Investigating the Kinetics of β 1 and β 2 Adrenoceptor Effector Recruitment using Luciferase Complementation Assays

Nanoluc‐based luciferase complementation assays (NanoBiT; Dixon et al, 2016) provide new approaches to measure G protein‐coupled receptor (GPCR) signalling, by assessing the kinetics of receptor subtype recruitment of effector proteins in real‐time. Here, we use NanoBiT systems to compare β adrenoceptor subtype (β 1 AR/β 2 AR), recruitment of β‐arrestin2 and an engineered Gα s GTPase domain sensor (mini Gα s ; mGα s )( Wan et al, 2018). HEK293T cells stably expressed SNAP‐tagged βAR (β 1 AR or β 2 AR) fused to C‐terminal 18 kDa LgBiT luciferase fragment, and the 11 amino acid SmBiT fused to either β‐arrestin2 or mGα s . NanoBiT assays were conducted in cells in 96 well plates in HBSS/0.1% BSA at 37°C. Cells were pre‐incubated with furimazine (5min; 37°C), before the addition of agonist. Luminescence was monitored for 31 min (BMG Pherastar Platereader, 37°C). Concentration response curves were fitted in GraphPad Prism v7, with pharmacology quoted as mean±s.e.m (n≥3) Both βAR subtypes stimulated transient β‐arrestin2 recruitment upon 10μM isoprenaline stimulation (Figure 1a). At 31 min, both receptor subtypes reported partial agonism of salbutamol and salmeterol, with increased potency and efficacy (relative to isoprenaline) at β 2 AR compared to β 1 AR (Figure 1c–d). 10μM Isoprenaline also stimulated recruitment of mGa s at each subtype, increasing rapidly between 0 and 5 min post agonist addition. Association of β 2 AR‐mGα s complexes was maintained stably throughout incubation, whilst at β 1 AR, mGα s recruitment continued to increase over incubation window, reaching 322.0±39.1% by 31 min (%10μM isoprenaline, at 3 min) (Figure 1b). NanoBiT complementation assays report real‐time recruitment of β‐arrestin2 and mGα s protein, at both β 1 AR and β 2 AR. Transient β‐arrestin2 recruitment was reported at both receptor subtypes responses distinguished full and partial agonists. Further studies of the continued βAR‐mGα s association will probe whether mGα s sensors remain stably associated with membrane or endosomal pools of adrenoceptors. Support or Funding Information PhD funded by Biotechnology and Biological Sciences Research Council (BBSRC).NanoBiT luciferase complementation monitors βAR recruitment of signalling effector proteins. Signalling timecourse of (a.) β‐arrestin2 and (b.) mGα s recruitment, at β 1 AR and β 2 AR. Concentration response curves of β‐arrestin2 recruitment, at (c.) β 1 AR and (d.) β 2 AR. Data points represent mean±s.e.m.; n≥3.Dixon A.S. , et al. ( 2016 ). ACS Chem. Biol. , 11 : 400 – 408Wan Q. , et al ( 2018 ). J. Biol. Chem. , 293 ( 19 ): 7466 – 7473