Methods of assaying receptor activity and constructs useful in such methods

Abstract

Described are methods of detecting G-protein coupled receptor (GPCR) activity in vivo and in vitro; methods of assaying GPCR activity; and methods of screening for GPCR ligands, G protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process. Constructs useful in such methods are described.

1 - 16 . (canceled) 
     
     
         17 . A method of assessing G protein coupled receptor (GPCR) pathway activity under test conditions, comprising:
 a) providing a test cell that expresses a GPCR, and contains a conjugate according to claim  1 ;   b) exposing the test cell to a known GPCR agonist under test conditions; and then   c) detecting translocation of said conjugate from the cytosol of the test cell to the membrane edge of the test cell; wherein the translocation of the detectable molecule in the test cell indicates activation of the GPCR pathway.   
     
     
         18 . A method according to  claim 17  wherein the test condition is the presence in the test cell of a test kinase. 
     
     
         19 . A method according to  claim 17  wherein the test condition is the presence in the test cell of a test G-protein. 
     
     
         20 . A method according to  claim 17  wherein the test condition is exposure of the test cell to a test ligand. 
     
     
         21 . A method according to  claim 17  wherein the test condition is coexpression in the test cell of a second receptor. 
     
     
         22 . A method for screening a β-arrestin protein or fragment thereof for the ability to bind to a phosphorylated GPCR, comprising:
 a) providing a cell that: i) expresses a GPCR; and ii) contains a conjugate according to claim  1 ;   b) exposing the cell to a known GPCR agonist; and then   c) detecting translocation of said conjugate from the cytosol of the cell to the membrane edge of the cell; wherein translocation of said conjugate in the test cell indicates β-arrestin protein binding to the phosphorylated GPCR.   
     
     
         23 . A method for screening a test compound for G protein coupled receptor (GPCR) agonist activity, comprising:
 a) providing a cell expressing a GPCR and containing a conjugate according to claim  1 ;   b) exposing the cell to the test compound; and then   c) detecting translocation of said conjugate from the cytosol of the cell to the membrane edge of the cell; wherein movement of said conjugate from the cytosol to the membrane edge of the cell after exposure of the cell to the test compound indicates GPCR agonist activity of the test compound.   
     
     
         24 . A method according to  claim 23  wherein the cell expresses a known GPCR. 
     
     
         25 . A method according to  claim 23  wherein the cell expresses an unknown GPCR. 
     
     
         26 . A method according to  claim 23  wherein the cell expresses an odorant GPCR. 
     
     
         27 . A method according to  claim 23  wherein the cell expresses a β-adrenergic GPCR. 
     
     
         28 . A method according to  claim 23  wherein said conjugate is optically detectable. 
     
     
         29 . A method according to  claim 23  wherein said conjugate comprises Green Fluorescent Protein. 
     
     
         30 . A method according to  claim 23  wherein the cell is a mammalian cell. 
     
     
         31 . A method according to  claim 23 , wherein the cell is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells and animal cells. 
     
     
         32 . A method according to  claim 23 , wherein the cell normally expresses a GPCR. 
     
     
         33 . A method according to  claim 23 , wherein the cell has been transformed to express a GPCR not normally expressed by such a cell. 
     
     
         34 . A method according to  claim 23 , where the test compound is in aqueous solution. 
     
     
         35 . A method according to  claim 23 , where the cells are deposited on a substrate. 
     
     
         36 . A method of screening a sample solution for the presence of an agonist to a G protein coupled receptor (GPCR); comprising:
 a) providing a cell expressing a GPCR and containing a conjugate according to claim  1 ;   b) exposing the cell to a sample solution; and then   c) detecting translocation of said conjugate from the cytosol of the cell to the membrane edge of the cell; wherein movement of said conjugate from the cytosol to the membrane edge of the cell after exposure of the cell to the sample solution indicates the sample solution contains an agonist for a GPCR expressed in the cell.   
     
     
         37 . A method according to  claim 36  wherein the cell expresses a known GPCR. 
     
     
         38 . A method according to  claim 36  wherein the cell expresses an unknown GPCR. 
     
     
         39 . A method according to  claim 36  wherein the cell expresses an odorant GPCR. 
     
     
         40 . A method according to  claim 36  wherein the cell expresses a β-adrenergic GPCR. 
     
     
         41 . A method according to  claim 36  wherein said conjugate is optically detectable. 
     
     
         42 . A method according to  claim 36  wherein said conjugate comprises Green Fluorescent Protein. 
     
     
         43 . A method according to  claim 36  wherein the cell is a mammalian cell. 
     
     
         44 . A method according to  claim 36 , wherein the cell is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells and animal cells. 
     
     
         45 . A method of screening a test compound for G protein coupled receptor (GPCR) antagonist activity, comprising:
 a) providing a cell expressing a GPCR, and containing a conjugate according to claim  1 ;   b) exposing the cell to a test compound;   c) exposing the cell to a GPCR agonist; and   d) detecting translocation of said conjugate from the cytosol of the cell to the membrane edge of the cell; where exposure to the agonist occurs at the same time as, or subsequent to, exposure to the test compound, and wherein movement of said conjugate from the cytosol to the membrane edge of the cell after exposure of the cell to the test compound indicates that the test compound is not a GPCR antagonist.   
     
     
         46 . A method according to  claim 45  wherein the cell expresses a known GPCR. 
     
     
         47 . A method according to  claim 45  wherein the cell expresses an odorant GPCR. 
     
     
         48 . A method according to  claim 45  wherein the cell expresses a β-adrenergic GPCR. 
     
     
         49 . A method according to  claim 45  wherein said conjugate is optically detectable. 
     
     
         50 . A method according to  claim 45  wherein said conjugate comprises Green Fluorescent Protein. 
     
     
         51 . A method according to  claim 45  wherein the cell is a mammalian cell. 
     
     
         52 . A method according to  claim 45 , wherein the cell is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells and animal cells. 
     
     
         53 . A method according to  claim 45 , where the cells are deposited on a substrate. 
     
     
         54 . A method of screening a test compound for G protein coupled receptor (GPCR) antagonist activity; comprising:
 a) providing a cell expressing a GPCR and containing a conjugate according to claim  1 ;   b) exposing the cell to a GPCR agonist so that translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell occurs;   c) exposing the cell to a test compound; where exposure to the agonist occurs prior to exposure to the test compound, and wherein movement of said conjugate from the membrane edge of the cell to the cytosol after exposure of the cell to the test compound indicates that the test compound has a GPCR antagonist activity.   
     
     
         55 . A method according to  claim 54  wherein said conjugate is optically detectable. 
     
     
         56 . A method according to  claim 54  wherein said conjugate comprises Green Fluorescent Protein. 
     
     
         57 . A method according to  claim 54  wherein the cell is a mammalian cell. 
     
     
         58 . A method according to  claim 54 , wherein the cell is selected from the group consisting of bacterial cells, yeast cells, fungal cells, plant cells and animal cells. 
     
     
         59 . A method according to  claim 54 , where the test compound is in aqueous solution. 
     
     
         60 . A method according to  claim 54 , where the cells are deposited on a substrate. 
     
     
         61 . A method of screening a cell for the presence of a G protein coupled receptor (GPCR); comprising:
 a) providing a test cell, said test cell containing a conjugate according to claim  1 ;   b) exposing the test cell to a solution containing a GPCR agonist; and   c) detecting translocation of said conjugate from the cytosol of the cell to the membrane edge of the cell; wherein movement of said conjugate from the cytosol to the membrane edge of the test cell after exposure of the test cell to the GPCR agonist indicates that the test cell contains a GPCR.   
     
     
         62 . A method according to  claim 61  wherein said conjugate is optically detectable. 
     
     
         63 . A method according to  claim 61  wherein said conjugate Green Fluorescent Protein. 
     
     
         64 . A method of screening a plurality of cells for those cells which contain a G protein coupled receptor (GPCR); comprising:
 a) providing a plurality of test cells, said test cells containing a conjugate according to claim  1 ;   b) exposing the test cells to a known GPCR agonist; and   c) detecting those cells in which said conjugate is translocated from the cytosol of the cell to the membrane edge of the cell; wherein movement of said conjugate from the cytosol to the membrane edge of a cell after exposure to the GPCR agonist indicates that the cell contains a GPCR for that known GPCR agonist.   
     
     
         65 . A method according to  claim 64  wherein said conjugate is optically detectable. 
     
     
         66 . A method according to  claim 64  wherein said conjugate comprises Green Fluorescent Protein. 
     
     
         67 . A method according to  claim 64  wherein the plurality of test cells are contained in a tissue. 
     
     
         68 . A method according to  claim 64  wherein, the plurality of test cells are contained in an organ. 
     
     
         69 . A method according to  claim 64  wherein step (b) comprises exposing the test cells to a plurality of known GPCR agonists. 
     
     
         70 - 83 . (canceled)