Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs

Abstract

The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and βarrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for G_s). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

Keywords: G protein activation; biochemistry; biosensor; chemical biology; effector membrane translocation assay; enhanced bystander bioluminescence resonance energy transfer; g protein-coupled receptor; high-throughput assay; human.

Figure 1.. EMTA ebBRET platform to monitor G protein activation and βarrestin recruitment.

(A) Schematic of the G protein Effector Membrane Translocation Assay (GEMTA) to monitor Gα protein activation. Upon receptor activation, RlucII-tagged effector proteins (Effector-RlucII) translocate towards and interact with active Gα subunits from each G protein family, leading to increased ebBRET. (B) Principle of the Effector Membrane Translocation Assay (EMTA) monitoring βarrestin recruitment to the plasma membrane (top) and Gα_s activation (bottom). Top; upon receptor activation, RlucII-tagged βarrestins (βarrestin-RlucII) translocate to the plasma membrane, thus increasing ebBRET with rGFP-CAAX. Bottom; Internalization of activated RlucII-tagged Gα_s (Gα_s-RlucII) following receptor stimulation decreases ebBRET with the membrane-anchored rGFP-CAAX.

Figure 2.. Validation of EMTA ebBRET-based platform to monitor Gα protein activation.

(A) Pharmacological validation of the Gα_i/o activation sensor. HEK293 cells were transfected with the D₂ receptor and the Gα_i/o family-specific sensor, along with each Gα_i/o subunit. Concentration-response curve using the Gα_i/o activation sensor, in the presence or absence of UBO-QIC (left) or PTX (right) inhibitors. Insets; E_max values determined from concentration-response curves of inhibitor-pretreated cells. (B) Pharmacological validation of the Gα_q/11 activation sensor. HEK293 cells were transfected with the GnRH receptor and the Gα_q/11 family-specific sensor, along with each Gα_q/11 subunit. Concentration-response curve using Gα_q/11 activation sensor, in the presence or absence of UBO-QIC (left) or PTX (right) inhibitors. Insets; E_max values determined from dose-response curves of inhibitor-pretreated cells. (C) Validation of the Gα_12/13 activation sensor. Cells were transfected with the CB₁ receptor and one of the Gα_12/13 activation sensors, along with the Gα₁₂ or Gα₁₃ subunits. Concentration-response curves of HEK293 cells (top) or the parental and devoid of G_12/13 (ΔG_12/13) HEK293 cells (bottom) using the PDZ-RhoGEF-RlucII/rGFP-CAAX (top and bottom left) or PKN-RBD-RlucII/rGFP-CAAX (bottom right) sensors, pretreated or not with UBO-QIC or PTX (top). (D) Pharmacological validation of the Gα_s activation sensor. HEK293 cells were transfected with the GPBA receptor and the Gα_s activation (left and central) or the EPAC (right) sensors. Left: Concentration-response curves using the Gα_s activation sensor in the presence or absence of UBO-QIC or PTX, inhibitors of Gα_q or Gα_i/o, respectively. Central: Concentration-response activation of the Gα_s sensor using CTX, a Gα_s activator. Right: Concentration-response curve using the EPAC sensor. Inset; E_max values determined from dose-response curves of inhibitors-pretreated cells. Data are expressed as BRET ratio for the concentration-response curves or expressed in % of respective control cells (E_max graphs) and are the mean ± SEM of 3 (A–C) or 4 (D) independent experiments performed in one replicate. Unpaired t-test (A–D): *p < 0.05 and ***p < 0.001 compared to control cells.

Figure 2—figure supplement 1.. Influence of endogenous G proteins.

Concentration-response curves elicited in parental (WT) HEK293 cells or devoid of G_s (ΔG_s), G_12/13 (ΔG_12/13), G_q/11 (ΔG_q/11), or G_i/o (ΔG_i/o) proteins, transfected with the indicated receptor (D₂, GnRHR, GIP, V₂, EP₃, or M₃) and one of the Gα_i/o, Gα_q/11, or Gα_12/13 activation sensors, along with the indicated Gα subunits. Mock condition corresponded to the response elicited in absence of heterologously expressed Gα subunits (i.e. endogenous G proteins effect). Data are the mean ± SEM of 3 -5 independent experiments performed in one replicate and are expressed as BRET² ratio. Data presented in (I) are the same as in (A–B), but with results expressed as % of maximal response elicited by endogenous G proteins (mock) in WT cells.

Figure 2—figure supplement 2.. Validation of EMTA ebBRET-based sensors selectivity for each Gα subunit families.

HEK293 cells were transfected with the ET_A receptor and Gα_i/o (A), Gα_q/11 (B), or Gα_12/13 (C) activation sensors along with each Gα subunit or control DNA (Mock) as control for response obtained with endogenous Gα proteins. Concentration-response curves in response to endothelin-1 are shown (left and central), as well as maximal responses obtained with each Gα subunit. Data are the mean ± SEM of 3 independent experiments performed in one replicate and are expressed as BRET² ratio. Unpaired t-test: ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 compared to Mock (without receptor) and one-way ANOVA test: *p < 0.05, **p < 0.01 and ***p < 0.001 compared to Mock + ET_A.

Figure 2—figure supplement 3.. Influence of G protein, GPCR or effector-RlucII level expression.

(A) Concentration-response curves elicited in HEK293 cells transfected with the B₂ receptor and one of the Gα_q/11, Gα_i/o, or Gα_12/13 activation sensors, along with increasing quantity of the indicated Gα subunits. Data represent a representative experiment (B) Concentration-response curves elicited in HEK293 cells transfected with increasing quantity of the M₃, D₂, or AT₁ receptors and the Gα_q/11, Gα_i/o, or Gα_12/13 activation sensors, along with the indicated Gα subunits. (C) Concentration-response curves elicited in HEK293 cells transfected with the ET_A receptor and increasing quantity of effector-RlucII (p63-RhoGEF for Gα_q/11, Rap1GAP for Gα_i/o or PDZ-RhoGEF for Gα_12/13), along with rGFP-CAAX and the indicated Gα subunits. Data are the mean ± SEM of 3 independent experiments performed in one replicate and are expressed in BRET² ratio.

Figure 2—figure supplement 4.. Kinetics of Gα proteins and βarrestins recruitment promoted by the ET_A receptor.

Kinetics of activation of the indicated pathways following stimulation with vehicle or Endothelin-1 in HEK293 cells expressing the ET_A receptor. Data are the mean ± SD of two replicates of a representative experiment from 3 independent experiments and are expressed in % of the respective basal response (determined before ligand addition at t = 0 sec).

Figure 2—figure supplement 5.. Comparison of EMTA platform and G protein activation BRET assay based on Gαβγ dissociation.

Concentration-response curves elicited in HEK293 cells transfected with the indicated receptor (D₂, GIP, PTH1, M₃, ET_A, B₁, FP, or Cys-LT₂) and one of the Gα_i/o, Gα_q/11, or Gα_12/13 EMTA activation sensors, along with the indicated Gα subunits, or the BRET-based Gαβγ dissociation sensors (Gα-RlucII and GFP10-Gγ₁ for Gα_q, Gα₁₂, and Gα₁₃ or GFP10-Gγ₂ for Gα_i1, Gα_i2, and Gα_oB, with untagged Gβ₁). Data are the mean ± SEM from 3-7 independent experiments performed in one replicate and results are expressed in % of the response obtained for cells treated with vehicle.

Figure 2—figure supplement 6.. Western blots of G protein level expression in cells transfected with the EMTA ebBRET platform.

G protein expression level detection in HEK293 cells transfected with the Gα_i/o, Gα_12/13, Gα_q/11, or Gα_s activation sensors along with the indicated Gα protein or control DNA (Mock). Representative immunoblots of 3 independent experiments are shown.

Figure 3.. Heatmaps illustrating the diversity of receptor-specific signaling signatures detected with the EMTA ebBRET platform.

(A) First, values within each pathway were normalized relative to the maximal response observed across all receptors (max = 1; left). These values were then normalized across pathways for the same receptor, with the highest-ranking pathway serving as the reference (max = 1; right). (B) Heatmap representation of double normalized E_max (left) and pEC₅₀ (right) data. Empty cells (grey) indicate no detected coupling. IUPHAR receptor names are displayed.

Figure 3—figure supplement 1.. Receptor-specific signaling signatures.

E_max values derived from concentration-response curves generated on 100 receptors using the 15 ebBRET-based assay are represented as radial graphs. A score of 0 indicates no coupling to a given pathway, whereas a score of 1 indicates a coupling. Receptors are rearranged according to the number of G protein families activated. Gα₁₅ has been considered apart from the G_q/11 family due to its promiscuous nature. See Supplementary file 3 that shows the concentration-response curves of the 100 receptors for the 15 different pathways.

Figure 3—figure supplement 2.. Detection of endogenous receptor-mediated responses with the EMTA ebBRET platform in HEK293 cells.

Comparison of concentration-response curves elicited by the indicated ligand for a specific pathway, following the stimulation of HEK293 cells expressing endogenous or heterologously expressed receptors. The data presented refer to the ligands where a signal was detected on non-transfected cells (endogenous expression) (See Supplementary file 3 for the curves on light gray and yellow background). Data are the mean ± SEM of at least 3 independent experiments performed in one replicate and expressed in % of the response obtained for cells treated with vehicle.

Figure 3—figure supplement 3.. Validation of G_12/13 and G₁₅ signaling for the newly characterized GPCRs.

(A) Validation of G_12/13-mediated signal using Rho and Ezrin activation sensors. HEK293 cells expressing FP or CysLT₂ receptors and the PKN-RBD-RlucII or MyrPB-Ezrin-RlucII/rGFP-CAAX sensors were pretreated or not with the Gα_q inhibitor YM-254890 and then stimulated with increasing concentrations of respective ligand. Data are the mean ± SEM from 3-5 independent experiments performed in one replicate and expressed in % of vehicle-treated cells. (B) Validation of Gα₁₅-mediated signal by measuring calcium production. Top: Kinetics of calcium release induced by the indicated ligand in HEK293 cells expressing the indicated receptor, alone or with Gα₁₅ subunit. For receptors that also couple to other G_q/11 family members, cells were pretreated with DMSO or the Gα_q inhibitor YM-254890. Bottom: The peak of calcium production obtained from kinetics were compared to the basal level of calcium (determined between 0 and 17 s). Data are the mean ± SEM from 5-7 independent experiments performed in one replicate and expressed in relative fluorescence unit (RFU). Two Way ANOVA test: *p < 0.05, **p < 0.01 and ***p < 0.001 compared to respective basal calcium level. ns: not significant.

Figure 4.. The EMTA ebBRET platform has a unique ability to uncover coupling selectivity between G protein families.

(A) Venn diagram showing the numbers of receptors coupled to each G protein family in the EMTA ebBRET biosensor assay. (B) Evaluation of receptors coupling promiscuity: number of receptors that couple to members of 1, 2, 3, or 4 G protein families. (C) Determination of G protein subunit coupling frequency: number of receptors that activate each Gα subunit. (D) Proportion of receptors recruiting βarrestins: number of receptors that do not recruit (-/-) or that recruit either (+/- or -/+) or both (+/+) βarrestin isotypes. All data are based on double normalized E_max values from Figure 3.

Figure 4—figure supplement 1.. G protein subtypes distribution across the 100 GPCRs profiled with the EMTA ebBRET-based platform.

(A) Number of receptors that can couple to 1–5 of the different subtypes from each G protein family. (B) % of receptors activating a specific G protein subtype (Y axis) that also activate another G protein subtype (X axis).

Figure 5.. Multiple applications using the EMTA ebBRET platform.

(A) Inverse agonist activity detection. Left: Gα_i2 activation in HEK293 cells transfected with the Rap1GAP-RlucII/rGFP-CAAX sensors with untagged Gα_i2 and increasing amount of A₁ receptor plasmid. Data are expressed in % of response obtained in control cells (0 ng of A₁) and are the mean ± SEM of 4–6 independent experiments performed in two replicates. One Way ANOVA test: ***p < 0.001 compared to control cells. HEK293 cells expressing the Gα_i2 activation sensor and control (Mock) or A₁ receptor plasmid were stimulated (10 min) with increasing concentrations of the indicated compound. Data are expressed in % of constitutive response obtained in vehicle-treated A₁ transfected cells and are the mean ± SEM of 4-6 independent experiments performed in one replicate. Right: Gα_z activation in HEK293 cells transfected with the Rap1GAP-RlucII/rGFP-CAAX sensors with untagged Gα_z and increasing amount of CB₁ receptor plasmid. Data are expressed in % of response obtained in control cells (0 ng of CB₁) and are the mean ± SEM of 4 independent experiments performed in one replicate. One Way ANOVA test: ***p < 0.001 compared to control cells. HEK293 cells expressing the Gα_z activation sensor and increasing amount of CB₁ receptor plasmid were directly stimulated (10 min) with increasing concentrations of the CB₁ inverse agonist rimonabant. Data are expressed as % of the response obtained in control cells (0 ng of CB₁) treated with vehicle and are the mean ± SEM of 4 independent experiments performed in one replicate. (B) Ligand-biased detection. Concentration-response curves of AT₁ for the endogenous ligand (Angiotensin II, AngII) and biased agonists [Sar1-Ile4-Ile8] AngII (SII), saralasin or TRV027. G protein and βarrestin2 signaling activity were assessed by EMTA platform. Data are expressed in % of maximal response elicited by AngII and are the mean ± SEM of 3–6 independent experiments performed in one replicate. (C) Functional selectivity of naturally occurring receptor variants. Concentration-response curves for WT or E/DRY motif Asp128Asn and Arg129His variants of GPR17 upon agonist stimulation in HEK293 cells co-expressing the indicated EMTA biosensor. Data are expressed in % of maximal response elicited by WT receptor and are the mean ± SEM of 3 independent experiments performed in one replicate.

Figure 5—figure supplement 1.. Modulation of ligand-promoted response detected by EMTA ebBRET platform by receptor constitutive activity.

(A) Concentration-response curves of Gα_i2 activation elicited by adenosine in HEK293 cells transfected with the Rap1GAP-RlucII/rGFP-CAAX sensors with untagged Gα_i2 and A₁ or A₃ receptors. Basal level of G_i2 activation detected by the GEMTA sensor in absence of heterologous receptor expression is represented by the interrupted line. Data are expressed as uBRET ratio and are the mean ± SEM of 4 independent experiments performed in one replicate. (B) Concentration-response curves of Gα_q activation elicited by serotonin in HEK293 cells transfected with the p63-RlucII/rGFP-CAAX sensors with untagged Gα_q and increasing amount of 5-HT_2C receptor plasmid. Data are expressed as BRET ratio and are the mean ± SEM of 4 independent experiments performed in one replicate.

Figure 6.. Detection of direct and indirect (trans) mechanisms of ligand polypharmacology using the G_z/G₁₅ biosensor.

(A) Test of the G_z/G₁₅ biosensor on a safety target panel. ebBRET signal was measured before and after stimulation with the indicated ligand in HEK293 cells transfected with the combined G_z/G₁₅ biosensor and one of the 24 receptors listed. (B) Cross-activation of D₂ and α_2AAR by other natural ligands. For the agonist mode read, HEK293 cells expressing D₂ or α_2AAR and either the Gα_i2, Gα_oB, or the βarrestin2 + GRK2 sensors were stimulated with increasing concentrations of the indicated ligand. For the antagonist mode read, cells were pretreated with increasing concentrations of the selective D₂ antagonist eticlopride or the selective α_2AAR antagonist WB4101 before stimulation with an EC₈₀ of the indicated ligand. Data are the mean ± SEM from 3-4 independent experiments performed in one replicate and expressed in % of the response elicited by dopamine or noradrenaline for D₂ and α_2AAR expressing cells, respectively. (C) Indirect (trans) activation of CB₁ by acetylcholine. For the agonist mode read, HEK293 cells expressing CB₁ and the Rap1GAP-RlucII/rGFP-CAAX sensors with untagged Gα_oB were stimulated with increasing concentrations of the indicated ligand. For the antagonist mode read, same cells were pretreated or not with increasing concentrations of the CB inverse agonist AM-630 (left) or the cholinergic antagonist atropine (central) before stimulation with an EC₈₀ of the indicated ligand. To evaluate the contribution of G_q/11-coupled receptor, cells were pretreated with the Gα_q inhibitor UBO-QIC and then stimulated with increasing concentrations of the indicated ligand (right). Data are the mean ± SEM from 3-5 independent experiments performed in one replicate and expressed in % of the response elicited by WIN55,212–2.

Figure 6—figure supplement 1.. Combined G_z/G₁₅ biosensor.

HEK293 cells transfected with the Rap1GAP-RlucII/p63-RhoGEF-RlucII/rGFP-CAAX sensors along with Gα_z and Gα₁₅ subunits and the indicated untagged receptor were stimulated with increasing concentrations of the indicated ligand. Data are the mean ± SEM from 3-5 independent experiments performed in one replicate and results are expressed in % of vehicle-treated cells.

Figure 6—figure supplement 2.. Validation of direct activation of α_2AAR by dopamine.

HEK293 cells expressing D₂ or α_2AAR and the Gα_i2 (A) or the Gα_oB (B) sensors were pretreated or not with the selective D₂-family antagonist eticlopride, before stimulation with increasing concentrations of dopamine. Data are the mean ± SEM from 2-4 independent experiments performed in one replicate and expressed in % of the response elicited by dopamine.

Figure 7.. Detection of endogenous receptor- and/or G protein-mediated responses in cells with the EMTA ebBRET platform.

Concentration-dependent activation of Gα_i2 protein by (A) endogenous S1P₁ receptor in iPSC-derived cardiomyocytes transfected with heterologous Gα_i2, (B) endogenous FPR2 in promyelocytic HL-60 cells transfected with heterologous Gα_i2, (C) endogenous FPR2 in promyelocytic HL-60 cells with endogenous G_i/o proteins and (D) endogenous PAR2 receptor in HEK293 cells with endogenous G_i/o proteins. In all cases, cells were co-transfected with the Rap1GAP-RlucII/rGFP-CAAX biosensor. Data are the mean ± SEM of 3-4 independent experiments performed in one replicate and are expressed as BRET² ratio in percentage of response induced by vehicle.

Author response image 1.

Author response image 2.