Fig. (1) GPCR LinkLight assay. a, The design of LinkLight assay technology. b, A schematic draw of GPCR-TEV and β-arrestin-pLuc interaction LinkLight assay. Molecules trigger the interaction of GPCR-TEV fusion protein and β-arrestin-pLuc fusion protein, resulting in the cleavage of the inactive permuted luciferase and reconstitution of an active luciferase. c, GPCR LinkLight assay with two different designs. ADRB2-TEV and Arr2-pLuc plasmids or Arr2-TEV and ADRB2-pLuc-V plasmids were co-expressed transiently in HEK293 cells to produce ligand-induced luciferase signals. d, Dose response curves of ligands in HEK293 cells stably expressing ADRB2-TEV and Arr2-pLuc fusion proteins. e, HEK293 cells stably expressing V2R-TEV and Arr2-pLuc-V fusion proteins and their response to the V2 agonist, Arg8-vasopressin. f, cAMP assay using HEK293 cells transiently expressing ADRB2-TEV fusion gene in response to agonists, partial agonists, and antagonists. g, Cre-Luc assay using CHO cells stably expressing CRE-Luc and transiently expressing ADRB2-TEV genes in response to isoproterenol. h, FLIPR assay using HEK293 cells stably expressing Ga16 and transiently expressing ADRB2-TEV genes in response to isoproterenol. All curves were generated by using GraphPad Prism 5 software. Data points are represented as mean ± SD from n=3. EC₅₀ values were derived from non-linear regression with the best-fit equation.