Fig. 3
Receptor signaling through the A1-A2AHet. Increases in cAMP percentage accumulation with respect to Fk-stimulated (a, b) or unstimulated (c) cells. A1-A2AHet-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 Gi- or Gs-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 (αiAH domain) and Glu238 (Ras domain) of Gi (in yellow), Asn112 (αsAH) and Asn261 (Ras) of Gs (green), Arg90 (αiAH) and Asn112 (αsAH) (dark red), and between the center of masses of the binding sites of the Gi-unbound A1R and Gs-unbound A2AR protomers (black) obtained from two independent molecular dynamics (MD) simulations of A1-A2AHet in complex with Gi and Gs in which αiAH was modelled in the closed conformation (Additional file 1: Figure S6C) and αsAH 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: Gi-bound A1R/red, Gi-unbound A1R/orange, Gs-bound A2AR/light green, Gs-unbound A2AR/dark green, α, β, and γ of Gi/Gs in dark gray/light gray/purple, respectively, TM4/light blue, TM5/gray, α-helical αiAH/green, and αsAH/yellow. g MD simulations could not be performed for open conformations of αsAH and αiAH due to steric clash