GPCR-G Protein-β-Arrestin Super-Complex Mediates Sustained G Protein Signaling

Abstract

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid β-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, β-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with β-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with β-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

Figure 1. Sustained Gs Signaling from Internalized Compartments by β₂AR, β₂V₂R, and V₂R

(A) Real-time cAMP measurements, using ICUE2-expressing HEK293 cells, in response to agonist stimulation of β₂AR (red), β₂V₂R (blue), and V₂R (black). For β₂AR and β₂V₂R, 1 μM ISO was used to stimulate cells. For V₂R, 100 nM AVP was used to stimulate cells. Surface expression of all GPCRs was matched. Data represent the mean ± SE of N = 3 experiments and n ≥ 90 cells. Area under the curve (AUC) was used to calculate the total cAMP response for each GPCR, and one-way ANOVA was performed to determine statistical differences relative to β₂AR (**p < 0.01; ****p < 0.0001) and β₂V₂R (##, p < 0.01) responses. (B) Schematic representation of the experimental design used to demonstrate sustained Gs activation and signaling from internalized GPCRs. (C) Real-time cAMP measurements utilized to demonstrate intracellular Gs signaling by GPCRs. Agonist-stimulated cAMP responses (100 nM ISO for β₂AR and β₂V₂R or 100 nM of desmopressin [DESMO] for V₂R) was antagonized at 10 min by the addition of 10 μM of cell-membrane-impermeable antagonist (CGP-12217 for β₂AR and β₂V₂R, or H-3192 for V₂R; shown in blue). The impact of cell-membrane-impermeable antagonists was measured relative to total antagonism caused by cell-membrane-permeable antagonists (ICI-118551 for β₂AR and β₂V₂R or SR121463 for V₂R). Data represent the mean ± SE of N = 3 experiments and n ≥ 87 cells. AUC was used to calculate the total cAMP response for each GPCR after the respective treatments. One-way ANOVA was performed to determine statistical differences among the antagonists compared to DMSO (*p < 0.05; ***p < 0.001; ****p < 0.0001) or compared to cell-membrane-impermeable antagonists (#, p < 0.05; ####, p < 0.0001). See also Figure S1.

Figure 2. Sustained Gs Activation from Internalized Compartments by β₂AR, β₂V₂R, and V₂R Assessed by BRET

(A) Schematic representation of the experimental design used to monitor agonist-promoted Gs activation, which leads to the separation of Gαs and Gβγ subunits, measured by BRET between RlucII-117-Gαs and GFP10-Gγ1. (B) BRET titration curves obtained using a constant amount of RlucII-Gαs and with increasing amounts of GFP10-Gγ1. BRET was measured 35 min following the addition of agonist or vehicle. Data are pooled from N = 4 experiments. (C) Relationship between the duration of agonist stimulation time and Gs activation response 20 min after agonist washout. Gs activity was determined by assessing the reduction in BRET signal between RlucII-117-Gαs and GFP10-Gγ1. Surface expression of all GPCRs was matched. Data are shown as a percent of BRET decrease observed in the unwashed condition (i.e., in the continuous presence of agonist) and represents the mean ± SE of N = 4–5 experiments. One-way ANOVA was performed to assess significant differences in Gs response by increasing agonist stimulation time versus pulse stimulation (0.5 min) (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). See also Figure S1.

Figure 3. Cellular Localization of SNAP-β₂V₂R Pre-labeled with SNAP-Surface 649 Fluorescent Substrate, mStrawberry-βarr2, and mEmerald-67-Gαs Visualized by Confocal Microscopy

(A) Cellular localization of SNAP-β₂V₂R (649), mStrawberry-βarr2, and mEmerald-67-Gαs prior to agonist addition (0 min) or 5 min and >20 min after 10 μM ISO treatment (100× objective, N = 4 experiments, n = 49 cells). (B) Representative endosome (orange dotted box) demonstrating co-localization of SNAP-β₂V₂R (649), mStrawberry-βarr2, and mEmerald-67-Gαs at >20 min post-ISO addition. (C) Line-scan analysis of representative endosomal fluorophore intensities. See also Figures S1, S2, and S3.

Figure 4. Interaction between βarr1/2 and Either Gαs or Gγ2 following Agonist Stimulation of β₂AR, β₂V₂R, or V₂R

(A) Schematic representation of the experimental design used to monitor agonist-promoted BRET between RlucII-67-Gαs (1), RlucII-Gγ2 (2), or RlucII-CD8 (3) and GFP10-βarr1/2. (B and D) BRET titration curves using a constant amount of RlucII-67-Gαs, RlucII-Gγ2, or RlucII-CD8 and increasing amounts of GFP10-βarr1 (B) or GFP10-βarr2 (D) monitored 20 min after agonist stimulation. Data are expressed as net BRET absolute values and represent the mean ± SE and are pooled from N = 3–5 experiments. Surface expression of all GPCRs was matched. (C and E) Kinetics of agonist-promoted BRET between GFP10-βarr1 (C) or GFP10-βarr2 (E) and RlucII-Gαs, RlucII-Gγ2, or RlucII-CD8 obtained for all three GPCRs. Each kinetic point represents the mean ± SE of ΔBRET between agonist-stimulated and vehicle-treated conditions (N = 3–10 experiments). Two-way ANOVA was performed to determine significant differences between CD8 condition and Gαs or Gγ2 for each time point (^a p < 0.05; ^b p < 0.01; ^c p < 0.001; ^d p < 0.0001). See also Figures S1 and S5.

Figure 5. Functionality and Capability of β₂V₂R-βarr1/2 Fusions to Activate Gs in HEK293 Cells

(A) Functional assessment of β₂V₂R-βarr1/2 fusions using radioligand competition binding experiments. Both agonist (ISO) and antagonist (ICI-118551) successfully competed off [¹²⁵I]-CYP at β₂V₂R, β₂V₂R-βarr1 and β₂V₂R-βarr2. Data represent the mean ± SE of N = 3–4 experiments. (B) Cellular localization of SNAP-β₂V₂R and SNAP-β₂V₂R-βarr1/2 fusions pre-labeled with SNAP-Surface 549 fluorescent substrate (549) using confocal microscopy (100× objective, N = 3 experiments, and n ≥ 16 cells). (C) Characterization of 1 μM ISO-stimulated ERK1/2 phosphorylation response at 10 min post-stimulation in mock, β₂V₂R, β₂V₂R-βarr1, and β₂V₂R-βarr2-transfected cells (N = 6 experiments). (D) ISO-stimulated Gs activation in mock (gray), β₂V₂R (black), β₂V₂R-βarr1 (red), and β₂V₂R-βarr2 (blue) transfected cells determined by BRET titration curves 30 min after stimulation (N = 4 experiments). (E) Real-time cAMP measurement, utilizing HEK293-ICUE2 cells, in response to ISO-stimulation of β₂V₂R (black), β₂V₂R-βarr1 (red), and β₂V₂R-βarr2 (blue). Data represent the mean ± SE of N = 3 experiments and n ≥ 93 cells. Surface expression of GPCRs was matched in all experiments. See also Figures S1 and S4.

Figure 6. In Vitro Formation and Functional Characterization of the Megaplex

(A) Coomassie-stained gels of representative coIP experiments of the megaplex by either M1 anti-FLAG beads (to pull-down FLAG-β₂V₂R; left) or protein A/G agarose beads (to pull-down Fab30; right) (N = 4 experiments). (B) Schematic presentation of the biochemical steps in G protein activation in the megaplex: (1) heterotrimeric G protein is recruited to the GPCR-βarr “tail” conformation to form the megaplex in an agonist-dependent manner; (2) activated receptor in the megaplex stimulates GDP-GTP exchange in the heterotrimeric G protein, causing activation and separation of the Gαs subunit; and (3) activated Gαs subunit has intrinsic GTPase activity causing hydrolysis of GTP to GDP and inorganic phosphate (Pi). (C) M1 anti-FLAG coIP experiment of BI-occupied β₂V₂R, Fab30 complex, or Cz-occupied β₂V₂R both with and without heterotrimeric Gs present. Gs binding was determined and quantified by western blot using an anti-Gαs antibody. Data represent the mean ± SE of N = 4 experiments. One-way ANOVA was performed with pairwise comparison to BI-β₂V₂R (****p < 0.0001). (D) M1 anti-FLAG coIP experiment with either BI-occupied β₂V₂R-Gs complex or megaplex in presence of control buffer, 20 μM GDP, or 20 μM GTPγS. Gαs subunit separation was determined and quantified by western blot by using an anti-Gαs antibody. Data represent the mean ± SE of N = 4 experiments. Two-way ANOVA was performed to assess significant differences between control buffer (*p < 0.05; **p < 0.01; ****p < 0.0001) and GDP (####, p < 0.0001). There were no statistical differences between the BI-occupied β₂V₂R-Gs complex and the megaplex. (E) Characterizing the ability of BI-occupied β₂V₂R (top) or Fab30 complex (bottom) to modulate GDP-GTP exchange and Gs activity via GTPase activity. Data represent the mean ± SE of N = 5–6 experiments. Two-way ANOVA was performed to test the effect of Gs presence at each receptor/complex concentrations (**p < 0.01; ****p < 0.0001), and one-way ANOVA tests the effect on Gs modulation by different receptor/complex concentrations (#, p < 0.05; ####, p < 0.0001). See also Figure S5.

Figure 7. Single-Particle EM Analysis of the (T4L) β₂V₂R-Gs-Nb35-βarr1-Fab30 Megaplex

(A) Representative EM image of negative-stained megaplex. (B) Representative class averages of the megaplex (135 total particle projections). (C) Class averages of the previously published (T4L) β₂AR-Gs-Nb35 complex and the (T4L)β₂V₂R-βarr1-Fab30 complex in the “tail” conformation (images reprinted and modified from Shukla et al., 2014; Westfield et al., 2011). Superimposition of these averages results in a density map identical to the one representing the megaplex. The scale bars in (A–C) correspond to 100, 10, and 10 nm, respectively. See also Figures S6 and S7.