Methods of assaying receptor activity and constructs useful in such methods
AssigneeDuke University
InventorsLawrence S. Barak, Marc G. Caron, Stephen S. Ferguson, Jie Zhang
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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.
Claims84
A method of assessing the effect of a test condition on G protein coupled receptor (GPCR) pathway activity, comprising:
- a) providing a cell that expresses a GPCR, and that contains a conjugate comprising a .beta.-arrestin protein and an optically detectable molecule;
- b) exposing the cell to an agonist for said GPCR under said test condition;
- c) detecting translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell;
- and then
- d) comparing any translocation detected in step (c) to that which occurs in the absence of said test condition;
- wherein increased translocation of the detectable molecule in the cell compared to that which occurs in the absence of said test condition indicates increased GPCR activation compared to activation that occurs in the absence of said test condition, and decreased translocation of the detectable molecule in the cell compared to that which occurs in the absence of said test condition indicates decreased GPCR compared to activation that occurs in the absence of said test condition.
A method for screening a .beta.-arrestin protein or fragment thereof for the ability to bind to phosphorylated GPCRs, comprising:
- a) providing a cell that:
- i) expresses at least one GPCR;
- and
- ii) contains a conjugate comprising a test .beta.-arrestin protein and an optically detectable molecule;
- b) exposing the cell to an agonist for said at least one GPCR;
- and then
- c) detecting translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell;
- wherein translocation of the detectable molecule in the cell indicates .beta.-arrestin protein binding to the phosphorylated GPCR.
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 comprising a .beta.-arrestin protein and an optically detectable molecule;
- b) exposing the cell to a test compound;
- and then
- c) detecting translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell;
- wherein movement of the detectable molecule 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.
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, the conjugate comprising a .beta.-arrestin protein and an optically detectable molecule;
- b) exposing the cell to a sample solution;
- and then
- c) detecting translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell;
- wherein movement of the detectable molecule 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.
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, the conjugate comprising a .beta.-arrestin protein and an optically detectable molecule;
- b) exposing the cell to a test compound;
- c) exposing the cell to an agonist for said GPCR;
- and
- d) detecting translocation of the detectable molecule from the cytosol 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 the detectable molecule 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 an antagonist for said GPCR.
A method for screening a test compound for G protein coupled receptor (GPCR) antagonist activity;
- comprising:
- a) providing a cell expressing a GPCR and containing a conjugate, the conjugate comprising a .beta.-arrestin protein and an optically detectable molecule;
- 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;
- and
- d) detecting translocation of the detectable molecule from the membrane edge of the cell to the cytosol of the cell;
- where exposure to the agonist occurs prior to exposure to the test compound, and wherein movement of the detectable molecule 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.
A method of screening a cell for the presence of a G protein coupled receptor (GPCR) responsive to a GPCR agonist, comprising:
- a) providing a cell, said cell containing a conjugate comprising a .beta.-arrestin protein and an optically detectable molecule;
- b) exposing the cell to a GPCR agonist;
- and
- c) detecting translocation of the detectable molecule from the cytosol to the membrane edge of the cell;
- wherein movement of the detectable molecule from the cytosol to the membrane edge of the cell after exposure of the cell to the GPCR agonist indicates that the cell contain a GPCR responsive to said agonist.
A method of screening a plurality of cells for those cells which contain a G protein coupled receptor (GPCR) responsive to a GPCR agonist, comprising:
- a) providing a plurality of cells, said cells containing a conjugate comprising a .beta.-arrestin protein and an optically detectable molecule;
- b) exposing the cells to a GPCR agonist;
- and
- c) detecting those cells in which the detectable molecule is translocated from the cytosol of the cell to the membrane edge of the cell;
- wherein movement of the detectable molecule from the cytosol to the membrane edge of a cell after exposure to the GPCR agonist indicates that the cell contains a GPCR for said GPCR agonist.
A substrate having deposited thereon a plurality of cells, said cells expressing at least one GPCR and containing a conjugate, the conjugate comprising a .beta.-arrestin protein and a detectable molecule selected from the group consisting of optically detectable molecules and immunologically detectable molecules.
A method of detecting G protein coupled receptor (GPCR) pathway activity in a cell expressing at least one GPCR and containing .beta.-arrestin protein conjugated to an optically detectable molecule, said method comprising detecting translocation of the detectable molecule from the cytosol of the cell to the membrane edge of the cell, wherein said translocation of the detectable molecule indicates activation of the GPCR pathway.
Dependencies are inferred from the claim text.