A Split Luciferase Complementation Assay for the Quantification of β-Arrestin2 Recruitment to Dopamine D₂-Like...

Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of β-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify β-arrestin2 recruitment to D_2long and D₃ receptors and measure time-resolved β-arrestin2 recruitment to the D_2long receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D_2longR and D₃R subtypes, whereas for the D_4.4R, no β-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the β-arrestin recruitment to the D_2longR and D₃R, as well as at the D₁R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/β-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists.