FIGURE 7.

Effect of intracellular Ca²⁺ on the molecular interactions between CaM, A_2A, and D₂ receptors. A, effect of Ca²⁺ levels on the A_2A-D₂ receptor heteromerization detected by BRET. BRET saturation curves were performed with HEK-293 cells co-expressing A_2ARRluc (0.6 μg of cDNA) and increasing amounts of D₂RYFP (0.3–4 μg of cDNA). Cells in HBSS buffer containing 1.26 mm CaCl₂ were untreated (black) or treated with 1 μm ionomycin for 2 (red), 5 (green) or 10 (blue) min before BRET determination. Both fluorescence and luminescence for each sample were measured to confirm similar donor expressions (about 100,000 luminescent units) while monitoring the increase acceptor expression (1,000–15,000 fluorescent units). The relative amount of acceptor is given as the ratio between the fluorescence of the acceptor (YFP) and the luciferase activity of the donor (Rluc). BRET data are expressed as means ± S.D. of four different experiments grouped as a function of the amount of BRET acceptor. mBU, milli-BRET units. B, effect of Ca²⁺ levels on the CaM-A_2A-D₂ receptor oligomerization detected by SRET. SRET saturation curves were performed in HEK-293 cells expressing A_2ARRluc, CaMGFP², and increasing amounts of D₂RYFP, as described in the legend to Fig. 6. Cells in HBSS buffer containing 1.26 mm CaCl₂ were treated with 1 μm ionomycin for 10 min before SRET determination as described in the legend to Fig. 6, and the SRET saturation curve was compared with the one obtained in the absence of ionomycin (dotted line; see Fig. 6).