AU685127B2 - Human calcium sensor - Google Patents

Description

WO 94/28019 PCT/SE9400483 1 HUMAN CALCIUM SENSOR BACKGROUND OF THE INVENTION The present invention relates to a cDNA clone encoding a human calcium sensor protein of parathyroid, placental, and kidney tubule cells.

In WO 88/03271 there is described monoclonal antiparathyroid antibodies identifying a parathyroid cell membrane-bound calcium receptor or sensor, crucially involved in calcium regulation of the parathyroid hormone (PTH) release The receptor function is essential for maintenance of normal plasma calcium concentrations, and reduced receptor expression within proliferating parathyroid cells of patients with hyperparathyroJdism (HPT) results in calcium insensitivity of the PTH secretion and variably severe hypercalcemia Reactivity with the antiparathyroid antibodies was also demonstrated for proximal kidney tubule cells and cytotrophoblast cells of the human placenta, and the cytotrophoblasts were demonstrated to exhibit an almost parathyroid-identical regulation of cytoplasmic calcium (Ca 2 The antibody-reactive structure was found to exert calcium sensing function also in the cytotrophoblasts, and as these cells constitute part of the syncytium, the calcium sensor was suggested to be actively involved in the calcium homeostasis of the fetus It was proposed that the antibody-reactive structure of the proximal kidney tubule cells exerts a similar calcium sensing function, and that the calcium sensor, thus, plays a more universal role in calcium regulation via different organ systems (1,7,9,10).

On HPT patients with hypercalcemia, surgery is performed to remove one or more of the parathyroid glands. It would be greatly desirable to have alternatives to this surgical procedure as HPT has proven to be a very common disorder and surgery is a relatively costly procedure and sometimes even entails some risks for the patients.

SUBSTITUTE

SHEET

The calcium sensor/receptor has been revealed as a 500 kDa single chain glycopr'otein However, the amino acid sequence as well as the corresponding DNA sequences thereof are hitherto unknown.

SUMMARY OF THE INVENTION 0 It is an object of the present invention to provide sufficient structural data of the calcium sensor/receptor to enable complete characterization thereof.

According to the invention there is provided a cDNA clone encoding the human 1 5 cailum sensor, comprising the following nucleotide sequence 27 AAA TAC OTA ATO CAG CCA GAT GOA ATA GCA GTG GAC TOG GTT GGA see 0 a* *to 000949 TAC TGG TCA

TAC

AAT

GAG

25

GOT

ACT

AGA

CAG

G

CGA

ATC

CJX~A

i) CTG

AGA

CCC

TCT

TG

GAO

GTC

TTA

CAA

ATC

TOO

000

CT

MOG

AAA

CC

CCA,

TTC

ATT

TAO

GGA

Trr

AGC

TCC

ATC

GAT

TGG

CTA

TG

ACT

AAG

OCA

TG

AAG

CAT

CAC

AGO

AAC

GTC AAO CTG ATT COG OTT ATO AAT WCO OTT GAO GAO AAG OAA ATA TOT AMA GAG CAA, OTC OTO TOO TTT ATA CTO 000

I

AAT

TOO

ATG

OGA

TOT

OTT

OCA

AAG

MA

AGA

OTT

GAG

COO

AAA

ACT

TTO

GAG

ATO

ATT

ATG

GMA

ACG

TAO

rG

CCA

81

COO

135

GAO

189

TOO

243

GAO

297

OAT

351

GMA

405

MOC

459

MOG

533

OTO

567

MAT

621

AGA

675

AGO

729

TOO

OTG

AOT

CC

TAT

ACC

COT

OGA

OTA

MAG

COT

ACC

AGO

ATT GAG GTG GOT A

GAO

GAO

TTG

ATA

TAO

GMA

OTO

TOA

GGA

ACT

TOO

CAA

TG

ATC

MA

AGO

GTA

OTG

GGA

GAG

OCA

GGA

OTO

MAT

TAT

rTG

TG

CT

CCC

TAO

TOT

CAC

GOT

MAG

OTT

GAO

OAT

GAO

AMA

TOO

AGO

GAT

GGA

OTT

GOT

GMA

TTO

OGA

ATO

CAA

OTO

TT

OCA

OGA

AGO

OAT

ATT

COT

GAG

ATC

ACT

TITr

MAT

ACT

TOO

000 000

MAT

CAT ATT 108 OA AGO GOJ62 216 MAA ATO 270 GAO OTT 324 TAO TG 378 OAT AGO 432 GMA GAO 486 AAA TTT 540 CMA OTT 594 AMA CAG 648 TOT CCC, 7 02 ATO GAM 756 TOO TAT M211

TO

1 I 1 783 G^T O1 GAG ACT GZ CTO COO MAA TOO MOG TOT COT AGC CGO TAO, ACC Further aspects of the present invention relate to the amino acid sequence corresponding to the above nucleotide sequence, and to the use of the nucleotide sequence for the isolation of the Complete nucleotide sequence of the calcium sensor.

1 0 In one embodiment, the present invention provides problems for identifying other novel calcium sensor protein.

A second object was to use said structural data to design novel treatment methods as well a3 compounds and compositions for treating calcium related 1 5 disorders, Two important human diseases associated with perturbations of the calcium ion .o homeoscasis are ihyperthyroidism and osteoporosis, Thus, in one embodiment cells expressing the calcium sensor protein or comprising the cDNA encoding 20 the calcium sensor protein of the present invention may be utilized in an assay to identify molecules which block or enhance the activity of the calcium sensor protein, These molecules will be useful in the treatment of mammalian pathological conditions associated with perturbations in the levels of PTH, SI:O vitamin D3 production, estrogen, osteoclast activity or osteoblast activity '0 25 (therefore, bone resorption and/or formation), calcium secretion and calcium ion homeostasis.

The present invention describes the 'solation and partial characterization of a *see cDNA clone encoding the calcium sensor/receptor in human placenta and Northern blots verifying the presence of the corresponding mRNA within the parathyroid and kidney. Close sequence similarity between the calcium sensor and a previously described rat Heyman nephritis antigen suggests that the common calcium sensor of the placenta, the WO 94/28019 PCT/SE94/00483 3 parathyroid and kidney tubule is related to this antigen, or represents its human homologue, and that it belongs to a family of large glycoproteins with receptor function and calcium binding ability.

BRIEF DESCRIPTION OF THE DRAWINGS Fig 1. Isolation by HPLC of peptides obtained after digestion of the calcium-sensor protein with Lys-C endoprotease (solid line).

Dashed line represents the chromatography of an identical reaction where the calcium-sensor was omitted. The flow rate was kept at 100 pl/min. Two peptide fractions which gave easily interpretable sequences are denoted by arrows.

Fig 2. Sequences of two Lys-C peptides (SEQ ID Nos. 1 and 2) isolated by HPLC of the calcium-sensor protein.

Fig 3. Partial nucleotide sequence (SEQ ID No. 3) and deduced amino acid sequence (SEQ ID No. 4) of thetcDNA clone, pCAS-2, encoding part of the calcium-sensor protein. Portions of the deduced amino acid sequence identical to the peptides 292 and 293 are underlined.

Fig 4. Alignment of the amino acid sequence of the calcium-sensor protein (SEQ ID No. 4) to corresponding portions of the Heymann antigen (HEYMANN, SEQ ID No. low density lipoprotein receptor (LDL-RC, SEQ ID No. and LDL related receptor protein (LDL- RRP, SEQ ID No. Stars denote residues identical between the calcium sensor protein and any of the other sequences. X denotes a position in the Heymann antigen sequence where identity has not been published.

Fig 5. Northern blot analysis of total RNA from parathyroid adenoma kidney liver placenta pancreas adrenal gland small gut Filters were hybridized with the 2.8 kb pCAS-2 insert probe, and reactions visualized by a WO 94/28019 PCT/SE94/00483 4 phosphorimager. Locations of 28S and 18S ribosomal RNA are indicated.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Unless indicated otherwise herein, the following terms have the indicated meanings.

The term "polypeptide" means a linear array of amino acids connected one to the other by peptide bonds between the a-amino and carboxy groups of adjacent amino acids.

"Substantially purified" is used herein to mean "substantially homogeneous", which is defined as a material which is substantially free of compounds normally associated with it in its natural state other proteins or peptides, carbohydrates, lipids). "Substantially purified" is not meant to exclude artificial or synthetic mixtures with other compounds. The term is also not meant to exclude the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification or compounding with a pharmaceutically acceptable preparation.

The term "biologically active polypeptide" means the naturally occurring polypeptide per se as well as biologically active analogues thereof, including synthetically produced polypeptides and analogues thereof, as well as natural and pharmaceutically acceptable salts and pharmaceutically acceptable derivatives thereof. The term "biologically active polypeptide" also encompasses biologically active fragments thereof, as well as "biologically active sequence analogues" thereof. Different forms of the peptide may exist. These variations may be characterized by difference in the nucleotide sequence of the structural gene coding for proteins of identical biological function.

The term "biologically active sequence analogue" includes nonnaturally occurring analogues having single or multiple amino

I

WO 94/28019 PCT/SE94/00483 acid substitutions, deletions, additions, or replacements. All such allelic variations, modifications, and analogues resulting in derivatives which retain one or more of the native biologically active properties are included within the scope of this invention.

In this application, nucleotides are indicated by their bases ising the following standard one-letter abbreviations: Juanine G Adenine A Thymine T Cytosine C Unknown N In this application, amino acid residues are indicated using the following standard one-letter abbreviations: Alanine A Cysteine C Aspartic Acid D Glutamic Acid E Phenylalanine F Glycine G Histidine H Isoleucine I Lys.i4e K Leucine L Methionine M Asparagine N Proline P Glutamine Q Arginine R Serine S Threonine T Valine V Tryptophan W Tyrosine Y Unknown X

~II

WO 94/28019 PCT/SE94/00483 6 The term "amino acid" as used herein is meant to denote the above recited natural amino acids and functional equivalents thereof.

This invention provides an isolated nucleic acid molecule encoding the calcium sensor protein and having a coding sequence comprising the sequence shown in Fig. 3 (SEQ ID No. 3).

Furthermore, this invention provides a vector comprising an isolated nucleic acid molecule encoding the calcium sensor protein.

Moreover, thG invention provides a method of preparing calcium sensor protein which comprises inserting a nuleic acid encoding calcium sensor in a suitable vector, inserting the resuting vector in a suitable host cell, recovering the calcium sensor protein produced by the resulting cell, and puryfying the calcium sensor protein so recovered. This method for prepararing calcium sensor protein uses recombinant DNA technology methods which are well known in the art.

The present invention also provides antisense nucleic acids which can be used to down regulate or block the expression of the calcium sensor protein either in vitro, ex vivo or in vivo. The down regulation of gene expression can be made at both translational or transcriptional levels. Antisense nucleic acids of the invention are more preferentially RNA fragments capable of specifically hybridizing with all or part of the sequence iEQ ID No. 3 or the corresponding messenger RNA. These antisense can be synthetic oligonucleotides prepared based on the sequence SEQ ID No. 3, optionally modified to improve their stability of selectivity, as disclosed for instance in EP 92574. They can also be DNA sequences whose expression in the cell produces RNA complementary to all or part of the calcium sensor protein mRNA.

These antisenses can be prepared expression of all or part of the sequence SEQ ID No. 3 in the opposite orientation (EP 140 308).

WO 94/28019 PCT/SE94/00483 7 Material and Methods Tissue specimens. Samples of human parathyroid glands were obtained at surgery of patients with primary HPT. Other human tissue specimens (kidney, epididymis, liver, pancreas, adrenal gland, small gut, spleen, lung and striated muscle) were sampled from organs removed at surgery. Human placental tissue was collected in conjunction with uncomplicated pregnancies at full term. All specimens were immediately quick-frozen in isopentane and stored at -70 0

C.

Isolation of the calcium sensor protein from human placenta. The 500 kDa calcium sensor protein was isolated and purified, from altogether 25 human placentas, by immunosorbent and ion exchange chromatographies, following a previously described protocol The procedure utilizes two different monoional antiparathyroid antibodies Ell and Gil, known to bind different epitopes of the calcium sensing protein; Ell has displayed no functional effect, while Gil efficiently blocks calcium regulation in both parathyroid and placental cells After purification, the calcium sensor protein preparation was subjected to gel chromatography on a Zorbax GF25 gel column (9.2 x 250 mm), prior to enzymatic digestion.

The biologically active calcium sensor protein of the present invention has been isolated as described. It can also be preparared by chemical synthesis of in a recombinant DNA biosystem. Biologically active fragments of the calcium sensor protein can also be prepared using synthetic or recombinant technologies which are known in the art.

Cleavage and sequence determination of isolated peptides.

Cleavage of the 500 kDa protein with endoprotease Lys C from Achromobacter lyticus generated peptides, which were subjected to separation on a Brownlee microbore C 4 column (2.2 x 30 mm), equilibrated in 5% acetonitrile in 0.02% trifluoroacetic acid.

A linear gradient of 5 to 60% acetonitrile in 0.02% trifluoroacetic acid was employed for peptide alution, monitored at 214 nm WO 94/28019 PCT/SE94/00483 8 using a Waters 990 diod-array detector (Millipore Corporation, Millford, Mass). Amino terminal sequences of the peptides were determined in an ABI 470A gas-phase sequenator, equipped with an ABI 120A PTH-amino acid chromatograph (Applied Biosystems, Foster City, Ca., USA).

Oligonucleotide synthesis. Oligonucleotides were synthesized using an ABI 381 oligonucleotide synthesizer (Applied Biosystems). The following oligonucleotide mixture was utilized as a probe for screening of the placental cDNA library: CCA ATA IAG CTG ATC CTC AAA GAT ATC IAG IGA ATA IGG ATT CAT IGC G G G G G G (SEQ ID No, 8) The following two oligonucleotides were synthesized for use in PCR reactions: GCG GAATTC GTA ATG CAA CCA GAC GG C G C T G G T T (SEQ ID No. 9) ATAGGAATC CTG ATC CTC AAA AAT ATC G T G G G

T

(SEQ ID No. The first nine nucleotides contain an EcoR I .and a BamH I site, respectively, and the remaining nucleotides correspond to amino acid residues 1 to 6 of peptide 293 and to residues 9 to 14 of peptide 292.

Screening of a placental cDNA library with a mixed oligonucleotide probe. A placental X gt 11 cDNA library (Clontech, Ca., USA) was plated out to a density of approximately 2 x 10 s plaques within a 20x25 cm agar plate. Replicate filters (Hybond-N+, Amersham) of ten plates were prehybridized in 5 x SSPE (SSPE; 120 mM NaC1, 8 mM NaH 2

PO

4 0.8 mM EDTA, pH 5 x Denhart's solution (12), SDS, 20pg/ml single stranded salmon sperm DNA (Sigma Chemical WO 94/28019 PCT/SE94/00483 9 Co., S:t Louis, Ohio). The mixed oligonucleotide probe, endlabeled with y-[ 32 P]-ATP and polynucleotide kinase (Amersham), was added to the hybridization mixture (30 x 106 cpm in 50 ml), and hybridization was carried out over night at 420 C. The filter was washed twice in 2 x SSPE and once in 0.1 x SSPE, exposed to an autoradiography screen and analysed by a phosphorimager (Molecular Dynamics, Image Count S.W, Sun Valley Ca).

PCR reaction. Part of the X gt 11 cDNA clone CAS-1 was amplified by PCR using two degenerated probes corresponding to portions of peptides 292 and 293. The following conditions were used: 170 ng template DNA, 1 pmol of each oligonucleotide mixture as primers, dNTP 3mM, Taq-polymerase 0.75 u. The reaction was carried out in pl of l0mM Tris-HC1, pH 8.0, 1.5 mM MgCl 2 50mM KC1 in a Perkin-Elmer 9600 PCR-machine (Perkin-Elmer, Norwalk, USA). Two cycles of denaturation at 940 C for 2 min, annealing at 470 C for 1 min and extension at 720 C for 1 min 30 sec were followed by 33 cycles of 940 C for 1 min, 540 C for 45 sec, 720 C for 1 min and a final extension at 720 C for 10 min.

Screening of a placental cDNA library with a PCR-fragment as probe. A placental I ZAP-II cDNA library, was screened with the PCR-fragment from the cDNA clone CAS-1 labeled by random priming as the probe. The screening was carried out as above. 2 x 106 plaques distributed on ten 20 x 25 cm agar plates were screened.

Nucleotide sequence determination. The insert of the phage clone CAS-2 was released from the phage vector in the Bluescript+ vector using a helper phage (Stratagene, La Jolla, Nucleotide sequence reactions were carried out according to the cycle sequencing procedure, utilizing a kit from Applied Biosystems.

Sequences were analyzed in an ABI 373 A DNA sequenator using the Data Collection Program VIII soft-ware (Applied Biosystems).

Data-base search. The EMBL-31 data-base in the Intelligenetics format (Intelligenetics Rel.5.4), was searched for sequence WO 94/28019 PCT/SE94/00483 similarities to the placental cDNA sequence using the FAST DB algorithm (13).

Immunostaining and Northern blot. Immunohistochemical studies were performed on acetone-fixed, 6 pm thick frozen sections, utilizing the monoclonal antiparathyroid antibodies Ell and Gil, at concentrAtions of 5 pg/ml, together with a mouse peroxidase antiperoxidese technique on human placental, parathyroid, kidney, and epididymis specimens as well as on the other human tissues see above Monoclonal antibodies to collagen-type II were used as negative controls (14).

Total RNA was extracted from tissue samples by the acid phenol/chloroform method. For Northern blot analysis approximately 10 pg of total RNA was electrophoresed in a 1.5%/37% agarose/formaldehyde gel, blotted onto nylon membranes (Qiabrane, Diagen GmbH, DUsseldorf, Germany) and probed with the 2.3 kb clone (see results) labeled by the random priming method. Hybridizations were performed at 42 0 C for 18-24 h in 50% formamide, 4 x saline sodium citrate (SSC; 300 mM NaCl, 30 mM Na-citrate, pH 2 x Denhart's solution, 10% dextran sulfate (Rabi-Pharmacia, Uppsala, Sweden) and 100 pg/ml salmon sperm DNA. Filters were washed at a final stringency of 1 x SSC/0.1% SDS for 30 min at 42 0 C, and exposed within a phosphorimager as above.

RESULTS

Isolation of the calcium sensor protein, peptide cleavage and sequence determination.

The calcium sensor protein was purified from placental tissue by means of lectin chromatography, immunosorbent chromatography utilizing the immobilized monoclonal anti-parathyroid antibodies, and finally ion exchange chromatography The same antibodies were used in a sandwich ELISA to monitor the purification In order to avoid contamination with low molecular peptides, the whole final preparation, consisting of 200 pg of the 500 kDa protein chain was made 6 M with regard to guanidine-HC1 and applied to a gel chromatography column, equilibrated with 2 M WO 94/28019 PCT/SE94/00483 11 guanidine-HCl, 0.1 M Tris-Cl, pH 8.5. The column was eluted with the same buffer. Virtually all protein material emerged close to the void volume at the expected position for a protein with a molecular mass of 500 kDa. Separate fractions containing this material were combined and endoproteinase Lys C (1 pg) was added.

The i~ ion was allowed to proceed over night at 37 0 C. The fr!, protein was reduced by incubation with 0.1% 8-merc ,t 1 l at 37 0 C for 30 min and subsequently alkylated with 4- .X,,dine at room temperature for 2 h. The peptide m.t-ras Was then applied to a reversed phase C 4 column equilibrated in 5% acetonitrile in 0.2% trifluoroacetic acid. Peptides were eluted by a linear gradient of 5 60% acetonitrile in 0.02% trifluoracetic acid (Fig Due to the large number of peptides, the elution pattern was >omplex. Several peptide fractions were sequenced in a gas phase sequenator and easily interpretable sequences were obtained for two fractions (Fig 2, SEQ ID Nos. 1 and 2).

Isolation of a cDNA clone encoding the 500 kDa calcium sensor.

An oligonucleotide mixture (48 bp) was constructed to encode amino acid residues 2 to 17 of the sequenced peptide 292. To reduce the complexity of the oligonucleotide mixture, five inosine bases were inserted at degenerated positions where no guidance could be obtained from the codon usage in humans. At nine positions, where two bases were possible, one of the bases was suggested with a likelihood exceeding 70% from codon usage, and was therefore used in the oligonucleotide mixture.

The mixed oligonucleotide was radioactively labelled and used as a probe to screen a human placental 1 gt 11 cDNA library.

Approximately 2 x 106 plaques were screened and a single positive clone, CAS-1, was found. The insert of this clone was estimated to 2.3 kb, by restriction mapping. To obtain a recognizable sequence of the clone in a rapid way, an attempt was made to PCR amplify part of the sequence using degenerated oliogonucleotides corresponding to part of peptides 292 and 293 as primers. A distinct DNA fragment of approximately 430 bp was obtained WO 94/28019 PCT/SE94/00483 12 assuming that the peptide 292 is located carboxy-terminal to peptide 293. The fragment was partially, sequenced using the oligonucleotide mixture corresponding to peptide 293 as the primer. In one reading frame from the obtained sequence, the sequence VGRHI could be deduced, in excellent agreement with the carboxyterminal 5 amino residues of peptide 293. To obtain a clone with a larger insert a human placental 1 ZAP-II cDNA library reported to contain clones with large inserts was screened with the PCR-fragment as the probe. From 2 x 106 plaques a single clone, CAS-2, was found. The insert of this clone, estimated to 2.8 kb, was released in the Bluescript vector, using a helper phage. Part of the insert of this clone, pCAS-2, was partially sequenced using synthetic oligonucleotides as primers (Fig 3, SEQ ID No. An open reading frame was found containing both peptide 292 and 293. There was perfect agreement between the peptide sequences and the predicted amino acid sequence (SEQ ID No. 4) from the cDNA clone.

The 500 kDa placental calcium sensor belongs to the LDL-receptor superfamily.

A search in a data-base with the available predicted amino acid sequence of the placental 500 kDa protein cDNA revealed that this protein was homologous to receptors belonging to the LDL-receptor superfamily. The highest similarity was found with the rat Heymann nephritis antigen Fig 4 shows an alignment of the available placental 500 kDa protein sequence to the sequence of the Heymann antigen (SEQ ID No. 5) as well as to two other members of the same protein superfamily, the LDL-receptor (SEQ ID No. 6) and the LDLreceptor-related protein (identical to the a 2 -macroglobulin receptor, 11,15,16, SEQ ID No. The sequence identity between the placental calcium-sensor and the Heymann antigen was estimated to 82% in the region available for comparison (236 amino acid residues), which is high, considering the fact that the two proteins are derived from different species.

WO 94/28019 PCT/SE94/00483 13 Immunohistochemistry and Northern blot.

The close similarity between the placental 500 kDa calcium-sensor chain and the rat Heymann nephritis antigen prompted the expanded immunohistochemical investigation of the present study. The antiparathyroid antibodies (Ell and Gil) were found to stain not only parathyroid, placental and proximal kidney tubule cells but also epididymal cells, as previously demonstrated for antibodies reactive with the Heymann antigen (17-20).

Northern blot analysis of total RNA (approximately 10 pg/lane) from human kidney, placenta and parathyroid glands with the identified 2.8 kb clone as the probe, revealed one major hybridizing RNA species of approximately 15 000 bases in all these tissues (Fig Human liver, pancreas, adrenal gland, and small gut (Fig 5) as well as spleen, lung and striated muscle (not shown) lacked hybridizing species.

Discussion The important role of the parathyroid as key regulator of the calcium homeostasis has been related to its exquisite capacity to sense and respond to variation in the extracellular Ca 2 ion concentration. Essential for recognition of changes in external calcium is a cation receptor or sensor of the parathyroid cell membrane, the presence of which was implicated by a series of in vitro studies on parathyroid cell regulation (9,10,21-24). The concept of a cell membrane receptor was further, substantiated when monoclonal antiparathyroid antibodies were found to recognize and interfere with the calcium sensing of parathyroid cells Another crucial piece of evidence was obtained when cytotrophoblast cells of the human placenta, selected by their reactivity with the antiparathyroid antibodies, displayed parathyroid-like sensing of changes in external calcium, a function which also could be blocked by one of the anti-parathyroid antibodies The calcium sensor of the placenta was subsequently isolated by immunosorbent and ion exchange chromatographies and shown to consist of a large glycoprotein of approximately 500 kDa molecular size It was also demonstrated by WO 94/28019 PCT/SE94/00483 14 immunoprecipitation that a protein of the same size reacted with the antiparathyroid antibodies within the parathyroid and kidney tubule cells (to be The parathyroid calcium sensor or receptor is known to have features in common with most other classical receptors for cellular activation, although it exhibits the unusual ability to bind and be activated by divalent cations. Cation binding triggers biphasic rise in [Cal 2 i] and concomittant activation of phospholipase C, possibly via a coupled G-protein, with a resulting accumulation of inositol phosphates An initial transient rise in [Ca"i] is due to inositoltrisphosphate (Ip 3)induced mobilization of Ca 2 from intracellular sources, while an ensuing steady-state elevation in [Ca 2 is caused by calcium gating through plasma membrane channels, possibly mediated by increase in inositol-tetraphosphate (Ip4) (9,10,23).

Sequence analysis of the obtained partial cDNA clone and data-base comparison of the deduced amino acid sequence showed that the placental calcium sensor protein belongs to the LDL-receptor superfamily of proteins, and available sequences showed close similarity with the rat Heymann nephritis antigen (11,15,16). This antigen was originally described in the rat as a 330 kDa glycoprotein (gp 330), present within the proximal kidney tubule brush border, and in placental and epididymal cells, but by special stai.ning techniques also demonstrated to occur sparsely on rat kidney glomerular cells, as well as on pneumocytes II in the lung and sporadic cells of the liver and small intestine (17-19). It has later been proposed that the molecular size of the protein was underestimated and actually should be in the range of 500 kDa The Heymann antigen has been revealed as the dominating antigen causing membranous, autoimmune glomerulonephritis in the rat after immunization with a crude tubular protein fraction (17,19). Using anti-gp 330 antibodies a protein with an estimated molecular size larger than 400 kDa has been identified in man The sequence similarity between the presently available portions of the human placental 500 kDa calcium sensor protein and 0 WO 94/28019 PCT/SE94/00483 the rat Heymann nephritis antigen, with 82% conformity over 236 amino acid residues, indicates that it may actually represent related forms of the calcium sensor protein in two different species. This view is supported by close similarities in tissue distribution of the two proteins, as revealed by the immunohistochemistry of the present study. The antibodies Ell and G11, reacting with the calcium sensor protein, thus stain parathyroid cells, proximal kidney tubule cells, placental cytotrophoblasts and also epididymal cells. Furthermore, we have recently reported staining with one of the antiparathyroid antibodies preferentially within coated pits and the base of the proximal tubule microvilli, which equals that previously described with antibodies against the gp 330 protein (19,26). A recognized glycoprotein of similar size within the tubule brush border, renal maltase, has been located mainly to microvillar membranes and not within the coated invaginations (18).

Thus far recognized members of the LDL-receptor superfamily, theQ) LDL- receptor, the LDL-receptor-related protein and the Heymann antigen, have been thought to function as receptors for proteins, but all exhibit functionally important Ca 2 ,-binding ability (16,27,28). Thus, the Ca 2 binding is necessary for the interaction of the LDL-receptor withapp-B 27). The LDL-receptor related protein (a,-macroglobulin receptor) is also known to bind Ca 2 which induces conformational changes, and Ca 2 is necessary for binding of activated a 2 -macroglobulin to the receptor (16).

Recently, the rat Heymann antigen was shown by a blotting tecnnique to interact with Ca 2 (28).

The Ca 2 binding motifs of the calcium sensor protein yet remain to be identified. The sensor protein (as well as the Heymann antigen) contains EGF-like modules, like other members of the LDLreceptor superfamily (11,16,27), which may represent putative Ca 2 binding sites. Thus, when present in the coagulation factors IX, X and protein C, each EGF-like module is known to bind one Calion (29-34), and the EGF-like modules have also been demonstrated to mediate Ca 2 "dependent protein/protein interaction Kinetic WO 94/28019 PCT/SE94/00483 16 data have suggested that the calcium sensor displays positive cooperativity in its interaction with Ca 2 a phenomenon which appears essential for the sigmoidal regulation of [Ca 2 i] and PTH release, with a steep relation within the physiological range of extracellular calcium The positive cooperativity should require multiple binding sites for Ca possibly resulting from the repetitive EGF-like modules, generally present in molecules of the LDL-receptor superfamily (11,16,27). However, Ca 2 binding to EGF-like domains are known to induce only minor, localized pertubations of the three-dimensional structure and it is possible that the calcium sensor contains also other Ca 2 l binding sites.

A 43 kDa membrane protein (ca-macroglobulin receptor-associated protein, or Heparin-binding protein) (28,36) is known to interact both with the LDL-receptor-related protein and with the rat Heymann antigen in a Ca 2 "dependent manner No physiological function has yet been assigned to this protein, but it appears also in tissues where the Heymann antigen and the LDL-receptorrelated proteins are not expressed An intriguing observation is the presence of a putative leucine-zipper motif in the aminoterminal part of the 43 kDa protein considering that such motifs have been suggested to influence the opening and closure of membrane ion channels Since the 43 kDa protein interacts with the Heymann antigen, it can be assumed to form a complex also with the calcium sensor protein in a Ca 2 dependent manner. Interaction with the 43 kDa protein might be important for the transmission of Ca 2 induced conformational changes within the extracellular portion of the molecule to the cell interior. It is also possible that additional proteins interact with the calcium sensor in a Ca 2 dependent manner, and that such an interaction is important for the modulation of the sensor response. The mechanisms by which an activated calcium sensor triggers further signalling to the cell interior is unknown, although we have in preliminary experiments utilized immunoprecipitation to isolate a phosphorylated form of the sensor protein in dispersed parathy- WO 94j28019 PCT/SE94/00483 17 roid cells loaded with 32 P]-ortophosphate (unpublished observation).

The calcium sensor protein of the placenta may be involved in maintenance of a feto-maternal Ca 2 gradient and placental Ca2 transport, possibly by mediating calcium regulation of the parathyroid hormone related peptide (PTHrP) production and/or 1,25

(OH)

2

D

3 metabolism Its presence already within the blastocyst (unpublished observation) may indicate a function also as adhesion molecule, or implicate involvement in differentiation or growth regulation, as suggested for the Heymann antigen The function of a calcium sensor within the kidney tubule brush border is less well explored. However, it should be noted that the enzyme 1-a-hydroxylase present in the placenta and proximal kidney tubule, is regulated by extracellular calcium, and the calcium sensor might accordingly regulate 1,25 (OH) 2

D

3 metabolism, but it may possibly also influence Ca 2 reabsorption from the glomerular filtrate The significance of the presence of the calcium sensor protein on epididymal cells, as well as rat pneumocytes, liver and intestinal cells as implicated by the distribution of the Heymann antigen (18,19), yet remains unknown. It has, however, been proposed that several cell types may exhibit Ca 2 sensing ability for regulation of various functions, separate from the general calcium homeostasis, either during development or In the differentiated state The association with autoimmune nephritis substantiates that the Heymann antigen is an immunogen molecule. This may have implication also in parathyroid disorder, as we have recently reported the presence of circulating parathyroid autoantibodies and induction of class II transplantation antigen in the pathological parathyroid tissue of patients with primary HPT. These findings suggested that autoimmune phenomena may be involved in HPT (39) and autoimmunity has also been implicated in the pathogenesis of rare idiopathic hypoparathyroidisi The availability of a cDNA clone for the calcium sensor should, enable extended studies on the pathophysiology in parathyroid disorder, and also in- WO 94/28019 PCTISE94/00483 18 vestigation of a possible genetic abberration affecting the calcium sensing function of the parathyroid and kidney tubule in kindreds with familial hypocalciuric hypercalcemia (FHH) (40,41).

The skilled person within this art realizes that the information obtainable from the nucleotide sequence of Fig. 3 can be used for isolating the complete DNA ard gene sequence encoding the calcium sensor. Preferably, an analysis of overlapping cDNA clones in conjunction with PCR techniques is used. The gene sequence can be obtained from the analysis of overlapping genomic cosmid and/or lambda phage clones.

WO 94/28019 PCTISE94/00483 19 SEQUENCE LIS7ING GENERAL INFORMATION; APPLICAN: Akerstrom, Gran Klareskog, Lars Juhlin, Claes Rask, Lars (ii) TITLE OF INNTIOI: Human Calcium Sensor (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Dr. Ludwig Brann Patentbyra Ab STREET: P.O. Box 1344, Drottninggatan 7 CITY, S-751 43 Uppoala COUNTRY: Sweden COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-IOS/MS-DOS SOFTWARE: Patentln Release i1.0, Version 41.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: PILIMG DATE:

CLASSIFICAT'ION:

(viii) A'ITORNEY/AGNT INFORMATION: NAME: Aldenback, Ulla (ix) TELECOMM2UNXICATION INFORMATIFINt TELEPHONE: 46 18 13 96 TELEFAX; 46 18 10 93 22 INFORMATION FOR SEQ ID NO;1: C i) SEQUENCE CHA)CTERISTICSs LENGTH: 17 amino acids TYPE: amino acid TQPOLOCY% linear (ii) MOLECULE TYPE, peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:lt Xaa Ala Hot Asn Pro Tyr Sor Lou Asp no~ Phe Glu Asp Gin Leu Tyr 1 5 R is Trp WO 94/28019 PCT/SE94/00483 INFORMATION FOR SEQ ID N012: SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids TYPE% amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Xaa Val Met Gin Pro Asp Gly Ile 1 0 Ala Xaa Asp Trp Val INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 804 base pairs TYPE: nucloic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYP4; cDNA (ix) FEA7R=: NAME/KEY: CDS LOCATIONi 1..804 (ad) SEQUENCE DESCRIPTION: SEQ ID NO:3t AM TAC OTA ATO CAG CCh GAT OA Lye Tyr Val Not Cln Pro Asp Gly 1 5 ATA OCA GTG Ile Ala Val 10 GAC TCG CT Asp Trp Val GOA AGO Gly Arg CAT AT? TAC His le Tyr CTT CAT COA Lou Asp G1l TCA GAT GTC AAG Ser Asp V4l Lys AAA CGC AT GAG Lys Arg Ile Olu CTh3 GT AAA Val Ala Lyn CT13GAC CAA Lou Asp Gin AGO TAC AGA Arg Tyr Arg MGW TOO L~ys Trp MAT CCC Ann Pro 55 CTG ATT TCC ACT Leu Ile Ser Thr CCA GCT Pro Ala GCT AT? GCT GTC Ala lie Ala Val AAA CTA COG CT? ATO TIC TOO ACT Lys Lu Gly LOu Met Pho Trp Thr

CAC

Agp 655 T'CC GGA AAC CA Trp Cly Lys Clu AAA ATC GAG TCT LIPS lie luI Sor CCC GGC Ala Trp A'TC AAT OGA GAO Het Asn Gly Clu GAC CCC AAC ATO CTG OTr 'PlC OAG Asp Arg Asn lie Lou Val Phe Glu GAO CTI Asp Lu COT TOO CCA G1y rp Pro ACT CCC OTT Thr Gly Lou WO 94/28019 WO 9428019PCTISE94OO483 'PCT ATC GAT Ser Ile Asp GAG GAO orr Glu Asp Val 115 TIC AMC MAT GAC LeU Asn Asn Asp ATC TAO TIGG Ile Tyr Trp ATT GAA ACC ATA 110 Glu Thr Ile TAT GhT ACT Tyr Asp Gly Thr AGT GAO TrC MAG Seir Asp Phe Lys GAT AGO ACA OTC Asp Axg Arg Val 125 ATC Tr GMA GAC Ile Phe GIu Asp ATT GCA Ile Ala 130 MAG GMA GCA A'IG MAC COT TAC AGC CMO Lys GIlU Alm Met Agn Pro Tyr $or Lou 135 CAG 71A TAO TOG Gin Lau Tlyr Trp 145 AAA TT1T GOG CMA Lys Phe Gly Gin ATA TOT lie 5cr 150 MAG GJ AAG wA Lys O1u Lys Gly OT.A TGO "NA CMA Va2 Trp Lys Gin MAG AMA GAG Lys Lys Ciii AA ACC Lys Thr 170 OTO OTA G2$ MAC Lou Val Val Asn CCT T GG Pro Trp 3.75 O'IC ACT CAA Lou Thr Gin CCC MAC CL 1 Pro Asn Lau 195 OA ATC TTT OAT Arg Ile Pho His C'TC AGA TIAO AAT Lau Arg Tyr Aen MAG TCA GYC, Ly~s Ser Val 190 CTG AGA CC'T Lou Axg Pro AAA CAG ATO Cys Lys Gin Ile AGC CAC C7O TGO Se' is Lou Cys OA GOA, dly Qlv 21.0 TAC AGC 11141 OCC ITOT Tyr gar Cya Ala 'Cya 215S CCC CMA GGC TCC Pro Oln Cly Ser MtI' ATA GAG CCC Pho Ile Glu Gly ACC ACT GAG Thr Thr Glu TMT OAT Cys Asp 230 GCA GCC ATC CMA Ala Ala Ile Ciii OCT ATC MAC CTG Pro le Asn LOU CCC COA TOO AGG Pro Pro Cya Arg TOC ATG CAC OA GCA AT TOO CyS a Iol ial Gly alY Ann Cys 24S 250 TAT TdITl GAT 'ryr Phe Asp CAG ACT Glii Thr 255 GAO CTC' CCC hoep Lou Pro TGO MAG TOT OCT Cyt3 Lys Cys Pro 14CC GO TAO ACC Sex? Oly Tryr Thr 265 rNPORMATION Ptig SEQ XD NOi4t SEQUENCE CHARACTERISTICS! LENGrHf: 268 amino acid6 TYPZ: amino acid TOPOLOGYt l.inear (ii) KOLEOULt TYPEt Protein (XI) SnQ0ENCE DESCRIPTION: SFrQ ID N0:4! Lyg Tyr Val Net Gin Pro Asp Gly Ile "la Val. Asp iTzP I S 10 Val Gly Arg is Ills lie 'Tyr Ttp Stir hSP Val Ly6 AMr LYa Artg Ile Ciii Val k1a Lyo 25 WO 94/28019 PCTISE94I0483 Lau Asp Gly Arg Tyr Axg Lys Trp 40 Leu le Ser 'hr Asp Lou Asp Gin Asp Arg Asn Ile Leu V Ser Ile Glu Asp Tie Ala 130 Gin Lou 145 Lys Phe Lei Thr Pro Asn Gay Gly 210 Ser Thr 225 Pro Pro ha Lou Tyr 100 Ile Glu Trp Gin Val 180 Cys lu Arg Lyn 260 Leu Giu Ala Ile Gly 165 Arg Lye Cy0 Cys Cys 245 Cyn

S

1

I

2 fal Aim ?ro Lys a I Phe snf Asn 1hr Ile Ot Asn 135 er Lye 50 ys Lys lo Ph.

in Ile la Cyn 215 sp Ala 30 Pro Ile Clu Asp Lys 120 Pro

GIU

Glu His Cy6 200 Pro Ala Lys Glu Anp Arg 105 Ty r Tyr Lys Lye Gln 185 Ser Gin lie Lou Ser Lou 90 I;te Asp Ser Gly Thr 170 LOu 14is dly Glu Ann 250 Gly Oly Lou Mot Phe Trp Thr Ala Trp Met hsn Gly Glu 75 Gly Trp Pro rhr Cly Lou Tyr Trp Ser Asp Ph Lys 110 31y Thx Azp Arg Arg Val 125 Lou Asp lie Phe Clu Asp 140 3lu Val Tp Lys Gln Asn 155 160 eu Val Vai Avn Pro Trp 175 krg Tyr Asn Lys Ser Val 190 Aju Cys Lou Liu Arg Pro 205 ;ar Sar Pho Ila Clu Gly 220 .ou Pro le Asn Leu Pro 135 240 ,ya Tyr Pho hop Glu Thr 255 Gly dly Pro Sor 265 INFORMATION FOR SEO ID N015: SEQUENCE CIIAIfCTERISTICS I LE11GT11: 269 amino acids TYPE: amino acid TOPOLOOY: linear (ii) MOLECULE WPEt protein (xi) SEQUENCE OESC1AIPTIONt SQ ID N*OtSt Xda X64 X&4 Xo.a Xaa Pro Asp Cly L.u iia Val Aap Trp Val Gly Ar 1 5 10 WO 94/28019 WO 9/28019PCT/SE94/00483 is ll1'.?

T

yr 'P1.p Ser Asp Ala Msn Ser Gin Atg le Ciu Val Ala Thr 25 Leu Pro Asp His S er G2u Ile Lyn 145 Asn Trp Xa Pro dly 225 ?o Asp Al a so Gin Arg Ile Asp Ile 130 Lou Lys Lou Xaa GlIy 210 So r Pro GilI Ala o ly Ser Asp ValI 115 Asrn Tyr Phe Thr Xaa 195 Gly Thr Pro Arg 110i Lys *Va I Tyr 200 Ile c1u Txp Oly Gin IS0 Xaa 'Ty r Val Cys Pro 260 Tr Ala Gin Lau Glu Ala Va I Lys Val1 Cys Ser Gin Ara 245 Lys 'Prp 40 Asn Pro Lyn 1ie Ser Glu Asp Asp Ile Lys 120 Lys Pro 135 Xaa Xaa Asn Lys Xaa Xaa r.in Val 200 Ala6 Cys 21i5 Kaa Xaa mot H~is Laeu Lys Ciu An Arg 105 Phe Xaa Giu 185 Cys Pro Giy Sor 265 le r.~eu Ser Leu 90 Val Asp Ser Xaa Lys 170 Xna.

Se r Gin Xaa Giy 250 Sor Thr Gly Ala 75 Gly 'Tyr G3Y Leu Xaa Val His Gly Xaa 235 An aly Thr Lieu Trp Trp 'rrp Thr Asp 140 Xaa Lou Xan Lou Se r 220 XaA Cy a Tyr Gin Met Met pro Ser Asp 12 Ile Xaa.

Val Xad Cys 205 Asp Pro Tyr Ser Lou Phe Msn Asn Asp 110 Arg Phe Kad Val Xaet 190 Lou Phe V)al Phe Asp Gin Trp Thr Cly (Oiu Gly Leu Ser Lys Arg Leu Giu Asp Arg Gin 160 Ann Pro 175 Xaa Xoo Lau Arg Val Thr Thr Mot 240 Asp Glu 255 Agn Glu Leu Lys Cys Lys Cys INFPORMATXON POfl SEQ XD N0161 SMQUENCE CHARACTERIMTCSt L=NTH* 280 amino acide TYPE: amino acid TOPOLOCY! linear (Ui) H=lCUUfl TYPE: protbin WO 94/28019 24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO;6: PCTSE94IOO483 Arg Asp Ile Can Ala Pro Asp Giy Leou Aa Val Asp Trp ie His Sex 1 5 10 Asn Thr Pro Asp Asp Th His Leo Glu 145 Ala Lou Gly Tyr Thr 225 Cy13 Val.

Ile Lys Arg Trp Ile Leo Ser Glu 130 Asp Aun Sor Val Lou 210 cys Arg Tyr Trp Gly Val Ala Ile Gly Thr Tyr Ser Asp Leu 100 le Ser 115 Asp Glu Lys Val Arg Lou Pro Glu 180 Asn Trp 195 Cys Lou Ala Cys Thr Glu Lou LyE 260 Thr Asp Lys Arg Val Val Pro Ala 70 Lou Val Lou Ser Ser Ile Lys Arg Phe Trp 150 Thr Gi! 165 Asp Mt cys Glu Pro Ala.

Pro Asp 230 Ala GLU 245 Val Ser Ser Lys Asp 55 Lys Thr Gly Asp Lou 135 Thr S3er Val Rrrg Pro 215

GIY

Ala Sor Vat Lou Gly Thr Val 25 Thr Lou Phe Arg Glu 40 Pro Val His Gly Phe Ile Lys Lys Oly Gly 75 Glu Aan Ile Gin Trp 90 Arg Leu TPyr Trp Val 105 Tyr Asn Gly Gly Ast 120 Ala His Pro Phw Ser 140 Asp Ile lie Asn Glu 155 Asp Vat Asr Leu Leo 170 Lou Phe Eis Asn Lou 185 Thr Thr Lou Sor Asn 200 Gin 1le Asn Pro His 220 Met Leo Lo Ala Ara 235 Ala Val Ala Thr Gln 250 Thr Ala Val Arg Thr 265 Ser 280 Ser Aet Met Leu Pro Asp Arg 125 Leu Ala Ala Thr Gly 205 Ser Asp clu Gln Val ly Tyr Asn Aen Ser 110 Lys laa Ile Glu Gin 190 Gly Pro met Thr His 270 Ala Ser Trp Gly Gly Lye Thr Val Phe Asn 175 Pro Cys Lys Arg Ser 255 rhr Asp Lys Thr val Ile Lou le Phe Ser 160 Lou Arg Gln Pho Ser 240 'Thr 'rhr Tht Arg Pro Val 275 Pro Asp Thr IITFORMATIO FOR SEQ ID NO:7: WO 94/28019 PCT/SE94100483 SEQUENCE CHARACTERISTICS: LENGTH: 281 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Gly Leu Ser Asn Pro Asp Gly Leu Ala Val Asp Trp Val Gly Gly is Asn Leu Leu Asn Pro Arg Asp Trp Set Arg Thr Leu Asp Tyr Leu Ser 130 Tyr Val 145 Lys Thr Pro Met Asn His Leu Ser 210 Leu Gly 225 Tyr Trp Giy Ala Ala Leu Gly Asp Ser Val Asp Tyr 100 Ile GI± 115 Gin Asp Tyr Trp Thr Gly Asp Lou 380 Pro Cys 195 Pro Oly Ser Asp 4 Cys Tyr His Ile Val' Phe Ile Thr Thr 165 His Lys Gly Gly Asp Arg Val Ser 70 Val Thr Ala Pro Asp 150 Asn Val Val Gly Arg 230 Lys Thr Asp 55 Lou Asp Glu Ser His 135 Trp Lys Phe Asn.

His 215 Thr cly Val 40 Val Tie Thr Arg Leu 120 Ile Glu Thr His Asn 200 Lys cys: Arg 25 Leu Gln Gly Lys Ii.

105 Asp Phe Thr Leu Ala 185 Gly Cys Val Asp Val Asn Arg Ile 90 Tyr cly Ala Lys Leu 170 Leu Gly 4 Ser Ile Ser jyr dly Trp Ala Asn Thr 140 Ile Ser Cln Ser Pro 220 Cyv Trp Glu Gly Leu Met Pro Asp Arg 125 Leu Asn Thr Pro Asn 205 Thr Thr Trp Val Leu Tyr Asp Asn Ala 110 His Phe Arg Leu Asp 190 Leu Asn Ala Lys Ser Arg Trp Gly Gly Arg Val Glu Ala His 175 Val Cys Phe Ser cys Lys Glu Thr Ser Leu Clu Val Asp His 160 Arg Pro Leu Tyr Gln 240 Asp Phe Val Cys Lys Asn Asp Lys Cys lie Pro Phe 245 250 Thr G].O Asp Asp Cys Gly Asp His Ser Asp Glu 260 265 Pro Pro Asp Cys Pro 270 WO 94/28019 WO 9428019PCT/SE94100483 26 GlU Phe Lys CyS Axg Pro Gly Gln Phe 275 280 INFORMATION FOR SEQ ID NOtS: SEQUENCE CHARACTERIST~tS: LENGTH. 48 base pairs TYPE: nucleic acid STRANDEDNEsS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acia (ix) FEATURE: NAMVEKEY modified-base LOCATION: 7 OTHER INFOPMATIONt /modj-ease= i (ix) FEATURE: NAME/KEY: modified~baise LOCATION: 28 OTHER INFORMATION.: /mod-base= i (ix) FEATUR2;- NAME/KEY: modified-base LOCATION: 31 OTHIERIn-FORMATION: /mod,.base= i (iX) FEATURE: NAME/KEY: xood~fied.base LOCATION: 37 OTRER INFORMAT'ION: /mod..basa= i (ixt) FEATURE% NAME/KEY: modified-.bace LOCATION: 46 OTUER INFORMATION: /modjbase= i (xi) SEQUENCE DESCRIPTION: SEQ XD NO: 0: CCARTANAGC TGRTCCICR AGATR'rCNAG NOARTANGGR TTCATNjC 4 INFORMATION FOR SEQ ID NO:-9.

SEQUENCE CHARACTERISTICS: LENGTH 26 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOOWi linear (ii) MOLECULE TYPE: other nucleic acid (xi) SEQUENCE DESCRIPTION: SEQ ID 00:9i GCGGAATrCG TNATGCJRCC NGAYGG 2 6 WO 94/28019 PCT/SE94100483 27 INFORMATION FOR SEQ ID NO:1O: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESSt single TOPOLOGY:, linear (ii) MOLECULE TYPE., other nucleic acid (xi) SEQUENCE DESCRIPTION; SEQ ID ATAGGAATCC TGRTCYTCRA ADATRTC 27 WO 94/28019 WO 9428019PCT/SE94100483 28 References 1.Juhlin, Holmdahl, Johansson, Rastad, Akerstrbm,.

Kiareskog, (1987) Proc. Natl. Acad. Sci. USA. 84, 2990- 2994.

2.Juhlin, Johansson, Holmdahl, Gylfe, Larsson, Rastad, Akerstr5m, Kiareskog, (1987) Biochem. IBiophys.

Res. Commun. 143, 570-574.

3.Juhlin, Kiareskog, Nygren, Gylfe, Ljurighall, S., Rastad, Akerstrbm, (1988) Endocrinol. 122, 2999-3001., 4.Juhlin, Akerstr~m, Klareskig, Gylfe, FPolmdahl, Johansson, Ljunghall, Larsson, Nygren, Rastad, (1988) World. J. Surg. 12, 552-558.

Juhlin, Akerstr6m, Kiareskog, Rask, L., Rastad, (1990) Cell Calcium. 11, 329-332.

6.Juhlin, Rastad, Kiareskog, Grinielius, Akerstrbm, (1989) Am. J. Pathol. 135, 321-328.

7.Juhlin, Lundgren, Johansson, Lorenzon, Rask, L., Larsson, Rastad, Akerstrzn, Kiareskog, (1990) J.

Biol. Chem. 265, 8275-8279.

8.Hellman, Ridefelt, Juhlin, Akerstrin, Rastad, Gylfe, (1992) Arch. Biochem. Biophys. 293, 174-180.

9.Akerstr~m, Rasltad, Ljunghall, Ridefelt, Juhlin, Gylfe, (1991) World. J. Surg. 15, 672-680.

l0.Brown, E. (1991) Phys. Rev. 71, 371-411.

l1.Raychowdury, Niles, J. McCluskey, R. Smith, J. A., (1989) Science. 244, 1163-1165.

12.Denhardt, (1966) Biochem. Biophys. Res. Commun. 23, 641- 646.

13.Pearson, W. Lipman, D. (1988) Proc. Natl. Acad. Sci.

USA. 85, 2444-2448.

14.Holmdahl, Rubin, Klareskog, Larsson, E.,I Wigzell, (1986) Arthritis. Rheum. 29, 400-410.

Davis, C. Brown, M. Schneider, W. J., Casey, M. L.,Goldstein, J. Russel, D. (1984) Cell. 39, 27- 38.

16.Herz, Haman, Rogne, Myklebost, Gausepohl, H., Stanley, K. K.,(1988) EMBO. J. 7, 4119-4127.

WO 94/28019 WO 9428019PCTISE94/00483 29 17.Chatelet, Brianti, Ronco, Roland, Verroust, (1986) Am. J. Pathol. 122, 500-511.

18.Chatelet, Brianti, Ronco, Roland, Verroust,. P~, (1986) Am. J. Pathol. 122, 512-519.

19.Kerjaschki, Farquhar M. (1984) in Nephrology ed Robinsson New York Springer-Verlag pp 560-574.

Horvat, Binder, Susani, Dekan, Ojha,' P. Hillermans, Ulrich, Doninn, (1987) Am.

J. Pathol. 129, 183-191.

21.Wallfelt, Larsson, Johansson, Rastad, Akerstr~ni, Ljunghall, Gylfe, (1985) Acta. Physiol. Scand.

124, 239-245.

22.Gylfe, Larsson, Johansson, Nygren, Rastad, J., Walifelt, Akerstr~m, G.,(1986) Febs. lett. 205, 132-136.

23.Nemeth, Scarpa, (1987) J. Biol. Chein. 262, 5188-5196.

24.Gylfe, Akerstrin, Juhlin, Klareskog, Rastad, (1990) In: Hormones and Cell Regulation. Eds: Dumnont, J.E., Nunex, King, John Libhey Eurotext Ltd, London pp 5-15.

Juhlin, Rastad, Kiareskog, Akerstrin, ,,Rask, Submitted.

26.Bjez-1'neroth, Juhlin, Akersttbm, Rastad, (1992) J. Submicrosc.Cytol. Pathol. 24, 179-186.

27.Brown, M. Goldstein, J. L (1986) Science. 232, 34-47.

28.Christensen, E. Glieinan, Moestrup, S. (1992) J.

Histochem. Cytochem.40, '1481-1490O.

29.Handford, P. Baron, Mayhew, Willis, Beasly, T., Brownlee, G. Campbell, 1. (1990) EMBO J. 9, 475-480.

L. Ke, Sweeny, Tam, 1. (1989) Biochem.

Biophys. Res. Commun. 160, 133-139.

31.Persson, Selander, Linse, Drakenberg, 6hlin, A. Stenflo,J., (1989) J. Biol. Chem. 264, 16897-16904.

32.6hlin, A. Linse, S.0 Stenflo, (1988) J. 6iol. Chemn.

263, 7411-7417.Urukawa, 33.6hlin, A. Landes, Bourdan, Oppenheimer, Wydro, Stenflo, J., (1988) 3. bio1. Chaem. 263, 19240-19248.

WO 94/28019 WO 9428019PCT/SE94/00483 34.Selander Sunnerhagen, Uliner, Persson, Teleman, Stenflo, Drakenberg, (1992) J. Biol. Chem. 267, 19642- 19649.

Fleming, R. Felion, R, Cherbas, Cherbas, Artavanis -Tsakonas, (1991) Cell. 67, 687-699.

36.Furukawa,T., Ozawa, Hvang, R. Muraznatsu, (1990) J.

Biochem. 108, 297-302.

37.McCormack, Campanelli, 1. Ramaswami, Mathew M. K., Tanoye, M. Iverson, Rudy, (1989) Nature. 340, 103.

38.Mendrick, D. Chung, D. Remcke, H. (1990) Exp. Cell.

Research. 188, 23-25.

39.Bjerneroth, (1992) Comprehensive summaries of Uppsala Disertations from the Faculty of Medicine 360, ISBN. 91-54-2928-9.

Attie, M. Levine, M. Spiegel, A. Downs, R. Lasker, R. (1981) Medicine 60, 397-~412.

41.Choo, Y-H. Brown, E. Levi, Crowe, G, Atkinson/ A. Arnqvist, H. Toss, Fuleihan, G, Seidman, J. X Seiap~fin, C. (1992) Nature Genetics. 1, 298-300.

Claims (3)

1. A cDNA clone encoding the hum~an calcium sensor, comprising the following nucleotiLde sequence AAA TAC TAC AAT GAG GGT AGT AGA CAG GGG CGA ATC CMA CTG TAC TGG AGA CCC TCT TOO GAC GTC TTA CAA ATC TGC GGC CCT GTA TCA AArO AMA GCC CCA TTC ATT TAC GGA AGC TCC ATC ATG GAT TGG CTA TGG ACT MAG GCA TG MAG CAT CAC AGC MAC CAG GTC CTG 0GG ATO GGC GAG MAG ATA AMA CAA CTC TTT CTG CCA MOG ATT CTT MAT CTT GAC GMA TCT GAG CTC TOO CCC GAT MAT TC ATG GGA TCT OTT GCA M.G AMA AGA CTT GAG CCC GGA A ACT TTC GAG ATC ATT ATG GMA ACG TAC CTO GGG CCA 27 ATA GCA 81 CGC ATT 135 GAC CTG 189 TOG ACT 243 GAC CGC 297 GAT TAT 351 GMA ACC 405 MAC CCT

4.59 MG GGA 513 CTG GTA 567 MAT MAG 621~ AGA COT 675 AGC ACC 729 TOO AGO GTG GAG GAC GAC MAC TTG ATA TAO GMA OTG TCA GGA ACT TGC GAC GTG CMA TGG ATC MAC AMA AGC GTA MAC GTG GGA GAG ATG TOG OTT OGA OCT AAA CTT CCA OCT OCT OGA CTG MAT TAT rTG TOG CCT CCC TAC TOT CAC MOG OTT GAC OAT GAO MA TOG MAC AOC OAT GGA GMA TTC COA 000 ATC CMA CTC OTT TOT GCA GGA AGO OAT ATT CT GAO ATC TTT MAT ACT TOC 0CC 0CC AAT 54 CAT ATT 108 OGA AGO 162 OCT OTO 216 AMA ATO 270 GAC CTT 324 TAC TG 378 OAT AGO 432 GMA GAO 486 MAA TTT 540 CMA OTT 594 AAA CAG 648 TOT COO 702 ATC GMA 756 TOO TAT 783 TTT OAT GAG ACT GAC OTC CCC AMA TGC MAG TOT COT AGO 000 TAC ACC WO 94/28019 PCT/SE94/00483 32 2. A calcium sensor protein comprising an amino acid sequence deduced from the nucleotide sequence according to claim 1. 3. Use of the nucleotide sequence according to claim 1 for the isolation of the complete nucleotide sequence of the calcium sensor. 4. Use of the nucleotide sequence according to claim 1 and/or the calcium sensor protein according to claim 2 to develep agonists and/or antagonists to calcium and other cations binding to said human calcium sensor. Use of the nucleotide sequence according to claim 1 and/or the calcium sensor protein according to claim 2 to develop agents interfering with other functions of said calcium sensor protein, for example in conjunction with immune reactions to said calcium sensor protein or molecules associated therewith.

6. An eucaryotic cell expressing the nucleotide sequence according to claim 1 for use in an assay to identify molecules which block or enhance the activity of said calcium sensor protein.