sAC is critical for endosome-generated cAMP in response to CRH. (A) Schematic representation of the experimental design. HT22-CRHR1-Epac-SH187 cells transfected with pcDNA3 (control) or DynK44A plus mCherry for 48 h were pretreated with vehicle (DMSO) or tmAC-specific (100 µM ddA) or sAC-specific (20 µM 2-HE) inhibitors and stimulated with 10 nM CRH. (B) Time course of FRET changes was measured in single cells. The cAMP response is expressed as the percentage of maximum FRET signal obtained in each condition assayed after stimulation (mean ± SEM, n = 7). Student’s t test was performed for each time point. P values in gray; P = 0.05 is indicated with a dotted line. (C and D) Effect of 2-HE on agonist-induced CRHR1 internalization analyzed by fluorescence flow cytometry (C) and cMyc-GFP-CRHR1 subcellular distribution (D). HT22-CRHR1 cells preincubated with vehicle (control) or 20 µM 2-HE were stimulated with 10 nM CRH for the indicated times. (C) Fluorescence measured at time 0 was defined as 100% (mean ± SEM, n = 4, 10,000 cells/condition). (D) Representative images obtained for each condition. Arrowheads point to internalized GFP clusters. Bars, 5 µM. (E) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells preincubated with vehicle (control) or sAC-specific (7.5 µM KH7) or endocytosis-specific (30 µM Dyngo-4a) inhibitors and stimulated with CRH. Results are expressed as the percentage of maximum pERK1/2 (mean ± SEM, n = 4). Two-way ANOVA followed by Tukey’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.001 with respect to control; #, P < 0.05 between indicated treatments.
