Pain signaling molecules

Abstract

The invention relates generally to novel genes expressed in normal but not Neurogenin-1-deficient animals. The invention relates specifically to a novel family of G protein-coupled receptors and a novel family of two-transmembrane segment proteins that are expressed in dorsal root ganglia, and a method of screening for genes specifically expressed in nociceptive sensory neurons.

What is claimed is:  
     
         1 . An isolated nucleic acid molecule selected from the group consisting of: 
 A) an isolated nucleic acid molecule comprising a sequence having at least 70% sequence identity to (1) a nucleic acid molecule that encodes the MrgD polypeptide of SEQ ID NO: 49, or (2) the complement of the nucleic acid molecule of (1); and    (B) an isolated nucleic acid molecule that hybridizes under stringent conditions to (1) a nucleic acid molecule that encodes the MrgD polypeptide of SEQ ID NO: 49, or (2) the complement of the nucleic acid molecule of (1).    
     
     
         2 . An isolated MrgD polypeptide selected from the group consisting of an polypeptide encoded by the isolated nucleic acid molecule of  claim 1  and the human MrgD polypeptide of SEQ ID NO: 35.  
     
     
         3 . The isolated nucleic acid molecule of  claim 1  operably linked to an expression control element.  
     
     
         4 . The isolated nucleic acid molecule of  claim 3  operably linked to a promoter element.  
     
     
         5 . A vector comprising the isolated nucleic acid molecule of  claim 4 .  
     
     
         6 . A host cell comprising the vector of  claim 5 .  
     
     
         7 . The host cell of  claim 6  wherein said host cell is a prokaryotic cell.  
     
     
         8 . The host cell of  claim 7  wherein said host cell is an  E. coli.    
     
     
         9 . The host cell of  claim 6  wherein said host cell is a eukaryotic cell.  
     
     
         10 . The host cell of  claim 9  wherein said host cell is a hamster embryonic kidney (HEK) cell.  
     
     
         11 . The host cell of  claim 9  wherein said host cell is a yeast cell.  
     
     
         12 . A method for producing an MrgD polypeptide comprising culturing the host cell of  claim 6  under conditions in which the protein encoded by said nucleic acid is expressed.  
     
     
         13 . A chimeric molecule comprising the MrgD polypeptide of  claim 2  fused to a heterologous amino acid sequence.  
     
     
         14 . The chimeric molecule of  claim 13  wherein said heterologous amino acid sequence is an epitope tag sequence.  
     
     
         15 . The chimeric molecule of  claim 13  wherein said heterologous amino acid sequence is an immunoglobulin constant domain sequence.  
     
     
         16 . An isolated antibody that specifically binds to an isolated MrgD polypeptide of  claim 2 .  
     
     
         17 . The isolated antibody of  claim 16  wherein said antibody is selected from the group consisting of a monoclonal antibody, an antibody fragment and a humanized antibody.  
     
     
         18 . The isolated antibody of  claim 16  wherein said antibody is selected from the group consisting of an agonist antibody and a neutralizing antibody.  
     
     
         19 . A composition of matter comprising an MrgD polypeptide of  claim 2  in admixture with a pharmaceutically acceptable carrier.  
     
     
         20 . A composition of matter comprising an anti-MrgD antibody of  claim 16  in admixture with a pharmaceutically acceptable carrier.  
     
     
         21 . An article of manufacture comprising: 
 a container;    an isolated MrgD polypeptide of  claim 2  in admixture with a pharmaceutically acceptable carrier; and    instructions for using the composition of matter to treat impaired sensory perception in a mammal.    
     
     
         22 . A method of identifying expression of an MrgD polypeptide of  claim 2  in a tissue sample obtained from a mammal comprising contacting said sample with an anti-MrgD antibody and determining binding of said antibody to the sample.  
     
     
         23 . The method of  claim 22  wherein said mammal is experiencing pain.  
     
     
         24 . The method of  claim 22  wherein the tissue sample is obtained from the dorsal root ganglion.  
     
     
         25 . A method of identifying a compound that can be used to alter pain perception in a mammal comprising the steps of: 
 a) contacting test compounds with at least a portion of an MrgD polypeptide of  claim 2;     b) identifying the test compounds that form complexes with the MrgD polypeptide;    c) measuring the effect of the test compounds identified in b) in an animal model of pain; and    d) identifying compounds that alter pain perception in the animal model as useful in altering pain perception in a mammal.    
     
     
         26 . The method of  claim 25  wherein the MrgD polypeptide is a native human MrgD polypeptide.  
     
     
         27 . The method of  claim 26  wherein the MrgD polypeptide comprises the amino acid sequence of SEQ ID NO: 35.  
     
     
         28 . The method of  claim 25  wherein the compound enhances the perception of pain.  
     
     
         29 . The method of  claim 25  wherein the compound decreases the perception of pain.  
     
     
         30 . The method of  claim 25  wherein at least one of the test compound or the MrgD polypeptide is attached to a solid support.  
     
     
         31 . The method of  claim 30  wherein said solid support is a microtiter plate.  
     
     
         32 . The method of  claim 25  wherein the MrgD polypeptide is present in a cell membrane.  
     
     
         33 . The method of  claim 25  wherein the MrgD polypeptide is present in a fraction of cell membrane prepared from cells expressing an MrgD polypeptide.  
     
     
         34 . The method of  claim 25  wherein the MrgD polypeptide is present in an immunoadhesin.  
     
     
         35 . The method of  claim 25  wherein the test compounds are selected from the group consisting of peptides, peptide mimetics, antibodies, small organic molecules and small inorganic molecules.  
     
     
         36 . The method of  claim 35  wherein the test compounds are peptides.  
     
     
         37 . The method of  claim 36  wherein the peptides are anchored to a solid support by specifically binding an immobilized antibody.  
     
     
         38 . The method of  claim 25  wherein the MrgD polypeptide is labeled.  
     
     
         39 . The method of  claim 25  wherein the test compounds are labeled.  
     
     
         40 . The method of  claim 25  wherein the test compounds are contained in a cellular extract.  
     
     
         41 . The method of  claim 40  wherein the cellular extract is prepared from cells known to express an MrgD polypeptide.  
     
     
         42 . The method of  claim 41  wherein said cellular extract is prepared from dorsal root ganglion cells.  
     
     
         43 . A method of identifying a compound that binds an MrgD polypeptide comprising the steps of: 
 a) contacting an MrgD polypeptide of  claim 2  or fragment thereof with a test compound and an RFamide peptide ligand under conditions where binding can occur; and    b) determining the ability of the test compound to interfere with binding of the RFamide peptide to the MrgD polypeptide.    
     
     
         44 . The method of  claim 43  wherein the MrgD polypeptide is a native human MrgD polypeptide.  
     
     
         45 . The method of  claim 43  wherein the MrgD polypeptide comprises the amino acid sequence of SEQ ID NO: 35.  
     
     
         46 . The method of  claim 43  wherein the MrgD polypeptide is contacted with the RFamide peptide prior to being contacted with the test compound.  
     
     
         47 . A method for identifying an MrgD agonist useful in altering sensory perception in a mammal comprising the steps of: 
 a) expressing an MrgD polypeptide of  claim 2  in a host cell capable of producing a second messenger response;    b) contacting the host cell with one or more test compounds;    c) measuring the second messenger response in the host cell; and    d) identifying compounds that increase the measured second messenger response as agonists that are useful in altering sensory perception in a mammal.    
     
     
         48 . The method of  claim 47  wherein the MrgD polypeptide is the human MrgD polypeptide of SEQ ID NO:35.  
     
     
         49 . The method of  claim 47  wherein said host cell is a eukaryotic cell.  
     
     
         50 . The method of  claim 49  wherein said host cell is a hamster embryonic kidney (HEK) cell.  
     
     
         51 . The method of  claim 50  wherein said HEK cell expresses Gα15.  
     
     
         52 . The method of  claim 47  wherein measuring a second messenger response comprises measuring a change in intercellular calcium concentration.  
     
     
         53 . The method of  claim 52  wherein said change in intercellular calcium concentration is measured with FURA-2 calcium indicator dye.  
     
     
         54 . The method of  claim 47  wherein measuring a second messenger response comprises measuring the flow of current across the membrane of the cell.  
     
     
         55 . The method of  claim 47  wherein said sensory perception is the perception of pain.  
     
     
         56 . A method for identifying an MrgD polypeptide antagonist useful in treating impaired sensory perception in a mammal comprising the steps of: 
 a) expressing an MrgD polypeptide of  claim 2  in a host cell capable of producing a second messenger response;    b) contacting the host cell with an RFamide peptide;    c) contacting the host cell with one or more test compounds;    d) measuring the second messenger response in the host cell; and    e) identifying compounds that alter the measured second messenger response to the RFamide peptide as antagonists that are useful in treating impaired sensory perception in a mammal.    
     
     
         57 . The method of  claim 56  wherein the MrgD polypeptide is the human MrgD polypeptide of SEQ ID NO:35.  
     
     
         58 . The method of  claim 56  wherein the impaired sensory perception is pain.  
     
     
         59 . A method of identifying an anti-MrgD agonist antibody useful in treating pain in a mammal comprising the steps of: 
 a) preparing candidate antibodies that specifically bind to an MrgD polypeptide of  claim 2;     b) expressing human MrgD (SEQ ID NO: 35) in a host cell known to be capable of producing a second messenger response;    c) contacting the host cell with a candidate antibody;    d) measuring the second messenger response in the host cell; and    e) identifying an antibody that increases the measured second messenger response as being an agonist antibody useful in treating pain in a mammal.    
     
     
         60 . The method of  claim 59  wherein the candidate antibodies specifically bind to human MrgD of SEQ ID NO:35.  
     
     
         61 . A method of treating pain in a mammal comprising administering to said mammal an agonist of the human MrgD polypeptide of SEQ ID NO:35.