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Fig. 5. | BMC Biology

Fig. 5.

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

Fig. 5.

Bioluminescence resonance energy transfer (BRET)-aided construction of a model consisting of Gi and Gs bound to an A1R-A2AR heterotetramer. a, b A1R-A2AR tetramer built using TM5/6 (a) or TM1 (b) inter-receptor interfaces modeled as in the structure of the μ opioid receptor [3]. TM helices 1, 4, and 5, involved in receptor dimerization, are highlighted in dark blue, light blue, and gray, respectively. nRluc8 and cRluc8 are shown in blue and nVenus and cVenus in dark yellow. c BRET and bimolecular fluorescence complementation experiments were performed in HEK-293T cells transfected with 1.5 μg of cDNA corresponding to A1R-cRluc8 and A2AR-nRluc8, and 1.5 μg of cDNA corresponding to A1R-nVenus and A2AR-cVenus. As the negative control, cells were transfected with 1 μg of cDNA corresponding to nRluc8 and 1.5 μg of cDNA corresponding to A2AR-nRluc8, A1R-nVenus, and A2AR-cVenus. Cells were treated for 16 h with medium (– toxins), 10 ng/ml of pertussis toxin (+ pertussis), or 100 ng/ml of cholera toxin (+ cholera) prior to BRET determination. The relative amount of BRET is given as in Fig. 4 and values are the mean ± standard error of the mean of three different experiments. Student’s t-test showed statistically significant differences with respect to the control (# p < 0.05, ## p < 0.01) and a significant effect in the presence of either toxin over BRET in the absence of toxins (*p < 0.05). A schematic representation at the top shows the protein to which the hemi luminescent or fluorescent proteins were fused. d Molecular model of the A1R-A2AR tetramer in complex with Gi and Gs. A1R bound to Gi is shown in red, Gi-unbound A1R is shown in orange, A2AR bound to Gs is shown in dark green, Gs-unbound A2AR is shown in light green, and the α, β-, and γ-subunits of Gi and Gs are shown in dark gray, light gray, and purple, respectively. Transmembrane helices 4 and 5 are highlighted in light blue and gray, respectively

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