Measuring α2-Adrenoceptor Arrestin Recruitment

Investigation of α_2AAR and α_2BAR stimulated β-arrestin-2 recruitment was performed applying an assay which is based on fragment complementation of β-galactosidase (PathHunter assay, DiscoverX, Birmingham, UK) as described (79 (link)). In detail, HEK293T cells stably expressing the enzyme acceptor (EA) tagged β-arrestin-2 were cotransfected with human α_2AAR or α_2BAR each fused to the ProLink-ARMS2-PKS2 fragment for enzyme complementation and GRK2 (cDNA Resource Center) at equal amounts and subsequently transferred into 384 well microplates (Greiner) after 1 day. After incubation for further 24 hours cells were incubated with test compounds for 60 min (α_2AAR) or 90 min (α_2BAR), arrestin recruitment was stopped by adding detection regent and the resulting chemoluminescence was monitored with a Clariostar plate microreader. Data were normalized relative to buffer (0%) and the maximum effect of norepinephrine (100%). Three to nine repeats for α_2AAR (three to six for α_2BAR) in duplicate were measured.