生物
荧光素酶
报告基因
G蛋白偶联受体
信号转导
细胞生物学
基因
计算生物学
基因表达
转染
遗传学
标识
DOI:10.1016/bs.mcb.2018.08.001
摘要
G protein-coupled receptors (GPCRs) play an active role in numerous cellular processes, from cell proliferation to differentiation, by modulating gene transcription through various signal transduction pathways. Transcriptional regulation coupled to reporter gene expression may be used to study both G protein-dependent and G protein-independent responses activated by GPCR ligands. Reporter genes are typically used to monitor changes in receptor-mediated cellular responses at the transcription/translation level. Genetic reporter assays are based on reporter gene expression in response to activation of specific signaling cascade, followed by monitoring the presence of the reporter protein by directly measuring its enzymatic activity. These optimized genes are expressed under the control of a response element to assess its transcriptional activity that can be readily detected by a luminescent signal. Firefly luciferase gene has been widely used as a genetic reporter that responds rapidly to modulation of a GPCR by agonists or antagonists. Luciferase assays have been successfully developed for deorphanization of GPCRs, high-throughput screening (HTS) applications for drug discovery and deciphering both canonical and non-canonical signaling of numerous GPCRs. The protocol outlined for STAT3-driven luciferase assay could be adapted with appropriate changes to any aspect of GPCR signaling.
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