D4 receptors form heteromers with α1B and β1 receptors in transfected cells.

(A) BRET saturation curves were performed in HEK-293T cells co-expressing a constant amount of D4-RLuc construct (2 µg of plasmid transfected) and increasing amounts of β1-YFP construct (0.4–5 µg plasmid transfected, red), α1B-YFP construct (0.4–5 µg of plasmid transfected, blue), or D1-YFP construct (1–4 µg of plasmid transfected, gray) or with cells co-expressing a constant amount of α1B-RLuc construct (3 µg of plasmid transfected) and increasing amounts of β1-YFP construct (0.4–5 µg of plasmid transfected, green). Both fluorescence and luminescence of each sample were measured prior to every experiment to confirm equal expression of Rluc construct (∼100,000 luminescence units) while monitoring the increase of YFP construct expression (2,000 to 40,000 fluorescence units). Milli BRET Units (mBU) are BRET ratio (see Materials and Methods)×1,000 and are expressed as means ± S.D. of five different experiments grouped as a function of the amount of BRET acceptor normalized with respect to the BRET donor (YFP/RLuc). (B and C) BRET was determined in HEK-293T cells expressing a constant amount of D4-RLuc construct (2 µg of plasmid transfected) and (B) α1B-YFP construct (4 µg of plasmid transfected) or (C) β1-YFP construct (4 µg of plasmid transfected) and increasing amounts (2–12 µg of plasmid transfected) of (B) α1B receptor (red) or β1 receptor (blue) or (C) β1 receptor (red) or α1B receptor (blue). Both fluorescence and luminescence of each sample were measured prior to every experiment to confirm that there were no changes in the expression of D4-RLuc, α1B-YFP, or β1-YFP constructs. BRET data (see above) are expressed as means ± S.D. of three different experiments. Significant differences with respect to cells not expressing α1B or β1 receptors were calculated by one-way ANOVA followed by a Dunnett's multiple comparison post hoc test (*p<0.05 and **p<0.01). (D) Confocal microscopy images of HEK-293T cells transfected with 1 µg of plasmid coding for D4-RLuc and 0.5 µg of plasmid coding for α1B-YFP or β1-YFP. Proteins were identified by fluorescence or by immunocytochemistry. D4-RLuc receptor is shown in red, α1B-YFP and β1-YFP receptors are shown in green, and co-localization is shown in yellow. Scale bar, 5 µm. (E and F) Co-immunoprecipitation of D4 and α1B or D4 and β1 receptors expressed in HEK-293T cells. Membranes from cells transfected with the indicated receptors were solubilized and processed for immunoprecipitation as described under Materials and Methods using goat anti-D4R, rabbit anti-α1 or goat anti-β1 receptor antibodies, or as negative controls (NC), goat anti-adenosine A2B receptor antibody (top in F) or rabbit anti-adenosine A1 receptor antibody (bottom in F). Solubilized membranes (E) and immunoprecipitates (F) were analyzed by SDS-PAGE and immunoblotted using rabbit anti-YFP, rabbit anti-α1, or goat anti-β1 antibody. IP, immunoprecipitation; WB, Western blotting (numbers are included to delineate the different lanes on the SDS-PAGE); MW, molecular mass.