Cross-communication between Gi and Gs in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain

Abstract

Background: G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A₁R or A_2AR) can form A₁R-A_2AR heteromers (A₁-A_2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (β-arrestin mediated) signaling. Adenosine has different affinities for A₁R and A_2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream G_i- and G_s-dependent signals. cAMP accumulation and β-arrestin recruitment assays have shown that, within the complex, activation of A_2AR impedes signaling via A₁R.

Results: We examined the mechanism by which A₁-A_2AHet integrates G_i- and G_s-dependent signals. A₁R blockade by A_2AR in the A₁-A_2AHet is not observed in the absence of A_2AR activation by agonists, in the absence of the C-terminal domain of A_2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A₁-A_2AHet, indicating that signaling mediated by A₁R and A_2AR is controlled by both G_i and G_s proteins.

Conclusions: We identified a new mechanism of signal transduction that implies a cross-communication between G_i and G_s proteins guided by the C-terminal tail of the A_2AR. This mechanism provides the molecular basis for the operation of the A₁-A_2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A₁R and A_2AR.

Keywords: BRET; C-terminal domain; GPCR; Heterotetramer; Molecular modeling.

Fig. 1

Effect of interference peptides on the A₁-A_2AHet structure determined by bimolecular fluorescence complementation (BiFC) assays. a–e BiFC assays were performed in HEK-293 T cells transfected with cDNAs (1 μg) for A_2AR-nYFP, A_2AR-cYFP, and non-fused A₁R (a) or A_2AR-cYFP and A₁R-nYFP (b–e). Cells were pre-treated for 4 h with medium (control, broken lines) or with 4 μM of A_2AR TM synthetic peptides (TM1 to TM7, green squares) or A₁R synthetic peptides (TM5 to TM7, orange squares). Subsequently, they were left untreated (a, b) or activated for 10 min with the A₁R agonist CPA (c, 100 nM), the A_2AR agonist CGS-21680 (d, 100 nM), or both (e). Fluorescence was read at 530 nm. Mean ± SEM (13 experiments/treatment). One-way ANOVA followed by a Dunnett’s multiple comparison test showed a significant fluorescence decrease over control values (*P < 0.05, **P < 0.01, ***P < 0.001). In each panel, there is a schematic representation of the BiFC pairs and conditions. (f) Schematic slice (left) and cartoon (right) representations of the A₁-A_2AHet built using the predicted experimental interfaces

Fig. 2

Effect of interference peptides on the A₁-A_2AHet structure determined by proximity ligation assay (PLA) confocal microscopy images (superimposed sections) in which A₁-A_2AHets appear as red spots. HEK-293 T cells expressing A₁R and A_2AR were treated for 4 h with medium (control) or 4 μM of indicated TM peptides of A_2AR; cell nuclei were stained with DAPI (blue); scale bars: 10 μm

Fig. 3

Receptor signaling through the A₁-A_2AHet. Increases in cAMP percentage accumulation with respect to Fk-stimulated (a, b) or unstimulated (c) cells. A₁-A_2AHet-expressed cells pre-treated with medium, PTX (10 ng/mL overnight) or CTX (100 ng/mL for 1 h) before adding medium, forskolin (Fk, 0.5 μM), CPA (100 nM) plus/minus forskolin, CGS-21680 (100 nM) plus/minus forskolin, or CPA + CGS-21680. b Same assays in the absence or presence of 0.5 μg of cDNA corresponding to G_i- or G_s-α-subunit-related minigenes. Mean ± SEM (7 experiments/group). One-way ANOVA followed by Bonferroni’s post-hoc test in panels a, b showed a significant effect over basal in samples treated with CGS-21680 or over forskolin in samples treated with CPA; in panel c, a significant effect is seen over basal (*P < 0.05, ***P < 0.001). d The dynamic mass redistribution analysis was plotted as pm shifts versus time (Representative experiment, performed in triplicate). e, f Distances between the Cα atoms of Arg90 (α_iAH domain) and Glu238 (Ras domain) of G_i (in yellow), Asn112 (α_sAH) and Asn261 (Ras) of G_s (green), Arg90 (α_iAH) and Asn112 (α_sAH) (dark red), and between the center of masses of the binding sites of the G_i-unbound A₁R and G_s-unbound A_2AR protomers (black) obtained from two independent molecular dynamics (MD) simulations of A₁-A_2AHet in complex with G_i and G_s in which α_iAH was modelled in the closed conformation (Additional file 1: Figure S6C) and α_sAH was modelled in closed (e) or open (f) conformation. The computed distances are depicted as double arrows in the adjacent schematic representations. Representative snapshots of the models are shown. Code: G_i-bound A₁R/red, G_i-unbound A₁R/orange, G_s-bound A_2AR/light green, G_s-unbound A_2AR/dark green, α, β, and γ of G_i/G_s in dark gray/light gray/purple, respectively, TM4/light blue, TM5/gray, α-helical α_iAH/green, and α_sAH/yellow. g MD simulations could not be performed for open conformations of α_sAH and α_iAH due to steric clash

Fig. 4

Effect of interference peptides on receptor signaling. a cAMP production was determined in HEK-293 T cells transfected with 0.4 μg of A₁R and A_2AR cDNAs. Cells were treated for 4 h with medium (control) or with 4 μM A_2AR TM synthetic peptides (TM1 to TM7, see Methods). Cells were unstimulated (basal, dotted line) or stimulated with forskolin (Fk, 0.5 μM, gray bars), with forskolin and the A₁R agonist CPA (100 nM, black bars), the A_2AR agonist CGS-21680 (100 nM, white bars), or with CPA and CGS-21680 (striped bars). Increases in cAMP percentage accumulation in relation to unstimulated cells. Mean ± SEM (7 experiments/condition). One-way ANOVA followed by Bonferroni’s post-hoc test showed a significant effect over basal in samples treated with CGS-21680 or CGS-21680 plus CPA, or over forskolin in samples treated with CPA (*P < 0.05, **P < 0.01, ***P < 0.001). One-way ANOVA followed by Bonferroni’s post-hoc test showed a significant effect over control in the absence of peptide (&P < 0.05, &&P < 0.01). b Intermolecular distances (depicted as double arrows in the adjacent schematic representation) were obtained from MD simulations of A₁-A_2AHet in complex with G_i and G_s (α_iAH and α_sAH were modeled in the open conformation, see Additional file 1: Figure S2B) in the presence of the TAT-fused TM6 peptide, which alters the heteromer interface between A₁R and A_2AR. A representative snapshot of the molecular model is shown, viewed from the intracellular site. The TAT-TM6 peptide is shown in purple, whereas the color code of the depicted proteins is as in Fig. 3

Fig. 5

A₁-A_2AHet as an adenosine concentration-sensing device. A₁-A_2AHet-expressed cells were treated for 4 h with medium (a) or with 4 μM of the synthetic A_2AR TM6 peptide (b). Cells were stimulated with forskolin (Fk, 0.5 μM, red broken line) and adenosine at increasing concentrations (30–3000 nM, black bars). cAMP levels were expressed as percentage over unstimulated cells (basal, 100%). Mean ± SEM of (7 experiments/condition). One-way ANOVA followed by a Dunnett’s multiple comparison tests showed statistical differences relative to cells stimulated only with forskolin (**P < 0.01, ***P < 0.001). Bottom panels show schemes that may provide an explanation of the results obtained at each adenosine concentration. (1) The higher affinity of adenosine for A₁R than for A_2AR is illustrated by the size of the black lines at the binding site (adenosine is shown as gray rectangles). (2) Adenosine-induced A₁R and A_2AR activation are depicted as arrows in pink and green, respectively, starting at the binding site of each receptor. (3) A₁R-induced G_i activation and A_2AR-induced G_s activation, with the corresponding decrease/increase of cAMP, are depicted as arrows in pink and green, respectively. The inhibitory effect of G_s on G_i-mediated signaling is shown as a red arrow. Width of arrows illustrates the magnitude of receptor or G protein activation or cross-talk. High adenosine concentrations increase the A_2AR binding (gray rectangle), the adenosine-induced A_2AR activation, the A_2AR-induced G_S activation (green arrows) and the cross-talk among G proteins (red arrow), while decreasing the A₁R-induced G_i activation (pink arrow) due to the cross-talk. In the presence of TM6 (in purple) the cross-talk among G proteins is lost, enabling simultaneous A₁R-induced G_i activation (pink arrow) and A_2AR-induced G_S activation (green arrow)

Fig. 6

Effect of interference peptides on recruitment of β-arrestin-2. a, b Receptor agonist-induced β-arrestin-2 recruitment was measured by BRET. HEK-293 T cells were transfected with the cDNAs for β-arrestin-2-Rluc (Arr-Rluc, 0.5 μg cDNA) and either A_2A-YFP (0.4 μg cDNA) and A₁R (0.4 μg cDNA) (a) or A₁-YFP (0.4 μg cDNA) and A_2AR (0.4 μg cDNA) (b). Cells were untreated (control) or treated for 4 h with 4 μM A_2AR TAT-TM synthetic peptides (TM4–7, see Methods) before addition of medium (basal, gray bars) or 100 nM of either the A₁R agonist CPA (black bars), the A_2AR agonist CGS-21680 (CGS, white bars), or both (striped bars). Positive BRET was expressed as milli-BRET units (see Methods). Mean ± SEM (7 experiments/condition). One-way ANOVA followed by Bonferroni’s post-hoc test showed a significant effect over basal in samples treated with CGS-21680 or over forskolin in samples treated with CPA (*P < 0.05, **P < 0.01, ***P < 0.001). One-way ANOVA followed by Bonferroni’s post-hoc test showed a significant effect of CPA + CGS-21680 over CGS-21680 treatments (&P < 0.05, &&P < 0.01). c Intermolecular distances between the center of masses of the N- and C-domains of the A₁R-bound arrestin and of the A_2AR-bound arrestin obtained from molecular dynamics (MD) simulations of A₁-A_2AHet in complex with two molecules of β-arrestin-2. d Intermolecular distances between the center of mass of the N- and C-domains of the A_2AR-bound arrestin and the Cα atom of Glu238 (RAS domain) of G_i obtained from MD simulations of A₁-A_2AHet in complex with G_i bound to A₁R and β-arrestin-2 bound to A_2AR. These intermolecular distances are depicted as double arrows in the adjacent representative snapshots of the molecular models. Arrestin is shown in gray, whereas the color code of the depicted proteins is as in Fig. 3

Fig. 7

Influence of A_2AR C-terminal domain over signaling properties of A₁-A_2AHets. a BRET in cells expressing constant A₁-Rluc amount (0.4 μg cDNA) and increasing (0.1–0.7 μg cDNA) amounts of A_2A^Δ40R-YFP or A_2A^ΔCTR-YFP. Mean of milli-BRET units ± SEM (n = 7). b BiFC assays (fluorescence measured at 530 nm) were performed in cells expressing (1 μg cDNA) A₁R-nYFP and A_2A^ΔCTR-cYFP and pre-treated for 4 h with medium or 4 μM A_2AR-TM peptides (TM1–7). Mean ± SEM (13 experiments/treatment). One-way ANOVA followed by a Dunnett’s multiple comparison test showed a significant fluorescence decrease over control values (***P < 0.001). c HEK-293 T cells expressing A₁R (0.4 μg cDNA) and A_2AR (0.3 μg cDNA), A_2A^Δ40R (0.3 μg cDNA), or A_2A^ΔCTR (0.3 μg cDNA) were unstimulated (basal, dotted line) or stimulated with forskolin (Fk, 0.5 μM, gray bars), with forskolin and CPA (100 nM, black bars), CGS-21680 (CGS, 100 nM, white bars), or with CPA + CGS-21680 (striped bars). cAMP percentage accumulation over unstimulated cells. Mean ± SEM (7 experiments/group). One-way ANOVA followed by Bonferroni’s post-hoc test: significant effect over basal in CGS-21680-stimulated samples or over forskolin-stimulated cells (**P < 0.01, ***P < 0.001) or CPA + CGS-21680 over CGS-21680-stimulated cells (&&&P < 0.001). d HEK-293 T cells expressing β-arrestin-2-Rluc (Arr-Rluc, 0.5 μg cDNA), A₁-YFP (0.4 μg) and A_2AR (0.3 μg), A_2A^Δ40R (0.3 μg), or A_2A^ΔCTR (0.3 μg). Cells stimulated with agonists as indicated. Mean ± SEM (7 experiments/condition). One-way ANOVA followed by the Bonferroni’s post-hoc test: significant differences over unstimulated cells (*P < 0.05, ***P < 0.001) or CPA-CGS-21680 over CGS-21680-stimulated cells (&P < 0.05, &&P < 0.01). e Molecular model of the A_2AR homodimer in complex with G_s. TMs involved in homodimerization: TM4/light blue and TM5/gray; color code of proteins is as in Fig. 3. C-tail of G_sα-subunit-unbound A_2AR protomer is near α_sAH (shown in closed conformation)