D₄ receptors form heteromers with α_1B 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), α_1B-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 α_1B-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) α_1B-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) α_1B receptor (red) or β₁ receptor (blue) or (C) β₁ receptor (red) or α_1B 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, α_1B-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 α_1B 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 α_1B-YFP or β₁-YFP. Proteins were identified by fluorescence or by immunocytochemistry. D₄-RLuc receptor is shown in red, α_1B-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 α_1B 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 A_2B 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.