An accessible and generalizable in vitro luminescence assay for detecting GPCR activation

ABSTRACTG-protein coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here we describe the design of a generalizable and accessibleIn vitroGPCR splitNanoLuc ligandTriggeredReporter (IGNiTR), having broad and diverse applications. IGNiTR leverages the interaction between a conformationspecific binder and agonist-activated GPCR to reconstitute a split nanoluciferase. We have demonstrated IGNiTR with three Gs-coupled GPCRs and a Gi-coupled GPCR with three classes of conformation-specific binders: nanobodies, miniG proteins, and G-protein peptidomimetics. IGNiTR demonstrated binding efficacy and potency values of various Dopamine Receptor D1 (DRD1) ligands that agree well with reported values. IGNiTR also allows the use of a synthetic G protein peptidomimetic, providing easily standardized reagents for characterizing GPCRs and ligands. We demonstrated three applications of IGNiTR: 1) characterizing GPCR functionality during Nanodisc-based reconstitution process; 2) highthroughput screening of ligands against DRD1; 3) detection of opioids for in the field applications. Due to its convenience, accessibility and consistency, IGNiTR will find extensive applications in GPCR ligand detection, screening and GPCR characterization.