Extended Data Fig. 2: GαsBP2 accelerates the re-association of Gβγ with Gαs after GPCR signaling termination.
From: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling
(A) mem-GαsBP2 does not affect Gαs-Gβγ association prior to GPCR stimulation, but accelerates Gβγ binding to Gαs after GPCR signal termination. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes. Graphs in the middle correspond to BRET measurements in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ and different amounts of mem-GαsBP2 WT or mem-GαsBP2 W11A, as indicated. Changes in BRET before β2AR stimulation compared to the condition without GαsBP2 (“ΔBRET (no GαsBP2)”) are shown in the graphs on the left, and real-time changes in BRET upon isoproterenol (agonist)/ alprenolol (antagonist) treatment compared to the unstimulated baseline (“ΔBRET (baseline)”) are shown in the graphs on the right. Mean ± S.E.M. (n = 4-5 independent experiments). Immunoblots on the right correspond to representative results of experiments confirming the expression of mem-GαsBP2 and BRET sensor components. (B) mem-GαsBP2 does not affect the efficacy or potency of isoproterenol on Gβγ responses. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes. Graphs correspond to BRET measurements in HEK293T cells co-expressing a BRET-based biosensor for free Gβγ and the indicated amounts of mem-GαsBP2 were stimulated with the indicated concentrations of isoproterenol. The changes in BRET upon isoproterenol stimulation compared to the unstimulated condition (“ΔBRET·103 (no agonist)”) are shown on the left graph, the maximal response (“maximum ΔBRET ·103”) is shown on the middle graph, and the negative logarithm of half-maximal effective concentration (pEC50) is shown on the right graph. Mean ± S.E.M. (n = 3 independent experiments). ns, p > 0.05 by one-way ANOVA corrected for multiple comparisons (Dunnett). (C) cyto-GαsBP2 accelerates Gβγ binding to Gαs after GPCR signal termination, whereas the GαsBP2 W11A mutant has no effect. Diagram on the left corresponds to the BRET-based assay to measure free Gβγ and the mechanism investigated with GαsBP2 probes Real-time changes in BRET compared to the unstimulated baseline (“ΔBRET (baseline)”) were measured in HEK293T cells expressing a BRET-based biosensor for free Gβγ co-transfected with 500 ng of plasmid DNA for cyto-GαsBP2 WT, mem-GαsBP2 WT, or their corresponding W11A mutants, upon isoproterenol (agonist)/ alprenolol (antagonist) treatment. Mean ± S.E.M. (n = 4 independent experiments). Immunoblots on the right correspond to representative results of experiments confirming the expression of GαsBP2 constructs and BRET sensor components.
