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<p>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<sub>q/11</sub>-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<sup>+</sup> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月26日

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  • Edward Wills,
  • Anjana Saji,
  • Jonathan Sumner,
  • Aman Khan,
  • Claudia M. Sisk,
  • Mona Shehata,
  • Benjamin Taylor,
  • Graham Ladds

摘要

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 Gαq/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.