FIGURE 1.

Consequences of GRK3 inactivation on the protein–protein interactions. HEK293 cells were transiently cotransfected with the following plasmid combinations: CB1R‐YFP + GRK3‐RLuc8 + empty plasmid pRK6 (2:1:2 ratio), CB1R‐SNAP + GRK3‐Rluc8 + Gβ‐flag + Gγ‐YFP (2:1:1:2 ratio) or CB1R‐YFP + β‐arrestin2‐Rluc + empty plasmid pRK6 (2:1:2 ratio). After 16 h, cells were pretreated for 30 min with cmpd101 (30 μM) prior to stimulation with the CB1R agonist WIN55212‐2 (WIN, 1 μM) where indicated. (a) Kinetic profiles of GRK3‐RLuc8 and Gγ‐YFP association dynamics in cmpd101 treated and nontreated cells. (b) Dose–response curves of GRK3‐RLuc8 and Gγ‐YFP association dynamics in cmpd101 treated and nontreated cells after CB1R stimulation with increasing concentrations of WIN. Twenty‐four hours after transfection, 5 μM coelenterazine h was added, cells were stimulated with increasing concentrations of WIN and the increase in BRET signal was measured 15 min after WIN application. (c) Kinetic profiles of GRK3‐RLuc8 recruitment by WIN‐activated CB1R‐YFP in HEK293 cells pretreated or not treated with cmpd101. (d) Kinetic profiles of β‐arrestin2‐Rluc recruitment by activated CB1R‐YFP in cmpd101 pretreated and non‐pretreated cells. Data represent the mean ± SEM of three experiments of independent cell preparations performed in three technical replicates. Data of graphs (a), (c), and were normalized against the maximal response of cmpd101 untreated cells. *p ≤ 0.05 (full statistical analysis is disclosed in Table S1)