NanoBiT–G protein dissociation assay

GPCR-induced G_s dissociation was measured by a NanoBiT–G protein dissociation assay (33), in which interaction between a Gα subunit and a Gβγ subunit was monitored by a NanoLuc-based enzyme complementation system called NanoBiT (Promega). Specifically, a NanoBiT-G_s protein consisting of Gα_s subunit fused with a large fragment (LgBiT) at the alpha helical domain and an N-terminally small fragment (SmBiT)-fused Gγ₂ subunit with a C68S mutation was expressed along with untagged Gβ₁ subunit, RIC8B and a test GPCR. HEK293A cells (Thermo Fisher Scientific) were seeded in a 6-well culture plate at a concentration of 2 × 10⁵ cells ml⁻¹ [2 ml per well in Dulbecco’s modified Eagle’s medium (DMEM; Nissui) supplemented with 10% fetal bovine serum (Gibco), glutamine, penicillin, and streptomycin] 1 day before transfection. Transfection solution was prepared by combining 5 μl (per well in a 6-well plate hereafter) of polyethylenimine Max solution (Polysciences; 1 mg ml⁻¹), 200 μl of Opti-MEM (Thermo Fisher Scientific) and a plasmid mixture consisting of 100 ng LgBiT-containing Gα_s subunit, 500 ng Gβ₁, 500 ng SmBiT-fused Gγ₂ (C68S) 100 ng, RIC8B and a test GPCR that was optimized to match an expression level. After incubation for 1 day, transfected cells were harvested with 0.5 mM EDTA-containing Dulbecco’s PBS, centrifuged and suspended in 2 ml of HBSS containing 0.01% bovine serum albumin (BSA; fatty acid–free grade; SERVA) and 5 mM HEPES pH 7.4 (assay buffer). The cell suspension was dispensed in a white 96-well plate at a volume of 80 μl per well and loaded with 20 μl of 50 μM coelenterazine (Carbosynth) diluted in the assay buffer. After 2 hours of incubation at room temperature, the plate was measured for baseline luminescence (Spectramax L, Molecular Devices) and a titrated test ligand (20 μl; 6X of final concentrations) were manually added. Ligands and their sources were as follows: (-)-isoproterenol hydrochloride (Sigma-Aldrich) for β₂AR; serotonin (5-hydroxytryptamine hydrochloride; FUJIFILM Wako Pure Chemical) for 5-HT₄; prostaglandin D₂ (Cayman Chemical) for DP; histamine dihydrochloride (FUJIFILM Wako Pure Chemical) for H₂R; arginine vasopressin (Peptide Institute) for V₂R; glucagon (Zealand Pharma) for GCGR; glucagon-like peptide 1 (7-37) (Peptide Institute) for GLP-1; PACAP27 (Peptide Institute) for PAC1; parathyroid hormone (1-34) (Peptide Institute) for PTH. The plate was immediately read at room temperature for the following 3 min at a measurement interval of 7 s with an accumulation time of 0.17 s per read. The kinetics luminescence counts were normalized to the initial count and fold-change signals over vehicle treatment were used to plot G protein dissociation response. G protein dissociation kinetics were calculated by fitting the normalized luminescent data to a one-phase dissociation model built in Prism 8 software (GraphPad Prism). Initial G protein dissociation speed was calculated by a formula of (Plateau – 1)*K where “Plateau” represents a saturated normalized luminescent counts whereas “K” denotes a rate constant in a unit that is reciprocal of time (sec). The resulting dissociation speed data were fitted to a three-parameter sigmoidal concentration-response curve (“Bottom” is fixed to a value of 0), from which an E_max value (“Top”) was used to represent G protein dissociation for a given experiment. When a sigmoidal curve did not converge due to a lack of saturating data points, a dissociation speed at the highest ligand concentration was used as an E_max value.