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

Abstract

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.

Figure 1.. Optimization workflow for the exemplar Gαq biosensor.

(a-c) RLuc8 donor positioning. (a) The inactive Gαq/Gβ1/γ2 crystal structure (PDB 3AH8) defined regions within the alpha-helical domain (red box) in close proximity to the N-terminus of the Gγ-subunit (green box). Twenty Gαq-RLuc8 chimeric proteins were generated between αA-αB and αB-αC helices. (b) Gαq-RLuc8 chimeras were evaluated in duplicate using the prototypic Gαq-coupled NT₁R neurotensin receptor. Performance was evaluated as fold-increase in dynamic range (Net BRET2) relative to the reference construct (insertion of RLuc8 after Lys97 in Gαq was named position 98). (c) The top five RLuc8 positions (119, 122, 123, 125, 126; boxed) were confirmed (N=3) and Gαq(125)-RLuc8 was chosen as the optimal chimeric donor (panel c, boxed). (d) Gγ-GFP2 optimization: the Gαq(125)-RLuc8 chimera was tested alongside each of 12 N-terminally fused Gγ-GFP2 constructs and a co-precipitated mixture of Gβ1–4 subunits. The Gγ9-GFP2 chimera provided the largest signal (N=3). (e) Gβ optimization: Gαq(125)-RLuc8 and Gγ9-GFP2 were used to screen each of four Gβ subunits (N=3). Stepwise optimization determined that Gαq(125)-RLuc8/Gβ3γ9-GFP2 was the optimal biosensor composition.

Figure 2.. Switch III in the Gα-subunit is a novel region for protein engineering.

(a) For challenging G proteins like Gα11, we identified a new site for RLuc8 insertion located between the β4-strand and α3 helix of the Ras-like domain (denoted Switch III, dashed box). The closest residue in the Gα11 model to the Gγ N-terminus was Glu241 (inset). (b) Schematic delineating the Switch III region and RLuc8 insertion sites for Gα11. (c) Donor optimization results for Gα11 Switch III RLuc8 insertions (N=1, two technical replicates; top 5 positions were 239, 241, 244, 245, 246 (boxed). (d) Gα11(246)-RLuc8 was confirmed as the optimal donor chimera (N=3). (e) Gγ13-GFP2 (boxed) was selected as the optimal BRET2 acceptor (N=3). (f) Gβ3 was selected as the optimal Gβ subunit, yielding Gα11(246)-RLuc8/Gβ3γ13-GFP2 as the final biosensor composition (N=3). Data presented as mean values ± SEM.

Figure 3.. Head-to-head comparisons of TRUPATH biosensors to first-generation BRET2 biosensors.

TRUPATH biosensors are shown in purple and first-generation biosensors are shown in black (published biosensors have green triangles; equivalent RLuc8 positioning was used for previously undisclosed G proteins). Scatter plots show fold difference in amplitude between comparator and TRUPATH biosensors (*two-tailed t-test, p<0.05). Additional comparisons are made in Supplemental Figure 18. (a) (Comparator) Gαi3(92)-RLuc8/Gβ1γ2-GFP2 < (TRUPATH) Gαi3(99)-RLuc8/Gβ3γ9-GFP2, p < 0.0001. (b) (Comparator) GαoA(92)-RLuc8/Gβ1γ2-GFP2 < (TRUPATH) GαoA(92)-RLuc8/Gβ3γ8-GFP2, p = 0.0007 (c) (Comparator) GαZ(92)-RLuc8/Gβ1γ2-GFP2 < (TRUPATH) GαZ(114)-RLuc8/Gβ3γ1-GFP2, p < 0.0001. (d) (Comparator) GαGustducin(92)-RLuc8/Gβ1γ2-GFP2 < (TRUPATH) GαGustducin(117)-RLuc8/Gβ3γ1-GFP2, p < 0.0001. (e) (Comparator) GαsS(100)-RLuc8/Gβ1γ1-GFP2 < (TRUPATH) GαsS(123)-RLuc8/Gβ3γ9-GFP2, p < 0.0001. (f) (Comparator) Gαq(98)-RLuc8/Gβ1γ1-GFP2 < (TRUPATH) Gαq(125)-RLuc8/Gβ3γ9-GFP2, p < 0.0001. (g) (Comparator) Gα11(98)-RLuc8/Gβ1γ1-GFP2 < (TRUPATH) Gα11(246)-RLuc8/Gβ3γ13-GFP2, p < 0.0001. (h) (Comparator) Gα15(101)-RLuc8/Gβ1γ2-GFP2 < (TRUPATH) Gα15(245)-RLuc8/Gβ3γ13-GFP2, p < 0.0001. (i) (Comparator) Gα12(115)-RLuc8/Gβ1γ2-GFP2 < (TRUPATH) Gα12(134)-RLuc8/Gβ3γ9-GFP2, p < 0.0001. (j) (Comparator) Gα13(107)-RLuc8/Gβ1γ2-GFP2 < (TRUPATH) Gα13(126)-RLuc8/Gβ3γ9-GFP2, p < 0.0001. Data presented as mean values ± SEM from three biological replicates. Raw values are reported in Supplementary Table 2.

Figure 4.. TRUPATH screens of prototypic and understudied GPCRs reveal varying degrees of transducer promiscuity.

Transducerome profiles of endogenous agonists for prototypic receptors (β₂AR β-adrenergic and NT₁R neurotensin) and understudied receptors (LPA₆ LPA and 5-HT₇ serotonin) demonstrate varying degrees of promiscuity. (a) Potency (Log EC₅₀) values are non-uniform across the transducerome for receptor-ligand pairs. (b) Relative amplitude (E_max or efficacy) of agonist-induced stimulation of TRUPATH biosensors is frequently non-uniform for a given receptor-ligand pair. Data presented as mean values ± SEM. Heat map values represent mean values. Mean values, standard error, and replicate numbers are reported in Supplementary Dataset 1. Statistically significant differences between efficacies and potencies are reported in Supplementary Datasets 2 and 3, respectively.

Figure 5.. TRUPATH screens of κ-opioid receptor (κOR) agonists reveal unappreciated transducer-selective effects on potency and efficacy.

TRUPATH heatmaps demonstrate how a panel of κOR agonists engage Gαi/o-class transducers with varying potency (a) and efficacy (b). Most ligands exhibit enhanced (GαZ) and diminished (GαGustducin) potencies relative to other G protein transducers. While many ligands activated all transducers with equal efficacy (Salvinorin A, U69,593, GR89,696, ML139, and RB64), others exhibited efficacy bias (BU74, dynorphin A, and diprenorphine). Heatmap colors represent mean Log EC50 and normalized efficacy values. Mean values, standard error, and N are reported in Supplementary Table 4. Statistical analyses of transducer-specific comparisons are reported in Supplementary Datasets 4 (efficacy) and 5 (potency).