Figure 3. Signaling of Gβγ from the plasma membrane to cellular organelles.

A, Schematic presentation of the BRET assay to monitor the translocation of Venus-Gβγ from the plasma membrane to organelles. GRK3ct-Nluc-HA sensor was recruited to early endosome (EE), mitochondria (Mit), Golgi apparatus (Golgi), and endoplasmic reticulum (ER) by tagging with rab5a, monoamine oxidase A (MoA), giantin, and PTP1B, respectively. Activation of G proteins with dopamine induces dissociation of Venus-Gβγ from the plasma membrane, and it binds with GRK3ct-Nluc-HA sensors at the destination. Graphics were adapted from Servier Medical Art (http://www.servier.com). B, Real-time monitoring of the translocation of Gβ₁γ₁. HEK293T/17 cells were transfected with D2R, Gα_oA, Venus 156-239-Gβ₁, and Venus 1-155-Gγ₁ together with different GRK3ct-Nluc-HA sensors individually. Dopamine (100 μM) was applied, and the BRET signal was followed across time. C, Translocation rates of Gβ₁γ₁ and Gβ₁γ₂ to organelles. D, Real-time monitoring of the translocation of Gβ₁γ₂. E, Effect of Gγ subunits on the translocation rates of Gβγ dimers to organelles. F, Comparison of the amount of Gβ₁γ₁ and Gβ₁γ₂ on each organelle by translocation. G, Effect of Gγ subunits on the amount of Gβγ translocated to organelles. Mean ± SEM from three independent experiments are shown as bar graphs (C, E, and F). Statistics: One-way ANOVA followed by Fisher’s LSD multiple-comparison post hoc test was carried out (n = 3 biological replicates using independent transfections) (C, E, and F): * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.