Figure 1.
D1–Gal1 and D5–Gal1 receptor heteromers in living cells. a, Confocal microscopy images of cells expressing (top to bottom) D5-Rluc (0.6 μg plasmid) and Gal1-YFP (1 μg plasmid), Gal2-Rluc (0.5 μg plasmid) and D5-YFP receptors (1 μg plasmid), D1-Rluc (0.5 μg plasmid) and Gal1-YFP (1 μg plasmid), and Gal2-Rluc (0.5 μg plasmid) and D1-YFP (1.3 μg plasmid) receptors. Proteins were identified by fluorescence or by immunocytochemistry. D5-Rluc, D1-Rluc, or Gal2-Rluc receptor immunoreactivity is shown in red; Gal1-YFP, D5-YFP, or D1-YFP receptor fluorescence in shown in green; and colocalization is shown in yellow. Scale bars, 5 μm. b, BRET experiments were performed with cells coexpressing D5-Rluc (400 ng plasmid; red) or D1-Rluc (300 ng plasmid; blue) and Gal1-YFP receptors (0.4 to 7 μg plasmid), Gal2-Rluc (300 ng plasmid) and D5-YFP receptors (0.5 to 5 μg plasmid; green) or D1-YFP receptors (0.5 to 4 μg plasmid; purple), or, as negative controls, D5-Rluc (600 ng plasmid; gray) or D1-Rluc (500 ng plasmid; orange) and CB1-YFP receptors (0.5 to 7 μg plasmid) or 5HT2B-Rluc (1 μg plasmid) and Gal1-YFP receptors (0.5 to 5 μg plasmid) (black). Both fluorescence and luminescence of each sample were measured before every experiment to confirm similar donor expressions (about 150,000 luminescent units) while monitoring the increase acceptor expression (10,000–70,000 fluorescent units). The relative amount of BRET is given as the ratio between the fluorescence of the acceptor minus the fluorescence detected in cells expressing only the donor and the luciferase activity of the donor. BRET data are expressed as the mean ± SD of 4–16 different experiments grouped as a function of the amount of BRET acceptor. At the top, a scheme corresponding to a BRET assay is shown.