PTH receptor and screening assay utilizing the same

Abstract

The invention provides a novel PTH receptor polypeptide, rΔNt, characterized by a deletion of the extracellular amino-terminus, ligand binding domain of the receptor. Additionally disclosed are nucleic acid molecules encoding the receptor. The receptor has a minimal domain for ligand binding and is useful in screening assays designed for the identification of agonists and antagonists of PTH receptor activity.

1 . An isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: 
 (a) a nucleotide sequence encoding the rΔNt receptor having the complete amino acid sequence at positions from about 1 to about 435 in SEQ ID NO:2;    (b) a nucleotide sequence encoding the rΔNt receptor having the amino acid sequence at positions from about 2 to about 435 in SEQ ID NO:2;    (c) a nucleotide sequence encoding the mature rΔNt receptor having the amino acid sequence at positions from about 23 to about 435 in SEQ ID NO:2;    (d) a nucleotide sequence encoding the rΔNt receptor having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-1136;    (e) a nucleotide sequence encoding the mature rΔNt receptor having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No PTA-1136;    (f) a nucleotide sequence encoding the rΔNt extracellular domain;    (g) a nucleotide sequence encoding the rΔNt transmembrane domain;    (h) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f) or (g).    
     
     
         2 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the complete nucleotide sequence in  FIG. 1  (SEQ ID NO:1).  
     
     
         3 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the nucleotide sequence in  FIG. 1  (SEQ ID NO:1) encoding the rΔNt receptor having the complete amino acid sequence in  FIG. 1  (SEQ ID NO:2).  
     
     
         4 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the nucleotide sequence in  FIG. 1  (SEQ ID NO:1) encoding the mature rΔNt receptor having the amino acid sequence in  FIG. 1  (SEQ ID NO:2).  
     
     
         5 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the complete nucleotide sequence of the cDNA clone contained in ATCC Deposit No. PTA-1136.  
     
     
         6 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the nucleotide sequence encoding the rΔNt receptor having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-1136.  
     
     
         7 . The nucleic acid molecule of  claim 1  wherein said polynucleotide has the nucleotide sequence encoding the mature rΔNt receptor having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-1136.  
     
     
         8 . 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) or (g) of  claim 1  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.  
     
     
         10 . The isolated nucleic acid molecule of  claim 1 , which encodes the rΔNt receptor extracellular domain.  
     
     
         11 . The isolated nucleic acid molecule of  claim 1 , which encodes the rΔNt receptor transmembrane domain.  
     
     
         12 . A method for making a recombinant vector comprising inserting an isolated nucleic acid molecule of  claim 1  into a vector.  
     
     
         13 . A recombinant vector produced by the method of  claim 12 .  
     
     
         14 . A method of making a recombinant host cell comprising introducing the recombinant vector of  claim 13  into a host cell.  
     
     
         15 . A recombinant host cell produced by the method of  claim 14 .  
     
     
         16 . A recombinant method for producing a rΔNt polypeptide, comprising culturing the recombinant host cell of  claim 15  under conditions such that said polypeptide is expressed and recovering said polypeptide.  
     
     
         17 . An isolated rΔNt polypeptide having an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: 
 (a) the amino acid sequence of the rΔNt polypeptide having the complete amino acid sequence at positions from about 1 to about 435 in SEQ ID NO:2;    (b) the amino acid sequence of the rΔNt polypeptide having the amino acid sequence at positions from about 2 to about 435 in SEQ ID NO:2;    (c) the amino acid sequence of the mature rΔNt polypeptide having the amino acid sequence at positions from about 23 to about 435 in SEQ ID NO:2;    (d) the amino acid sequence of the rΔNt polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-1136; and    (e) the amino acid sequence of the mature rΔNt polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. PTA-1136.    
     
     
         18 . An isolated antibody that binds specifically to a rΔNt receptor polypeptide of  claim 17 .  
     
     
         19 . A method of screening for agonists or antagonists of PTH receptor activity comprising: 
 (a) contacting a test compound to cells expressing a PTH receptor; and    (b) measuring the biological response of said cells.