Luciferase Reporter Assay System for Deciphering GPCR Pathways

Abstract

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 Ca(2+) 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 m(3) muscarinic receptor and endogenous β(2)-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.

Keywords: GPCR; Luciferase; Reporter assays; profile of receptor/G protein coupling..

Fig. (1)

Schematic diagram showing major GPCR signaling pathways. Upon stimulation, G_s coupled receptors activate adenylyl cyclase (AC) resulting in an increase of cAMP; G_i-coupled receptors inhibit AC, and the βγ subunits activate ERK pathway; G_q coupled receptors activate phospholipase C (PLC) to produce inositol trisphosphate (IP3) and diacylglycerol (DAG), which in turn increases intracellular calcium concentration (Ca²⁺); G₁₂-coupled receptors activate small G protein RhoA GTPase.

Fig. (2)

Destabilized luciferase genes increase the reporter response and reduce the assay time to reach maximum induction. luc2, firefly luciferase; luc2P, firefly luciferase with hPEST sequence; luc2CP, firefly luciferase with hCL1 and hPEST sequences. HEK293 cells transiently expressing various versions of SRE- or SRF-RE- reporters (luc2, luc2P or luc2CP) were serum starved, induced with 20% FBS plus 10ng/ml PMA for SRE (2B, 2D), or with 20% FBS for SRF-RE (2C, 2E) for 2-8 hours. Cells not induced after serum starvation were used as control (uninduced). Firefly luciferase activity (RLU) were determined using One-Glo assay system and measured on the GloMax 96 multiplate luminometer. (2B, 2C) Firefly luciferase activity (RLU) and fold of induction measured after six hours of induction. (2D, 2E) Time course of fold of induction of SRE- and SRF-RE measured every two hours for 8 hours after induction.

Fig. (3)

GPCR assays for G_s, G_i, G_q, and G₁₂ coupled receptors in 384-well format. HEK293 cells used are either stably expressing D₁ dopamine receptor and CRE-luc2P (A), m3 muscarinic receptor with NFAT-RE-luc2P reporter (B), SRF-RE-luc2P reporter (test endogenous EDG receptor) (D), or transiently expressing m₄ muscarinic receptor with SRE-luc2CP reporter (C). Cells were induced with various agonists in 384-well format. Agonists used (time of induction): 1 µM dopamine (4 hours); 1 µM muscarine chloride (8 hours); 10 µM acetylcholine (2 hours); 10 µM LPA (6 hours); Firefly luciferase activity was measured using One-Glo assay system. Fold of induction and Z’ values were determined as described in “Materials and Methodology”.

Fig. (4)

Profiling of receptor/G protein coupling. HEK293 cells transiently expressing various luciferase reporters as indicated: CRE-luc2P (A), NFAT-RE-luc2P (B), SRE-luc2P (C) or SRF-RE-luc2P (D) were induced with carbachol (for exogenously introduced m3 muscarinic receptor) or isoproterenol (for endogenous β₂-adrenergic receptor) for six hours in 96-well format. Firefly luciferase activity was measured using One-Glo assay system.

Fig. (5)

Potency ranking of agonists and antagonists for m3 muscarinic receptor using NFAT-RE- and SRE- reporters. HEK293 cells were transiently transfected with NFAT-RE-lluc2Pl (A, B) or SRE-luc2P (C, D) along with m₃ muscarinic receptor (M₃R)-lRenillal fusion for twenty-four hours. Firefly luciferase activity was measured after 6 hours induction with addition of 1:3 serial dilutions of various agonists (A, C) or antagonists in the presence of 100 nM muscarine chloride (B, D) using the Dual-Glo assay system in 96-well format. Fold of induction and % of control were determined as described in “Materials and Methodology”.