Fig. 4.

Orientation of a G protein in a receptor homodimer. Bioluminescence resonance energy transfer (BRET) saturation experiments were performed in HEK-293T cells transfected with 2 μg of cDNA corresponding to the α-subunit of G_s fused to Rluc and increasing amounts of A_2AR-YFP (0.1–0.5 μg) cDNA. a BRET measurements in cells pretreated for 16 h with medium (black line) or with 100 ng/ml of cholera toxin (red line). Both fluorescence and luminescence of each sample were measured before every experiment to confirm similar donor expressions (approximately 50,000 bioluminescence units) while monitoring the increase in acceptor expression (1000–10,000 fluorescence units). miliBRET unit (mBU) values are the mean ± standard error of the mean of four to five different experiments grouped as a function of the amount of BRET acceptor. A scheme of the placement of donor and acceptor BRET moieties is provided (top). b Molecular model of the A_2AR-G_s complex. Rluc (blue) is attached to the N-terminal αN helix of G_s (gray), and YFP (yellow) is attached to the C-terminal domain of A_2AR (light green) (see Additional file 9: Figure S9 for details). c Arrangement of A_2AR homodimers modeled via the TM4/5 interface as observed in the oligomeric structure of β₁-AR [4]. The A_2AR protomer bound to α_s is shown in light green, whereas the second A_2AR-YFP protomer is shown in dark green. The molecular model in panel c (BRET between Rluc in G_s α subunit and YFP in a second A_2AR protomer; center-to-center distance between Rluc and YFP of 6.5 nm), in contrast to the model shown in panel B (BRET between Rluc in G_s α subunit and YFP in the G-protein bound A_2AR protomer; center-to-center distance between Rluc and YFP of 8.3 nm), would favor the observed high-energy transfer (see panel a) between α_s-Rluc and A_2AR-YFP