Compositions and methods for the assay of G-protein coupled receptors and their ligands
OwnerMatthew Hsuinventor
Life of this patent
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Abstract
A cell comprising one or more endogenous promiscuous G-proteins and an exogenous nucleic acid sequence that encodes a G-protein coupled receptor (GPCR), which optionally comprises a calcium release activated calcium channel (CRAC channel) provides features and improvements that significantly improve membrane level expression of the GPCR, increase signal responses and duration of signal, and decrease background in GPCR-related assay applications.
Claims45
A cell comprising one or more endogenous promiscuous G-proteins and an exogenous nucleic acid sequence that encodes a G-protein coupled receptor (GPCR), wherein said one or more G-proteins and said GPCR are expressed at high levels.
pHS vector, deposited at the American Type Culture Collection (ATCC) as Accession Number PTA-6986.
A cell transfected with pHS vector, deposited at the American Type Culture Collection (ATCC) as Accession Number PTA-6986.
A method of identifying an agent that increases activity of a G-protein coupled receptor (GPCR) comprising:
- 1) combining:
- a) a cell that comprises one or more endogenous promiscuous G-proteins and an exogenous nucleic acid sequence that encodes a G-protein coupled receptor (GPCR), wherein said one or more G-proteins and said GPCR are expressed at high levels;
- and
- b) a test agent;
- and
- 2) detecting activity of said GPCR,
- wherein an increase in activity of said GPCR, relative to a control, indicates that said test agent increases activity of said GPCR.
A method of identifying a ligand of a G-protein coupled receptor (GPCR) comprising:
- 1) combining:
- a) a cell that comprises one or more endogenous promiscuous G-proteins and an exogenous nucleic acid sequence that encodes a G-protein coupled receptor (GPCR), wherein said one or more G-proteins and said GPCR are expressed at high levels;
- and
- b) a test ligand;
- and
- 2) detecting activity of said GPCR,
- wherein an increase in activity of said GPCR, relative to a control, indicates that said test ligand is a ligand of said GPCR.
A method of identifying an agent that modulates activity of a G-protein coupled receptor (GPCR) comprising:
- 1) combining:
- a) a cell that comprises one or more endogenous promiscuous G-proteins and an exogenous nucleic acid sequence that encodes a G-protein coupled receptor (GPCR), wherein said one or more G-proteins and said GPCR are expressed at high levels;
- b) an agent that activates said GPCR;
- and
- c) a test agent;
- and
- 2) detecting activity of said GPCR,
- wherein an alteration in activity of said GPCR, relative to a control, indicates that said test agent modulates activity of said GPCR.
A method of expressing a G-protein coupled receptor (GPCR) in a cell, comprising transfecting said cell with a nucleic acid sequence encoding said GPCR, wherein said cell comprises one or more endogenous promiscuous G-proteins, and wherein said one or more G-proteins and said GPCR are expressed at high levels.
A method of measuring an alteration in intracellular calcium in a cell comprising
- 1) combining:
- a) a cell that comprises one or more endogenous promiscuous G-proteins and an exogenous nucleic acid sequence that encodes a G-protein coupled receptor (GPCR), wherein said one or more G-proteins and said GPCR are expressed at high levels;
- and
- b) an agent that activates said GPCR;
- and
- 2) measuring intracellular calcium in said cell.
A method of coupling a G-protein coupled receptor (GPCR) to the PLCβ pathway comprising
- 1) combining:
- a) a cell that comprises one or more endogenous promiscuous G-proteins and an exogenous nucleic acid sequence that encodes a G-protein coupled receptor (GPCR), wherein:
- i) said one or more G-proteins and said GPCR are expressed at high levels;
- and
- ii) said one or more G-proteins are selected from the group consisting of a G protein of the Gαi/0 family, a G protein of the Gαs family and a G protein of the Gα12 family;
- and
- b) an agent that activates said GPCR,
- under conditions in which said GPCR is activated.
Dependencies are inferred from the claim text.