Prokineticin polypeptides, related compositions and methods

Abstract

The invention provides isolated polypeptides that stimulate gastrointestinal smooth muscle contraction, including human prokineticin 1 and human prokineticin 2 polypeptides, and functional fragments and modifications thereof. Also provided are methods of stimulating gastrointestinal smooth muscle contraction in a mammal, by administering to the mammal an effective amount of a prokineticin polypeptide. The invention also provides nucleic acid molecules encoding a prokineticin polypeptide, and antibodies that selectively bind a prokineticin polypeptide. Further provided are methods of identifying a prokineticin receptor ligand, agonist or antagonist.

What is claimed is:  
     
         1 . An isolated polypeptide that stimulates gastrointestinal smooth muscle contraction, comprising an amino acid sequence at least 80% identical to the sequence of human prokineticin 1 (SEQ ID NO:3), said sequence comprising the N-terminal 6 amino acids of SEQ ID NO:3, the 10 conserved cysteine residues of SEQ ID NO:3, and from 0 to 9 of the 9 C-terminal amino acids of SEQ ID NO:3.  
     
     
         2 . The isolated polypeptide of  claim 1 , wherein amino acid residues that differ from residues in SEQ ID NO:3 are conservative substitutions thereof.  
     
     
         3 . The isolated polypeptide of  claim 1 , wherein amino acid residues that differ from residues in SEQ ID NO:3 consist of the corresponding residues from SEQ ID NO:6.  
     
     
         4 . The isolated polypeptide of  claim 3 , comprising SEQ ID NO:13.  
     
     
         5 . The isolated polypeptide of  claim 1 , comprising amino acids 1-77 of SEQ ID NO:3.  
     
     
         6 . The isolated polypeptide of  claim 1 , comprising SEQ ID NO:3.  
     
     
         7 . The isolated polypeptide of  claim 1 , comprising a 6XHis tag.  
     
     
         8 . The isolated polypeptide of  claim 1 , which is detectably labeled.  
     
     
         9 . An isolated peptide comprising at least 10 contiguous amino acids of SEQ ID NO:3, wherein said peptide is immunogenic.  
     
     
         10 . A pharmaceutical composition, comprising the isolated polypeptide of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         11 . A method of stimulating gastrointestinal smooth muscle contraction in a mammal, comprising administering to said mammal an effective amount of the polypeptide of  claim 1 .  
     
     
         12 . A nucleic acid molecule encoding the polypeptide of  claim 1 .  
     
     
         13 . An expression vector containing the nucleic acid molecule of  claim 12  operatively linked to a promoter of gene expression.  
     
     
         14 . A host cell comprising the expression vector of  claim 13 .  
     
     
         15 . A method of preparing the isolated polypeptide of  claim 1 , comprising culturing the host cell of  claim 14  so as to express said polypeptide, substantially purifying said polypeptide, and refolding said polypeptide.  
     
     
         16 . An antibody that selectively binds the polypeptide of  claim 1 .  
     
     
         17 . An isolated polypeptide that stimulates gastrointestinal smooth muscle contraction, comprising an amino acid sequence at least 80% identical to the sequence of human prokineticin 2 (SEQ ID NO:6), said sequence comprising the N-terminal 6 amino acids of SEQ ID NO:6, the 10 conserved cysteine residues of SEQ ID NO:6, and from 0 to 4 of the 4 C-terminal amino acids of SEQ ID NO:6.  
     
     
         18 . The isolated polypeptide of  claim 17 , wherein amino acid residues that differ from residues in SEQ ID NO:6 are conservative substitutions thereof.  
     
     
         19 . The isolated polypeptide of  claim 17 , wherein amino acid residues that differ from residues in SEQ ID NO:6 consist of the corresponding residues from SEQ ID NO:3.  
     
     
         20 . The isolated polypeptide of  claim 19 , comprising SEQ ID NO:14.  
     
     
         21 . The isolated polypeptide of  claim 17 , comprising amino acids 1-77 of SEQ ID NO:6.  
     
     
         22 . The isolated polypeptide of  claim 17 , comprising SEQ ID NO:6.  
     
     
         23 . The isolated polypeptide of  claim 17 , comprising a 6XHis tag.  
     
     
         24 . The isolated polypeptide of  claim 17 , which is detectably labeled.  
     
     
         25 . An isolated peptide comprising at least 10 contiguous amino acids of SEQ ID NO:6, wherein said peptide is immunogenic.  
     
     
         26 . A pharmaceutical composition, comprising the isolated polypeptide of  claim 17  and a pharmaceutically acceptable carrier.  
     
     
         27 . A method of stimulating gastrointestinal smooth muscle contraction in a mammal, comprising administering to said mammal an effective amount of the polypeptide of  claim 17 .  
     
     
         28 . A nucleic acid molecule encoding the polypeptide of  claim 17 .  
     
     
         29 . An expression vector containing the nucleic acid molecule of  claim 17  operatively linked to a promoter of gene expression.  
     
     
         30 . A host cell comprising the expression vector of  claim 29 .  
     
     
         31 . A method of preparing the isolated polypeptide of  claim 17 , comprising culturing the host cell of  claim 30  so as to express said polypeptide, substantially purifying said polypeptide, and refolding said polypeptide.  
     
     
         32 . An antibody that selectively binds the polypeptide of  claim 17 .  
     
     
         33 . A method of identifying a prokineticin receptor ligand, comprising contacting a preparation comprising prokineticin receptor with one or more candidate compounds, and identifying a compound that specifically binds to said receptor, said compound being characterized as a prokineticin receptor ligand.  
     
     
         34 . The method of  claim 33 , wherein said preparation is an intestinal smooth muscle preparation or membrane preparation thereof.  
     
     
         35 . The method of  claim 33 , wherein said preparation is a cell line or membrane preparation thereof.  
     
     
         36 . The method of  claim 35 , wherein said cell line is M2A7 (ATCC CRL-2500).  
     
     
         37 . The method of  claim 33 , wherein the ability of said ligand to selectively agonize or antagonize prokineticin receptor signaling is further determined.  
     
     
         38 . The method of  claim 37 , wherein said signaling is determined in a cell line.  
     
     
         39 . The method of  claim 38 , wherein said cell line is M2A7 (ATCC CRL-2500).  
     
     
         40 . The method of  claim 37 , wherein said signaling is determined by monitoring calcium mobilization.  
     
     
         41 . The method of  claim 33 , wherein the ability of said ligand to modulate smooth muscle contractility is further determined.  
     
     
         42 . A method of identifying a prokineticin receptor agonist, comprising contacting a preparation comprising a prokineticin receptor with one or more candidate compounds, and identifying a compound that selectively promotes production of a prokineticin receptor signal, said compound being characterized as a prokineticin receptor agonist.  
     
     
         43 . The method of  claim 42 , wherein said preparation is a cell line.  
     
     
         44 . The method of  claim 43 , wherein said cell line is M2A7 (ATCC CRL-2500).  
     
     
         45 . The method of  claim 42 , wherein said signaling is determined by monitoring calcium mobilization.  
     
     
         46 . The method of  claim 42 , wherein the ability of said agonist to modulate smooth muscle contractility is further determined.  
     
     
         47 . A method of identifying a prokineticin receptor antagonist, comprising contacting a preparation comprising a prokineticin receptor with one or more candidate compounds in the presence of a prokineticin, and identifying a compound that selectively inhibits production of a prokineticin receptor signal, said compound being characterized as a prokineticin receptor antagonist.  
     
     
         48 . The method of  claim 47 , wherein said prokineticin comprises an amino acid sequence selected from the group consisting of amino acids 1-77 of SEQ ID NOS:3 and amino acids 1-77 of SEQ ID NO:6.  
     
     
         49 . The method of  claim 47 , wherein said preparation is a cell line.  
     
     
         50 . The method of  claim 49 , wherein said cell line is M2A7 (ATCC CRL-2500).  
     
     
         51 . The method of  claim 47 , wherein said signaling is determined by monitoring calcium mobilization.  
     
     
         52 . The method of  claim 47 , wherein the ability of said antagonist to modulate smooth muscle contractility is further determined.