G-protein-coupled receptors (GPCRs) remain major drug targets, despite our incomplete understanding of how they signal through 16 non-visual G-protein signal transducers (collectively named the transducerome) to exert their actions. To address this gap, we have developed an open-source suite of 14 optimized bioluminescence resonance energy transfer (BRET) Gαβγ biosensors (named TRUPATH) to interrogate the transducerome with single pathway resolution in cells. Generated through exhaustive protein engineering and empirical testing, the TRUPATH suite of Gαβγ biosensors includes the first Gα15 and GαGustducin probes. In head-to-head studies, TRUPATH biosensors outperformed first-generation sensors at multiple GPCRs and in different cell lines. Benchmarking studies with TRUPATH biosensors recapitulated previously documented signaling bias and revealed new coupling preferences for prototypic and understudied GPCRs with potential in vivo relevance. To enable a greater understanding of GPCR molecular pharmacology by the scientific community, we have made TRUPATH biosensors easily accessible as a kit through Addgene.
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All data that were generated or analyzed during this study are included in this Article and its Supplementary Information files or are available from the corresponding authors upon reasonable request. All TRUPATH sensors are available to academic and non-profit institutions as a kit through Addgene (https://www.addgene.org/).
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We thank M. Bouvier for the gift of the Gβ1 and Gγ2-GFP2 constructs, T. Kenakin for insights regarding quantitative pharmacology and J. Aubé for the gift of ML139. We also thank A. Inoue for the donation of the HEK293ΔG cells. Due to space constraints we could not include all citations; for this we apologize.
R.H.J.O., J.F.D., J.G.E., B.L.R. and R.T.S. are inventors of the TRUPATH technology and could receive royalties. These relationships have been disclosed to and are under management by UNC-Chapel Hill.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Tables 1–8, Figs. 1–24 and Note.
TRUPATH transducerome profiling analysis of β2AR, NT1R, LPAR6 and 5-HT7.
Statistical analysis of transducer-specific efficacy (Emax) values reported in Supplementary Data Set 1.
Statistical analysis of transducer-specific potency (log EC50, M) values reported in Supplementary Data Set 1.
Comparisons of ligand efficacy (Emax, % maximal transducer response from Supplementary Table 4.2) between transducers, within each drug.
Comparisons of ligand potency (log EC50, M; from Supplementary Table 4.1) between transducers, within each drug.
Comparisons of agonist transduction coefficients (log (Emax/EC50) from Supplementary Table 4.3) between transducers, within each drug.
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Olsen, R.H.J., DiBerto, J.F., English, J.G. et al. TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome. Nat Chem Biol (2020). https://doi.org/10.1038/s41589-020-0535-8