a, Kinetic ligand-binding assay using ROX-exendin-4 as the fluorescent probe. TT-OAD2 is only able to partially displace the probe and with slower kinetics relative to exendin-4 that shows complete displacement of the probe with rapid kinetics. b, cAMP accumulation studies using GLP-1 and TT-OAD2 as the agonist in wild-type HEK293 cells and HEK293 cells in which G s/olf (ΔG s ) or all G i/o/z (ΔG i/o/z ) have been depleted using CRISPR–Cas9. c, HEK293A cells transiently transfected with the GLP-1R and the NanoBit constructs for Gα s and Gα i2 (Gα-LgBIT, Gγ 2 -SmBIT). Luminescence signal was assessed over time (0–20 min) in the presence of increasing concentrations of GLP-1 and TT-OAD2. Concentration response curves are expressed as AUC (0–20 min) for each concentration and normalized to the negative response observed by GLP-1 at 1 μM. d, Agonist-induced changes in trimeric G s protein conformation. Ligand-induced changes in BRET were measured in plasma membrane preparations performed in kinetic mode until kinetic equilibrium was reached for vehicle or increasing concentrations of GLP-1 (left) and TT-OAD2 (right). The addition of GTP dissociated the trimeric G protein complex stabilized by GLP-1-occupied and TT-OAD2-occupied GLP-1R. e, Agonist-induced changes in trimeric G i2 protein conformation. Left, ligand-induced changes in BRET were measured in plasma membrane preparations performed in kinetic mode until kinetic equilibrium with a saturating concentration of GLP-1 and TT-OAD2. The BRET signal decreased in the presence of GTP, which suggests that GTP dissociated the G i2 protein complex stabilized by GLP-1-occupied and TT-OAD2-occupied GLP-1R. Quantification of the plateau (middle) and the rate of ligand-induced conformational change (right) for each agonist (1 μM GLP-1 and 10 μM TT-OAD2) was calculated by applying a one-phase association curve to the kinetic data with values from each individual experiment show in black circles. f, Concentration–response curves of production in live HEK293 cells expressing the GLP-1R and an EPAC BRET biosensor in the presence of different concentrations of GLP-1 and TT-OAD2. Left, cAMP response taken 25 min after ligand addition. Right, area under the curve (AUC) analysis of the response calculated as AUC across the full kinetic trace for each ligand concentration (from data in Fig. 2d). Data are mean + s.e.m. of 4–6 independent experiments performed in duplicate or triplicate.