BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and β-Arrestins

Abstract

The histamine H₁ receptor (H₁R) is a G protein-coupled receptor (GPCR) and plays a key role in allergic reactions upon activation by histamine which is locally released from mast cells and basophils. Consequently, H₁R is a well-established therapeutic target for antihistamines that relieve allergy symptoms. H₁R signals via heterotrimeric G_q proteins and is phosphorylated by GPCR kinase (GRK) subtypes 2, 5, and 6, consequently facilitating the subsequent recruitment of β-arrestin1 and/or 2. Stimulation of a GPCR with structurally different agonists can result in preferential engagement of one or more of these intracellular signaling molecules. To evaluate this so-called biased agonism for H₁R, bioluminescence resonance energy transfer (BRET)-based biosensors were applied to measure H₁R signaling through heterotrimeric G_q proteins, second messengers (inositol 1,4,5-triphosphate and Ca²⁺), and receptor-protein interactions (GRKs and β-arrestins) in response to histamine, 2-phenylhistamines, and histaprodifens in a similar cellular background. Although differences in efficacy were observed for these agonists between some functional readouts as compared to reference agonist histamine, subsequent data analysis using an operational model of agonism revealed only signaling bias of the agonist Br-phHA-HA in recruiting β-arrestin2 to H₁R over G_q biosensor activation.

Keywords: BRET assay; G protein; GPCR; GPCR kinase; H1R; biased signaling; histamine; β-arrestin.

Figure 1

H₁R activates heterotrimeric G_q proteins, InsP₃ production and Ca²⁺ release in response to histamine (HA). BRET measurements in HEK293T cells transiently co-expressing H₁R (0.5–1 µg plasmid/dish) in combination with the heterotrimeric G_q activation sensor (2.5 µg plasmid/dish) (A,B), InsP₃ (C,D) or Ca²⁺ (E,F) sensor (4.0–4.5 µg plasmid/dish) in real time upon stimulation with 10 µM histamine (A,C,E) or after 20-min stimulation with increasing concentrations histamine (B,D,F). InsP₃ and Ca²⁺ cells were first stimulated with vehicle (VEH) or histamine (HA) at t = 0 min followed by a second injection of vehicle (VEH) or 10 µM mepyramine (MEP) at t = 10 min, indicated by the numbered arrows 1 and 2, respectively (C–E). Data are shown as mean ± SD from at least three independent experiments performed in triplicate.

Figure 2

Histamine (HA)-induced β-arrestin1/2 recruitment to H₁R. BRET measurements in HEK293T cells transiently co-expressing H₁R-Rluc8 (0.5 µg plasmid/dish) in combination with β-arrestin1-eYFP or β-arrestin2-mVenus (4.0 µg plasmid/dish) in real time upon stimulation with 10 µM histamine (A) or after 1-h stimulation with increasing concentrations histamine (B). Data are shown as mean ± SD from at least four independent experiments performed in triplicate. Area under the curve (AUC) of 1-h BRET traces in response to 10 µM histamine following 30-min pretreatment of the cells with vehicle (control), 3 µM cmpd101, or 1 µM UBO-QIC. AUC of the BRET experiments are presented as mean ± SD from three independent experiments performed in triplicate, with scatter plots (gray dots) showing the individual AUC values (C). Statistical differences (p < 0.05) compared to vehicle-pretreated (i.e., control) cells were determined using one-way ANOVA with Dunnett’s multiple comparison test and are indicated by an asterisk.

Figure 3

Histamine (HA)-induced modulation in H₁R interaction with GRKs. BRET measurements in HEK293T cells transiently co-expressing H₁R-Rluc8 (0.5 µg plasmid/dish) in combination with GRK2-mVenus, GRK3-mVenus, GRK5-mVenus, or GRK6-mVenus (4.0 µg plasmid/dish) in real time upon stimulation with 10 µM histamine (A) or after 1-h stimulation with increasing concentrations histamine (B). Data are shown as mean ± SD from at least three independent experiments performed in triplicate. Area under the curve (AUC) of 1-h BRET traces in response to 10 µM histamine following 30-min pretreatment of the cells with vehicle (control) or 1 µM UBO-QIC. AUC of the BRET experiments are presented as mean ± SD from three independent experiments performed in triplicate, with scatter plots (gray dots) showing the individual AUC values (C). No statistical differences (p < 0.05) compared to vehicle-pretreated (i.e., control) cells are observed using one-way ANOVA with Dunnett’s multiple comparison test.

Figure 4

Histamine (HA)-induced internalization of H₁R. BRET measurements in HEK293T cells transiently co-expressing H₁R-Rluc8 (0.5 µg plasmid/dish) in combination with the early endosome marker Venus-Rab5a (2 µg plasmid/dish) in real time upon stimulation with 10 µM histamine (A). Data are shown as mean ± SD from at least three independent experiments performed in triplicate. Area under the curve (AUC) of 1-h BRET traces in response to 10 µM histamine following 30-min pretreatment of the cells with vehicle (control), 1 µM UBO-QIC, 3 µM cmpd101, or 0.16 µM sucrose, or co-transfection with dominant negative dynamin mutant K⁴⁴A or β-arrestin1/2 siRNA. AUC of the BRET experiments are presented as mean ± SD from three independent experiments performed in triplicate, with scatter plots (gray dots) showing the individual AUC values (B). Statistical differences (p < 0.05) compared to vehicle-pretreated (i.e., control) cells were determined using one-way ANOVA with Dunnett’s multiple comparison test and are indicated by an asterisk.

Figure 5

Concentration response curves of BRET-based responses in HEK293T cells upon stimulation with various H₁R agonists. BRET measurements in HEK293T cells transiently co-expressing H₁R (0.5–1.0 µg plasmid/dish) in combination with G_q activation sensor (2.5 µg plasmid/dish) (A), InsP₃ (B) or Ca²⁺ (C) sensor (4.0 µg plasmid/dish), or H₁R-Rluc8 (1.0 µg plasmid/dish) in combination with GRK2-mVenus (D), GRK3-mVenus (E), GRK5-mVenus (F), GRK6-mVenus (G), β-arrestin1-eYFP (H), or β-arrestin2-mVenus (I) (4.0 µg plasmid/dish), upon stimulation with increasing concentrations agonist for 20 (A–C)or 60 min (D–I). Data are shown as mean ± SD from at least three independent experiments performed in triplicate.

Figure 6

Biased activities of agonists in BRET-based H₁R responses in transfected HEK293T cells. The pEC₅₀ (A) and intrinsic activity (B) values obtained from the concentration response curves in Figure 5 (Supplementary Tables S1 and S2). Intrinsic activities were calculated versus reference full agonist histamine. Transduction ratio (Log(τ/K_A) (C), relative effectiveness (ΔLog(τ/K_A) (D) and Log bias factor (ΔΔLog(τ/KA) (E) as calculated after analyzing the concentration response curves in Figure 5 using the operational model of agonism to retrieve the transduction ratio (Log(τ/K_A; Supplementary Table S3), followed by normalization to the reference full agonist histamine (ΔLog(τ/K_A; Supplementary Table S4) and comparison of the various functional readouts to the G_q activation response (ΔΔLog(τ/K_A; Supplementary Table S5). Data are shown as mean ± SD from at least three independent experiments performed in triplicate. Statistical differences (p < 0.05) between relative effectiveness of each ligand in the tested responses were determined using Welch ANOVA with Dunnett’s T3 multiple comparison test and are indicated by an asterisk.

Figure 7

Activities of agonists in endogenous H₁R-mediated Ca²⁺ response in HeLa cells. Concentration response curves of Ca²⁺ mobilization measured 20–30 s after stimulation of HeLa cells with agonists (A). The pEC₅₀ (B), intrinsic activity (C), transduction ratio (Log(τ/K_A) (D), and relative effectiveness (ΔLog(τ/K_A) as compared to reference agonist histamine (E) of agonist-induced Ca²⁺ mobilization in HeLa cells are shown as mean ± SD from at least three independent experiments performed in triplicate.