Objective

BACKGROUND. G protein coupled receptors (GPCRs) signal through multiple intracellular effectors, such as G proteins and β-arrestins. Some ligands are suggested to preferentially activate one signalling pathway over another, creating signalling bias. There is increasing evidence that the kinetics of agonist binding and signalling influence bias (Herenbrink CK et al, 2016), highlighting the need for novel methods to measure receptor signalling dynamics. Here, NanoLuc based luciferase complementation assays (NanoBiT®) (Dixon AS et al, 2016) were used to monitor the recruitment of β-arrestin2 (βARR2) and a ‘mini’ G_αs protein (mG_αs) to the β₂ adrenoceptor (β₂AR).

METHODS. HEK293T cells stably expressing SNAP-tagged β₂AR with C-terminally fused 18 kDa ‘LgBiT’ luciferase fragment and the 11 amino acid ‘SmBiT’ fragment fused to the N-terminal of either βARR2 or mG_αs were seeded into white 96 well plates. After 24hrs, NanoBiT assays were conducted in HBSS/0.1% BSA at 37°C. Cells were pre-incubated with furimazine, followed by agonist stimulation. Luminescence was monitored throughout agonist stimulation (31min, BMGPherastar2, 37°C). Concentration response curves were fitted in GraphPad Prism v7, with pEC₅₀ and R_MAX values as mean±S.E.M (n=4/5).

RESULTS. 10µM isoprenaline-induced βARR2 recruitment peaked at 3 minutes, whilst mG_αs recruitment rapidly increased between 0-3 minutes and was sustained throughout the incubation. The potency of isoprenaline for βARR2 recruitment increased 10-fold between 3 and 31 minutes (pEC50 values; 7.12±0.06 vs 8.11±0.08, respectively), whilst mGαs increased 3-fold between 3 and 31 minutes (pEC50 values; 7.55±0.13 vs 8.08±0.11, respectively). At 31 minutes, partial agonism was observed for salbutamol and salmeterol in both βARR2 (74.8±8.3 and 48.3±5.5, respectively. %10µM isoprenaline) and mG_αs (90.2±9.6 and 70.3±4.8, respectively. %10µM isoprenaline) recruitment.

DISCUSSION. NanoBiT® complementation reports rapid kinetics of mG_αs and βARR2 at β₂ adrenoceptors, reflecting pharmacology of full and partial agonists. Further investigation of GPCR-mG_αs response will identify whether this reflects continued associations with cell surface or endosomal located receptors.

- Herenbrink C.K., Sykes D.A., et al. (2016). Nature communications, 7:10842.

- Dixon A.S., Schwinn M.K., et al. (2016). ACS Chem Biol, 11:400-408