Fig. 7
Influence of A2AR C-terminal domain over signaling properties of A1-A2AHets. a BRET in cells expressing constant A1-Rluc amount (0.4 μg cDNA) and increasing (0.1–0.7 μg cDNA) amounts of A2AΔ40R-YFP or A2AΔCTR-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 A2AΔCTR-cYFP and pre-treated for 4 h with medium or 4 μM A2AR-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 A2AR (0.3 μg cDNA), A2AΔ40R (0.3 μg cDNA), or A2AΔCTR (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 A2AR (0.3 μg), A2AΔ40R (0.3 μg), or A2AΔCTR (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 A2AR homodimer in complex with Gs. TMs involved in homodimerization: TM4/light blue and TM5/gray; color code of proteins is as in Fig. 3. C-tail of Gsα-subunit-unbound A2AR protomer is near αsAH (shown in closed conformation)