Skip to main content
Fig. 2 | BMC Biology

Fig. 2

From: Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs

Fig. 2

Influence of G proteins on A1R and A2AR homodimerization and heterodimerization. B Bioluminescence resonance energy transfer (BRET) saturation curves were performed in HEK-293T cells 48 h post-transfection with (a, c) 0.3 μg of cDNA corresponding to A1R-Rluc and increasing amounts of A1R-YFP (0.1–1.5 μg cDNA) or GHS1a-YFP (0.25–2 μg cDNA) as negative control (a, purple line), without (a) or with (c) 0.15 μg of cDNA corresponding to A2AR; (b, d) 0.2 μg of cDNA corresponding to A2AR-Rluc and increasing amounts of A2AR-YFP (0.1–1.0 μg cDNA) or GHS1a-YFP (0.25–2 μg cDNA) as negative control (b, purple line), without (b) or with (d) 0.5 μg of cDNA corresponding to A1R; (e) 0.3 μg of cDNA corresponding to A1R-Rluc and increasing amounts of A2AR-YFP (0.1–1.0 μg cDNA); and (f) 0.5 μg of cDNA corresponding to A1R (except control blue curves that were obtained in cells not expressing A1R), 2 μg of cDNA corresponding to Gi-Rluc, and increasing amounts of A2AR-YFP (0.1–0.5 μg cDNA). In panels a, b, and e, cells were also transfected with 0.5 μg of cDNA corresponding to the Gi-related (orange curves) or Gs-related (blue curves) minigenes. Cells were treated for 16 h with medium (black curves), with 10 ng/ml of pertussis toxin (green curves), or with 100 ng/ml of cholera toxin (red curves) prior to BRET determination. To confirm similar donor expressions (approximately 100,000 bioluminescence units) while monitoring the increase in acceptor expression (1000–40,000 fluorescence units), the fluorescence and luminescence of each sample were measured before energy transfer data acquisition. MiliBRET unit (mBU) values are the mean ± standard error of the mean of four to six different experiments grouped as a function of the amount of BRET acceptor. In each panel (top) a cartoon depicts the proteins to which Rluc and YFP were fused and the presence or not of partner receptors and/or Gs or Gi proteins [schemes in c to f are not intended to illustrate on stoichiometry because the predominant form in cells expressing the two receptors was the heterotetramer containing two A1 and two A2A receptors (see “Results”)]

Back to article page