A nanoluciferase complementation-based assay for monitoring β-arrestin2 recruitment to the dopamine D(3) 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 D(3) receptor (D(3)R) 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 D(3)R agonist FAUC-73. We also investigate the influence of exogenous GRK2 on β-arrestin2 recruitment to the D(3)R. We find that, in contrast to the D(2)R and D(4)R, the potency of dopamine to induce arrestin recruitment to D(3)R is not significantly influenced by GRK2 overexpression. In further agreement with a lack of GRK2 regulation of D(3)R signalling and again contrary to the D(2)R and D(4)R, we do not observe dopamine-induced recruitment of GRK2 to D(3)R. Conversely, dopamine concentration-dependently decreases the interaction between GRK2 and D(3)R. Additionally, we examine both the Ser-9 and Gly-9 variants of the human D(3)R, 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 D(3)R pharmacology in living cells.