Additional file 5: Figure S5. of Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs

BRET assays in cells expressing fusion proteins containing hemi-Rluc8 and hemi-Venus moieties fused to adenosine receptors or containing the ghrelin GHS1a receptor instead of one of the adenosine receptors. (A) Saturation BRET curve in HEK-293T co-transfected with 1.5 μg of the two cDNAs corresponding to A1R-cRLuc8 and A2AR-nRLuc8 and with increasing amounts of cDNAs corresponding to A1R-nVenus and A2AR-cVenus (equal amounts of the two cDNAs). BRETmax was 35 ± 2 mBU and BRET50 was 16 ± 3 mBU. BRET in cells expressing cRluc8 instead of A1R-cRluc8 gave a linear, non-saturable signal. (B) Comparison of BRET responses using complementary and non-complementary pairs, or replacing one adenosine receptor with the ghrelin GHS1a (gn) receptor. Data are mean ± SD of three different experiments grouped as a function of the amount of BRET acceptor. ***p < 0.001 with respect to BRET in cells expressing adenosine receptors and hemi-Rluc8 and hemi-Venus proteins. (TIF 398 kb)