Cell-based assays for G-protein-coupled receptor-mediated activities
AssigneeAtto Bioscience, Inc.
InventorsYong Yao, Liang Cao
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Abstract
Disclosed are compositions and methods for their use, such as in identifying G-protein-coupled receptors, ligands and compounds that modulate the activities of G-protein-coupled receptors. The compositions and methods employ cyclic nucleotide-gated channels and fluorescence dyes in detecting changes of intracellular cAMP levels in response to the stimulation of G-protein-coupled receptors. Activation of the G-protein-coupled receptors can be detected in a variety of assays, including cell-based imaging assays with fluorescence microscopes and high throughput assays with multi-well plates and fluorescence plate readers.
Claims55
A method of detecting activity of a G protein-coupled receptor (GPCR) in response to ligand binding, comprising:
- (a) expressing the GPCR in a cell from an exogenous nucleic acid molecule;
- (b) expressing in the cell a mutant cyclic nucleotide-gated (CNG) channel comprising at least one mutation that makes the channel more sensitive to cAMP than a channel that does not comprise the mutation;
- (c) exposing the cell to at least one membrane potential dye that produces a fluorescent signal in response to cell depolarization;
- (d) exposing the cell to said ligand;
- and
- (e) measuring detectable fluorescent signal from the dye in the cell indicative of activity of the channel, wherein activity of the channel indicates activity of the GPCR in response to said ligand.
A method according to 1, wherein the dye is a voltage sensitive dye.
A method of identifying a putative ligand for a G protein coupled receptor, comprising:
- (a) contacting a cell with a compound wherein the cell expresses the receptor and at least one cyclic nucleotide-gated (CNG) channel, wherein the receptor is not endogenous to the cell and the CNG channel is a mutant CNG channel that has been engineered to increase the channel sensitivity to cAMP;
- (b) exposing the cell to at least one membrane potential dye that produces a fluorescent signal in response to cell depolarization;
- and
- (c) measuring detectable fluorescent signal from the dye in the cell indicative of activity of the CNG channel, wherein activation of the CNG channel indicates that the compound is a putative ligand for the receptor.
A method according to 20, wherein the dye is a voltage potential sensitive dye.
A method of identifying a putative agent that modulates an activity mediated by a G protein-coupled receptor (GPCR) comprising:
- (a) contacting a cell with the agent and a ligand for the GPCR wherein the cell expresses the GPCR and at least one cyclic nucleotide-gated (CNG) channel and, wherein the CNG channel is a mutant CNG channel that has been engineered to increase the channel sensitivity to cAMP;
- (b) exposing the cell to at least one membrane potential dye that produces a fluorescent signal in response to cell depolarization;
- and
- (c) measuring detectable fluorescent signal from the dye in the cell indicative of activity of the CNG channel, wherein a putative agent is identified when the activity in the presence of the agent is greater or lesser than the activity in the absence of the agent.
A method according to 43, wherein the dye is a voltage sensitive dye.
Dependencies are inferred from the claim text.