Figure 5. cAMP response to CRH depends on endocytosis. (A) Agonist-induced CRHR1 internalization was analyzed by fluorescence flow cytometry at the indicated time points after stimulation by 10 nM CRH in HT22-CRHR1 cells. Fluorescence at time 0 was defined as 100% (mean ± SEM, n = 4, 10,000 cells/condition). ***, P < 0.001; **, P < 0.01 with respect to basal by one-way analysis of variance followed by Tukey’s test. HT22-CRHR1-Epac-SH187 cells were stimulated at time 0 with CRH (B and D) or forskolin (C and E). (B and C) Cells were transfected with pcDNA3 (control) or DynK44A 48 h before stimulation. (D and E) Cells were preincubated with vehicle (control) or 30 µM Dyngo-4a 15 min before stimulation. Time course of FRET changes was measured in single cells (mean ± SEM, n = 4). Student’s t test was performed for each time point. P values in gray; P = 0.05 is indicated with a dotted line.
Figure 5.

cAMP response to CRH depends on endocytosis. (A) Agonist-induced CRHR1 internalization was analyzed by fluorescence flow cytometry at the indicated time points after stimulation by 10 nM CRH in HT22-CRHR1 cells. Fluorescence at time 0 was defined as 100% (mean ± SEM, n = 4, 10,000 cells/condition). ***, P < 0.001; **, P < 0.01 with respect to basal by one-way analysis of variance followed by Tukey’s test. HT22-CRHR1-Epac-SH187 cells were stimulated at time 0 with CRH (B and D) or forskolin (C and E). (B and C) Cells were transfected with pcDNA3 (control) or DynK44A 48 h before stimulation. (D and E) Cells were preincubated with vehicle (control) or 30 µM Dyngo-4a 15 min before stimulation. Time course of FRET changes was measured in single cells (mean ± SEM, n = 4). Student’s t test was performed for each time point. P values in gray; P = 0.05 is indicated with a dotted line.

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