Fig. 2.
BRET measurements of β-arrestin 2 recruitment to dopamine D2L receptor in living cells. (A) BRET titration curve for the β-arrestin 2 recruitment to D2LR. BRET was measured in cells expressing a fixed amount of Rluc-tagged D2LR and increasing amounts of YFP-tagged β-arrestin 2, treated (with dopamine 1 μM ▾ or quinpirole 1 μM (■) or not (♦). Specificity of the BRET signal between D2LR and the β-arrestin 2 was controlled by measuring BRET between Rluc-tagged D2LR and YFP alone, treated or not with dopamine and quinpirole (see Fig. S2). (B) Kinetic of β-arrestin 2 recruitment to D2LR after addition, at time 0, of quinpirole 1 μM (■) or PBS (○). As a negative control, similar experiments were carried out on cells transiently expressing only Rluc-tagged D2LR. (C) Dose-response curve of quinpirole for β-arrestin 2 recruitment to D2LR. Cells coexpressing Rluc-tagged D2LR and YFP-tagged β-arrestin 2 were stimulated with dopamine (○) or quinpirole (■). Results are expressed in Net BRET as described in the Methods section. Data represent the mean ± SEM of 3 to 7 independent experiments each performed in duplicate.