Fig. 5
A1-A2AHet as an adenosine concentration-sensing device. A1-A2AHet-expressed cells were treated for 4 h with medium (a) or with 4 μM of the synthetic A2AR 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 A2AR is illustrated by the size of the black lines at the binding site (adenosine is shown as gray rectangles). (2) Adenosine-induced A1R and A2AR activation are depicted as arrows in pink and green, respectively, starting at the binding site of each receptor. (3) A1R-induced Gi activation and A2AR-induced Gs activation, with the corresponding decrease/increase of cAMP, are depicted as arrows in pink and green, respectively. The inhibitory effect of Gs on Gi-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 A2AR binding (gray rectangle), the adenosine-induced A2AR activation, the A2AR-induced GS activation (green arrows) and the cross-talk among G proteins (red arrow), while decreasing the A1R-induced Gi 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 Gi activation (pink arrow) and A2AR-induced GS activation (green arrow)