AU735346B2 - Isoforms of the human vitamin D receptor - Google Patents

Description

WO 99/16872 PCT/AU98/00817 ISOFORMS OF THE HUMAN VITAMIN D RECEPTOR Field of the Invention:- The present invention relates to isolated polynucleotide molecules which encode novel isoforms of the human Vitamin D receptor (hVDR) or variant transcripts for hVDR. The polynucleotide molecules may be utilised in, for example, methods of screening compounds for VDR agonists and/or antagonists.

Background of the Invention:- The active hormonal form of vitamin D, 1,25-dihydroxyvitamin

D

3 (1.25(OH) 2 has a central role in calcium and phosphate honmeostasis, and the maintenance of bone. Apart from these calcitropic effects, 1,25-(OH) 2

D:

has been shown to play a role in controlling cell growth and differentiation in many target tissues. The effects of are mediated by a specific receptor protein, the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcriptional regulators which also includes steroid, thyroid and retinoid receptors as well as a growing number of orphan receptors. Upon binding hormone the VDR regulates gene expression by direct interaction with specific sequence elements in the promotor regions of hormone responsive target genes. This transactivation or repression involves multiple interactions with other protein cofactors, heterodimerisation partners and the transcription machinery.

Although a cDNA encoding the human VDR was cloned in 1988 little has been documented characterising the gene structure and pattern of transcription since that time. The regulation of VDR abundance is one potentially important mechanism for modulating 1.25-(OH) 2

D

3 responsiveness in target cells. It is also possible that VDR has a role in nontranscriptional pathways, perhaps via localization to a non-nuclear compartment and/or interaction with components of other signalling pathways. However, the question of how VDRs are targetted to different cell types and how they are regulated remains unresolved. There have been many reports in the literature describing translational or transcriptional control of VDR levels, both homologously and heterologously, mostly in non-human systems.

WO 99/16872 PCT/AU98/00817 2 A recent study showed that in the kidney, alternative splicing of human VDR transcripts transcribed from a GC rich promotor generates several transcripts which vary only in their 5' UTRs. The present inventors have now identified further upstream exons of the VDR gene which generate 5' variant transcripts, suggesting that the expression of the VDR gene is regulated by more than one promoter. A subset of these transcripts is expressed in a restricted tissue-specific pattern and further variant transcripts have the potential to encode an N-terminally variant protein. These results may have implications for understanding the actions of in different tissues and cell types, and the possibility that N-terminally variant VDR proteins may be produced has implications for altered activities such as transactivation function or subcellular localisation of the receptor protein.

Furthermore, these variants, by their level, tissue specificity, subcellular localisation and functional activity, may yield targets for pharmaceutical intervention. The variants may also be useful in screening potential analogs and/or antagonists of vitamin D compounds.

Disclosure of the Invention:- In a first aspect, the invention provides an isolated polynucleotide molecule encoding a human Vitamin D receptor (hVDR) isoform, said polynucleotide molecule comprising a nucleotide sequence which includes sequence that substantially corresponds or is functionally equivalent to that of exon Id of the human VDR gene.

Exon Id (referred to as exon lb in the Australian Provisional Patent Specification No. P09500) is a 96 bp exon located 296 bp downstream from exon la The sequence of exon Id is: AAGGAGCGATTGGCTGTCGATGGTGCTCAGAACTGCTGGAGTGGAGG3' (SEQ ID NO: 1).

The nucleotide sequence of the polynucleotide molecule of the first aspect of the invention, preferably does not include sequence corresponding to that of exon la, exon If and/or exon le. However, the nucleotide sequence of the polynucleotide molecule of the first aspect of the invention, may or WO 99/16872 PCT/AU98/00817 3 may not include sequence that substantially corresponds or is functionally equivalent to that of exon lb and/or exon Ic.

Preferably, the polynucleotide molecule of the first aspect comprises a nucleotide sequence which includes; sequence that substantially corresponds or is functionally equivalent to that of exons id, Ic and 2-9 and encodes a VDR isoform of approximately 477 amino acids, (ii) sequence that substantially corresponds or is functionally equivalent to that of exons Id and 2-9 and encodes a VDR isoform of approximately 450 amino acids, or (iii) sequence that substantially corresponds or is functionally equivalent to that of exons id and 2-9 and further includes a 152 bp intronic sequence, and encodes a truncated VDR isoform of approximately 72 amino acids.

Most preferably. the polynucleotide molecule of the first aspect of the invention comprises a nucleotide sequence substantially corresponding to that shown as SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

In a second aspect, the invention provides an isolated polynucleotide molecule encoding a human Vitamin D receptor (hVDR), said polynucleotide molecule comprising a nucleotide sequence which includes sequence that substantially corresponds to that of exon If and/or le of the human VDR gene.

Exon If is a 207bp exon located more than 9kb upstream from exon la bp upstream from exon lc(8). The sequence of exon If is:

CAGAGACGGACGGACGCAGGGGCCCGGCCCAAGGCGAGGG

AGAACAGCGGCACTAAGGCAGAAAGGAAGAGGGCGGTGTG

TTCACCCGCAGCCCAATCCATCACTCAGCAACTCCTAGAC

GCTGGTAGAAAGTTCCTCCGAGGAGCCTGCCATCCAGTCGT

GCGTGCAG3' (SEQ ID NO: Exon le is a 157 bp exon located 1826bp upstream from exon la The sequence of exon le is: 4 t.

WO 99/16872 PCT/AU98/00817 4

AGAAGATCTGGGTCCAGTAGCTCTGACACTCCTCAGCTGT

AGAAACCTTGACAACTCTGCACATCAGTTGTACAATGGAA

CGGTATTTTTTACTCTTCATGTCTGAAAAGGCTATGATAA

AGATCAA3' (SEQ ID NO: 6) The nucleotide sequence of the polynucleotide molecule of the second aspect of the invention, preferably does not include sequence corresponding to that of exon la, Id or lb. However, the nucleotide sequence of the polynucleotide molecule of the second aspect of the invention, may or may not include sequence that substantially corresponds or is functionally equivalent to that of exon 1c.

Preferably, the nucleotide molecule of the second aspect comprises a nucleotide sequence which includes sequence that substantially corresponds or is functionally equivalent to that of exons if and 2-9.

Most preferably, the polynucleotide molecule of the first aspect of the invention comprises a nucleotide sequence substantially corresponding to that shown as SEQ ID NO: 7.

The polynucleotide molecule of the first or second aspects may be incorporated into plasmids or expression vectors (including viral vectors), which may then be introduced into suitable host cells bacterial, yeast, insect and mammalian host cells). Such host cells may be used to express the VDR or functionally equivalent fragment thereof encoded by the isolated polynucleotide molecule.

Accordingly, in a third aspect, the present invention provides a host cell transformed with the polynucleotide molecule of the first or second aspect.

In a fourth aspect, the present invention provides a method of producing a VDR or a functionally equivalent fragment thereof, comprising culturing the host cell of the first or second aspect under conditions enabling the expression of the polynucleotide molecule and, optionally, recovering the VDR or functionally equivalent fragment thereof.

Preferably, the host cell is of mammalian origin. Preferred examples include NIH 3T3 and COS 7 cells.

t WO 99/16872 PCT/AU98/00817 In a preferred embodiment, the VDR or functionally equivalent fragment thereof is localised to a cell membrane or other subcellular compartment as distinct from a nuclear localisation.

The polynucleotide molecules of the first aspect of the invention encode novel VDR isoforms which may be of interest both clinically and commercially. By using the polynucleotide molecule of the present invention it is possible to obtain VDR isoform proteins or functionally equivalent fragments thereof in a substantially pure form.

Accordingly. in a fifth aspect, the present invention provides a human VDR isoform or functionally equivalent fragment thereof encoded by a polynucleotide molecule of the first aspect, said VDR isoform or functionally equivalent fragment thereof being in a substantially pure form.

In a sixth aspect. the present invention provides an antibody or antibody fragment capable of specifically binding to the VDR isoform of the fourth aspect.

The antibody may be monoclonal or polyclonal. however, it is presently preferred that the antibody is a monoclonal antibody. Suitable antibody fragments include Fab, and scFv.

In an eighth aspect. the present invention provides a non-human animal transformed with a polynucleotide molecule according to the first or second aspect of the invention.

In a seventh aspect, the invention provides a method for detecting agonist and/or antagonist compounds of a VDR isoform of the fourth aspect, comprising contacting said VDR isoform, functionally equivalent fragment thereof or a cell transformed with and expressing the polynucleotide molecule of the first aspect, with a test compound under conditions enabling the activation of the VDR isoform or functionally equivalent fragment thereof, and detecting an increase or decrease in the activity of the VDR isoform or functionally equivalent fragment thereof.

An increase or decrease in activity of the receptor or functionally equivalent fragment thereof may be detected by measuring changes in interactions with known cofactors SRC-1, GRIP-1 and TFIIB) or unknown cofactors through use of the yeast dual hybrid system).

In a ninth aspect, the present invention provides an oligonucleotide or polynucleotide probe comprising a nucleotide sequence of 10 or more nucleotides, the probe comprising a nucleotide sequence such that the probe WO 99/16872 PCT/AU98/0081 7 6 specifically hybridises to the polynuicleo tide miolecule of the first or second aspect under high stringency conditions (Sambrook et Molecular- clonfing: a laboratorly manual, Second Edition, Gold Sprinig Harbor Laboratory Press).

Preferably, the probe is labelled.

In a tenth aspect. the present invention provides anl antisense polynucleotide molecule comprising a nucleotide sequence capable of specifically hybridising to ailn mNA mi-olecule which encodes a VDR encoded by the polynucleotide molecule of the first or second aspect., so as to prevent translation of the inRNA molecule.

Such antisense polynucleotide molecules may include a ribozyme region to catalytically iniactivate mRNA to which it is hybridised.

The polynucleotide molecule of the first or second aspect of thle invention mnay be a dominant negative mutant which encodes a genie product causing an altered phenotype by, for examiple, reducing or eliminating the activity of endogenous VDR.

In anl eleventh aspect, the inventioni provides anl isolated polynucleotide molecule comprising a nucleotide sequence substantially corresponding or, at least, showing 750/ (preferably 85% or, even more preferably, sequence identity to:

CGGACGGACGCAGGGGCGCGGCCCAJ\GGCGAGGGAGACAGCGGCACTA

AGGCAGAAAGGAAGAGGGCGGTGTGTTCACCCGGAGCGCAXTCGATCAC

TCAGcAATc~rCTAGACGGTGGTAGAAAGTTCGTCCGAGGAGCCTGCCATG GAGTCGTGCGTGCAG 3'(exon 1f) (SEQ ID NO: (ii) s 'AGGCAGCATGAAACAGTGGGATGTGCAGAGAGAJAGATCTGGGTC

CATGTTAATCCGTTAAACTAACCGAA

CAGTTGTACAATGGAACGGTATTTTTTACTCTTATGTCTGAAAGGCTA

TGATAAAGATCAA3' (exoni le) (SEQ ID NO: or (iii) s 'GTTTCCTTCTTCTGTCGGGGCGCCTTGGCATGGAGTGGAGGATA

AGAAAAGGAGCGATITGGCTGTCGATGGTGCTCAGAACTGCTGGAGTGGA

GG3' (exon 1d) (SEQ ID NO: 1).

WO 99/16872 PCT/AU98/00817 7 The polynucleotide molecules of the eleventh aspect may be useful as probes for the detection of VDR variant transcripts and as such may be useful in assessing cell or tissue-specific expression of variant transcripts.

The terms "substantially corresponds" and "substantially corresponding" as used herein in relation to nucleotide sequences is intended to encompass minor variations in the nucleotide sequence which due to degeneracy in the DNA code do not result in a substantial change in the encoded protein. Further, this term is intended to encompass other minor variations in the sequence which may be required to enhance expression in a particular system but in which the variations do not result in a decrease in biological activity of the encoded protein.

The term "functionally equivalent" as used herein in relation to nucleotide sequences encoding a VDR isoform is intended to encompass nucleotide sequence variants of up to 5% sequence divergence retaining 95% or more sequence identity) which encode VDR isoforms of substantially equivalent biological activity(ies) as said VDR isoform.

The term "functionally equivalent fragment" as used herein in respect of a VDR isoform is intended to encompass functional peptide and polypeptide fragments of said VDR isoform which include the domain or domains which bestow the biological activity characteristic of said VDR isoform.

The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated step, component or feature or group of steps, components or features with or without the inclusion of a further step, component or feature or group of steps, components or features.

The invention will hereinafter be further described by way of the following non-limiting example and accompanying figures.

Brief description of the figures:- FIG.1. Human VDR gene locus. Four overlapping cosmid clones were isolated from a human lymphocyte genomic library (Stratagene) and directly sequenced. Clone J5 extends from the 5' flanking region to intron 2; AE, from intron lb to intron 5; D2, from intron 3 to the 3' UTR: WE, from intron 6 through the 3'-flanking region. Sequence upstream of exon If was obtained by WO 99/16872 PCT/AU98/00817 8 anchored PCR from genomic DNA. Structure of hVDR transcripts.

Transcripts 1-5 originate from exon la. Transcript 1 corresponds to the published cDNA Transcripts 6-10 originate from exon Id and transcripts 11-14 originate from exon If. Boxed numbers indicate the major transcript (based on the relative intensities of the multiple PCR products) within each exon-specific group of transcripts generated with a single primer set. While all transcripts have a translation initiation codon in exon 2, exon Id transcripts have the potential to initiate translation upstream in exon id, with transcripts 6 and 9 encoding VDR proteins with extended N termini. (C) N-terminal variant proteins encoded by novel hVDR transcripts. Transcript 1 corresponds to the published cDNA sequence and encodes the 427-aa hVDR protein. Transcripts 6 and 9 code for a protein with an extra 50 aa or 23 aa, respectively, at the N-terminal. The 23 aa of the hVDR A/B domain are shown in bold.

FIG. 2. RT-PCR analysis of expression of variant hVDR transcripts. Exon la transcripts (220 bp. 301 bp, 342 bp, 372 bp, and 423 bp). (B) Exon Id transcripts (224 bp, 305 bp, 346 bp, 376 bp. and 427 bp). Exon If transcripts (228 bp, 309 bp, 387 bp, and 468 bp). RT-PCR was carried out with exon la-, id-. or If-specific forward primers and a common reverse primer in exon 3. The sizes of the PCR products and the pattern of bands are similar in A and B by virtue of the identical splicing pattern of exon la and Id transcripts and the fact that primers were designed to generate PCR products of comparable sizes. All tissues and cell lines are human in origin.

FIG. 3. Functional analysis of sequence-flanking exons la and Id and exon If in NIH 3T3 (solid bars) and COS 7 cells (open bars).

The parent vector pGL3basic was used as a promoterless control, and a promoter-chloramphenicol acetyltransferase (CAT) gene reporter construct was cotransfected as an internal control for transfection efficiency in each case. The activity of each construct was corrected for transfection efficiency and for the activity of the pGL3basic empty vector control and expressed as a percentage of the activity of the construct la(-488,+ SEM of at least three separate transfections. Exon la and Id flanking constructs are defined in relation to the transcription start site of exon WO 99/16872 PCT/AU98/00817 9 la, designated 11, which lies 54 nt upstream of the published cDNA Exon If flanking constructs are defined relative to the exon If transcription start site, designated 11. Transcription start sites were determined by the termini of the longest RACE clones. The open box corresponds to the GC-rich region.

FIG 4. Provides the nucleotide sequence of novel exons detected by 5' RACE: exon Ib (SEQ ID NO: exon if (SEQ ID NO: 5) [Plf is indicated by an arrow above the sequence], exon le (SEQ ID NO: exon Id (SEQ ID NO: 1) [in-frame ATG codons are highlighted and Pld is indicated by an arrow above the sequence]. Intronic sequences are shown in lower case.

Canonical splice site consensus sequences are indicated in bold. The transcription start sites for exons If and Id were determined by the 5' termini of RACE clones. No intron sequence is shown 3' to exon If as cosmid clone J5 terminated in the intron between exons If and le.

FIG 5. Provides the nucleotide sequence corresponding to transcript 6 (see figure 1) (SEQ ID NO: together with the predicted amino acid sequence (SEQ ID NO: 9) of the encoded protein. Nucleotides 1-96 correspond to exon Id; nucleotides 97-1463 correspond to exons Ic to the stop codon in exon 9 (or nucleotides -83-1283 of the hVDR cDNA FIG 6. Provides the nucleotide sequence corresponding to transcript 9 (see figure 1) (SEQ ID NO: together with the predicted amino acid sequence (SEQ ID NO: 10) of the encoded protein. Nucleotides 1-96 correspond to exon Id; nucleotides 97 1382 correspond to exon 2 to the stop codon in exon 9 (or nucleotides -2 1283 of the hVDR cDNA FIG 7. Provides the nucleotide sequence corresponding to transcript 10 (see figure 1) (SEQ ID NO: together with the predicted amino acid sequence (SEQ ID NO: 11) of the encoded protein. Nucleotides 1-96 correspond to exon Id; nucleotides 97-244 correspond to exon 2; nucleotides 245-396 correspond to intronic sequence immediately 3' to exon 2; nucleotides 397- 1534 correspond to exons 3 to the stop codon in exon 9 (or nucleotides 146- 1283 of the hVDR cDNA WO 99/16872 PCT/AU98/00817 FIG 8. Provides the nucleotide sequence corresponding to transcript 11 (see figure 1) (SEQ ID NO: together with the predicted amino acid sequence (SEQ ID NO: 12) of the encoded protein. Nucleotides 1-207 correspond to exon if; nucleotides 208-1574 correspond to exon Ic to the stop codon in exon 9 (or nucleotides -83-1283 of the hVDR cDNA Example:- EXPERIMENTAL

PROCEDURES

Isolation and Characterisation of Genomic Clones A human lymphocyte cosmic library (Stratagene, La Jolla, Ca) was screened using a 2.1kb fragment of the hVDR cDNA encompassing the entire coding region but lacking the 3'UTR, a 241 bp PCR product spanning exons 1 to 3 of the human VDR cDNA, and a 303 bp PCR product spanning exons 3 and 4 of the hVDR cDNA, following standard colony hybridisation techniques. DNA probes were labelled by nick translation (Life Technologies, Gaithersburg, MD) with 32 P] dCTP. Positively hybridising colonies were picked and secondary and tertiary screens carried out until complete purification. Cosmid DNA from positive clones was purified (Qiagen), digested with different restriction enzymes and characterised by Southern blot analysis using specific [y32 P]ATP labelled oligonucleotides as probes.

Cosmid clones were directly sequenced using dye-termination chemistry and automated fluorescent sequencing on an ABI Prism. 377 DNA Sequencer (Perkin-Elmer, Foster City, Ca). Sequence upstream of the most 5' cosmid was obtained by anchored PCR from genomic DNA using commercially available anchor ligated DNA (Clontech, Palo Alto, Ca).

Rapid Amplification of cDNA 5-prime Ends Alternative 5' variants of the human VDR gene were identified by using commercially prepared anchor-ligated cDNA (Clontech) following the instructions of the manufacturer. Two rounds of PCR using nested reverse primers in exons 3 and 2 (P 1: 5'ccgcttcatgcttcgcctgaagaagcc-3', P2: 5'-tgcagaattcacaggtcatagcattgaag-3') were carried out on a Corbett FTS- 4000 Capillary Thermal Sequencer (Corbett Research, NSW, Australia). After 26 cycles of PCR, 2% of the primary reaction was reamplified for 31 cycles.

WO 99/16872 PCT/AU98/00817 11 The PCR products were cloned into PUC18 and sequenced by the dideoxy chain termination method.

Cell-Culture The embryonal kidney cell line, HEK-293, an embryonic intestine cell line, Intestine-407 and WS 1, a foetal skin fibroblast cell line were all cultured in Eagle's MEM with Earle's BSS and supplemented with either heat-inactivated FBS, 15% FBS or 10% FBS with non-essential amino acids, respectively. The osteosarcoma cell lines MG-63 and Saos-2 were cultured in Eagle's MEM with nonessential amino acids and 10% heat-inactivated FBS and McCoy's 5a medium with 15% FBS. respectively. The breast carcinoma cell line T47D and the colon carcinoma cell lines LIM 1863 and COLO 206F were cultured in RPMI medium supplemented with 0.2 IU bovine insulin/ml and 10% FBS, 5% FBS or 10% FBS, respectively. LIM 1863 were a gift from R.H. Whitehead HK-2 kidney proximal tubule cells were grown in keratinocyte-serum free medium supplemented with 5ng/ml recombinant EGF, 40ug/ml bovine pituitary extract. BC1 foetal osteoblast-like cells were kindly donated by R. Mason and were grown in Eagle's MEM with 5% FBS and 5mg/L vitamin C. Unless otherwise stated all cell lines were obtained from the American Type Culture Collection (Manassas, VA).

Reverse Transcriptase-PCR

(RT-PCR).

Total RNA extracted from approximately 1.5 x 10 cells, from leukocytes prepared from 40 ml blood, or from human tissue using acidphenol extraction was purified by using a guanidium isothiocyanate-cesium chloride step gradient. First-strand cDNA was synthesized from 5 .tg of total RNA primed with random hexamers (Promega) using Superscript II reverse transcriptase (Life Technologies). One-tenth of the cDNA (21p) was used for subsequent PCR, with 36 cycles of amplification, using exon-specific forward primers (exon la: corresponding to nucleotides 1-21 of hVDR cDNA exon id: 5'-GGCTGTCGATGGTGCTCAGAAC-3'; exon if: 5'-AAGTTCCTCCGAGGAGCCTGCC-3'); and a common reverse primer in exon 3 [corresponding to nucleotides 301- 280 of hVDR cDNA All RT-PCRs were repeated multiple times by using RNA/cDNA prepared at different times from multiple sources. Each PCR included an appropriate cDNA-negative control, and additional controls WO 99/16872 PCT/AU98/00817 12 included RT-negative controls prepared alongside cDNA and RNA/cDNA prepared from VDR-negative cell lines. PCR products were separated on 2% agarose and visualized with ethidium bromide staining.

Functional Analysis of hVDR Gene Promoters.

Sequences flanking exons la, Id, and If (see Fig. 1A) were PCRamplified by using Pfu polymerase (Stratagene) and cloned into the pGL3basic vector (Promega) upstream of the luciferase gene reporter.

Promoter-reporter constructs were transfected into NIH 3T3 and COS 7 cells by using the standard calcium phosphate-precipitation method. Cells were seeded at 2.3±2.5 x 10" per 150-cm 2 flask the day before transfection. Several hours before the precipitates were added the medium was changed to DMEM with 2% charcoal-stripped FBS. Cells were exposed to precipitate for 16 h before subculturing and were harvested 24 h later. The parent vector pGL3basic was used as a promoterless control in these experiments and a simian virus 40 promoter-chloramphenicol acetyltransferase (CAT) gene reporter construct was cotransfected as an internal control for transfection efficiency in each case. The activity of each construct was corrected for transfection efficiency and for the activity of the pGL3 basic empty vector control and expressed as a percentage of the activity of the construct la(-488,+ 75). Luciferase and CAT assays were carried out in triplicate, and each construct was tested in transfection at least three times.

RESULTS

Identification of Alternative 5' Variants of the hVDR Gene.

Upstream exons were identified in human kidney VDR transcripts by RACE (exons If, le, Id. and Ib) and localized by sequencing of cosmid clones (Fig. 1A). To verify these results and to characterize the structure of the 5' end of the VDR gene, exon-specific forward primers were used with a common reverse primer in exon 3 to amplify specific VDR transcripts from human tissue and cell line RNA (Fig. 1B). The identity of these PCR products was verified by Southern blot and by cloning and sequencing. Five different VDR transcripts originating from exon la were identified. The major transcript (transcript 1 in Fig. 1B) corresponds to the published cDNA sequence Three less-abundant forms 3, and 4 in Fig. 1B) arise from WO 99/16872 PCT/AU98/0017 13 alternative splicing of exon Ic and a novel 122-bp exon lb into or out of the final transcript. These three variant transcripts were described recently by Pike and colleagues A fifth minor variant was identified (5 in Fig. 1B) that lacks exons lb and Ic, but includes an extra 152 bp of intronic sequence immediately 3' to exon 2, potentially encoding a truncated protein as a result of an in-frame termination codon in intron 2.

Four more transcripts were characterized that originate from exon If, a novel 207-bp exon more than 9 kb upstream from exon la. The major ifcontaining transcript (11 in Fig.1B) consists of exon If spliced immediately adjacent to exon Ic. Three less-abundant variants (12, 13, and 14 in Fig. 1B) arise from alternative splicing of exon Ic and a novel 159-bp exon le into or out of the final transcript. All these hVDR variants differ only in their UTRs and encode identical proteins from translation initiation in exon 2.

Of considerable interest, another five hVDR transcripts were identified that originate from exon ld, a novel 96-bp exon located 296 bp downstream from exon la. The major exon id-containing transcript (6 in Fig. 1B) utilizes exon id in place of exon la of the hVDR cDNA. Three minor variants 8, and 9 in Fig. 1B) arise from alternative splicing of exons lb and Ic into or out of the transcript, analogous to the exon la-containing variants 2, 3, and 4. A fifth minor variant transcript (10 in Fig. 1B) lacks exons lb and Ic, but includes 152 bp of intron 2 analogous to the exon la-containing transcript and also potentially encodes a truncated protein. Two of these exon Idcontaining hVDR transcripts encode an N-terminal variant form of the hVDR protein. Utilization of an ATG codon in exon Id, which is in a favorable context and in-frame with the major translation start site in exon 2. would generate a protein with an additional 50 aa N-terminal to the ATG codon in exon 2 in the case of variant 6 or 23 aa in the case of variant 9 (Fig.1C).

The relative level of expression of the different transcripts is difficult to address with PCR since relatively minor transcripts may be amplified.

However, Southern blots of PCR products from the linear range of PCR amplification indicated that equivalent amounts of PCR product were accumulated after 26 cycles for exon la transcripts compared with 30 cycles for exon id transcripts, suggesting that id abundance is about 5% of that of la transcripts. This is consistent with the frequency of clones selected and sequenced from RACE analysis of two separate samples of kidney RNA: la (21/27;78%), Id (2/27; and If (4/27; RT-PCR with exon la-, Id-, or WO 99/16872 PCT/AU98/00817 14 If-specific forward primers and reverse primers in exons 7 or 9, followed by cloning and sequencing, suggests that these 5' variant transcripts are not associated with differences at the 3' end of the transcript.

Exon-Intron Organization of the hVDR Gene.

Overlapping cosmid clones were isolated from a human lymphocyte genomic library and characterized by hybridization to exon-specific oligonucleotide probes (Fig. 1A). The exon-intron boundaries of the hVDR gene were determined by comparison of the genomic sequence from cosmid clones with the cDNA sequence. Upstream exons were localized in the VDR gene by sequencing cosmid clones, which extend approximately 7 kb into the intron between exons le and if, enabling verification of both their sequence and the presence of consensus splice donor/acceptor sites. Sequence upstream of exon If was obtained by anchored PCR from genomic DNA by using commercially available anchor-ligated DNA (CLONTECH). In total, the liVDR gene spans more than 60 kb and consists of at least 14 exons (Fig. 1A).

Tissue-Specific Expression of hVDR Transcripts.

The pattern of expression of variant hVDR transcripts was examined by RT-PCR in a variety of cell lines and tissues with exon la-, Id-, or If-specific forward primers and a common reverse primer in exon 3. Exon la and Id transcripts (Fig. 1B, variants 1-10) were coordinately expressed in all RNA samples analyzed (Fig. 2 A and Exon If transcripts (Fig. 1B, variants 11- 14), however, were detected only in RNA from human kidney tissue (two separate samples), human parathyroid adenoma tissue, and an intestinal carcinoma cell line, LIM 1863 (Fig. 2C). Interestingly. these represent major target tissues for the calcitropic effects of vitamin D.

Functional Analysis of hVDR Gene Promoters.

Promoter activities of the 5' flanking regions of exons la, id, and if were examined in NIH 3T3 and COS 7 cells (Fig. Sequences flanking exon la exhibited high promoter activity in both cell lines (Fig. 3A). Maximum luciferase expression of 36- and 54-fold over the empty vector was attained for construct la(-488,+ 75) in NIH 3T3 and COS 7 cells, respectively. This activity could be attributed largely to a GC-rich region containing multiple consensus Spl-binding motifs lying within 100 bp immediately adjacent to WO 99/16872 PCT/AU98/00817 the transcription start site. This region alone, upstream of a luciferase reporter [construct la(-94,+ accounted for 43% of the maximum activity observed in NIH 3T3 cells and 86% of the maximum observed in COS 7 cells.

The removal of this GC-rich region [construct la(-29,+ 75)] reduced luciferase activity to only 13% of the maximum in NIH 3T3 and 19% in COS 7 cells.

Despite the fact that VDR transcripts that originated from exon Id were identified, distinct promoter activity was not associated with sequences within 300 bp of exon Id [constructs ld(+87,+424) and ld(+244,+424)]; rather, the sequence immediately adjacent to exon Id mav contain a suppressor element (Fig. 3A). Construct la-ld(-846,+470). spanning the flanking regions of both exons la and id, resulted in only 42% and 60% of the activity of la(-898.+75) in NIH 3T3 and COS 7 cells, whereas the 3' deletion of 227 bp restored luciferase activity to 65% and 97% of the activity of la(-898,+75), respectively. Similarly. the 5' truncated construct la-id 94,+470), spanning the 5' flanking regions of both la and Id. resulted in only and 40% of the activity of la(-94,+ 75), while a further 3' deletion of 227 bp restored luciferase activity to 69% and 91% of the activity of la(-94,+ in NIH 3T3 and COS 7 cells. It is possible that transcription from exons la and Id is driven by overlapping promoter regions rather than from two distinct promoters, as has been described for the mouse androgen receptor gene.

Sequence upstream of exon If showed significant promoter activity in NIH 3T3 cells of 22% of that of the most active construct, la(-488,+ 75), or 9fold over pGL3basic [construct lf(-1168,+58)] (Fig. 3B). A shorter construct [lf(-172,+58)] had similar activity, with evidence of a suppressor element (between nucleotides -278 and +172) able to repress luciferase activity by Interestingly, the same constructs were not active in COS 7 cells. This cell line-specific activity of exon If flanking sequences may reflect a requirement for tissue- or cell-specific protein factors.

Identification of VDR isoforms in whole cell lysates The existence of a VDR isoform including exons Id and Ic has been confirmed in cell lysates from multiple human, monkey, rat and mouse cell lines derived from kidney, intestine, liver and bone, by immunoprecipitation (using the anti-VDR 9A7 rat monoclonal antibody; Affinity Bioreagents Inc., WO 99/16872 PCT/AU98/00817 16 Golden, Colorado) followed by Western blot analysis. The ld- and Ic-exonspecific antibodies detected the same band in all inmmunoprecipitations.

DISCUSSION

The present inventors have identified 5' variant transcripts of the hVDR that suggest the existence of alternative promoters. These transcripts may not have been discriminated in previous Northern analyses because of their similarity in size. Transcription initiation from exons la or If and alternative splicing generate VDR transcripts that vary in their 5' UTRs but encode the same 427-aa protein. Transcription initiation from exon id and alternative splicing generate hVDR transcripts with the potential to encode variant proteins with an additional 50 or 23 aa at the N terminus. There was no evidence that these 5' variants are associated with differences at the 3' end of the transcript. Although isoforms are common in other members of the nuclear receptor superfamily, the only evidence for isoforms of the hVDR is a common polymorphism in the triplet encoding the initiating methionine of the 427-aa form of the VDR that results in initiation of translation at an alternative start codon beginning at the 10th nucleotide down-stream, encoding a protein truncated by 3 aa at the N terminus Similarly, two forms of the avian VDR. differing in size by 14 aa, are generated from a single transcript by alternative translation initiation and in the rat a dominantnegative VDR is generated by intron retention Heterogeneity in the 5' region is a common feature of other nuclear receptor genes. Tissue-specific alternative-promoter usage generates multiple transcripts of the human estrogen receptor a (ERa). the human and rat mineralocorticoid receptors, and the mouse glucocorticoid receptor (GR), which differ in their 5' UTRs but code for identical proteins. However, other members of the nuclear receptor superfamily have multiple, functionally distinct isoforms arising from differential promoter usage and/or alternative splicing. The generation of N-terminal variant protein isoforms has been described for the progesterone receptor peroxisome proliferatoractivated receptor and the retinoid and thyroid receptors. Some receptor isoforms exhibit differential promoter-specific transactivation activity. The N-terminal A/B regions of many nuclear receptor proteins possess a ligand-independent transactivation function (AF1). An AF1 WO 99/16872 PCT/AU98/00817 17 domain has been demonstrated for the thyroid receptor bl (TRbl), ER. GR, PR, PPARg, and the retinoid receptors. The activity of the AF1 domain has been shown to vary in both a tissue- and promoter-specific manner. The Nterminal A/B region of nuclear receptors is the least-conserved domain across the family and between receptor subtypes, varying considerably both in length and sequence. The VDR, however, is unusual as its N-terminal A/B region is much shorter than that of other nuclear receptors, with only 23 aa N-terminal to the DNA-binding domain, and deletion of these residues seems to have no effect on VDR function. This region in other receptors is associated with optimal ligand-dependent transactivation and can interact directly with components of the basal transcription complex. Two stretches of basic amino acid residues, RNKKR and RPHRR, in the predicted amino acid sequences of the variant hVDR N termini (Fig. 1C) resemble nuclear localization signals. An N-terminal variant VDR protein therefore might exhibit different transactivation potential, possibly mediated by different protein interactions, or may specify a different subcellular localization. The tissue-specific expression of exon If-containing transcripts is mediated by a distal promoter more than 9 kb upstream of exons la and Id. Exon If transcripts were detected only in kidney tissue, parathyroid adenoma tissue, and an intestinal cell line, LIM 1863. It is interesting that these tissues represent major target tissues for the calcitropic effects of vitamin D. The absence of If-containing transcripts in two other kidney cell lines, HK-2 (proximal tubule) and HEK-293 (embryonal kidney). as well as one other embryonal intestinal cell line, Intestine-407, suggests that the expression of If transcripts is cell type-specific. The cell line-specific activity of exon If flanking sequences in promoter reporter assays may reflect a requirement for tissue- or cell-specific protein factors to mediate expression from this promoter.

This study has demonstrated that expression of the human VDR gene, which spans more than 60 kb and consists of 14 exons. is under complex transcriptional control by multiple promoters. The expression of multiple exon If transcripts is mediated by utilization of a distal tissue-specific promoter. Transcription from a proximal promoter. or promoters, generates multiple variant hVDR transcripts, two of which code for N-terminal variant proteins. Multiple, functionally distinct isoforms mediate the tissue- and/or developmental-specific effects of many members of the nuclear receptor WO 99/16872 PCT/AU98/00817 18 superfamily. Although the actual relative abundance of the various transcripts and their levels of translation in vivo have not yet been characterized, the results suggest that major variant isoforms of the hVDR exist. Differential regulation of these hVDR gene promoters and of alternative splicing of variant VDR transcripts may have implications for understanding the various actions of 1,25-(OH),D in different cell types, and variant VDR transcripts may play a role in tissue specific VDR actions in bone and calcium homeostasis.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are. therefore, to be considered in all respects as illustrative and not restrictive.

WO 99/16872 PCT/AU98/0081 7 19 References:- 1. Baker, A.R. et.al. (1988) Proc. Natl. Acad. Sci. USA 85. 3294-3298 2. Miyamoto, K. et.al. (1997) Mol. Endocrinol. 11, 1165-1179 3. Whitehead, R.I~et.al. (1987) Cancer Res. 47, 2683-2689 4. Slater, M. et al. (1994) Am. J. Physiology 267, E990-1001 Saijo, TYet.aI. (1991) Amj. Hui. Genet. 49, 668-673 6. Lu, Z. et.al. (1997) Arch. Biochein. Biophys. 339. 99-106 7. Ebihara, K. et.al. (1996) Mol.Cell.Biol. 16, 3393-3400 WO 99/16872 WO 9916872PCT/AU98/0081 7 Sequence fisfings:- <110> Garvan Institute of Medical Research Title of the Invention: Isoforms of the Human Vitamin D Receptor <130> 91317 <140> <141> <160> 12 <170> Patentln Ver. SEQ ID NO: 1 <211> 96 <212> DNA <213> Homo sapiens <400> 1 gtttccttct ggctgtcgat tctgtcgggg cgccttggca tgqagtggaq gaataaqaaa agqagcgatt ggtgctcaga actgctggag tggagg 96 SEQ ID NO: 2 <211> 1413 <212> DN4A <213> H-omno sapiens <400> 2 gtttccttct ggctgtcgat tgaqacctca tggaggcaat cccggatctg gtgaaggctq ccttcaacgq tcaaacgctg agaggaagcg ggcccaagct agacctacga atggtggagg actcctcctc gcttctccaa tqtcccagct tcattggctt tactgctgaa tggacgacat ccaaagccgg agaagctgaa cagatcqtcc acacactgca ccaagatgat accgctgcct tgtttggcaa tctgtcggqg ggtgctcaga cagaagagca ggcggccagc tggggtgtqt caaaggcttc ggactgccgc tgtggacatc ggagatgatc gtctgaggag ccccacctac gagccatcct c *tcctgctca tctggatcto ctccatgctcj tgctaagatg gtcaagtgcc gtcctggacc acacagcctg cttgcatgag tggqgtgcag cjacgtacatc ccagaagcta ctccttccag tgagatctcc cqccttqgca actgctggag cccctgqgct acttccctgc ggagaccgag ttcaggcgaa atcaccaagg qgcatgatga ctgaagcgga cagcagcgca tccgacttct tccaggccca gatcactgta agtgaagaag ccccacctgg ataccaggat attgaggtca tgtggcaacc gagctgattg gaggagcatg gacgccgcgc cgctgccgcc gccgacctgc cctgagtgca tga tgqagtggag tggaggaagc ccacttacct ctgaccctgg ccactggctt gca tgaagcg acaaccgacg aggagttcat aqgaggagga tcattgccat gccagttccg act ccagaca tcacctcttc attcagatga ctga cctggt tcagagacct tca tqttgcg aagactacaa agcccctcat tcctgctcat tgattgaggc acccqccccc qcagcctcaa gcatgaagct gaataagaaa ctttqggtct gccccctgct agactttgac tcacttcaat gaaggcacta ccactgccag tctgacagat ggccttgaag actgctggac gcctccagtt cactcccagc agacatqatq cccttctqtg cagttacagc cacctctgag ctccaatgag gtaccgcgtc caaqttccag ggccatctgc catccaqgac gggcagccac tqaggagcac aacgcccctt a gga gcqat t qaagtgtctg ccttcaggga cggaacgtgc gctatgacct ttcacctgcc gcctgccggc gaggaagtgc gacagtctgc gcccaccata cgtgtqaatg ttctctgggg gactcgtcca accctagagc atccaaaaqg gaccagatcg tccttcacca agtgacgtqa gtgggactqa atcgtctccc cgcctgtcca ctgctctatg tccaagcagt gtgctcgaag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1463 WO 99/16872 WO 99/ 6872PCT/AU98/0081 7 SEQ ID NO: 3 <211> 1382 <212> DNA <213> Homo sapiens <400> 3 gtttccttct ggctgtcgat cttccctgcc gagaccgagc tcagqcgaag tcaccaagga gcatgatgaa tgaagcggaa agcagcgcat ccgacttctg ccaggcccaa atcactgtat gtgaagaaga cccacctggc taccaggatt ttgaggtcat gtggcaacca agctgattga aggagcatgt acgccgcgct gctgccgcca ccgacctgcg ctgagtgcag ga tctgtcgggg ggtgctcaga tgaccctgga cactggcttt catgaagcjq caaccgacgc ggagttcatt ggaggaggag cattgccata ccagttccgg ctccagacac cacctcttca ttcagatgac tqacctggtc cagagacctc catqttgcqc agactacaag gcccctcatc cctgctcatg gattgaggcc Cccgcccccg cagcctcaat catgaagcta cgccttggca actgctggag ga ctttgacc cacttcaatg aaggcactat ca ctgccagg ctgacagatg gccttgaagg ctgctggacg cctccaqttc actcccagct gacatgatgg ccttctgtga agttacagca a cctctgagg t ccaatgagt taccgcgtca aagttccagg gccatctgca atccaggacc gjgcagccacc gaggagcact a cgccccttg tggagtggag tggaggggat ggaacgtgcc ctatga cctq tczacctgccc cctgccggct aggaagtgca acagtctqcg cccaccataa gtgtgaatga tctctgggga actcgtccag ccctagagct tccaaaaggt accagatcqt ccttcaccat gtgacgtgac tgggactgaa tcgtctcccc gcctgtccaa tgctctatgc ccaagcaqta tgctcgaagt gaataagaaa ggaggcaatg ccggatctgt tgaaggctqc cttcaacggg caaacgctgt gacjgaagcqg qcccaagctg gacctacgac tgqtgqaggq ctcctcctcc cttctccaat gtcccagctc cattqqcttt actgctgaag ggacgacatg caaaqccgga gaagctgaac agatcgtcct cacactgcag caagatgatc ccgctgcctc gtttggcaat aggaqcgatt gcggccagca ggggtgtgtg aaaggcttct qactgccgca qtggacatcg gagatgatcc tctgaggagc cccacctact agccatcctt tcctgctcag ctggatctga tccatgctgc gctaagatga tcaaqtgcca tcctggacct cacagcctgq ttgcatgagg ggggtgcagg acgtacatcc cagaagctag tccttccagc qagatctcct 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1382 SEQ ID NO: 4 <211> 1534 <212> DNA <213> Homo sapiens <400> 4 gtttccttct ggctgtcgat cttccctqcc gagaccgagc tcaggtgagc tttccatgaa tgcggcgctc attcacctgc ggcctgccgg tgaggaagtg ggacagtctg cgcccaccat t cqtqtgaat cttctctqggg ggactcgtcc gaccctagag catccaaaag gqaccagatc gtccttcacc cagtgacgtq ggtgggactg catcgtctcc tctgtcgggg ggtgctcaga tgaccctgga cactggcttt ccccctccca gggagccctt acagccacag cccttcaacg ctcaaacgct cagaggaagc cggcccaagc aagacctacg gatqgtggag qactcctcct agcttctcca ctgtcccagc gtcattggct gtactgctga atggacgaca accaaagccg aaqaagctga ca gat cgt c cgccttggca actgctggag gactttgacc ca ct tcaat g ggctctcccc gcatttttca gagcaggag gggactgccg gtgtggacat gggagatgat tgtctgagga accccaccta ggagccatcc cctcctgctc atctggatct tctccatgct ttgctaagat agtcaagtgc tgtcctggac gacacagcct acttgcatga ctggggtgca tggagtggag tggaggggat ggaacgtgcc ctatgacctg agtggaaagg catctccttc gtcttggcga catcaccaag cggcatgatg cctgaagcgg gcagcagcgc ctccgacttc ttccaqqccc agatcactgt gagtgaagaa gccccacctg gata ccagga cattgaggtc ctgtggcaac ggagctgatt ggaggagcat ggacgccgcg gaa taagaaa ggaggcaatg ccggatctgt tgaagqctgc gagggagaaq cttacaatgt agcatgaagc gacaaccgac aaggagttca aaggaggagg atcattgcca tqccagttcc aactccagac atcacctctt gattcagatg gctgacctgg ttcagaqacc atcatgttgc caagactaca gagcccctca qtcctqctca ctqattgagg aggagcgatt gcggccagca ggggtgtqtg aaaggcttct aagcaaggtq ccatgqaaca ggaaggcact gccactgcca ttctgacaga aggccttgaa tactgctgqa ggcctccagt acactcccag cagacatgat acccttctgt tcagttacag tcacctctga gctccaatga agtaccgcgt tcaagttcca tggccatctg ccatccagga 120 180 240 300 360 420 480 540 600 660 720 780 840 900.

960 1020 1080 1140 1200 1260 1320 WO 99/16872 WO 99/ 6872PCT/AU98/0081 7 ccgcctgtcc cctgctctat ctccaagcag tgtgctcgaa aacacactgc agacgtacat qccaagatga tccagaagct taccgctgcc tctccttcca gtgtttggca atgagatctc ccgctgccgc cacccgcccc cgggcagcca 1380 agccgacctg cqcagcctca atgaqgagca 1440 gcctgagtgc agcatgaagc taacgcccct 1500 ctq a 1534 SEQ ID NO: <211> 207 <212> DNA <213> Homo sapiens <400> tgcgaccttg gcggtagcc. tqgggacagg ggtgaggcca gagacggacg gacgcagggg cccggcccaa qgcqaggqag aacagcggca ctaagqcaqa aacjqaagagg gcggtgtgtt 120 cacccgcagc ccaatccatc actcagcaac tcctagacgc tggtaqaaaq ttcctccqag 180 gagcctgcca tccagtcqtg cgtgcaq 207 SEQ ID NO: 6 <211> 157 <212> DNA <213> Homo sapiens <400> 6 aqqcagcatg cctcagctgt tactcttcat aaacagtggg agaaaccttg gtctgaaaag atgtgcagag agaagatctg ggtccagtag ctctqacact acaactctgc acatcagttg tacaatggaa cggtattttt 120 gctatgataa agatcaa 157 SEQ ID NO: 7 <211> 1574 <212> DNA <213> Homo sapiens <400> 7 tgcgaccttg cccggcccaa cacccgcagc gagcctgcca acaqaagagc tggcqgccag qtggggtgtg gcaaaggctt gggactgccg gtqtggacat gggagatgat tgtctgagga accccaccta ggagccatcc cctcctgctc atctggatct tctccatgct ttgctaagat agtcaagtgc tgtcctggac gacacagcct acttgcatga ctggggtqca agacgtacat tccagaagct gcggtgagcc ggcgagggag ccaatccatc tccagtcgtg acccctgggc cacttccctg tggagaccga cttcaggcga catcaccaag cggcatgatg cctgaagcgg gcagcagcgc ctccgacttc ttccaggccc aqatcactgt gagtgaagaa gccccacctg gataccagga cattgaggtc ctgtggcaac gqagctgatt ggaggagcat ggacgccgcg ccgctgccgc aqccgacctg tggggacagg aacagcggca actcagcaac cgtgcagaag tccacttacc cctgaccctg gccactggct agcatgaagc gacaaccgac aaggagttca aaggaggagg atcattgcca tgccagttcc aactccagac atcacctctt gattcagatg gctgacctgg ttcagagacc atcatgttgc caagactaca gaqcccctca gtcctgctca ctgattgagg cacccgcccc cgcagcctca ggtgaggcca ctaaggcaga tcctagacgc cctttgggtc tgccccctgc gagactttga ttcacttcaa ggaaggcact gccactgcca ttctgacaga aggccttgaa tactgctgga ggcctccagt acactcccag caga ca tgat acccttctgt tcagttacag tcacctctga gctccaatga agtaccgcgt tcaagttcca tggccatctg ccatccagga cgggcagcca atgaggagca gagacggacg aaggaagagg tggtagaaag tgaagtgtct tccttcaggg ccggaacgtg tgctatgacc attcacctgc ggcctgccgg tgaggaagtg ggacagtctg cgcccaccat tcgtgtgaat cttctctqggg ggactcgtcc ga ccctagag catccaaaag ggaccagatc gtccttcacc cagtgacgtg ggtgggactg catcgtctcc ccgcctgtcc cctgctctat ctccaagcag gacgcagggq gcggtgtgtt ttcctccgag gtgagacctc atggaggcaa ccccggatct tgtgaaggct cccttcaacg ctcaaacgct cagaggaagc cggcccaagc aagacctacg gatggtggag gactcctcct agcttctcca ctgtcccagc gtcattggct gtactgctga atgqacqaca accaaagccg aagaagctga ccagatcgtc aacacactgc gccaagatga taccgctgcc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 WO 99/16872 WO 9916872PCT/AU98/0081 7 23 tctccttcca gcctqagtgc agcatgaagc taacqcccct tgtgctcgaa gtgtttggca 1560 atgagatctc ctga 1574 SEQ ID NO: 8 <211> 122 <212> DNA <213> Homo sapiens <400> 8 qgctcctgaa cctagcccag ctggacggag aaatggactc tagcctcctc tgatagcctc atgccaggcc ccgtgcacat tgctttgctt gcctccctca atcctcatag cttctctttg 120 gg 122 SEQ ID NO: 9 <211> 477 <212> PRT <213> Homno sapiens <400> 9 Met Glu Trp Arg Asn 1 Ar g Pro Ser Asp Al a Phe As n Cys Leu 145 Lys Glu Tyr Al a Arg Met Asp Gly Arg Asp 115 Leu Asp Gi u Gin Pro 195 Gi y Arg Giu Arg Phe Arg 100 Cys Lys Giu Glu Ar g 180 Thr 5 Val1 Al. a Al a As n His Ser Arg Arg Gi u Ala 165 Ile Tyr Lys Glu Pro Met Val1 70 Phe Met Ile Cys Val1 150 Leu Ile Ser Lys Glu Le u Al a Pro As n Ly s Th r Val 135 Gin Lys Al a Asp Arg Al a Gi y Al a Arg Al a Arg Lys 120 Asp Ar g Asp Ile Phe 200 Ser Phe 25 Ser Ser Ilie Met Lys 105 Asp Ile Lys Ser Leu 185 Cys Leu Glu Leu Leu Val1 Gl u Phe Arg Met 140 Met Pro Al a Ar g Met Ser Pro Asp Gi y Cys Cys 110 Cys Glu Leu Leu His 190 Pro Val1 Val1 Al a Pro Asp Lys Pro Gin Phe Lys Ser 175 Lys Val1 Leu Ar g Pro Gl y Ar g Gi y Phe Al a Ile Arg 160 Gi u Thr Arg WO 99/16872PC/U8017 PCT/AU98/00817 Val Thr 225 Ile Leu Gin Gin Thr 305 Ile Thr Al a Gi y Al a 385 Leu Ile Met Lys Gly Ph e Ser Gi u 260 Met Ile Asp Arg As n 340 Ser Lys Ile Aila Arq 420 Lys Ar g Gi y Ser Asp 245 Asp Leu Gly Gin Ser 325 Gin Leu Leu Val1 Ile 405 His Leu Cys Gi y Gly 230 Met Ser Pro Phe Ile 310 As n Asp Giu As n Ser 390 Gin Pro Al a Leu Ser 215 Asp Met Asp His Al a 295 Val Gi u Tyr Leu Leu 375 Pro Asp Pro Asp Ser 455 His Ser Asp Asp Leu 280 Lys Leu Ser Lys Ile 360 His Asp Arg Pro Le u 440 Phe Pro Ser Ser Pro 265 Al a Met Leu Phe Tyr 345 Giu Glu Arg Leu Gi y 425 Ar g Gin Se r Ser Ser 250 Ser Asp Ile Lys Th r 330 Arg Pro Gi u Pro Ser 410 Ser Ser Pro Pro 220 Cys Phe Thr ValI ci y 300 Ser Asp Ser Ile His 380 Va 1 Th r Leu As n Cys 460 As n Ser Ser Leu Ser 285 Phe Al a Asp Asp Lys 365 Vali Gin Leu Leu Gi u 445 Ser Ser Asp As n Giu 270 Tyr Ar g Ile Met Val1 350 Phe Leu Asp Gin Tyr 430 Giu Met His Cys 240 Asp Ser Ile Leu Val1 320 Trp Lys Val1 Met Al a 400 Tyr Ly s Se r Leu Thr Pro Leu Val Leu Giu Val Phe Gly Asn Glu Ile Ser 465 470 475 WO 99/16872 WO 9916872PCT/AU98/0081 7 SEQ ID NO: <211> 450 <212> PRT <213> Homo sapiens <400> Met Glu Trp Arg Asn

I

Arg Leu Val1 Giu Phe Arg Met Met Pro 145 Ala Ar g Pro Cys Phe 225 Thr Val1 Thr Pro Cys Gly Tb r His Lys Ile 130 Lys His Pro As n Ser 210 Ser Leu Ser Al a Asp Gi y Cys Cys Cys Giu 115 Leu Leu His Pro Ser 195 Asp As n Giu Tyr 5 Val1 Gly Arg Gly Ph e Al a Ile Arg Giu Thr 165 Arg His Cys Asp Ser 245 Ile Lys Giu Asp Al a Phe 70 As n Cys Leu Lys Giu 150 Tyr Val1 Thr Ile Leu 230 Gin Gin Lys Gly Phe Thr 55 Phe Gi y Ar g Tb r Gi u 135 Gin Asp As n Pro Thr 215 Ser Leu Lys Arg Met Asp 40 Gi y Ar g Asp Leu Asp 120 Glu Gin Pro Asp Ser 200 Ser Giu Ser Val1 Ser Giu Ar g Phe Arg Cys Lys 105 Giu Gi u Ar g Thr Gi y 185 Phe Se r Gi u Met Ile 265 Asp 10 Al a As n His Ser Arg 90 Arg Giu Al a Ile Tyr 170 Gly Ser Asp Asp Leu 250 Giy T rp Met Val1 Ph e Met 75 Ilie Cys Val1 Leu Ile 155 Ser Gi y Gi y Met Ser 235 Pro Phe Le u Al a Pro As n Lys Th i.

Val1 Gin Ly s 140 Al a Asp Se r Asp Met 220 Asp His Al a Ser Al a Arg Al a Arg Lys Asp Arg 125 Asp Ile Phe His Ser 205 Asp Asp Leu Lys Val1 Thr Cys Thr Al a As n Gly A r c Leu Leu Gin 175 Ser S ex Ser Ser Asp 255 Ile Leu Ser Gly Cys Leu Arg Met Giu Arg Asp 160 Phe Arg Ser Ser Val1 240 Leu Pro Gly Phe Arg 275 Asp Leu Thr Ser Giu Asp Gin Ile Val 280 Leu Leu Lys Ser 285 WO 99/16872PC/U8087 PCT/AU98/00817 Ser Ala Ile Giu Val Ile 290 Asp Asp Met Ser Trp Thr 305 310 Ser Asp Val Thr Lys Ala 325 Ile Lys Phe Gin Val Gly 340 His Val Leu Leu Met Ala 355 Val Gin Asp Ala Ala Leu 370 Thr Leu Gin Thir Tyr Ile 385 30 Leu Leu Tyr Ala Lys Met 405 Asn Giu Giu His Ser Lys 420 Cys Ser Met Lys Leu Thr 435 Ile Ser 450 SEQ ID NO: 11 <211> 72 <212> PRT <213> Homo sapiens <400> 11 Met Giu Trp Arg Asn Lys 1 5 Arg Thr Ala Gly Vai Giu Leu Pro Asp Pro Giy Asp Val Cys Gly Asp Arg Ala Glu Gly Cys Lys Gly Phe Met 295 Cys Gi y Leu Ile Ile 375 Arg Ilie Gin Pro Leu Gly His Lys Cys 360 Gi u Cys Gin Tyr Leu 440 Arg As n Ser Lys 345 Ilie Al a Ar g Lys Ar g 425 Val1 As n Asp 315 Glu As n Ser Gin Pro 395 Al a Leu Glu Giu 300 Tyr Leu Leu Pro Asp 380 Pro Asp Ser Val1 Phe Tyr Giu Gi u 350 Arg Leu Gi y Arg Gin 430 Gi y Th r Arg Pro 335 Giu Pro Ser Ser Ser 415 Pro As n Met Val 320 Leu Giu Gly As n His 400 Leu Giu Giu Lys Arg Ser Asp Trp Leu Ser Met Vai Leu 10 Giy Met Glu Ala Met Ala Ala Ser Thr Ser 25 Phe Asp Arg Asn Val Pro Arg Ile Cys Gly 40 Thr Gly Phe His Phe Asri Ala Met Thr Cys 55 Phe Arg WO 99/16872PC/U/087 PCT/AU98/00817 SEQ ID NO: 12 <211> 427 <212> PRT <213> Hoio sapiens <400> 12 Met Glu Ala Met Ala 1 Asp Gi y Arg Asp Leu Asp Giu Gin Pro 145 Asp Ser Ser Glu Ser 225 Val1 Gi u Arg Phe Arg Cys Lys Glu Glu Arg 130 Thr Gi y Phe Ser Gi u 210 Met Ile Asp As n His Ser Arg Arg Glu Al a 115 Ile Tyr Gly Ser Asp 195 Asp Leu Giy Gin Val1 Phe Metr Ile Cys Val1 100 Leu Ile Ser Gly Gi y 180 Met Ser Pro Phe Ile 260 5 Pro As n Lys Thr Val1 Gin Lys Al a Asp Ser 165 Asp Met Asp His Al a 245 Val1 Al a Ar g Al a Ar g Lys 70 Asp Arg Asp Ile Phe 150 His Ser Asp Asp Leu 230 Lys Leu Set Ile Met Lys 55 Asp Ile Lys Ser Leu 135 Cys Pro Se r Ser Pro 215 Al a Met Leu Thr Cys Th r 40 Al a As n Gi y Arg Leu 120 Leu Gin Ser Ser Ser 200 Ser Asp Ile Ly s Set Gi y 25 Cys Leu Ar g Met Giu 105 Arg Asp Phe Ar g Set 185 Set Val1 Leu Pro Ser 265 Leu Val1 Glu Phe Arg Met 90 Met Pro Al a Arg Pro 170 Cys Phe Th r Val1 Gi y 250 Ser Pro Cys Gi y Tb r His 75 Lys Ile Lys His Pro 155 Asn Set Set Leu Ser 235 Phe Al a Asp Gly Cys Cys Cys Gi u Le u Leu His 140 Pro Set Asp As n Glu 220 Tyr Arg Ile Pro Asp Lys Pro Gin Phe Lys Ser 125 Lys Val Arg His Leu 205 Leu Ser Asp Glu Gi y Arg Gi y Phe Al a Ile Arg 110 Gi u Thr Arg His Cys 190 Asp Set Ile Leu Val1 270 Asp Al a Phe As n Cys Le u Lys Gi u Tyr Val1 Thr 175 Ile Le u Gin Gin Thr 255 Ile Phe Thr Ph e Gly Arg Tb r Giu Gin Asp As n 160 Pro Thr Set Le u Lys 240 Set Met Leu Arg Set Asn Glu Ser Phe Thr Met Asp Asp Met Ser Trp Tht Cys 280 285 WO 99/16872 WO 9916872PCT/AU98/0081 7 Gi y His 305 Lys Cys Giu Cys Gin 385 Tyr Leu Asn 290 Ser Lys Ile Al a Arg 370 Lys Arg Val Gin Leu Leu Val1 Ile 355 His Le u Cys Leu Asp Giu As n Ser 340 Gin Pro Al a Leu Gi u 420 Tyr Leu Leu 325 Pro Asp Pro Asp Ser 405 ValI Lys Ile 310 His Asp Arg Pro Leu 390 Phe Phe Tyr 295 Gi u Giu Arg Leu Gly 375 Arq Gin Gly Arg Pro Glu Pro Sex 360 Ser Ser Pro Asn Val Leu Giu Gi y 345 As n His Leu Gi u Glu 425 Ser Ile His 330 Val Thr Leu As n Cys 410 Ile Asp Lys 315 Vai Gin Leu Leu Glu 395 Set Ser ValI 300 Ph e Leu Asp Gin Tyr 380 Gi u Met Th r Gin Leu Ala Thr 365 Al a His Lys Lys Val Met Al a 350 Tyr Lys Set Leu Al a Gi y Al a 335 Leu Ile Met Lys Thr 415

Claims (20)

1. An isolated polynucleotide molecule encoding a human Vitamin D receptor (hVDR) isoform, said polynucleotide molecule comprising a nucleotide sequence which includes sequence that substantially corresponds or is functionally equivalent to that of exon id of the human VDR gene.

2. A polynucleotide molecule according to claim 1, wherein said nucleotide sequence further includes sequence that substantially corresponds or is functionally equivalent to that of exon lb and/or exon Ic.

3. A polynucleotide molecule according to claim 1. wherein the nucleotide sequence includes: sequence that substantially corresponds or is functionally equivalent to that of exons id, Ic and 2-9 and encodes a VDR isoform of approximately 477 amino acids, (ii) sequence that substantially corresponds or is functionally equivalent to that of exons Id and 2-9 and encodes a VDR isoform of approximately 450 amino acids, or (iii) sequence that substantially corresponds or is functionally equivalent to that of exons Id and 2-9 and further includes a 152bp intronic sequence and encodes a truncated VDR isoform of approximately 72 amino acids.

4. A polynucleotide molecule according to claim 1. wherein the nucleotide sequence substantially corresponds to that shown as SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. An isolated polynucleotide molecule encoding a human Vitamin D receptor (hVDR), said polynucleotide molecule comprising a nucleotide sequence which includes sequence that substantially corresponds or is functionally equivalent to that of exon If and/or le of the human VDR gene.

6. A polynucleotide molecule according to claim 5, wherein the nucleotide sequence further includes sequence that substantially corresponds or is functionally equivalent to that of exon Ic. WO 99/16872 PCT/AU98/00817

7. A polynucleotide molecule according to claim 5. wherein the nucleotide sequence includes sequence that substantially corresponds or is functionally equivalent to that of exons If and 2-9.

8. A polynucleotide molecule according to claim 5, wherein the nucleotide sequence substantially corresponds to that shown as SEQ ID NO: 7.

9. A plasmid or expression vector including a polynucleotide molecule according to any one of the preceding claims. A host cell transformed with a polynucleotide molecule according to any one of claims 1-8 or a plasmid or expression vector according to claim 9.

11. A host cell according to claim 10. wherein the cell is a mammalian cell.

12. A host cell according to claim 10, wherein the cell is a NIH 3T3 or COS 7 cell.

13. A method of producing a VDR or VDR isoform or functionally equivalent fragments thereof, comprising culturing a host cell of any one of claims 10-12 under conditions enabling the expression of the polynucleotide molecule and, optionally, recovering the VDR or VDR isoform or functionally equivalent fragments thereof.

14. A method according to claim 13, wherein the VDR or VDR isoformn or functionally equivalent fragments thereof are expressed onto the host cell membrane or other sub-cellular compartment. A human Vitamin D receptor (hVDR) isoform or functionally equivalent fragment thereof encoded by a polynucleotide molecule according to any one of claims 1-4, said hVDR isoform or functionally equivalent fragment thereof being in a substantially pure form. WO 99/16872 PCT/AU98/008 17 31

16. An antibody or antibody fragment capable of specifically binding to a VDR isoform according to claim

17. A non-human animal transformed with a polynucleotide molecule according to any one of claims 1-8.

18. A method for detecting agonist and/or antagonist compounds of a VDR isoform of claim 15, comprising contacting said VDR isoform, functionally equivalent fragment thereof or a cell transformed with and expressing a polynucleotide molecule according to any one of claims 1-4, with a test compound under conditions enabling the activation of the VDR isoform or functionally equivalent fragment thereof, and detecting an increase or decrease in the activity of the VDR isoform or functionally equivalent fragment thereof.

19. An oligonucleotide or polynucleotide probe comprising a nucleotide sequence of 10 or more nucleotides, the probe comprising a nucleotide sequence such that the probe specifically hybridises to a polynucleotide molecule according to any one of claims 1-8 under high stringency conditions. An antisense polynucleotide molecule comprising a nucleotide sequence capable of specifically hybridising to a mRNA molecule which encodes a VDR or VDR isoform encoded by a polynucleotide molecule according to any one of claims 1-8, so as to prevent translation of the mRNA molecule.

21. An isolated polynucleotide molecule comprising a nucleotide sequence showing greater than 75% sequence identity to: CGGACGGACGCAGGGGCCCGGCCCAAGGCGAGGGAGAACAGCGGCACTA AGGCAGAAAGGAAGAGGGCGGTGTGTTCACCGCAGCCCAATCCATCAC TCAGCAACTCCTAGACGCTGGTAGAAAGTTCCTCCGAGGAGCCTGCCATC CAGTCGTGCGTGCAG3' (SEQ ID NO: WO 99/1 6872PC/U8087 PCT/AU98/00817 32 (ii) GAGTFAGCTGTGACAGTGCTCAGCTGTAGAAACT'TGAGAAGTCTGCACAT CAGTFTGTACAATGGAACGGTATTTTTTACTCTTCATGTCTGAAAGGCTA TGATAAAGATCAA3' (SEQ ID NO: or (iii) AGAAAAGGAGCGATTGGGTGTGGATGGTGCTGAGAACTGCTGGAGTGGA GG3' (SEQ ID NO: 1)

22. An isolated polynucleotide molecule comprising a nucleotide sequence showing greater than 85% sequence identity to: CGGACGGACGGAGGGGGGCGGCCAAGGCGAGGGAGAACAGCGGCACTA AGGCAGAAAGGAAGAGGGCGGTGTGTTCACCCGCAGCCCAATCCATCAC TCAGCAACTCGTAGACGCTGGTAGAAAGTTCCTCCGAGGAGCCTGCCATC CAGTCGTGCGTGGAG3" (SEQ ID NO: (ii) CAGTAGCTCTGAGACTCCTCAGCTGTAGAJ\JCGTTGACAACTCTGCACAT CAGTTGTACAATGGAACGGTATTTTTTACTCTTCATGTCTGAAGGCTA TGATAAAGATCAA3' (SEQ ID NO: or (iii) AGAAAAGGAGCGATTGGGTGTCGATGGTGGTGAGAACTGCTGGAGTGGA GG3'(SEQ ID NO: 1).

23. An isolated polvilucleotide molecule comprising a nucleotide sequence showing greater than 95% sequence identity to: 5 'TGCGACCTTGGCGGTGAGCCTGGGGACAGGGGTGAGGCCAGAGA CGGACGGACGCAGGGGCCCGGCCCAAGGCGAGGGAGAAJCAGCGGCACTA AGGCAGAAAGGAAGAGGGCGGTGTGTTCACCCGCAGCCCAJATCCATCAC TCAGCAACTCCTAGACGCTGGTAGAAAGTTCCTCCGAGGAGCCTGCCATC CAGTCGTGCGTGCAG3' (SEQ ID NO: WO 99/16872 PTAJ808 PCT/AU98/00817 33 (ii) CAGTAGCTCTGACACTGC'l'CAGCTGTAGA AAcc'Fi'GACAACTCTGCACAT CAGTTGTACAATGGAACGGGTATTTTTITAcTrCTTCATGTGCTGAAAAGGCTA TrGATAAAGATCAA3' (SEQ ID NO: or (iii) 5 'GTTTCCTTGTTCTGTCGGGGCGCCTTGGCATGGAGTGGAGGAATA AGAAAAGGAGCGAT'I'GGCTGTGGATGGTGCTCAGAACTGCTGGAGTGGA GG3' (SEQ ID NO: 1)

24. An isolated polynuicleotidle molecule comprising nucleotide sequence substantially corresponding to: CGGACGGACGGAGGGGCCGGCCCAAGGCGAGGGAGAACAGCGGCACTA AGGCAGAAAGGAAGAGGGGGTGTGTTCACCCGCAGCGCAATGGATCAC TCAGCAACTGCTAGACGCTGGTAGAAAGTTCCTGGGAGGAGGCTGCCATC CAGTCGTGCGTGGAG3' (SEQ ID NO: (ii) 5 'AGGCAGGATGAAACAGTGGGATGTGGAGAGAGAAGATCTGGGTC GAGTAGCTCTGACACTGCTCAGCTGT'AGAAAGGTTGACAACTGTGCACAT CAGTTGTACAATGGAAGGGTATTTTTTACTCTTCATGTCTGAAAAGGCTA TGATAAAGATCAA3' (SEQ ID NO: or (iii) 5 'GT'1TCCTTCTTFCTGTCGGGGGGCCTTGGCATGGAGTGGAGGAATA AGAAAAGGAGCGATTGGCTGTCGATGGTGCTCAGAACTGCTGGAGTGGA GG3' (SEQ ID NO: 1)