Establishment of a chimeric reporting system for the universal detection and high-throughput screening of G protein-coupled receptors

G proteins, further divided into four subfamilies (Gs, Gq, G12 and Gi) based on their Gα subunits, are the primary components activated by G protein-coupled receptors (GPCRs). Current GPCR assays are limited to the evaluation of selective Gα signaling and do not allow comprehensive screening for orphan GPCRs without a known coupled Gα. Therefore, our aim was to design a chimeric reporting system that covers responses from all Gα subfamilies simultaneously. Because Gs activates cAMP response element (CRE)-driven genes whereas Gq and G12 activate serum response element (SRE)-driven genes, we therefore incorporated 2× CRE and 5× SRE (2CRE5SRE) into a promoter for driving luciferase expression. To further report Gi signals, a 2CRE5SRE-driven chimeric Gqi, in which the C-terminus of Gq is replaced by that of Gi, was integrated to switch the responses of Gi-coupled GPCRs to the Gq signaling. The novel reporter system showed a strong signal amplification when activated by neuromedin U receptor 1 (mainly activates Gq), neuromedin U receptor 2 (mainly activates Gi) or luteinizing hormone receptor (mainly through the Gs and Gq pathways). In addition, 293T cells stably carrying our reporter construct showed a similar sensitivity to the radioactive cAMP assay when revealing the constitutive signal from gain-of-function mutants of luteinizing hormone receptor. To our knowledge, this is the first reporting system capable of covering the Gs, Gq, G12 and Gi signals and revealing the phenomena of constitutively active GPCRs. Such a universal platform will benefit future high-throughput screening and drug designs for any GPCR.