GPCR Activation Monitoring by BRET

Gq BRET probes (Gq-RLucII, Gβ1, Gγ1-GFP10) and Gi BRET probes (Gi1-RLuc8, Gβ3, Gγ9-GFP10) were generated according to previous reports.^{53 (link),78 (link)} G protein activation BRET assay was performed as previously described.^{53 (link)} Briefly, HEK293T cells were transiently co-transfected with WT or chimeric AT1 or AT2 plasmid together with Gq or Gi BRET probes. Twenty-four hours after transfection, cells were distributed into a 96-well microplate (Corning Inc., Corning, NY, USA) for additional 24 h and then preincubated with or without COMP protein or losartan for 30 min, followed by stimulation with an increasing amount of AngII for 2 min. BRET² between RLucII and GFP10 or between RLuc8 and GFP10 was measured after the addition of the substrate coelenterazine 400a (5 μM, Interchim) using a Mithras LB940 multimode reader (Berthold Technologies). The BRET² signal was calculated as the ratio of emission of GFP10 (510 nm) to RLucII or RLuc8 (400 nm).