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Fig. 4. BRET between AC-RLuc and either GFP-G₂ or YFP-Gß₁₂. (A) HEK 293 cells were transfected with recombinant plasmids to express AC-RLuc and GFP-G₂. In addition, cells expressed ß₂AR, G_s and/or Gß₁ as indicated. BRET was significantly increased when G_s and Gß₁ were co-expressed with GFP-G₂ (**P<0.005 between groups as indicated). Isoproterenol caused a significant increase in BRET in all cells that expressed exogenous ß₂AR (P<0.005). (B) HEK 293 cells were transfected to express ß₂AR, G_s and AC-RLuc as well as the indicated tagged G-protein subunits (YFP_1-158-Gß₁, YFP_159-238-G₂ and GFP-G₂) and their untagged counterpart (i.e. Gß₁ or G₂). For some samples, isoproterenol caused a significant increase in BRET (*P<0.01 and **P<0.005 between groups as indicated). As a positive control, cells were transfected to express a protein consisting of YFP fused to the C-terminus of RLuc (RLuc-YFP). Transfections were performed as described in the Materials and Methods except that 0.1 µg of recombinant plasmid containing the cDNA for AC-RLuc was used per 10 cm² tissue culture well. The data represent the mean ± s.d. for a representative experiment. (C) HEK 293 cells stably expressing the ß₂AR were transiently transfected to co-express G_s, Gß₁, GFP-G₂ and AC-RLuc (or CD8-RLuc as a negative control). The amount of plasmid encoding GFP-G₂ varied as represented by the ratio of fluorescence/luminescence whereas that for all the other plasmids was kept constant. Cells were treated (filled symbols) or not (open symbols) with 10 µM isoproterenol before assaying BRET. Significant differences were determined by a paired t-test for three or more experiments.

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