Monkey calcium sensing receptor

Cynomolgous Monkey Calcium-Sensing Receptor polypeptides and polynucleotides and method for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for screening for compounds that either agonize or antagonize Cynomolgous Monkey Calcium-Sensing Receptor. Such compounds are expected to be useful in treatment of human diseases, including, but not limited to: infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; diabetes, obesity; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; urinary retention; osteoporosis; angina pectoris; myocardial infarction; stroke; ulcers; asthma; allergies; benign prostatic hypertrophy; migraine; vomiting; psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, depression, delirium, dementia, and severe mental retardation; and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome.

EXAMPLES 
 Example 1 Mammalian Cell Expression The receptors of the present invention are expressed in either human embryonic kidney 293 (HEK293) cells or adherent dhfr CHO cells. To maximize receptor expression, typically all 5′ and 3′ untranslated regions (UTRs) are removed from the receptor cDNA prior to insertion into a pCDN or pCDNA3 vector. The cells are transfected with individual receptor cDNAs by lipofectin and selected in the presence of 400 mg/ml G418. After 3 weeks of selection, individual clones are picked and expanded for further analysis. HEK293 or CHO cells transfected with the vector alone serve as negative controls. To isolate cell lines stably expressing the individual receptors, about 24 clones are typically selected and analyzed by Northern blot analysis. Receptor mRNAs are generally detectable in about 50% of the G4 18-resistant clones analyzed. 
 Example 2 Ligand bank for binding and functional assays A bank of over 600 putative receptor ligands has been assembled for screening. The bank comprises: transmitters, hormones and chemokines known to act via a human seven transmembrane (7TM) receptor; naturally occurring compounds which may be putative agonists for a human 7TM receptor, non-mammalian, biologically active peptides for which a mammalian counterpart has not yet been identified; and compounds not found in nature, but which activate 7TM receptors with unknown natural ligands. This bank is used to initially screen the receptor for known ligands, using both functional (i.e. calcium, cAMP, microphysiometer, oocyte electrophysiology, etc, see below) as well as binding assays. 
 Example 3 Ligand Binding Assays Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format. The purified ligand for a receptor is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards its receptor. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell receptor sources. For these assays, specific receptor binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding. 
 Example 4 Functional Assay in Xenopus Oocytes Capped RNA transcripts from linearized plasmid templates encoding the receptor cDNAs of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures. In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocyte) are injected in a 50 nl bolus using a microinjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response to agonist exposure. Recordings are made in Ca2&plus; free Barth's medium at room temperature. The Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands. 
 Example 5 Microphysiometric Assays Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell. The acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process. The pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, Calif.). The CYTOSENSOR is thus capable of detecting the activation of a receptor which is coupled to an energy utilizing intracellular signaling pathway such as the G-protein coupled receptor of the present invention. 
 Example 6 Extract/Cell Supernatant Screening A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be included within the ligand banks as identified to date. Accordingly, the 7TM receptor of the invention is also functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify natural ligands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified. 
 Example 7 Calcium and cAMP Functional Assays 7TM receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimulation or inhibition. Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombinant receptors are loaded with fura 2 and in a single day >150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization. Similarly, HEK 293 cells expressing recombinant receptors are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor. 1 SEQUENCE INFORMATION SEQ ID NO:1 ATGGCATTTTATTGCTGCTTCTGGGTCCTCTTGGCACTCACCTGGCACACCTCTGCCTATGGGCCCGACCAGCG AGCCCAAAAGAAAGGGGACATTATACTTGGCGGGCTCTTTCCTATTCATTTTGGAGTAGCAGCTAAAGATCAAG ATCTCAAATCAAGGCCGGAGTCTGTGGAATGCATCAGGTATAATTTCCGTGGGTTTCGCTGGTTACAGGCTATG ATATTTGCCATAGAGGAGATAAACAGCAGCCCAGCCCTTCTTCCCAACTTGACGCTGGGATACAGGATATTTGA CACTTGCAACACCGTTTCTAAGGCCTTGGAAGCCACCCTGAGTTTTGTTGCTCAAAACAAAATTGATTCTTTGA ACCTTGATGAGTTCTGCAACTGCTCAGAGCACATTCCCTCTACGATTGCTGTGGTGGGAGCAACTGGCTCAGGC GTCTCCACGGCAGTGGCAAATCTGCTGGGGCTCTTCTACATTCCCCAGGTCAGTTACGCCTCCTCCAGCAGACT CCTTAGCAACAAGAATCAATTCAAGTCCTTCCTCCGAACCATCCCCAATGATGAGCACCAGGCCACTGCCATGG CAGACATCATTGAGTATTTCCGCTGGAACTGGGTGGGCACAATTGCAGCTGATGATGACTATGGGCGGCCAGGG ATTGAGAAGTTCCGAGAGGAAGCTGAGGAAAGGGACATCTGCATTGACTTCAGTGAACTCATCTCCCAGTACTC TGATGAGGAAGAGATCCAGCATGTGGTGGAGGTGATTCAAAATTCCACGGCCAAAGTAATCGTGGTTTTCTCCA GTGGCCCAGACCTTGAGCCCCTCATCAAGGAGATTGTCCGGCGCAATATCACAGGCAAGATCTGGCTGGCCAGT GAGGCCTGGGCCAGCTCCTCCCTGATCGCCATGCCCGAGTACTTCCACGTGGTTGGAGGCACCATTGGATTTGC TCTGAAGGCTGGGCAGATCCCAGGCTTTCGGGAATTCCTGAAGAAGGTCCATCCCAGGAAGTCTGTCCACAATG GTTTTGCCAAGGAGTTTTGGGAAGAAACATTTAACTGCCACCTCCAAGAAGGTGCCAAAGGACCTTTACCTGTG GACACCTTTCTGAGAGGTCACGAAGAAAGTGGAGGCAGGTTTAGCAACAGTTCGACAGCCTTCCGACCCCTCTG TACAGGGGATGAGAACATCAGCAGTGTTGAGACCCCTTACATAGATTACACACATTTACGGATATCCTACAATG TGTACTTAGCAGTCTACTCCATTGCCCATGCCTTGCAAGATATATATACCTGCTTACCTGGGAGAGGGCTCTTC ACCAACGGCTCCTGTGCAGACATCAAGAAAGTTGAGGCGTGGCAGGTCCTGAAGCACCTACGGCACCTAAACTT TACCAACAATATGGGGGAGCAGGTGACCTTTGATGAGTGTGGTGACCTGGTGGGGAACTATTCCATCATCAACT GGCACCTCTCCCCAGAGGATGGCTCCATCGTGTTTAAGGAAGTCGGGTATTACAACGTCTATGCCAAGAAGGGA GAAAGACTCTTCATCAACGAGGAGAAAATCCTGTGGAGTGGGTTCTCCAGGGAGGTGCCCTTCTCCAACTGCAG CCGAGACTGTCTGGCAGGGACCAGGAAAGGGATCATTGAGGGGGAGCCCACCTGCTGCTTTGAGTGTGTGGAGT GTCCTGATGGGGAGTACAGCGATGAGACAGATGCCAGTGCCTGTAACAAGTGCCCAGATGACTTCTGGTCCAAT GAGAACCACACCTCCTGCATTGCCAAGGAGATCGAGTTTCTGTCGTGGACAGAGCCCTTTGGGATTGCACTCAC CCTCTTTGCCGTGCTGGGCATTTTCCTGACAGCCTTTGTGCTGGGTGTGTTTATCAAGTTCCGCAACACGCCCA TCGTCAAGGCCACGAACCGAGAGCTCTCCTACCTTCTCCTCTTCTCCCTGCTCTGCTGCTTCTCCAGCTCCCTG TTCTTCATTGGGGAGCCCCAGGACTGGACTTGCCGCCTGCGCCAACCGGCCTTTGGCATCAGTTTCGTGCTCTG CATCTCGTGCATCCTGGTGAAAACCAACCGTGTCCTCCTGGTGTTTGAGGCCAAGATCCCCACCAGCTTCCATC GCAAGTGGTGGGGGCTCAACCTGCAGTTTCTGCTGGTTTTCCTCTGCACCTTCATGCAGATTGTCATCTGTGTG ATCTGGCTCTACACCGCGCCCCCCTCAAGCTACCGCAACCACGAGCTGGAGGATGAGATCATCTTCATCACGTG CCACGAGGGCTCACTCATGGCCCTGGGCTTCCTGATCGGCTACACCTGCCTGCTGGCTGCCATCTGCTTCTTCT TTGCCTTCAAGTCCCGGAAGCTGCCAGAGAACTTCAATGAAGCCAAGTTCATCACCTTCAGCATGCTCATCTTC TTCATTGTCTGGATCTCCTTCATTCCAGCCTATGCCAGCACCTACGGCAAGTTTGTCTCTGCCGTGGAGGTGAT TGCCATCCTGGCAGCCAGCTTTGGCTTGCTGGCGTGCATCTTCTTCAACAAGATCTACATCATTCTCTTCAAGC CATCCCGCAACACCATCGAGGAGGTGCGTTGCAGCACCGCAGCTCACGCTTTCAAGGTGGCTGCCCGGGCCACG CTGCGCCGCAGCAACGTCTCCCGCAAGCGGTCCAGCAGCCTTGGGGGCTCCACTGGATCCACCCCCTCCTCCTC CATCAGCAGCAAGAGCAACAGCGAAGACCCATTCCCACAGCCCGAGAGGCAGAAGCAGCAGCAGCCGCTGGCCC TAACCCAACAAGAGCAGCAGCAGCAGCCCCTGACCCTCCCACAGCAGCAACAATCGCAGCAGCAGCCAAGATGC AAGCAGAAGGTCATCTTCGGCAGTGGCACGGTCACCTTCTCACTGAGCTTCGATGAGCCTCAGAAGAACGCCAT GGCCCACAGGAATTCTACACACCAGAACTCCCTGGAGGCCCAGAAAAGCAGCGATACGCTGGCCCGACACCAGG CATTACTCCCGCTGCAGTGCGGGGAAGCGGACTCAGATCTGAGCGTCCAAGAAACAGGTCTGCAAGGACCAGTG GGTGGAGACCACCGGCCAGAGGTGGAGGTCCCTGAAGAGTTGTCCCCAGCACTTGTAGTGTCCAGTTCACAGAG CTTTGTCATCAGCGGTGGAGGAAGCACTGTTACAGAAAATGTACTGCATTCATAA SEQ ID NO:2 MAFYCCFWVLLALTWHTSAYGPDQPAQKKGDIILGGLFPIHFGVAAKDQDLKSRPESVECIRYNFRGFRWLQAM IFAIEEINSSPALLPNLTLGYRIFDTCNTVSKALEATLSFVAQNKIDSLNLDEFCNCSEHIPSTIAVVGATGSG VSTAVANLLGLFYIPQVSYASSSRLLSNKNQFKSFLRTIPNDEHQATAMADIIEYFRWNWVGTIAADDDYGRPG IEKFREEAEERDICIDFSELISQYSDEEEIQHVVEVIQNSTAKVIVVFSSGPDLEPLIKEIVRRNITGKIWLAS EAWASSSLIANPEYFHVVGGTIGFALKAGQTPGFREFLKKVHPRKSVHNGFAKEFWEETFNCHLQEGAKGPLPV DTFLRGHEESGGRFSNSSTAFRPLCTGDENISSVETPYIDYTHLRISYNVYLAVYSIAHALQDIYTCLPGRGLF TNGSCADIKKVEAWQVLKHLRHLNFTNNMGEQVTFDECGDLVGNYSIINWHLSPEDGSIVFKEVGYYNVYAKKG ERLFINEEKILWSGFSREVPFSNCSRDCLAGTRKGIIEGEPTCCFECVECPDGEYSDETDASACNKCPDDFWSN ENHTSCIAKEIEFLSWTEPFGIALTLFAVLGIFLTAFVLGVFIKFRNTPIVKATNRELSYLLLFSLLCCFSSSL FFIGEPQDWTCRLRQPAFGISFVLCISCILVKTNRVLLVFEAKIPTSFHRKWWGLNLQFLLVFLCTFMQIVICV IWLYTAPPSSYRNHELEDEIIFTTCHEGSLMALGFLTGYTCLLAAICFFFAFKSRKLPENFNEAKFITFSMLIF FIVWISFIPAYASTYGKFVSAVEVIAILAASFGLLACTFFNKIYTILFKPSRNTIEEVRCSTAAHAFKVAARAT LRRSNVSRKRSSSLGGSTGSTPSSSISSKSNSEDPFPQPERQKQQQPLALTQQEQQQQPLTLPQQQQSQQQPRC KQKVIFGSGTVTFSLSFDEPQKNAMAHRNSTHQNSLEAQKSSDTLARHQALLPLQCGEADSDLSVQETGLQGPV GGDHRPEVEVPEELSPALVVSSSQSFVISGGGSTVTENVLHS