Reporter gene systems are used widely for studying gene regulation in response to GPCRs. There are many reporter genes available, including alkaline phosphatase, β-galactosidase, β-lactamase, green fluorescent protein and luciferase. Among them, luciferase is the reporter of choice especially in high-throughput screening due to its sensitivity, broad dynamic range, lack of endogenous activity and inherently low interference from compound libraries (2). The pGL4 Vectors containing minimal promoter and Rapid Response™ luciferase have been shown to improve the responsiveness of cAMP response element to Gs-coupled receptor. We further constructed SRE and SRF-RE reporter in various pGL4 Vectors and transiently expressed these reporters in HEK293 cells. By adding protein degradation sequences hPEST and hCL to the C-terminus of luciferase, both SRE- and SRF-RE reporters gave a larger induction response (75- to 120-fold) with a lower basal activity than did traditional luciferase without degradation signals (Figure 2). In addition, the destabilized luciferase response was rapid, reaching a maximum induction in 4–6 hours.
The pGL4.33[luc2P/SRE/Hygro] Vector and pGL4.34[luc2P/SRF-RE/Hygro] Vector contain SRE and SRF-RE, respectively, upstream of the minimal promoter and Rapid Response™ luciferase. SRE is known to respond to ternary complex factor (TCF)-dependent MAP kinase pathway, while SRF-RE, a mutant form of SRE lacking TCF binding domain, is designed to respond to SRF-dependent and TCF-independent pathways, such as RhoA activation. GPCRs, particularly those coupled with Gi and Gq can activate the MAPK pathway and induce transcriptional activation of SRE, while GPCRs coupled with G12 family are known to activate Rho guanine nucleotide exchange factors (RhoGEFs), which leads to activation of RhoA and transcriptional activation of SRF-RE (3).
HEK293 cells transiently transfected with pGL4.33 and pGL4.34 reporter vectors show a large dynamic range of luciferase expression upon activation of GPCR (Figure 3). Cells expressing m3 muscarinic receptor (Gq-coupled) and SRE reporter showed 50-fold induction after six hours of treatment with various agonists. Among the agonists, muscarinic chloride was the most potent, with an EC50 of 19nM, followed by carbachol, then pilocarpine. Two antagonists were tested in the presence of carbachol: scopolamine with an IC50 of 0.07nM was much more potent than pirenzipine. Similarly, double transfection of SRF-RE with thromboxane A2 receptor (G12-coupled) showed a dose-response curve with agonists (120-fold induction) and antagonists. The potency ranking for agonist is I-BOP (EC50 =0.16nM) > U46619 = U44069 and for antagonist, as BM-531 > SQ-29548. The orders of potency ranking are consistent with previous reports, indicating that results from luciferase reporter assay reflect biologically relevant conditions (4–5).