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

Abstract

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.

Keywords

• Luminescence measurements
• Luciferase
• HEK 293 cells
• G protein-coupled receptor kinase