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 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.

Apela Regulates Fluid Homeostasis by Binding to the APJ Receptor to Activate Gi Signaling

“…In addition, we monitored apela induction of the phosphorylation of p42/44 ERKs, known to be downstream of Gi/Go signaling (23). As shown in Fig.…”

Multi-functional norrin is a ligand for the LGR4 receptor

“…As shown in supplementary material Fig. S2, norrin is not capable of stimulating different G proteins (Gs, Gi, Gq and G12) mediated by LGR4, 5 or 6 (as determined by CRE-, SRE-, NFAT-and SRF-RE reporter assays) (Cheng et al, 2010). Because LGR4, 5 and 6 are known to mediate Wnt signaling (de Lau et al, 2011), we further tested the ability of norrin to activate Wnt signaling mediated by these receptors.…”

FTY720 Phosphate Activates Sphingosine-1-Phosphate Receptor 2 and Selectively Couples to Gα12/13/Rho/ROCK to Induce Myofibroblast Contraction

“…Activation of Rho/ROCK Signaling by FTY720-P via S1P 2 ROCK signaling (Liu and Wu, 2004;Cheng et al, 2010). Since, in contrast to S1P, FTY720-P did not activate calcium signaling in CHO-S1P 2 cells, we conclude that FTY720-P, at least in CHO-S1P 2 cells, behaved as a biased agonist that selectively activated Ga 12/13 /Rho/ROCK signaling in the absence of the other second messenger release.…”

“…The second messenger experiments in this study suggested an absence of cAMP and calcium modulation by FTY720-P through S1P 2 receptors. Furthermore, impedance experiments suggested a selective activation of the Ga 12/13 /Rho/ ROCK pathway by FTY720-P. To confirm the activation of the Ga 12/13 /Rho/ROCK pathway by FTY720-P and S1P in CHO-S1P 2 cells, we employed a Ga 12/13 /Rho/ROCK pathwayspecific reporter gene assay measuring transcriptional activation of firefly luciferase under control of the serum response factor response element (SRF-RE) (Mao et al, 1998;Liu and Wu, 2004;Cheng et al, 2010;Zhang and Xie, 2012). After transient transfection of the CHO-S1P 2 cells with the reporter gene construct, cells were stimulated with different concentrations of FTY720-P, S1P, ponesimod, positive control LPA, or vehicle (Fig.…”