Extended Data Fig. 7: Gβγ favors the association of Gαs with membranes and mem-GαsBP2 prevents the cytosolic translocation of active Gαs upon GPCR stimulation.
From: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling
(A) Gβγ facilitates the association of Gαs with the plasma membrane. Diagram on the left corresponds to the BRET-based assay to measure the association of Gαs with membranes. Graphs in the middle correspond BRET measurements in HEK293T cells co-expressing plasma membrane anchored Venus (Venus-KRas), nanoluciferase-fused Gαs (Gαs-Nluc), and different amounts of Gβγ, as indicated, in the presence or absence of 100 nM isoproterenol stimulation as indicated. Results are presented as the ratio between luminescence at 535 nm and 450 nm (“BRET ratio (535/450 nm)”). Mean ± S.E.M. (n = 4 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of Venus-KRas, Gαs-Nluc, and Gβγ. (B) mem-GαsBP2 prevents the translocation of Gαs from membranes to the cytosol upon the GPCR stimulation. Diagram on the left corresponds to the BRET-based assay to measure the association of Gαs with membranes. Graphs in the middle correspond BRET measurements in HEK293T cells co-expressing plasma membrane anchored Venus (Venus-KRas), nanoluciferase fused Gαs (Gαs-Nluc), and mem-GαsBP2 WT or mem-GαsBP2 W11A, as indicated. Changes in BRET compared to the unstimulated baseline (“ΔBRET·103 (baseline)”) are shown in the kinetic curves on the right graph, whereas the bar graph on the right corresponds to the quantification of ΔBRET before or after isoproterenol stimulation for 10 min. Mean ± S.E.M. (n = 4 independent experiments). Immunoblots on the left right correspond to representative results of experiments confirming the expression of Venus-KRas, Gαs-Nluc, and mem-GαsBP2.
