AU6902194A - Human calcium sensor - Google Patents

Description

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 (1,2). The receptor function is essential for maintenance of normal plasma calcium concentrations, and reduced receptor expression within proliferating parathyroid cells of patients with hyperparathyroidism (HPT) results in calcium insensitivity of the PTH secretion and variably severe hypercalcemia (3-6). 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 [Ca²⁺i] (7,8). 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 (7,8). 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. The calcium sensor/receptor has been revealed as a 500 kDa single chain glycoprotein (7). However, the amino acid sequence as well as the corresponding DNA sequences thereof are hitherto unknown.

SUMMARY OF THE INVENTION

Therefore, an object of the present invention was to provide sufficient structural data of the calcium sensor/receptor to enable complete characterization thereof.

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

Another object was to use said structural data to design novel treatment methods as well as compounds and compositions for treating calcium related disorders.

Two important human diseases associated with perturbations of the calcium ion homeostasis are hyperthyroidism and osteoporosis. Thus, in one embodiment cells expressing the calcium sensor protein or comprising the cDNA encoding 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, vitamin, D3 production, estrogen, osteoclast activity or osteoblast activity (therefore, bone resorption and/or formation), calcium secretion and calcium ion homeostasis.

The present invention describes the isolation and partial characterization of a 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 Heymann nephritis antigen (11), suggests that the common calcium sensor of the placenta, the 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 μl/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 the cDNA 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. 5), low density lipoprotein receptor (LDL-RC, SEQ ID No. 6), and LDL related receptor protein (LDL-RRP, SEQ ID No. 7). 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 (1), kidney (2), liver (3), placenta (4), pancreas (5), adrenal gland (6), small gut (7). Filters were hybridized with the 2.8 kb pCAS-2 insert probe, and reactions visualized by a 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 α-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 (e.g., 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 non-naturally occurring analogues having single or multiple amino 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 using the following standard one-letter abbreviations:

Guanine 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

Lysine 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 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, the 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 SEQ 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). 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°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 (7). The procedure utilizes two different monoclonal antiparathyroid antibodies (1,7), E11 and G11, known to bind different epitopes of the calcium sensing protein; Ell has displayed no functional effect, while G11 efficiently blocks calcium regulation in both parathyroid and placental cells (1,7). After purification, the calcium sensor protein preparation was subjected to gel chromatography on a Zorbax GF25 gel column (9.2 × 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 C₄ column (2.2 × 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 elution, monitored at 214 nm 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. 10)

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 λ gt 11 cDNA library (Clontech, Ca., USA) was plated out to a density of approximately 2 × 10⁵ plaques within a 20x25 cm agar plate. Replicate filters (Hybond-N+, Amersham) of ten plates were prehybridized in 5 × SSPE (SSPE; 120 mM NaCl, 8 mM NaH₂PO₄, 0.8 mM EDTA, pH 7.4), 5 × Denhart's solution (12), 0.5% SDS, 20μg/ml single stranded salmon sperm DNA (Sigma Chemical Co., S:t Louis, Ohio). The mixed oligonucleotide probe, endlabeled with γ-[³²P]-ATP and polynucleotide kinase (Amersham), was added to the hybridization mixture (30 × 10⁶ cpm in 50 ml), and hybridization was carried out over night at 42° C. The filter was washed twice in 2 × SSPE and once in 0.1 × 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 λ 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 20 μl of 10mM Tris-HCl, pH 8.0, 1.5 mM MgCl₂, 50mM KCl in a Perkin-Elmer 9600 PCR-machine (Perkin-Elmer, Norwalk, USA). Two cycles of denaturation at 94° C for 2 min, annealing at 47° C for 1 min and extension at 72° C for 1 min 30 sec were followed by 33 cycles of 94° C for 1 min, 54° C for 45 sec, 72° C for 1 min and a final extension at 72° C for 10 min.

Screening of a placental cDNA library with a PCR-fragment as probe. A placental λ 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 × 10⁶ plaques distributed on ten 20 × 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, Ca.). 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 similarities to the placental cDNA sequence using the FAST DB algorithm (13).

Immunostaining and Northern blot. Immunohistochemical studies were performed on acetone-fixed, 6 μm thick frozen sections, utilizing the monoclonal antiparathyroid antibodies E11 and G11, at concentrations of 5 μg/ml, together with a mouse peroxidase antiperoxidase technique on human placental, parathyroid, kidney, and epididymis specimens as well as on the other human tissues - see above (1,7). 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 μg of total RNA was electrophoresed in a 1.5%/37% agarose/formaldehyde gel, blotted onto nylon membranes (Qiabrane, Diagen GmbH, Dϋsseldorf, Germany) and probed with the 2.3 kb clone (see results) labeled by the random priming method. Hybridizations were performed at 42°C for 18-24 h in 50% formamide, 4 × saline sodium citrate (SSC; 300 mM NaCl, 30 mM Na-citrate, pH 7.0), 2 × Denhart's solution, 10% dextran sulfate (Kabi-Pharmacia, Uppsala, Sweden) and 100 μg/ml salmon sperm DNA. Filters were washed at a final stringency of 1 × SSC/0.1% SDS for 30 min at 42°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 (1,7). The same antibodies were used in a sandwich ELISA to monitor the purification (7). In order to avoid contamination with low molecular peptides, the whole final preparation, consisting of 200 μg of the 500 kDa protein chain (7), was made 6 M with regard to guanidine-HCl and applied to a gel chromatography column, equilibrated with 2 M 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 μg) was added. The digestion was allowed to proceed over night at 37°C. The fragmented protein was reduced by incubation with 0.1% ß-mercaptoethanol at 37°C for 30 min and subsequently alkylated with 4-vinyl pyridine (0.3%) at room temperature for 2 h. The peptide mixture was then applied to a reversed phase C₄ 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 1). Due to the large number of peptides, the elution pattern was complex. 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 λ gt 11 cDNA library. Approximately 2 × 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 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 λ ZAP-II cDNA library reported to contain clones with large inserts was screened with the PCR-fragment as the probe. From 2 × 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. 3). 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 (11). 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 LDL-receptor-related protein (identical to the α₂-macroglobulin receptor, 11,15,16, SEQ ID No. 7). 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. 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 (E11 and G11) 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 μg/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 5). 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 Ca²⁺ 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 (1-6). 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 (7,8). 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 (7). It was also demonstrated by immunoprecipitation that a protein of the same size reacted with the antiparathyroid antibodies within the parathyroid and kidney tubule cells (to be published, (25).

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 [Ca²⁺i] and concomittant activation of phospholipase C, possibly via a coupled G-protein, with a resulting accumulation of inositol phosphates (2,5,9,10). An initial transient rise in [Ca²⁺i] is due to inositoltrisphosphate (Ip 3)-induced mobilization of Ca²⁺ from intracellular sources, while an ensuing steady-state elevation in [Ca²⁺i] 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 staining 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 (20). 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 ( 20) . The sequence similarity between the presently available portions of the human placental 500 kDa calcium sensor protein and 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 E11 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, the 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 Ca²⁺-binding ability (16,27,28). Thus, the Ca²⁺ binding is necessary for the interaction of the LDL-receptor with apo-B (27). The LDL-receptor related protein (α₂-macroglobulin receptor) is also known to bind Ca²⁺, which induces conformational changes, and Ca^2* is necessary for binding of activated α₂-macroglobulin to the receptor (16). Recently, the rat Heymann antigen was shown by a blotting technique to interact with Ca²⁺ (28).

The Ca²⁺ 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 LDL-receptor superfamily (11,16,27), which may represent putative Ca²⁺ binding sites. Thus, when present in the coagulation factors IX, X and protein C, each EGF-like module is known to bind one Ca²⁺-ion (29-34), and the EGF-like modules have also been demonstrated to mediate Ca²⁺dependent protein/protein interaction (35). Kinetic data have suggested that the calcium sensor displays positive cooperativity in its interaction with Ca²⁺, a phenomenon which appears essential for the sigmoidal regulation of [Ca²⁺i] and PTH release, with a steep relation within the physiological range of extracellular calcium (9,10). The positive cooperativity should require multiple binding sites for Ca²⁺, possibly resulting from the repetitive EGF-like modules, generally present in molecules of the LDL-receptor superfamily (11,16,27). However, Ca²⁺ binding to EGF-like domains are known to induce only minor, localized pertubations of the three-dimensional structure (32), and it is possible that the calcium sensor contains also other Ca²⁺ binding sites.

A 43 kDa membrane protein (α₂-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 Ca²⁺dependent manner (28). No physiological function has yet been assigned to this protein, but it appears also in tissues where the Heymann antigen and the LDL-receptor-related proteins are not expressed (28). An intriguing observation is the presence of a putative leucine-zipper motif in the aminoterminal part of the 43 kDa protein (36), considering that such motifs have been suggested to influence the opening and closure of membrane ion channels (37). 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 Ca²⁺dependent manner. Interaction with the 43 kDa protein might be important for the transmission of Ca²⁺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 Ca²⁺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 roid cells loaded with [³²P]-ortophosphate (unpublished observation).

The calcium sensor protein of the placenta may be involved in maintenance of a feto-maternal Ca²⁺ gradient and placental Ca²⁺ transport, possibly by mediating calcium regulation of the parathyroid hormone related peptide (PTHrP) production and/or 1,25 (OH)₂D₃ metabolism (8). 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 (38). 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-α-hydroxylase present in the placenta and proximal kidney tubule, is regulated by extracellular calcium, and the calcium sensor might accordingly regulate 1,25 (OH)₂D₃ metabolism, but it may possibly also influence Ca²⁺ reabsorption from the glomerular filtrate (7-9). 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 Ca²⁺ sensing ability for regulation of various functions, separate from the general calcium homeostasis, either during development or in the differentiated state (10).

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 hypoparathyroidism (10). The availability of a cDNA clone for the calcium sensor should, enable extended studies on the pathophysiology in parathyroid disorder, and also in 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 and 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.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Akerstrom, Goran

Klareskog, Lars

Juhlin, Claes

Rask, Lars

(ii) TITLE OF INVENTION: Human Calcium Sensor

(iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Dr. Ludwig Brann Patentbyra Ab

(B) STREET: P.O. Box 1344, Drottninggatan 7

(C) CITY: S-751 43 Uppsala

IE) COUNTRY: Sweden

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Aldenback, Ulla

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 46 18 13 9635

(B) TELEFAX: 46 18 10 93 22

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Xaa Ala Met Asn Pro Tyr Ser Leu Asp Ile Phe GLu Asp Gln Leu Tyr

1 5 10 15

Trp (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Xaa Val Met Gln Pro Asp Gly Ile Ala Xaa Asp Trp Val

1 - 5 10

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 804 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..804

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

AAA TAC GTA ATG CAO CCA GAT GGA ATA GCA GTG GAC TGG GTT GGA AGG 48 Lys Tyr Val Met Gln Pro Asp Gly Ile Ala Val Asp Trp Val Gly Arg

1 5 10 15

CAT ATT TAC TGG TCA GAT GTC AAG AAT AAA CGC ATT GAG GTG GCT AAA 96 His Ile Tyr Trp Ser Asp Val Lys Asn Lys Arg Ile Glu Val Ala Lys

20 25 30

CTT GAT GGA AGG TAC AGA AAG TGG CTG ATT TCC ACT GAC CTG GAC CAA 144 Leu Asp Gly Arg Tyr Arg Lys Trp Leu Ile Ser Thr Asp Leu Asp Gln

35 40 45

CCA GCT GCT ATT GCT GTG AAT CCC AAA CTA GGG CTT ATG TTC TGG ACT 192 Pro Ala Ala Ile Ala Val Asn Pro Lys Leu Gly Leu Met Phe Trp Thr

50 55 60

GAC TGG GGA AAG GAA CCT AAA ATC GAG TCT GCC TGG ATG AAT GGA GAG 240 Asp Trp Gly Lys Glu Pro Lys Ile Glu Ser Ala Trp Met Asn Gly Glu

65 70 75 80

GAC CGC AAC ATC CTG GTT TTC GAG GAC CTT GGT TGG CCA ACT GGC CTT 288 Asp Arg Asn Ile Leu Val Phe Glu Asp Leu Gly Trp Pro Thr Gly Leu

85 90 95 TCT ATC GAT TAT TTG AAC AAT GAC CGA ATC TAC TGG AGT GAC TTC AAG 336 Ser Ile Asp Tyr Leu Asn Asn Asp Arg Ile Tyr Trp Ser Asp Phe Lys

100 105 110

GAG GAC GTT ATT GAA ACC ATA AAA TAT GAT GGG ACT GAT AGG AGA GTC 384 Glu Asp Val Ile Glu Thr Ile Lys Tyr Asp Gly Thr Asp Arg Arg Val

115 120 125

ATT GCA AAG GAA GCA ATG AAC CCT TAC AGC CTG GAC ATC TTT GAA GAC 432 Ile Ala Lys Glu Ala Met Asn Pro Tyr Ser Leu Asp Ile Phe Glu Asp

130 135 140

CAG TTA TAC TGG ATA TCT AAG GAA AAG GGA GAA GTA TGG AAA CAA AAT 480 Gln Leu Tyr Trp Ile Ser Lye Glu Lys Gly Glu Val Trp Lys Gln Asn

145 150 155 160

AAA TTT GGG CAA GGA AAG AAA GAG AAA ACG CTG GTA GTG AAC CCT TGG 528 Lys Phe Gly Gln Gly Lys Lys Glu Lys Thr Leu Val Val Asn Pro Trp

165 170 175

CTC ACT CAA GTT CGA ATC TTT CAT CAA CTC AGA TAC AAT AAG TCA GTG 57δ Leu Thr Gln Val Arg Ile Phe His Gln Leu Arg Tyr Asn Lys Ser Val

180 185 190

CCC AAC CTT TGC AAA CAG ATC TGC AGC CAC CTC TGC CTT CTG AGA CCT 624 Pro Asn Leu Cys Lys Gln Ile Cys Ser His Leu Cys Leu Leu Arc? Pro

195 200 205

GGA GGA TAC AGC TGT GCC TGT CCC CAA GGC TCC AGC TTT ATA GAG GGG 672 Gly Gly Tyr Ser Cys Ala Cys Pro Gln Gly Ser Ser Phe Ile Glu Gly

210 215 220

AGC ACC ACT GAG TGT GAT GCA GCC ATC GAA CTG CCT ATC AAC CTG CCC 720 Ser Thr Thr Glu Cys Asp Ala Ala Ile Glu Leu Pro Ile Asn Leu Pro

225 230 235 240

CCC CCA TGC AGG TGC ATG CAC GGA GGA AAT TGC TAT TTT GAT GAG ACT 768 Pro Pro Cys Arg Cys Met His Gly Gly Asn Cys Tyr Phe Asp Glu Thr

245 250 255

GAC CTC CCC AAA TGC AAG TGT CCT AGC GGC TAC ACC 804

Asp Leu Pro Lys Cys Lys Cys Pro Ser Gly Tyr Thr

260 265

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 268 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Lys Tyr Val Met Gln Pro Asp Gly Ile Ala Val Asp Trp Val Gly Arg

1 5 10 15

His Ile Tyr Trp Ser Asp Val Lys Asn Lys Arg Ile Glu Val Ala Lys

20 25 30 Leu Asp Gly Arg Tyr Arg Lys Trp Leu Ile Ser Thr Asp Leu Asp Gln

35 40 45

Pro Ala Ala Ile Ala Val Asn Pro Lys Leu Gly Leu Het Phe Trp Thr

50 55 60

Asp Trp Gly Lys Glu Pro Lys Ile Glu Ser Ala Trp Met Asn Gly Glu 65 70 75 80

Asp Arg Asn Ile Leu Val Phe Glu Asp Leu Gly Trp Pro Thr Gly Leu

85 90 95

Ser Ile Asp Tyr Leu Asn Asn Asp Arg Ile Tyr Trp Ser Asp Phe Lys

100 105 110

Glu Asp Val Ile Glu Thr Ile Lys Tyr Asp Gly Thr Asp Arg Arg Val

115 120 125

Ile Ala Lys Glu Ala Met Asn Pro Tyr Ser Leu Asp Ile Phe Glu Asp

130 135 140

Gln Leu Tyr Trp Ile Ser Lys Glu Lys Gly Glu Val Trp Lys Gin Asn 145 150 155 160

Lys Phe Gly Gln Gly Lys Lys Glu Lyε Thr Leu Val Val Asn Pro Trp

165 170 175

Leu Thr Gln Val Arg Ile Phe His Gln Leu Arg Tyr Asn Lys Ser Val

180 185 190

Pro Asn Leu Cys Lys Gln Ile Cys Ser His Leu Cys Leu Leu Arg Pro

195 200 205

Gly Gly Tyr Ser Cys Ala Cys Pro Gln Gly Ser Ser Phe Ile Glu Gly

210 215 220

Ser Thr Thr Glu Cys Asp Ala Ala Ile Glu Leu Pro Ile Asn Leu Pro 225 230 235 240

Pro Pro Cys Arg Cys Met His Gly Gly Asn Cys Tyr Phe Asp Glu Thr

245 250 255

Asp Leu Pro Lys Cys Lys Cys Pro Ser Gly Tyr Thr

260 265

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 269 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Xaa Xaa Xaa Xaa Xaa Pro Aep Gly L«u Ala Val Asp Trp Val Gly Arg 1 5 10 15 His Ile Tyr Trp Ser Asp Ala Asn Ser Gln Arg Ile Glu Val Ala Thr 20 25 30

Leu Asp Gly Arg Tyr Arg Lys Trp Leu Ile Thr Thr Gln Leu Asp Glr

35 40 45

Pro Ala Ala Ile Ala Val Asn Pro Lys Leu Gly Leu Met Phe Trp Thr 50 55 60

Asp Gln Gly Lys Gln Pro Lys Ile Glu Ser Ala Trp Met Asn Gly Glu 65 70 75 80

His Arg Ser Val Leu Val Ser Glu Asn Leu Gly Trp Pro Asn Gly Leu

85 90 95

Ser Ile Asp Tyr Leu Asn Asp Asp Arg Val Tyr Trp Ser Asp Ser Lys

100 105 110

Glu Asp Val Ile Glu Ala Ile Lys Tyr Asp Gly Thr Asp Arg Arg Leu

115 120 125 Ile Ile Asn Glu Ala Met Lys Pro Phe Ser Leu Asp Ile Phe Glu Asp 130 135 140

Lys Leu Tyr Trp Val Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Gln 145 150 155 160

Asn. Lys Phe Gly Lys Glu Asn Lye Glu Lys Val Leu Val Val Asn Pro

165 170 175

Trp Leu Thr Gln Val Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

180 185 190

Xaa Xaa Xaa Xaa Cys Lys Gln Val Cys Ser His Leu Cys Leu Leu Arg

195 200 205

Pro Gly Gly Tyr Ser Cys Ala Cys Pro Gln Gly Ser Asp Phe Val Thr 210 215 220

Gly Ser Thr Val Gln Cys Xaa Xaa Xaa Xaa Xaa Xaa Pro Val Thr Met 225 230 235 240

Pro Pro Pro Cys Arg Cys Met His Gly Gly Asn cys Tyr Phe Asp Glu

245 250 255

Asn Glu Leu Pro Lys Cys Lys Cys Ser Ser Gly Tyr Ser

260 265

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 280 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCEIPTION: SEQ ID NO:6:

Arg Asp Ile Gln Ala Pro Asp Gly Leu Ala Val Asp Trp Ile His Ser 1 5 10 15

Asn Ile Tyr Trp Thr Asp Ser Val Leu Gly Thr Val Ser Val Ala Asp

20 25 30

Thr Lys Gly Val Lys Arg Lys Thr Leu Phe Arg Glu Asn Gly Ser Lys

35 40 45

Pro Arg Ala Ile Val Val Asp Pro Val Hie Gly Phe Met Tyr Trp Thr 50 55 60

Asp Trp Gly Thr Pro Ala Lys Ile Lys Lys Gly Gly Leu Asn Gly Val 65 70 75 80

Aep Ile Tyr Ser Leu Val Thr Glu Asn Ile Gln Trp Pro Asn Gly Ile

85 90 95

Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr Trp Val Asp Ser Lys Leu

100 105 110

His Ser Ile Ser Ser Ile Asp Tyr Asn Gly Gly Asn Arg Lys Thr Ile

115 120 125

Leu Glu Asp Glu Lys Arg Leu Ala His Pro Phe Ser Leu Ala Val Phe 130 135 140

Glu Asp Lys Val Phe Trp Thr Asp Ile Ile Asn Glu Ala Ile Phe Ser 145 150 155 160

Ala Asn Arg Leu Thr Gly Ser Asp Val Asn Leu Leu Ala Glu Asn Leu

165 170 175

Leu Ser Pro Glu Asp Met Val Leu Phe His Asn Leu Thr Gln Pro Arg

180 185 190

Gly Val Asn Trp Cys Glu Arg Thr Thr Leu Ser Asn Gly Gly Cys Gln

195 200 205

Tyr Leu Cys Leu Pro Ala Pro Gln Ile Asn Pro His Ser Pro Lys Phe 210 215 220

Thr Cys Ala Cys Pro Asp Gly Met Leu Leu Ala Arg Asp Met Arg Ser 225 230 235 240

Cys Leu Thr Glu Ala Glu Ala Ala Val Ala Thr Gln Glu Thr Ser Thr

245 250 255

Val Arg Leu Lys Val Ser Ser Thr Ala Val Arg Thr Gln His Thr Thr

260 265 270

Thr Arg Pro Val Pro Asp Thr Ser

275 280

(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 281 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Thr Gly Leu Ser Asn Pro Asp Gly Leu Ala Val Asp Trp Val Gly Gly 1 5 10 15

Asn Leu Tyr Trp Cys Aep Lys Gly Arg Asp Thr Ile Glu Val Ser Lys

20 25 30

Leu Asn Gly Ala Tyr Arg Thr Val Leu Val Ser Ser Gly Leu Arg Glu

35 40 45

Pro Arg Ala Leu Val Val Asp Val Gln Asn Gly Tyr Leu Tyr Trp Thr 50 55 60

Asp Trp Gly Asp His Ser Leu Ile Gly Arg Ile Gly Met Asp Gly Ser

65 70 75 80

Ser Arg Ser Vαl Ile Vαl Asp Thr Lys Ile Thr Trp Pro Asn Gly Leu

85 90 95

Thr Leu Asp Tyr Val' Thr Glu Arg Ile Tyr Trp Ala Asp Ala Arg Glu

100 105 110

Asp Tyr Ile Glu Phe Ala Ser Leu Asp Gly Ser Asn Arg His Val Val

115 120 125

Leu Ser Gln Asp Ile Pro His Ile Phe Ala Leu Thr Leu Phe Glu Asp 130 135 140

Tyr Val Tyr Trp Thr Asp Trp Glu Thr Lys Ser Ile Asn Arg Ala His 145 150 155 160

Lys Thr Thr Gly Thr Asn Lys Thr Leu Leu Ile Ser Thr Leu His Arg

165 170 175

Pro Met Asp Leu His Val Phe His Ala Leu Arg Gln Pro Asp Val Pro

180 185 190

Asn His Pro Cys Lys Val Asn Asn Gly Gly Cys Ser Asn Leu Cys Leu

195 200 205

Leu Ser Pro Gly Gly Gly His Lys Cys Ala Cys Pro Thr Asn Phe Tyr 210 215 220

Leu Gly Ser Asp Gly Arg Thr Cys Val Ser Asn Cys Thr Ala Ser Gln 225 230 235 240

Phe Val Cys Lys Asn Asp Lys Cys Ile Pro Phe Trp Trp Lys Cys Asp

245 250 255

Thr Glu Asp Asp Cys Gly Asp His Ser Asp Glu Pro Pro Asp Cys Pro

260 265 270 Glu Phe Lys Cys Arg Pro Gly Gln Phe

275 280

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

( ix) FEATURE:

(A) NAME/KEY : modified_base

(B) LOCATION: 7

(D) OTHER INFORMATION: /mod_base= i

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 28

(D) OTHER INFORMATION : / mod_base= i

( ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 31

(D) OTHER INFORMATION: /mod_base= i

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 37

(D) OTHER INFORMATION: /mod_base= i

(ix) FEATURE:

(A) NAME/KEY: modified_base

(B) LOCATION: 46

(D) OTHER INFORMATION: /mod_base= i

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

CCARTANAGC TGRTCCTCRA AGATRTCNAG NGARTANGGR TTCATNGC 48

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

GCGGAATTCG TNATGCARCC NGAYGG 26 (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

ATAGGAATCC TGRTCYTCRA ADATRTC 27

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Claims (6)

1. A cDNA clone encoding the human calcium sensor, comprising the following nucleotide sequence

27 54

AAA TAC GTA ATG CAG CCA GAT GGA ATA GCA GTG GAC TGG GTT GGA AGG CAT ATT

81 108

TAC TGG TCA GAT GTC AAG AAT AAA CGC ATT GAG GTG GCT AAA CTT GAT GGA AGG

135 162

TAC AGA AAG TGG CTG ATT TCC ACT GAC CTG GAC CAA CCA GCT GCT ATT GCT GTG

189 216

AAT CCC AAA CTA GGG CTT ATG TTC TGG ACT GAC TGG GGA AAG GAA CCT AAA ATC

243 270

GAG TCT GCC TGG ATG AAT GGA GAG GAC CGC AAC ATC CTG GTT TTC GAG GAC CTT

297 324

GGT TGG CCA ACT GGC CTT TCT ATC GAT TAT TTG AAC AAT GAC CGA ATC TAC TGG

351 378

AGT GAC TTC AAG GAG GAC GTT ATT GAA ACC ATA AAA TAT GAT GGG ACT GAT AGG

405 432

AGA GTC ATT GCA AAG GAA GCA ATG AAC CCT TAC AGC QTG GAC ATC TTT GAA GAC

459 486

CAG TTA TAC TGG ATA TCT AAG GAA AAG GGA GAA GTA TGG AAA CAA AAT AAA TTT

513 540

GGG CAA GGA AAG AAA GAG AAA ACG CTG GTA GTG AAC CCT TGG CTC ACT CAA GTT

567 594

CGA ATC TTT CAT CAA CTC AGA TAC AAT AAG TCA GTG CCC AAC CTT TGC AAA CAG

621 648

ATC TGC AGC CAC CTC TGC CTT CTG AGA CCT GGA GGA TAC AGC TGT GCC TGT CCC

675 702

CAA GGC TCC AGC TTT ATA GAG GGG AGC ACC ACT GAG TGT GAT GCA GCC ATC GAA

729 756

CTG CCT ATC AAC CTG CCC CCC CCA TGC AGG TGC ATG CAC GGA GGA AAT TGC TAT

783

TTT GAT GAG ACT GAC CTC CCC AAA TGC AAG TGT CCT AGC GGC TAC ACC

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.

5. 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 senson 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.