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TRUPATH, an open-source biosensor platform for interrogating the GPCR transducerome


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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Fig. 1: Optimization workflow for the exemplar Gαq biosensor.
Fig. 2: Switch III in the Gα-subunit is a novel region for protein engineering.
Fig. 3: Head-to-head comparisons of TRUPATH biosensors to first-generation BRET2 biosensors.
Fig. 4: TRUPATH screens of prototypic and understudied GPCRs reveal varying degrees of transducer promiscuity.
Fig. 5: TRUPATH screens of κOR agonists reveal unappreciated transducer-selective effects on potency and efficacy.

Data availability

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 (


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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.

Author information

Authors and Affiliations



R.H.J.O., J.F.D., J.G.E., B.L.R. and R.T.S. developed the concept and designed the experiments. R.H.J.O. and J.F.D. performed the bulk of the molecular biology and experimentation, with A.M.G., B.E.K., S.T.S., T.C., J.D.M., A.C.G. and R.T.S. contributing to the in vitro pharmacology assays. J.G.E. established the cloning strategy and helped with construct generation. R.H.J.O., J.F.D., J.G.E., B.L.R. and R.T.S. wrote and edited the manuscript. This work was supported by NIH grants (nos. R37DA035764, U24DK116195 and RO1MH112205 to B.L.R., KO1MH109943 to R.T.S. and F31NS093917 to R.H.J.O.) and the Michael Hooker Distinguished Professorship to B.L.R.

Corresponding authors

Correspondence to Bryan L. Roth or Ryan T. Strachan.

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Competing interests

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.

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Supplementary information

Supplementary Information

Supplementary Tables 1–8, Figs. 1–24 and Note.

Reporting Summary

Supplementary Dataset 1

TRUPATH transducerome profiling analysis of β2AR, NT1R, LPAR6 and 5-HT7.

Supplementary Dataset 2

Statistical analysis of transducer-specific efficacy (Emax) values reported in Supplementary Data Set 1.

Supplementary Dataset 3

Statistical analysis of transducer-specific potency (log EC50, M) values reported in Supplementary Data Set 1.

Supplementary Dataset 4

Comparisons of ligand efficacy (Emax, % maximal transducer response from Supplementary Table 4.2) between transducers, within each drug.

Supplementary Dataset 5

Comparisons of ligand potency (log EC50, M; from Supplementary Table 4.1) between transducers, within each drug.

Supplementary Dataset 6

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 16, 841–849 (2020).

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