PTH1R and PTH3R receptors, methods and uses thereof

Abstract

The present invention relates to novel parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP) receptors (PTH1R and PTH3R) isolated from zebrafish. The receptors of the present invention share homology with previously identified parathyroid hormone (PTH)/parathyroid related protein (PTHrP) receptors. Isolated nucleic acid molecules are provided encoding the zebrafish PTH1R and PTH3R receptors. PTH1R and PTH3R receptor polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of PTH1R and PTH3R receptor activity and to diagnostic and therapeutic methods.

1 - 21 . (canceled)  
     
     
         22 . An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: 
 (a) a nucleotide sequence encoding a parathyroid hormone (PTH)/PTH-related peptide (PfHrP) receptor (PTH3R receptor) having the amino acid sequence from positions 1 to 542 in SEQ ID NO:4;    (b) a nucleotide sequence encoding the PTH3R receptor having the amino acid sequence from positions 2 to 542 in SEQ ID NO:4;    (c) a nucleotide sequence encoding the mature PTH3R receptor having the amino acid sequence from positions 22 to 542 in SEQ ID NO:4;    (d) a nucleotide sequence encoding the PTH3R receptor having the complete amino acid sequence encoded by the cDNA clone deposited with the ATCC as deposit PTA-915;    (e) a nucleotide sequence encoding the mature PTH3R receptor having the amino acid sequence encoded by the cDNA clone deposited with the ATCC as deposit PTA-915;    (f) a nucleotide sequence encoding the PTH3R extracellular domain, said extracellular domain having the amino acid sequence of amino acids 22 to 145 in SEQ ID NO:4;    (g) a nucleotide sequence encoding the PTH3R transmembrane domain, said transmembrane domain having the amino acid sequence of amino acids 146 to 402 in SEQ ID NO:4;    (h) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f) or (g).    
     
     
         23 . The nucleic acid molecule of  claim 22  wherein said polynucleotide has the complete nucleotide sequence in  FIG. 1D  (SEQ ID NO:3).  
     
     
         24 . The nucleic acid molecule of  claim 22  wherein said polynucleotide has the nucleotide sequence in  FIG. 1D  (SEQ ID NO:3) encoding the PTH3R receptor having the complete amino acid sequence in  FIG. 2B  (SEQ ID NO:4).  
     
     
         25 . The nucleic acid molecule of  claim 22  wherein said polynucleotide has the nucleotide sequence in  FIG. 1D  (SEQ ID NO:3) encoding the mature PTH3R receptor having the amino acid sequence in  FIG. 2B  (SEQ ID NO:4).  
     
     
         26 . The nucleic acid molecule of  claim 22  wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone deposited with the ATCC as deposit PTA-915.  
     
     
         27 . The nucleic acid molecule of  claim 22  wherein said polynucleotide has the nucleotide sequence encoding the PTH3R receptor having the complete amino acid sequence encoded by the cDNA clone deposited with the ATCC as deposit PTA-915.  
     
     
         28 . The nucleic acid molecule of  claim 22  wherein said polynucleotide has the nucleotide sequence encoding the mature PTH3R receptor having the amino acid sequence encoded by the cDNA clone deposited with the ATCC as deposit PTA-915.  
     
     
         29 . An isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence identical to a nucleotide sequence in (a), (b), (c), (d), (e), (f), (g), or (h) of claim  221   wherein said stringent hybridization conditions comprise overnight incubation at 42° C. in a solution comprising: 50% forrnamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at 65° C.:    and wherein said polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.    
     
     
         30 . An isolated nucleic acid molecule comprising a polynucleotide which encodes an epitope-bearing portion of a PTH3R receptor comprising an amino acid sequence of between 7 and 30 amino acids from the amino acid sequence in (a), (b), (c), (d), (e), (f), or (g) of  claim 22 .  
     
     
         31 . The isolated nucleic acid molecule of  claim 22 , which encodes the PTH3R receptor extracellular domain.  
     
     
         32 . The isolated nucleic acid molecule of  claim 22 , which encodes the PTH3R receptor transmembrane domain.  
     
     
         33 . A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of  claim 22  into a vector.  
     
     
         34 . A recombinant vector produced by the method of  claim 33 .  
     
     
         35 . A method of making a recombinant host cell comprising introducing the recombinant vector of  claim 34  into a host cell.  
     
     
         36 . A recombinant host cell produced by the method of  claim 35 .  
     
     
         37 . A recombinant method for producing a PTH3R polypeptide, comprising culturing the recombinant host cell of  claim 36  under conditions such that said polypeptide is expressed and recovering said polypeptide.  
     
     
         38 - 47 . (canceled)