FIGURE 6.

BRET assays showing direct recruitment of β-arrestins to CCK2R stimulated by full agonists but not partial agonists. A, BRET titration curve for the β-arrestin recruitment to CCK2R was measured in HEK 293 cells expressing a constant amount of CCK2R-Rluc and increasing amounts of β-arrestin2-YFP, stimulated by 0.1 μm CCK or not stimulated. Net BRET is expressed as a function of the acceptor/donor ratio and is measured 300 s after the addition of CCK. Specificity of the BRET signal between CCK2R-Rluc and β-arrestin2-YFP was confirmed in experiments showing an absence of BRET signal expressing both CCK2R-Rluc and free YFP (results not shown). B, kinetics of β-arrestin2 recruitment to CCK2R after treatment by full agonists CCK and gastrin (0.1 μm), partial agonists PD135,158 and JB93,242 (10 μm), and inverse agonist JB93,182 (10 μm). C, dose-response curves for CCK and gastrin stimulation of β-arrestin recruitment to CCK2R. D, dose-response curve for inhibition by PD135,158 of CCK-stimulated recruitment of β-arrestin to CCK2R. HEK cells were stimulated for 300 s by 0.1 μm CCK in the absence or in the presence of increasing concentrations of PD135,158 before BRET measurements. Results are expressed as net BRET as described under “Experimental Procedures.” Data are the mean ± S.E. (error bars) of 4–6 independent experiments, each performed in duplicate.