Extended Data Fig. 1: Domain structure and optimization of PAGERTF.
From: Synthetic GPCRs for programmable sensing and control of cell behaviour
a, Domain structures of PAGERTF (without antagonist) and Arrestin-TEVp. b, Schematic showing how furimazine instead of light can be used to uncage LOV-TEVcs in PAGERTF. This is based on our previous SPARK tools14,15. NanoLuc luciferase fused to arrestin-TEVp is recruited to activated PAGERTF and uncages the LOV domain via BRET when furimazine (NanoLuc’s substrate) is present. Uncaging leads to TEVcs cleavage by TEVp and TF translocation to the nucleus. Alternatively, these same PAGERTF constructs can be uncaged using blue light. c, Schematic and experimental timeline for PAGERTF assay. d–f, Optimization of PAGERTF construct. SalB dose response curves of PAGERTF constructs with various truncations in KORD (d), various linkers or RAA mutation in KORD (e), and various signal peptides (f). RAA refers to V360A/R361A mutations (-RVR- to -RAA-) in the KORD gene. IL2SP, hGHSP, and InsSP are Interleukin-2 (IL2), human growth hormone (hGH) and insulin signal peptides, respectively. Full length KORD (V360A/R361A) with an IL-2 signal peptide was chosen as the optimal PAGERTF construct and used in all subsequent experiments. Plots are mean ± s.d. for 3 technical replicates per condition, representing n = 2 independent experiments. g, Crystal structure of the Kappa-opioid receptor (KOR) in complex with dynorphin A (PDB: 8F7W)13. Dynorphin binds KOR with its N-terminus buried in the orthosteric binding pocket. h, List of candidate peptide antagonists screened in PAGERTF in Fig. 1c. i, Domain structure of α-GFP PAGERTF including an N-terminal antagonist and TEVcs in the extracellular linker between KORD and nanobody.
