Jey Cheng, Denise Garvin, Pete Stecha, Keith Wood and Frank Fan
Promega Corporation, Madison, WI
The G12 subfamily of heterotrimeric G protein has been the subject of intense interest in both basic science and drug discovery research due to its involvement in regulating cell proliferation, cell migration and metastatic invasion, where not many drug-screening assay for G12-linked pathway are available. Bioluminescent reporter assays are frequently used in high-throughput screening due to their inherently high sensitivity, wide dynamic range and low susceptibility to compound interference. Here we have incorporated the technical advancement of luciferase reporter genes and vectors to build a novel SRF-RE luciferase construct for RhoA signaling pathways. The large dynamic range of SRF-RE luciferase reporter assays allows efficient screening for GPCR modulators for G12 pathways. The dual-luciferase GPCR assay system, wherein the second plasmid expresses the target GPCR and Renilla luciferase as internal control, improves data quality by providing simultaneous monitoring of nonspecific effects such as cytotoxicity.