Assays for sensory modulators using a sensory cell specific G-protein beta subunit

Abstract

The invention identifies nucleic acid and amino acid sequences of sensory specific G-protein beta subunits that are specifically expressed in sensory cells, antibodies to such subunits, methods of detecting such nucleic acids and proteins, and methods of screening for modulators of sensory cell specific beta subunits.

1 . A method for identifying a compound that modulates taste signaling in taste cells, the method comprising the steps of: 
 (i) contacting the compound with a taste cell specific G-protein beta polypeptide, the polypeptide comprising greater than 90% amino acid sequence identity to an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5; and    (ii) determining the functional effect of the compound upon the polypeptide.    
     
     
         2 . (canceled)  
     
     
         3 . The method of  claim 1 , wherein the functional effect is a chemical effect.  
     
     
         4 . The method of  claim 1 , wherein the functional effect is a physical effect.  
     
     
         5 . The method of  claim 1 , wherein the functional effect is determined by measuring changes in intracellular cAMP, cGMP, IP3, DAG, or Ca 2+ .  
     
     
         6 . The method of  claim 5 , wherein the changes in intracellular cAMP or cGMP are measured using immunoassays.  
     
     
         7 . The method of  claim 1 , wherein the functional effect is determined by measuring binding of radiolabeled GTP to a G protein comprising the polypeptide, or to the polypeptide.  
     
     
         8 . The method of  claim 1 , wherein the functional effect is determined by measuring changes in intracellular Ca 2+ .  
     
     
         9 . The method of  claim 1 , wherein the polypeptide is expressed in a cell or cell membrane.  
     
     
         10 . The method of  claim 9 , wherein the functional effect is determined by measuring changes in the electrical activity of the cell or the cell membrane expressing the polypeptides.  
     
     
         11 . The method of  claim 10 , wherein the changes in the electrical activity are measured by an assay selected from the group consisting of a voltage clamp assay, a patch clamp assay, a radiolabeled ion flux assay, and a fluorescence assay using voltage sensitive dyes.  
     
     
         12 . The method of  claim 9 , wherein the cell is a eukaryotic cell.  
     
     
         13 . The method of  claim 1 , wherein functional effect is determined by measuring changes in the level of phosphorylation of taste cell specific proteins.  
     
     
         14 . The method of  claim 1 , wherein the functional effect is determined by measuring changes in transcription levels of taste cell specific genes.  
     
     
         15 . The method of  claim 1 , wherein the polypeptide is linked to a solid phase.  
     
     
         16 . The method of  claim 15 , wherein the polypeptide is covalently linked to a solid phase.  
     
     
         17 . The method of  claim 1 , wherein the polypeptide is recombinant.  
     
     
         18 . The method of  claim 1 , wherein the polypeptide is from a human, a mouse or a rat.  
     
     
         19 . The method of  claim 1 , wherein the polypeptide comprises an amino acid sequence of SEQ ID NO:3 or SEQ ID NO:5.  
     
     
         20 . A method for identifying a compound that modulates taste signaling in taste cells, the method comprising the steps of: 
 (i) expressing a taste cell specific G-protein beta polypeptide in a host cell, wherein the G-protein beta polypeptide comprises greater than 90% amino acid sequence identity to a polypeptide having a sequence of SEQ ID NO:3 or SEQ ID NO:5;    (ii) expressing a promiscuous G-protein alpha polypeptide and a taste cell specific G-protein coupled receptor in the host cell,    (iii) contacting the host cell with the compound that modulates taste signaling in taste cells; and    (iv) determining changes in intracellular calcium levels in the host cell, thereby identifying the compound that modulates taste signaling in taste cells.    
     
     
         21 . The method of  claim 20  wherein the promiscuous G-protein alpha polypeptide is Gα15.  
     
     
         22 . The method of  claim 20  wherein the promiscuous G-protein alpha polypeptide is Gα14.  
     
     
         23 . The method of  claim 20  wherein the taste cell specific G-protein coupled receptor is G-protein coupled receptor B3.  
     
     
         24 . The method of  claim 20  wherein the taste cell specific G-protein coupled receptor is G-protein coupled receptor B4.  
     
     
         25 . The method of  claim 20  wherein the host cell is HEK-293.  
     
     
         26 . The method of  claim 20  wherein the G-protein beta polypeptide comprises an amino acid sequence of SEQ ID NO:3.  
     
     
         27 . The method of  claim 20  wherein the G protein beta polypeptide acid sequence of SEQ ID NO:5.