Noncanonical scaffolding of Gαi and β-arrestin by G protein-coupled receptors

Abstract

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are common drug targets and canonically couple to specific G_α protein subtypes and β-arrestin adaptor proteins. G protein-mediated signaling and β-arrestin-mediated signaling have been considered separable. We show here that GPCRs promote a direct interaction between G_αi protein subtype family members and β-arrestins regardless of their canonical G_α protein subtype coupling. G_αi:β-arrestin complexes bound extracellular signal-regulated kinase (ERK), and their disruption impaired both ERK activation and cell migration, which is consistent with β-arrestins requiring a functional interaction with G_αi for certain signaling events. These results introduce a GPCR signaling mechanism distinct from canonical G protein activation in which GPCRs cause the formation of G_αi:β-arrestin signaling complexes.

Fig. 1.. G protein:β-arrestin:GPCR complex formation.

(A) Arrangement of luciferase fragments and mKO acceptor fluorophore on G protein (LgBiT), β-arrestin-2 (mKO), and V₂R (smBiT). HEK 293T cells were transiently transfected and stimulated with agonist or vehicle. (B) BRET ratio of G_αs-LgBiT:β-arrestin-mKO:V₂R-smBiT after AVP (500 nM) treatment. After AVP treatment, an increase in the BRET ratio was observed in cells expressing β-arrestin-mKO but not in cells expressing cytosolic (untagged) mKO. (C) Quantification of G_αs-LgBiT:β-arrestin-mKO:V₂R-smBiT complex formation in cells treated with either vehicle or AVP at a single 5-min time point. (D) Similar experiment to panel (B), except testing the ability of G_αi to form a complex. BRET ratio of G_αi-LgBiT:β-arrestin-mKO:V₂R-smBiT after treatment with AVP. After AVP treatment, an increase in the BRET ratio was observed in cells expressing β-arrestin-mKO, but not in cells expressing cytosolic mKO [similar to (B)]. (E) G_αi-LgBiT:β-arrestin-mKO:V₂R-smBiT formation at a single 5-min time point. (F) Rearrangement of BRET components; G_αi protein (LgBiT), β-arrestin (smBiT), and V₂R (mKO). (G) BRET ratio of G_αi-LgBiT:β-arrestin-smBiT:V₂R-mKO after AVP treatment. Rearrangement of BRET tags increased the observed signal compared with (D). (H) Five-minute quantification of G_αi-LgBiT:β-arrestin-smBiT:V₂R-mKO complexes . (I) Similar experiment to (G), except testing the ability of G_αi-LgBiT:β-arrestin-smBiT to form a complex with the β₂AR-mKO as opposed to the V₂R-mKO. After isoproterenol (10 μM) treatment, an increase in the BRET ratio was observed in cells expressing β₂AR-mKO but not in cells expressing cytosolic mKO. (J) Five-minute quantification of G_αi-LgBiT:β-arrestin-smBiT:β₂AR-mKO complexes. For kinetic experiments, *P < 0.05 by two-way ANOVA, Fisher’s post hoc analysis with a significant difference between treatments; for 5-min quantification, *P < 0.05 by Student’s two-tailed t test. For (B) to (E), n = 3 per condition; for (G) to (J), n = 4 per condition. Graphs show mean ± SEM. Cyto, cytoplasmic.

Fig. 2.. Confocal microscopy of G_αi:β-arrestin:V₂R complexes.

Confocal microscopy analysis of AVP-induced complexes of G_αi:β-arrestin:V₂R in HEK 293T cells transfected with mVenus-tagged G_αi, mKO-tagged β-arrestin-2, and Mars1-tagged V₂R. (A) Single cells preceding treatment (basal), at 5 min, or at 30 min. Localization of G_αi-mVenus:β-arrestin-mKO:V₂R-Mars1 was observed at 5 min, with less at 30 min. Scale bars, 5 mm. (B) Inset of images in (A). Scale bars, 1 mm. (C) Line scan analysis of the 5-min time point demonstrating colocalization of fluorophores after AVP (100 nM) treatment. (D) Line-scan analysis of the 30-min time point. Data are representative of 10 (basal), 20 (5 min), or 15 (30 min) fields of view from three independent experiments.

Fig. 3.. Canonically G_αs-coupled V₂R forms G_αi:β-arrestin complexes.

(A) Canonical G protein signaling of the V₂R. (B) cAMP generation of the V₂R. (C) Canonical β-arrestin-2-smBiT recruitment after AVP (500 nM) treatment of the V₂R-LgBiT. (D) Arrangement of luciferase fragments on G protein (LgBiT) and β-arrestin (smBiT). (E) Only G_αi-LgBiT formed complexes with β-arrestin-2-smBiT after AVP (500 nM) treatment in cells overexpressing the V₂R. (F) Concentration-response on G_αi-LgBiT:β-arrestin-2-smBiT association in cells overexpressing the V₂R. (G) G_αi-LgBiT:β-arrestin-2-smBiT complex formation is sensitive to PTX pretreatment. Data are normalized to maximal signal within each replicate. (H) Loss of PTX sensitivity on G_αi-LgBiT:β-arrestin-2-smBiT by mutation of the ADP-ribosylation site (C352). (I) Pretreatment for 30 min with a membrane-permeable (SR121463, 10 μM) or a membrane-impermeable (H3192, 10 μM) V₂R antagonist on G_αi-LgBiT:β-arrestin-2-smBiT complex formation. (J) V₂R-LgBiT association with β-arrestin-2-smBiT after either membrane-permeable SR121463 or membrane-impermeable H3192 V₂R antagonists. (K) G_αi-LgBiT association with β-arrestin-2-smBiT after membrane-permeable (SR121463) or membrane-impermeable (H3192) V₂R antagonists. (L) GST-β-arrestin-2 association with purified G_αi. For (A), TGF-α shedding assay was conducted in Δ3G HEK 293T cells. All other experiments were conducted in WT HEK 293T cells overexpressing the indicated assay components. For (A) and (E), *P < 0.05 by two-way ANOVA, with a main effect of G_αs or G_αs, respectively, versus other G_α subunits. For (G) and (H), *P < 0.05 by two-way ANOVA, main effect of PTx treatment. For (I), *P < 0.05 by two-way ANOVA, main effect of vehicle relative to either antagonist. For (J) and (K), *P < 0.05 by two-way ANOVA, Bonferroni post hoc test of vehicle versus either antagonist; #P < 0.05 by two-way ANOVA, Bonferroni post hoc test of SR121463 versus H3192. For (A) to (C), (E), (F), and (H) to (J), n = 3 per condition; for (K), n = 6 to 9 per condition; for (G), n = 8 per condition. (L) is representative of three pulldown experiments. Graphs show mean ± SEM.

Fig. 4.. GPCRs form G_αi:β-arrestin complexes irrespective of canonical G protein coupling.

(A to E) Canonical G protein signaling at (A) β₂AR, (B) CXCR3, (C) D₁R, (D) D₂R, and (E) NTS₁R assessed with the TGF-α shedding assay. (F to J) G_αi:β-arrestin complex formation at (F) β₂AR (10 μM isoproterenol), (G) CXCR3 (1 μM VUF10661), (H) D₁R (500 nM dopamine), (I) D₂R (500 nM dopamine), and (J) NTS₁R (10 nM neurotensin). *P < 0.05 by two-way ANOVA, main effect of G_αi subtype. For (A) to (E), n = 4 per condition. For (F), n = 3 to 6 per condition; for (G), n = 3 to 4 per condition; for (H), n = 4 per condition; for (I), n = 3 to 4 per condition; for (J), n = 3 biological replicates per condition. Graphs show mean + SEM. Iso, isoproterenol.

Fig. 5.. G_αi:β-arrestin scaffolds form functional complexes with ERK.

(A) Arrangement of split luciferase components and mKO on G_α protein (LgBiT), β-arrestin (smBiT), or ERK (mKO). (B) BRET of G_αi-LgBiT, β-arrestin-smBiT, and ERK2-mKO in cells overexpressing untagged V₂R treated with AVP (500 nM). (C) Representative immunoblot of phospho and total ERK1/2 in Δ3G HEK 293T cells pretreated with PTx (200 ng/ml) and/or βarr1/2 siRNA stimulated with either vehicle or AVP (500 nM). (D) Quantification of ERK immunoblots in Δ3G HEK 293T cells. “100%” represents the maximal signal of phospho ERK/total ERK in the control, vehicle-treated condition. (E) Representative immunoblot of β-arrestin, G_αi, phospho-ERK, and total ERK1/2 in Δ4G HEK 293T cells (lacking all G proteins) transfected with the indicated amounts of G_αi and/or βarr1/2. “100%” represents the signal of phospho ERK/total ERK in the control siRNA, absent PTx, AVP-stimulated condition. (F) Quantification of ERK immunoblots in Δ4G HEK 293T cells. *P< 0.05, ***P < 0.001, two-way ANOVA with Bonferroni post hoc compared with the no-treatment, control siRNA group. The net BRET ratio of cytosolic mKO control was subtracted from the net BRET ratio of ERK-mKO to yield an adjusted BRET ratio that is the ordinate of (B). Immunoblots are representative of four to five separate experiments. For (B), n = 5; for (D), n = 4; and for (F), n = 5 per condition. NT, no transfection of either G_αi or β-arrestin. Graphs show mean ± SEM.

Fig. 6.. Cell migration to the β-arrestin–biased ligand TRV120023 requires both G_αi and β-arrestins.

(A) BRET assay quantifying recruitment of β-arrestin-2-YFP to AT₁R-RlucII after treatment with either AngII or TRV120023. (B and C) Canonical G protein signaling through the TGF-α shedding assay at the AT₁R after treatment with either the endogenous ligand AngII (B) or TRV120023 (C). (D) Representative images of the four TRV120023 migration conditions in HEK 293T cells stably expressing AT_IR. (E) Quantification of PTx pretreatment (200 ng/ml) and/or βarr1/2 siRNA on TRV120023-induced migration for the experiment shown in (D). (F) Split luciferase assay for monitoring G_αi-LgBiT:β-arrestin-smBiT association after treatment of AT₁R with either AngII or TRV120023. (G) Cartoon schematic of three described GPCR transduction effectors. *P < 0.05, two-way ANOVA with Bonferroni post hoc test compared with the no-treatment, control siRNA group. #P < 0.05, two-way ANOVA with Bonferroni post hoc test compared with control siRNA, PTX-pretreated group. For (A), n = 4; for (B) and (C), n = 4 to 5; for (E) and (F), n = 4 replicates per condition. Graphs show mean ± SEM.