FIGURE 5.
CaM interaction with A2A-D2 receptor heteromers detected by BRET competition experiments. BRET competition experiments were performed with HEK-293 cells expressing donor and acceptor constructs to give submaximal BRET values. A, cells were co-transfected with the cDNA corresponding to A2ARRluc (0.6 μg) and CaMYFP (0.4 μg) and increasing amounts of D2R cDNA as competitor (0.3–2.5 μg), whose expression was monitored by Western blotting (bottom) mBU, milli-BRET units. B, cells were cotransfected with the cDNA corresponding to A2ARRluc (0.6 μg) and D2LRYFP (1.5 μg) and increasing amounts of cDNA of CaMGFP2 as competitor (0.2–1.5 μg), whose expression was monitored by measuring the GFP2 fluorescence (1,000–20,000 fluorescence units), as described under “Experimental Procedures.” To determine the YFP fluorescence in BRET experiments, the spectral signature was considered to control the GFP2 contribution to the detection channel (28, 29). Cells were cotransfected with the cDNA corresponding to D2LRRluc (1 μg; C) or D2SRRluc (0.8 μg; D) and CaMYFP (0.4 μg) and increasing amounts of cDNA of A2AR as competitor (black bars) or A1R as negative control (white bars), whose expression was monitored by dot blotting (results not shown). At the top of each panel, a scheme depicts the expressed proteins in BRET competition assays.