Enhancement of a nuclear factor of activated T cells (NFAT) reporter for the study of G protein-coupled receptors

Abstract Cell-based assays are fundamental to G protein-coupled receptor (GPCRs) drug discovery. As the field strives to increase the use of physiological cell types with endogenous receptor expression, enhancing the sensitivity of simple-to-use assays unlocks new screening modalities. Here, we enhanced the responsivity of a Nuclear Factor of Activated T cells response element (NFAT-RE) reporter, by concatenating the IL-2 promoter-derived triplicate-binding sites, to produce three nano-luciferase reporter constructs termed NFAT 2X, NFAT 3X and NFAT 4X. Our enhanced reporters demonstrate larger maximal Fold Induction (FI) when co-expressed with both primarily and secondarily Gα q/11 -coupled GPCRs. This pattern was maintained when stimulating endogenous GPCRs in a panel of immortalised cell lines (HEK293, HeLa, A549, and HEK293T) and allowed us to observe Sphingosine-1-Phosphate (S1P)-mediated signalling in primary human CD8 + T cells via CRISPR/Cas9 knock-in. Our NFAT-reporter T cells demonstrate the reporters potential for use in bi-allelic expression systems and primary cell types.