Extended Data Fig. 3: Temporally resolved inhibition of Gαs signaling in cells via chemically controlled GαsBP2 probes.
From: Inhibitory probes for spatiotemporal analysis of Gαs protein signaling
(A) Rapamycin-induced recruitment of GαsBP2 to membranes blocks GPCR-stimulated AC activity. Diagram on the left depicts the recruitment of a GαsBP2 probe to the plasma membrane upon rapamycin stimulation to block Gαs-AC signaling. Graphs with kinetic traces in the middle correspond to luminescence-based cAMP measurements in HEK293T cells expressing FKBP-GαsBP2 and membrane-anchored FRB, or mem-GαsBP2 upon stimulation with isoproterenol in the presence or absence of rapamycin pre-treatment, as indicated. The bar graph corresponds to the quantification of the area under the curve (AUC) from the kinetic response curves. Mean ± S.E.M. (n = 4 independent experiments). ns, p > 0.05, ***p < 0.005 by paired, two-tailed Student’s t-test. Immunoblots on the right correspond to representative results of experiments confirming the expression of GαsBP2 constructs, membrane-anchored FRB, and G proteins. (B) Acute, rapamycin-induced recruitment of GαsBP2 to membranes rapidly inhibits the kinetics GPCR-stimulated AC activity. Experiments were carried out as in (A), except that rapamycin was added at different times relative to the addition of isoproterenol, as indicated by the color coding and the diagram on the left. Grey traces correspond to the isoproterenol stimulated control with no addition of rapamycin. Mean ± S.E.M. (n = 3 independent experiments). (C) Abscisic acid (ABA)-induced recruitment of GαsBP2 to membranes blocks GPCR-stimulated AC activity. Diagram on the left the recruitment of a GαsBP2 probe to the plasma membrane upon ABA stimulation to block Gαs-AC signaling. Graphs with kinetic traces in the middle correspond to luminescence-based cAMP measurements in HEK293T cells expressing PYLcs-GαsBP2 and membrane-anchored ABIcs, or mem-GαsBP2 upon stimulation with isoproterenol in the presence or absence of ABA pre-treatment, as indicated. The bar graph corresponds to the quantification of the area under the curve (AUC) from the kinetic response curves. Mean ± S.E.M. (n = 3 independent experiments). ns, for p > 0.05, **p < 0.01, by paired, two-tailed Student’s t-test. Immunoblots on the right correspond to representative results of experiments confirming the expression of GαsBP2 constructs, membrane-anchored ABIcs, and G proteins.
