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