaagatcat tgctgcgcat tcacaggtag ccctctgaqc ctgctggcct ggacatgggt gtggagagtg ttaaaaggcc gaaqggacca ttcgcttcct ttcccctcct gataqtgaat cctcaqcctg gtcctgaaca at tgqt tcaa a aagaggccca tqtacactgt agtcggaqgt tgtcatgaca gctgaggtgc ccaggcctgc cgacgaggcc gactcagcca gatqqacgct gccaggggtg agaagctgcc gctgcggggg aqagatcttc catcagcttt cctgctgaag ggaqactgga cttccagcaa gctgcatgag aqgtgtgctg gtcctacatt ggctatgctc ccaggacata ctgagcggct cgccactccc gtctccctag gccccccacc cactgacctq ctgtggtctq agcgaccaag qagtcttttc cttccgagct tcgtgggatg caagccaagt aggcagcgqc gtggacccca gaagcaaacc gaggacacaq ccccaaatct tgtgaaggat cccttccgga.

cgcctgcgca.

gtggaggaga ctgggagtqc cagatgaaaa cttagcagtg aagtggaqcc gaqgatggca tccctgctgc gccaaagtca ggggccgctt acctgggagt cttctactg gaggagtatq cagcaccgcg qaatqcaatc accgagctcc cacccctttg gcccttqqqt gggccaaqac ggaattcctg cccagttcag t agqtcagga qggagaaatc gataggccat attgctacct qctttgtggg ggaagaggaa gttcacaqtg tccttggtaa qgggagaagt tggaggtgag agt ctgttcc qccgtgtatg gcaagggctt agggcqcctg agtgcctqqa ggcgggcctt aggggctgac cctttgacac gctgcgaqtt aggtccggaa.

gtgtctggaa cccacatqgc tctcctactt tcgagctgtg gtggccggct agcccatgct tgctgatqca tggtgqacca ggccccaqcc qcagcatcaa ctacgcccct gacacctccg aqatqqacac ctatqacaqc tctgtaggga ccatcagaga cctcaqatcc ctgggqtcta ctaatagtcc ctcaaggcct gcactgcctt aaaaaagcaa agctactcct cqgagcaaag acccaaagaa tggaaagccc tgqggacaag tttcaggagg cgagatcacc gagcgqcatq gatcaagcgg agaggagcag taccttctcc gccagagtct agatctgtgc ctacaaaccc tgacatgtca caqqgacttg tcaactgaga gtcctactgc gaaattccac qgccatctcc gctgcaggag tgctcataqg tgctcagcac catgcaggag aqaggcagcc tgccaagagc tggctagcat gtgaaqccac gqcaaggttg cactaaaqtq tgcccacata tgtctcccac gtactcatcg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 0@ S

S

*0.5.0 0 S S S S SO -18gcagqtgcat caaatgtcag atttgaacac ctcaqatata caatttgqat gtggatgctg agtctgtagg gtagqtctgt tgctctgtga tgcacct tat ccttgtttat ataaggcatt atcaaggcaa aaaaaaaaaa gagtatctgt aagcttggca attattaagc gatcctgagc caaaaggaga agctgtgatg aqcaagggca ttgccacttg caaggctacq atttctgtgt agccacttgt ccacacctaa aaggaattaa aaaaaaaaaa gggagtcctc tqacctcatt actgataata tcacagagtt aaatgataag gcaggcactg caaactgcag atgggqcctq ctgacaatca acacatctat gaqtaaaaat gaactagttt ataatgtact aaaaa tagagagatg ccggccacat ggtagcctgc tataqttaaa tgacaaaagc ggtacccaag ctgtqagtqc ggtttgttcc gttaaacaca tctcaaagct ttttttgcat tgggaaatgt tttggctaaa agaagccaqg cattctgtgt tgtggggtat aaaacaaaca agcacaaqqa tgaaggttcc gtgtgtgtga tggggctgga ccggagaaga aaagggtatg tttcacaaat agccctgggt aaaaaaaaaa aggcctgcac ctctgcatcc acagcattga gaaacacaaa atttccctgt cgaggacatg tttggtgtag atgctgggta accatttaca aaaqtgcctg tat actt tat ttaatgtcaa aaaaaaaaaa 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2905 <210> 2 <211> 434 <212> PRT <213> Homo sapiens <400> 2 Met Glu Val Arg Pro 1 5 Lys Giu Ser Trp As n 10 His Ala Asp Phe Val His

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S S 0 Cys Giu Asp Giu Giu Val 35 Giu Ser Val Pro Gly 25 Lys Pro Ser Val Asn Ala Asp Asp Lys Ala Gly Gly Pro Gin Cys Arg Val Cys Gly Thr Gly 50 Tyr His Phe Asn Val1 55 Met Thr Cys Giu Gly Cys Lys Gly Phe Phe Arg Arg Ala Met Arq Asn Ala Arg Arg Cys Pro Phe Lys Gly Ala Cys Glu Ile Thr Arg Lys Thr 90 Arg Arg Gin Cys Gin Ala Cys Arg Leu Met Ser Asp 115 Arg 100 Lys Cys Leu Giu Ser 105 Gly Met Lys Lys Giu Met Ile 110 Lys Arg Lys Giu Ala Val Giu Giu 120 Arq Arg Ala Leu Ile 125

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Lys Ser 130 Giu Arg Thr Gly Thr 135 Gin Pro Leu Gly Gin Gly Leu Thr Giu 145 Giu Gin Arq Met Ile Arg Giu Leu Met 155 Asp Ala Gin Met Lys 160 Thr Phe Asp Thr Thr 165 Phe Ser His Phe Lys 170 Asn Phe Arg Leu Pro Gly 175 Val Leu Ser Gly Cys Giu Leu Pro GiuAlPrSe 18519 Ser Leu Gin Ala Pro Ser 190 -UL- PU 1 4 19 Arg Leu Tyr 225 Pro Phe Ile Asn Ser 305 Glu Glu Arg Phe Ala 385 Arg Glu Lys 210 Lys His Ala Ser Thr 290 Tyr Pro Glu Pro Ala 370 His Ser Glu 195 Val Pro Met Lys Leu 275 Val Cys Met Glu Gly 355 Ile Arg Ile Ala Ser Pro Ala Val 260 Leu Phe Leu Leu Tyr 340 Val Thr Phe Asn Ala Leu Ala Asp 245 Ile Lys Asn Glu Lys 325 Val Leu Leu Leu Ala 405 Lys Gin Asp 230 Met Ser Gly Ala Asp 310 Phe Leu Gin Lys Phe 390 Gin Trp Leu 215 Ser Ser Tyr Ala Glu 295 Thr His Met His Ser 375 Leu His Ser 200 Arg Gly Thr Phe Ala 280 Thr Ala Tyr Gin Arg 360 Tyr Lys Thr Gin Gly Gly Tyr Arg 265 Phe Gly Gly Met Ala 345 Val Ile Ile Gin Val Glu Lys Met 250 Asp Glu Thr Gly Leu 330 Ile Val Glu Met Arg 410 Arg Asp Glu 235 Phe Leu Leu Trp Phe 315 Lys Ser Asp Cys Ala 395 Leu Lys Gly 220 Ile Lys Pro Cys Glu 300 Gin Lys Leu Gin Asn 380 Met Leu Asp 205 Ser Phe Gly Ile Gin 285 Cys Gin Leu Phe Leu 365 Arg Leu Arg Leu Val Ser Ile Glu 270 Leu Gly Leu Gin Ser 350 Gin Pro Thr Ile Cys Trp Leu Ile 255 Asp Arg Arg Leu Leu 335 Pro Glu Gin Glu Gin 415 Ser Asn Leu 240 Ser Gin Phe Leu Leu 320 His Asp Gin Pro Leu 400 Asp a

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5454 <400> 3 tgaattcgtg tgacagtcac tgcacagtgc aqgtgagacc ctgttcctgg gtgtatgtgg agggcttttt gcgcctgcqa gcctggagag gggccttgat ggctgacaqa ttgacactac gcgagttgcc tccggaaaga tctggaacta acatggctga cctacttcaq aqctqtgtca gccggctgtc ccatgctgaa tgatgcaggc tggaccagct cccagcctgc gcatcaatgc cgcccctcat acctccgaga tggacactgc tgacagctg gtaggqagtg tcagagaggc cagatcccac gggtctatgc atagtcctgt aaggcctgta agccagqagg tctgtgtctc ggggtataca acaaacagaa acaaggaatt aggttcccga tgtgtqattt qgctggaatg gagaagaacc gqqtatgaaa cacaaat tat cct qggt t ta aaaaaaaaaa ggcctgctgg caggactcac tgcqgctgag caaagaaagc aaagcccagt ggacaaggcc caggagggcc gatcacccgg cggcatgaag caagcggaaq ggagcagcgg cttctcccat agagtctctg tctgtgctct caaaccccca catgtcaacc gqacttgccc actgaqattc ctactgcttg attccactac catctccctc gcaggagcaa tcataggttc tcagcacacc gcaggagttg ggcagccaga caagagccga ctagcattcc aagccacaga aaggttgccc taaagtqtca ccacataccc ctcccacttc ctcatcggca cctgcaccaa tgcatccatt gcattgactc acacaaacaa tccctgtgtg ggacatgaqt ggtgtaggta ctqqqtatgc atttacatgc gtgcctqcct actttatata atgtcaaatc aaaaaaaaaa gttagtgctg cacttcaagg ttggcttcaa t gga acc at g gtcaacgcag actggctatc atgaaacgca aagacccggc aaggagatga aaaagtgaac atgatqatca ttcaagaatt caggccccat ttgaaqgtct gccgacagtg tacatgttca atcgagqacc aacacagtgt gaagacactg atgctqaaga ttctccccag ttcgccatta ttgttcctga cagcggctgc ttcqgcatca cccaqagccc caatgccctg t caggaagga ctcttacgtg tttcctttta aggtqtggaa acgtttgttc ccactcgttc ggtgcatgaq atqtcaqaag tqaacacatt agatatagat tttggatcaa gatgctgagc ctgtaggagc ggtctqtttg tctqtgacaa accttatatt tqtttatagc aqqcattcca aaggcaaaag aaaaaaaaaa gcagcccccc aggqgtccct accatccaag ctgactttgt atgagqaagt act tcaatqt acgcccggct gacagtgcca tcatgtccga qgacaqggac qggagctgat tccggctgcc cgagggaaga ctctqcagct gcgggaaaga aaggcat cat agatctccct tcaacqcgga caggtggct t agctgcagct accgcccag ctctgaagtc agatcatggc tgcgcatcca caggtagctg tctgagccgc ctggcctgtc catgggtgcc gagagtgcac aaaggccctg gggaccaagc gcttcctgag ccctcctctt tatctgtggg cttggcatga.

attaagcact cctgagctca aaggagaaaa.

tqtgatgqca aaggqcacaa ccacttgatg qgctacgctg tctqtgtaca cacttgtqaq cacctaagaa gaattaaata aaaaaaaaaa tgaggccaag cagagcacct aggcccagaa acactgtgag cggaggtccc cat gacatgt gaggtgcccc ggcctqccgc cgaggccgtg tcagccactg qqacgctcag aqgggtgctt agctgccaag gcgqqgggag gatcttctcc cagctttgcc gctgaagggg gactggaacc ccaqcaactt qcatgaggag tgtgctgcag ctacattqaa tatgctcacc gqacatacac agcggctgcc cactcccggg tccctaggga ccccaccccc tgacctgtag tggtctgggg gaccaaqgat tcttttcatt ccgagctgct agtcctctag cctcattccg gataatagqt cagagtttat tgataagtga qgcactgggt actgcagctg ggcctggqt acaatcaqtt catctattct taaaaatttt ctaqttttg atgtactttt aa gacagcagca gccatacccc 120 gcaaacctgg 180 gacacagagt 240 caaatctgcc 300 gaaggatgca 360 ttccggaagg 420 ctgcgcaagt 480 gaqqagaggc 540 ggagtgcagg 600 atgaaaacct 660 agcagtggct 720 tggagccagg 780 gatqqcaqtg 840 ctgctqcccc 900 aaagtcatct 960 gccgctttcg 1020 tgggagtgtg 1080 ctactggagc 1140 gagtatgtgc 1200 caccgcqtgg 1260 tgcaatcggc 1320 gagctccgca 1380 ccctttgcta 1440 cttgggtgac 1500 ccaagacaga 1560 attcctgcta 1620 agttcagtct 1680 gtcaggacca 1740 agaaatccct 1800 aggccatctg 1860 gctacctcta 1920 ttgtgggctc 1980 agagatgaga 2040 gccacatcat 2100 agcctgctqt 2160 agttaaaaaa 2220 caaaagcagc 2280 acccaagtga 2340 tgagtgcgtg 2400 ttgttcctqg 2460 aaacacaccg 2520 caaagctaaa 2580 tttqcatttt 2640 gaaatgtaqc 2700 qgctaaaaaa 2760 2802 0

S.

9 S. 0 4 4* 21 <210> 4 <211> 473 <212> PRT <213> Homo sapiens <400> 4 Met Thr Val Thr Arg Thr His His Phe Lys Glu Gly Ser Leu Arg Ala 1 5 10 Pro Ala Ile Pro Leu His Ser Ala Ala Ala Glu Leu Ala Ser Asn His 25 Pro Arg Gly Pro Glu Ala Asn Leu Glu Val Arg Pro Lys Glu Ser Trp 40 Asn His Ala Asp Phe Val His Cys Glu Asp Thr Glu Ser Val Pro Gly 55 Lys Pro Ser Val Asn Ala Asp Glu Glu Val Gly Gly Pro Gin Ile Cys 70 75 Arg Val Cys Gly Asp Lys Ala Thr Gly Tyr His Phe Asn Val Met Thr 90- Cys Glu Gly Cys Lys Gly Phe Phe Arg Arg Ala Met Lys Arg Asn Ala 100 105 110 Arg Leu Arg Cys Pro Phe Arg Lys Gly Ala Cys Glu Ile Thr Arg Lys 115 120 125 Thr Arg Arg Gin Cys Gin Ala Cys Arg Leu Arg Lys Cys Leu Glu Ser 66 130 135 140 Gly Met Lys Lys Glu Met Ile Met Ser Asp Glu Ala Val Glu Glu Arg 145 150 155 160 Arg Ala Leu Ile Lys Arg Lys Lys Ser Glu Arg Thr Gly Thr Gin Pro 165 170 175 Leu Gly Val Gin Gly Leu Thr Glu Glu Gin Arg Met Met Ile Arg Glu 6 180 185 190 Leu Met Asp Ala Gin Met Lys Thr Phe Asp Thr Thr Phe Ser His Phe 195 200 205 6 Lys Asn Phe Arg Leu Pro Gly Val Leu Ser Ser Gly Cys Glu Leu Pro 210 215 220 Glu Ser Leu Gin Ala Pro Ser Arg Glu Glu Ala Ala Lys Trp Ser Gin 225 230 235 240 Val Arg Lys Asp Leu Cys Ser Leu Lys Val Ser Leu Gin Leu Arg Gly 245 250 255 Glu Asp Gly Ser Val Trp Asn Tyr Lys Pro Pro Ala Asp Ser Gly Gly ~C l~1L' 22 Lys Glu Ile Phe Ser Leu Leu Pro His Met Ala Asp Met Ser Thr Tyr 275 280 285 Met Phe Lys Gly Ile Ile Ser Phe Ala Lys Val Ile Ser Tyr Phe Arg 290 295 300 Asp Leu Pro Ile Glu Asp Gin Ile Ser Leu Leu Lys Gly Ala Ala Phe 305 310 315 320 Glu Leu Cys Gin Leu Arg Phe Asn Thr Val Phe Asn Ala Glu Thr Gly 325 330 335 Thr Trp Glu Cys Gly Arg Leu Ser Tyr Cys Leu Glu Asp Thr Ala Gly 340 345 350 Gly Phe Gin Gin Leu Leu Leu Glu Pro Met Leu Lys Phe His Tyr Met 355 360 365 Leu Lys Lys Leu Gin Leu His Glu Glu Glu Tyr Val Leu Met Gin Ala 370 375 380 Ile Ser Leu Phe Ser Pro Asp Arg Pro Gly Val Leu Gin His Arg Val 385 390 395 400 Val Asp Gin Leu Gin Glu Gin Phe Ala Ile Thr Leu Lys Ser Tyr Ile 405 410 415 Glu Cys Asn Arg Pro Gin Pro Ala His Arg Phe Leu Phe Leu Lys Ile 420 425 430 Met Ala Met Leu Thr Glu Leu Arg Ser Ile Asn Ala Gin His Thr Gin 435 440 445 Arg Leu Leu Arg Ile Gin Asp Ile His Pro Phe Ala Thr Pro Leu Met 450 455 460 Gin Glu Leu Phe Gly Ile Thr Gly Ser 465 470 *o *ee

Claims (14)

1. A nucleic acid molecule encoding a VDRR polypeptide, said VDRR polypeptide comprising a DNA-binding domain (DBD) and a ligand-binding domain (LBD), wherein said DNA-binding domain (DBD) has about 65% amino acid sequence similarity with the DBD of hVDR; and about 71% amino acid sequence similarity with the DBD of xONR1; or, wherein the said ligand-binding domain (LBD) has about 42% amino acid sequence similarity with the LBD of hVDR; and about 54% amino acid sequence similarity with the LBD of xONRI.

2. The nucleic acid molecule according to claim 1 wherein the said DNA binding domain comprises about 77 amino acids with 9 cysteine residues.

3. The nucleic acid molecule according to claim 1, having a sequence which is the same or S* substantially the same as that given in Fig. 1 (SEQ ID NO: 1) or Fig. 7 (SEQ ID NO: 3).

4. A nucleic acid molecule encoding a VDRR polypeptide, said nucleic acid molecule 20 having a sequence which is the same or substantially the same as that given in Fig. 1 (SEQ ID NO: 1) or Fig. 7 (SEQ ID NO: 3).

5. The nucleic acid molecule according to any one of claim 1 to 4, having the sequence given in Fig. 1 (SEQ ID NO: 1) or Fig. 7 (SEQ ID NO: or alleles thereof.

6. The nucleic acid molecule according to any one of claims 1 to 5, having the sequence given in Fig. 1 (SEQ ID NO: 1) or Fig. 7 (SEQ ID NO: 3).

7. A nucleic acid probe comprising at least 14 contiguous nucleotides of the nucleic acid sequence given in Fig. 1 (SEQ ID NO: 1) or Fig. 7 (SEQ ID NO: 3) or the complement thereof, wherein the probe is capable of detecting the nucleic acid molecule of any one of claims 1 to 6 or the complement thereof. z 8. A method for identifying clones encoding a VDRR polypeptide, said method comprising S screening a genomic or cDNA library with a nucleic acid probe according to claim 7 24 under low stnngency hybrdization conditions, and identitying clones which display a substantial degree of hybridization to said probe.

9. A vector comprising the nucleic acid molecule according to any one of claims I to 6. The vector according to claim 9 which is an expression vector.

11. A cell containing a nucleic acid molecule according to any one of claims I to 6.

12. A cell containing a vector according to claim 9 or

13. A process for recombinant production of a VDRR polypeptide, said process comprising expressing the nucleic acid according to any one of claims 1 to 6 in a suitable host cell.

14. The process according to claim 12, wherein the host cell is eukaryotic. An isolated or recombinant human, VDRR polypeptide having an amino acid sequence which is the same or substantially the same as that given in Fig. 4 (SEQ ID NO: 2) or Fig. 8 (SEQ ID NO: 4).

16. A pharmaceutical formulation comprising an isolated or recombinant VDRR polypeptide according to claim 15, and one or more therapeutically acceptable excipients. 25 17. A method for identifying a ligand to a VDRR polypeptide according to claim S: by a cell-based reporter assay, transgenic-animal reporter assay or in vitro-binding assay. *o P:\OPER\Fas\22284(41)-sp.doc-)2/1)2/O l

18. A method for treating metabolic, proliferative or inflammatory conditions comprising introducing into a human a nucleic acid vector according to claim 9 or encoding for expression of a VDRR polypeptide, wherein said nucleic acid vector is capable of transforming a cell in vivo and expressing said polypeptide in said transformed cell. DATED this 5 t h day of February 2001 Pharmacia AB by DAVIES COLLISON CAVE Patent Attorneys for the Applicants o