Luciferase Reporter Assay System for Deciphering GPCR Pathways
Zhijie Cheng, Denise Garvin, Aileen Paguio, Pete Stecha, Keith Wood, Frank Fan*
Identifiers and Pagination:Year: 2010
First Page: 84
Last Page: 91
Publisher Id: CCGTM-4-84
Article History:Received Date: 21/8/2010
Revision Received Date: 25/10/2010
Acceptance Date: 25/10/2010
Electronic publication date: 21/12/2010
Collection year: 2010
open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.5/), which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited.
The G protein coupled receptors (GPCR) represent the target class for nearly half of the current therapeutic drugs and remain to be the focus of drug discovery efforts. The complexity of receptor signaling continues to evolve. It is now known that many GPCRs are coupled to multiple G-proteins, which lead to regulation of respective signaling pathways downstream. Deciphering this receptor coupling will aid our understanding of the GPCR function and ultimately developing drug candidates. Here, we report the development of four homogenous bioluminescent reporter assays using improved destabilized luciferases and various response elements: CRE, NFAT-RE, SRE, and SRF-RE. These assays allowed measurement of major GPCR pathways including cAMP production, intracellular Ca2+ mobilizations, ERK/MAPK activ-ity, and small G protein RhoA activity, respectively using the same reporter assay format. We showed that we can decipher G protein activation profiles for exogenous m3 muscarinic receptor and endogenous β2-adrenergic receptors in HEK293 cells by using these four reporter assays. Furthermore, we demonstrated that these assays can be readily used for potency rankings of agonists and antagonists, and for high throughput screening.