(A) The GLB (5 μM) and cAMP (50 μM Fsk plus 100 μM IBMX) responses of WT (N = 10), D32A/R53A (N = 7), R447A (N = 12), R466A (N = 8), and R819A (N = 8) Epac2 biosensors in HEK293T cells. Values are depicted as mean ± s.d. ***P <1E-08. (B) Time course depicting the diminished GLB-induced response of the R447A mutant (□) Epac2 biosensor compared to the WT (■) Epac2 biosensor. (C) Emission spectrum of the R447A biosensor excited at 435 nm in the basal state (■), after GLB [1 μM (
); 10 μM (
); 100 μM (
)] addition, and after cAMP (500 μM) addition (
). (D) Molecular docking studies identify a putative SU binding site where ACT hydrogen bonds with R447 of Epac2 and makes Van der Waals contacts with F374.