Figure 4. Mechanisms of Gβγ signaling deactivation.

A and B, Subcellular localization of Gα_oA and β₁γ₁ and Gβ₁γ₂ with or without prolonged stimulation of D2R. Transfected cells were stimulated with 100 μM dopamine for 10 min (bottom). Immunocytochemistry was performed with anti-Gα_o antibody. The subcellular localization of Gα_oA and Venus-Gβγ were visualized with confocal microscopy. C, Schematic presentation of the BRET assay to monitor the deactivation of Venus-Gβγ on the plasma membrane and ER. D, Real-time monitoring of the deactivation of Gβ₁γ₁ and Gβ₁γ₂ on the plasma membrane and ER. HEK293T/17 cells were transfected with D2R, Gα_oA, and Venus 156-239-Gβγ with masGRK3ct-Nluc-HA or GRK3ct-Nluc-HA-PTP1B. To accelerate GTP hydrolysis rate, RGS7, Gβ_5S, and R7BP were also transfected. The transfected cells were stimulated with 100 μM dopamine for 35 sec (Gγ₁ on PM, Gγ₂ on PM, and Gγ₁ on ER) and 10 min (Gγ₂ on ER) to activate G protein. Then, 100 μM haloperidol was applied to inhibit the activity of D2R. E, The deactivation rates of Gβ₁γ₁ and Gβ₁γ₂ on the plasma membrane (top) and ER (bottom). Statistics: One-way ANOVA followed by Fisher’s LSD multiple-comparison post hoc test was carried out (n = 3 biological replicates using independent transfections): * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. F, Effects of Gβγ on G protein deactivation rate on the plasma membrane. Deactivation rates of Go when complexed with 48 different Gβγ dimers are reported as heatmaps. One-way ANOVA followed by Fisher’s LSD multiple-comparison post hoc test was carried out (n = 3 biological replicates using independent transfections). Statistically insignificant data (P > 0.05) are colored in gray.