A nanoluciferase complementation-based assay for monitoring β-arrestin2 recruitment to the dopamine D₃ receptor

Luciferase complementation assays have emerged as a simple means of monitoring receptor-effector interactions in living cells in a time-resolved manner. Here, we describe a nanoluciferase complementation assay capable of reporting on β-arrestin2 recruitment to the human dopamine D₃ receptor (D₃R) upon its activation in intact HEK293T cells. Using this assay in time-resolved experiments, we detect differences in arrestin response termination rates between the endogenous agonist dopamine and the synthetic D₃R agonist FAUC-73. We also investigate the influence of exogenous GRK2 on β-arrestin2 recruitment to the D₃R. We find that, in contrast to the D₂R and D₄R, the potency of dopamine to induce arrestin recruitment to D₃R is not significantly influenced by GRK2 overexpression. In further agreement with a lack of GRK2 regulation of D₃R signalling and again contrary to the D₂R and D₄R, we do not observe dopamine-induced recruitment of GRK2 to D₃R. Conversely, dopamine concentration-dependently decreases the interaction between GRK2 and D₃R. Additionally, we examine both the Ser-9 and Gly-9 variants of the human D₃R, which, according to some earlier reports, differ in terms of dopamine affinity and functional potency. However, we find no difference in the concentration-response relationships between these two variants, neither when arrestin recruitment nor GRK2 interactions are studied. In summary, the present report demonstrates the utility of nanoluciferase complementation for studying D₃R pharmacology in living cells.