Signal profiling of the β1AR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by β1AR and β2AR

Abstract

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β₁-adrenergic receptor (β₁AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the βAR. These include coupling to G_z and G₁₂ pathways. The signalling cascade linking the β₁AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both G_s and G₁₂ are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.

Figure 1

Identification of the Gα protein involved in β₁AR signalling. (a) Schematic representation of the Gα-Rluc/Gγ-GFP biosensor used to identify the Gα proteins involved in β₁AR signalling. (b–k) HA-β₁AR HEK 293 stable cell lines were transfected with a constant amount of Gα-Rluc (BRET donor) and untagged Gβ₁, along with increasing amounts of Gγ₁-GFP construct (BRET acceptor). Cells were stimulated (red curves) or not (black curves) with 1 μM isoproterenol and BRET values collected. NetBRET values were calculated by subtracting the background BRET signal detected in cells expressing the Rluc-fused constructs alone (donor-Rluc) from the BRET values obtained in cells expressing the energy donor and acceptor (donor-Rluc and acceptor-GFP). BRET titration curves were generated for 10 different Gα subunits: Gα_s (b), Gα_q (c), Gα₁₂ (d), Gα₁₃ (e), Gα_i1 (f), Gα_i2 (g), Gα_i3 (h), Gα_z (i), Gα_oA (j) and Gα_oB (k). Values represent mean ± SEM of 3 independent experiments performed in triplicate. Responses for Gα_s, Gα_i2, Gα_z and Gα₁₂ were further analyzed in subsequent sections.

Figure 2

Gα_s activation and cAMP production induced by the β₁AR. (a) Schematic representation of the Gα_s-Rluc/Gγ₁-GFP biosensor used to study the Gα_s induced β₁AR signalling. (b) Schematic representation of the cAMP biosensor used to study the cAMP increase induced by the β₁AR Gα_s activation. HEK 293 cells were transfected with (c,e) Gα_s-Rluc, Gγ₁-GFP and untagged Gβ₁ or with the (d,f) EPAC biosensor, along with the β₁AR. Kinetic curves represent time course of (c) Gα_s activation (vehicle and isoproterenol, n=3) or (d) cAMP accumulation (vehicle and isoproterenol, n=3) expressed as absolute BRET ratio. Concentration-responses curves were generated for (e) Gα_s activation and (f) cAMP accumulation following β₁AR activation by the indicated ligands. Data were normalized to maximal isoproterenol response, which was taken as 100%, and are expressed as mean ± SEM values. Detail of the number of experiments, maximal responses, pEC₅₀ values and statistical comparisons of curve parameters for different ligands are provided in Supplementary Tables S1 and S2.

Figure 3

Gα_i2-induced activation by the β₁AR and β₂AR. (a) Schematic representation of the Gα_i2-Rluc/Gγ₁-GFP biosensor used to study the Gα_i induced β₁AR and β₂AR signalling. HEK 293 cells were transfected with (b-e) Gα_i2-Rluc, Gγ₁-GFP and untagged Gβ₁, along with (b-c,e) β₁AR or (d-e) β₂AR. (b,d) Kinetics curves represent time course of Gα_i2 activation by (b) β₁AR (vehicle and isoproterenol, n=3) or (d) β₂AR (vehicle and isoproterenol, n=3) expressed as absolute BRET ratios. (c) Concentration-responses curves for Gα_i2 activation following β₁AR activation by indicated ligands. (e) Concentration-responses curves for Gα_i2 activation following isoproterenol-induced activation of β₁AR or β₂AR (n=2). Data were normalized to maximal isoproterenol response, which was take as 100%, and are expressed as mean ± SEM values. Detail (c) of the number of experiments, maximal responses, pEC₅₀ values and statistical comparisons of curve parameters for different ligands are provided in Supplementary Tables S1 and S2.

Figure 4

Gα_z-induced activation by the β₁AR and β₂AR. (a) Schematic representation of the Gα_z-Rluc/Gγ₁-GFP biosensor used to study the Gα_z induced βAR signalling. HEK 293 cells were transfected with Gα_z-Rluc, Gγ₁-GFP and untagged Gβ₁, along with (b,d) β₁AR or (c,e) β₂AR. Kinetic curves represent time course of Gα_z activation by (b) β₁AR (vehicle n = 1; isoproterenol n = 2) or (c) β₂AR (vehicle and isoproterenol, n = 2) expressed as absolute BRET ratios. Concentration-responses curves for Gα_z activation following (d) β₁AR or (e) β₂AR activation by indicated ligands. Data were normalized to maximal isoproterenol response, which was take as 100%, and are expressed as mean ± SEM values. Details of the number of experiments, maximal responses, pEC₅₀ values and statistical comparisons of curve parameters for (d) β₁AR activation by different ligands are provided in Supplementary Tables S1 and S2. For (e) β₂AR activation, n = 3 for all ligands.

Figure 5

Gα₁₂-induced activation by the β₁AR. (a) Schematic representation of the Gα₁₂-Rluc/Gγ₁-GFP biosensor used to study the Gα₁₂ induced βAR signalling. (b) Schematic representation of the Gα₁₂-Rluc/p115-RhoGef-GFP (p115-GFP) biosensor used to study the Gα₁₂ induced βAR signalling. HEK 293 cells were transfected with (c,e,g) Gα₁₂-Rluc, Gγ₁-GFP and untagged Gβ₁ or with (d,f) Gα₁₂-Rluc, p115-GFP and untagged Gγ₁ and Gβ₁, along with β₁AR or (g) β₂AR. Kinetic curves represent time course of (c) Gα₁₂ activation (vehicle and isoproterenol n = 3) or (d) Gα₁₂-p115 biosensor activation (vehicle n = 2, isoproterenol n = 3), expressed as absolute BRET ratio. Concentration-responses curves for (e) Gα₁₂ activation or (f) Gα₁₂-p115 biosensor activation following β₁AR activation by indicated ligands. (g) Concentration-responses curves for Gα₁₂ activation following isoproterenol-induced β₁AR or β₂AR stimulation (n = 6). Data were normalized to maximal isoproterenol response (100%), and are expressed as mean ± SEM values. Detail (e,f) of the number of experiments, maximal responses, pEC₅₀ values and statistical comparisons of curve parameters for (e,f) β₁AR activation by different ligands are provided in Supplementary Tables S1 and S2.

Figure 6

PKN-recruitment based biosensor to monitor receptor-induced activation of Rho signalling for TPαR and β₁AR. (a) Schematic representation of the PKN-recruitment based biosensor. (b) Schematic representation of the mode of action of p115-RGS-CAAX construct (P115-CAAX) as a dominant negative construct for G protein signalling. HEK 293 cells were transfected with PKN-RlucII and rGFP-CAAX along with either (c–f) HA-TPαR or (g,h) β₁AR, in the presence or absence of (d) constitutively active RhoA mutant (Q63L), (e) Gα₁₂, Gα₁₃ or their constitutively active (CAM) versions (Gα₁₂CA Q231L and Gα₁₃CA Q226L) or (f-h) p115-CAAX. (c) Treatment with the TP antagonist SQ 29,548 inhibits PKN recruitment following TPαR activation by U46619. 0 ng represents the activity of endogenously expressed receptor. Data are expressed as ΔBRET signal and are the mean ± SEM (n = 3). Statistical comparisons for antagonistic effect were done using two-way ANOVA followed by post-hoc comparison with Sidak’s test. (d,e) PKN recruitment to the plasma membrane upon U46619-induced TPαR activation in the presence or absence of (d) constitutively active RhoA mutant (Q63L) or (e) Gα₁₂, Gα₁₃ or their CAM versions. Data are expressed as BRET signal and are mean ± SEM ((d) n = 6, (e) n = 3). Statistical comparisons were done using two-way ANOVA followed by post-hoc comparison with Tukey’s test. #### p < 0.0001 compared to (d) Q63L or (e) TPαR Vehicle Mock. (f) Inhibition of PKN recruitment upon U46619-induced TPαR activation in the presence of the dominant negative p115-CAAX construct or the Gq inhibitor YM-254890. Data expression and statistical comparisons were done as in (d,e). n = 4, #### p < 0.0001 compared to -p115-CAAX. (g) Kinetics of PKN recruitment upon isoproterenol-induced β₁AR activation in the presence of the dominant negative p115-CAAX (representative of n = 3). (h) β₁AR-mediated PKN recruitment in the presence of p115-CAAX. Data are expressed as the area under the curve (AUC), calculated from 30 sec kinetics of 1 µM isoproterenol stimulation, and are the mean ± SEM (n = 4). #### p < 0.0001 compared to -p115-CAAX. ns: non-significant.

Figure 7

Calcium response promoted by β₁AR or β₂AR activation. (a) Schematic representation of the luminescence Obelin biosensor used to detect calcium. (b) Schematic representation of the mode of action of p115-RGS-CAAX (p115-CAAX) construct as a dominant negative construct for the inhibition of G_12/13 signalling. HEK 293 cells stably expressing HA-β₁AR were transfected with Obelin. (c) Kinetics of calcium response by indicated ligands (representative, isoproterenol, n = 10, other ligands n = 3) and expressed as relative luminescence units. (d) Concentration-responses curves for Ca²⁺ mobilization following β₁AR activation by indicated ligands. Data were normalized to maximal isoproterenol response (100%), and expressed as mean ± SEM values. Detail of the number of experiments, maximal responses, pEC₅₀ values and statistical comparisons of curve parameters for different ligands are provided in Supplementary Tables S1 and S2. (e) HEK 293 cells were transiently transfected with β₁AR or β₂AR along with the obelin biosensor, with or without the p115-CAAX inhibitor. Data were normalized to maximal A23187 response (100%), determined from the area under the curve (AUC), and are expressed as the mean ± SEM values (n = 3). Statistical comparisons were done using two-way ANOVA followed by post-hoc comparison with Tukey’s test. (f) Parental HEK 293 or ΔGαs cells were transfected with β₁AR or β₂AR, along with obelin, with or without Gα_s. Data normalization and statistical analysis were done as described in (e) (n = 3).

Figure 8

β-arrestin2 recruitment by β₁AR. (a) Schematic representation of the βarr2 biosensor. HEK 293 cells were transfected with β₁AR-GFP along with β-arrestin2-Rluc. (b) Kinetic curves represent time course of β-arrestin2 recruitment (vehicle and isoproterenol n = 3) expressed as absolute BRET ratio. (c) Concentration-responses curves for β-arrestin2 recruitment following β₁AR activation by indicated ligands. Data were normalized to maximal isoproterenol response, which was taken as 100%, and are expressed as mean ± SEM values. Detail of the number of experiments, maximal responses, pEC₅₀ values and statistical comparisons of curve parameters for different ligands are provided in Supplementary Tables S1 and S2.