Universal G-protein coupled receptor reporter constructs
View Patent ↗The present invention provides universal G protein-coupled receptor (GPCR) reporter constructs. The constructs comprise a serum response element, a cAMP response element, and a multiple response element. These reporter constructs are able to detect activities of all GPCRs examined. Further provided by the present invention are host cells harboring a universal GPCR reporter construct of the invention, as well as assays for detecting modulators of GPCRs using the host cells.
1. A polynucleotide vector comprising a multiple response element (MRE), a cAMP response element (CRE) and a serum response element (SRE).
2. The vector of claim 1 , wherein the SRE comprise a sequence of CCXXXXXXGG (SEQ ID NO: 1) wherein X is A or T.
3. The vector of claim 1 , wherein the SRE is from human c-fos gene.
4. The vector of claim 1 , wherein the SRE comprises a sequence of CCATATTAGG (SEQ ID NO: 5).
5. The vector of claim 1 , further comprising a reporter gene operably linked to the MRE, the CRE, and the SRE.
6. The vector of claim 5 , wherein the reporter gene is a luciferase gene.
7. A host cell comprising a polynucleotide vector that comprises a multiple response element (MRE), a cAMP response element (GRE), and a serum response element (SRE).
8. The host cell of claim 7 which is human embryo kidney 293 (HEK-293) cell stably transfected with the vector.
9. The host cell of claim 7 , further comprising an exogenous G protein coupled receptor.
10. The host cell of claim 9 , wherein the G protein coupled receptor is encoded by a polynucleotide introduced into the host cell.
11. The host cell of claim 7 , further comprising a reporter gene operably linked to the MRE, the GRE, and the SRE.
12. A method for identifying a modulator of a G protein coupled receptor (GPCR), comprising (i) contacting a test agent with a host cell comprising the GPCR and a universal GPCR reporter vector, and (ii) identifying a change of expression level of a reporter gene from the vector relative to expression level of the reporter gene in the absence of the test agent; thereby identifying a modulator of the GPCR; wherein the GPCR reporter vector comprises a MRE, a GRE, and a SRE that are operably linked to the reporter gene.
13. The method of claim 12 , wherein the GPCR is heterologous to the host cell.
14. The method of claim 13 , wherein the GPCR is expressed from a second vector that has been introduced into the cell.
15. The method of claim 12 , wherein the GPCR is a Gi-coupled receptor, a Gs-coupled receptor, or a Gs-coupled receptor.
16. The method of claim 12 , wherein the modulator is an agonist of the GPCR.
17. The method of claim 12 , wherein the modulator is an antagonist of the GPCR.
18. The method of claim 12 , wherein the host cell is HEK-293 cell.
19. The method of claim 12 , wherein the reporter gene is a luciferase gene.