Extended Data Fig. 5: Inhibition of isoproterenol-induced transcriptional responses by different GαsBP2 constructs.

(A) Design of different plasma membrane-anchored GαsBP2 constructs. HEK293T cells transfected with GαsBP2-mem constructs were stained as indicated and visualized by confocal fluorescence microscopy. Scale bars = 10 µm. (B) GαsBP2-mem blocks the isoproterenol-stimulated cAMP response as efficiently as mem-GαsBP2. Diagram on the top left corresponds the mechanism of cAMP regulation investigated with GαsBP2 probes. Graphs with kinetic traces on the bottom correspond to luminescence-based cAMP measurements in HEK293T cells expressing the indicated constructs. Mean ± S.E.M. (n = 3 independent experiments). Immunoblots on the top right correspond to representative results of experiments confirming the expression of GαsBP2 constructs. (C) GαsBP2-mem blocks the isoproterenol-induced gene transcription as efficiently as mem-GαsBP2. Bar graphs on the bottom correspond to relative mRNA levels of PCK1 or FOS in HEK293T cells transfected with GαsBP2-mem or mem-GαsBP2 in the presence or absence of isoproterenol stimulation, as indicated. Mean ± S.E.M. (n = 4 independent experiments); ns, p > 0.05, *p < 0.05, by one-way ANOVA corrected for multiple comparisons (Dunnett). (D) Diagram on the top shows two potential scenarios of Gαs-mediated regulation of gene expression triggered by β2AR. Bar graphs on the bottom correspond to relative mRNA levels of PCK1, FOS, or DUSP1 in HEK293T cells transfected with mem-GαsBP2, endo-GαsBP2, or both constructs simultaneously in the presence or absence of isoproterenol stimulation, as indicated. Mean ± S.E.M. (n = 4 independent experiments); ns, p > 0.05, *p < 0.05, **p < 0.01 by one-way ANOVA corrected for multiple comparisons (Dunnett).

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