Fig. 7: Allosteric effect of the G protein on peptide affinity and the peptide on Gs activation.
From: Dynamics of GLP-1R peptide agonist engagement are correlated with kinetics of G protein activation
a Equilibrium competition binding assays assessing the ability of GLP-1, oxyntomodulin, exendin-4 and exendin-P5 to compete for the probe Rox-Ex4, in HEK293 cells overexpressing the GLP-1R in the presence of endogenous Gα proteins (blue), the absence of Gα proteins (red) and when Gαs is overexpressed (no endogenous Gα proteins) (green). Data are presented as % specific binding with 100% binding defined as total probe binding in the absence of competing ligand and non-specific (0%) binding determined as probe binding in the presence of 1 μM exendin-4. Data are means ± s.e.m. of 7 independent experiments performed in duplicate. b HEK293A cells are transiently transfected with the GLP-1R and the NanoBit constructs for Gαs (Gα-Lgbit, Gγ2-Smbit). Left; Luminescence signal was assessed over time (0–20 min) in the presence of saturating concentrations of GLP-1 (1 μM), exendin-4 (1 μM), oxyntomodulin (10 μM) and exendin-P5 (10 μM) and responses were normalised to the max loss of luminescence observed with GLP-1. Data shown are mean ± s.e.m. of four independent experiments performed in triplicate. Right; Quantification of the rate of G protein dissociation (luminescence change) for each agonist was calculated by applying a one-phase decay curve to the kinetic data with values from each individual experiment shown in circles with the mean ± s.e.m. of the four individual experiments. c Agonist-induced changes in trimeric Gs protein conformation. Left; Ligand-induced changes in BRET were measured in plasma membrane preparations performed in a kinetic mode in the presence of saturating concentrations of GLP-1 (1 μM), exendin-4 (1 μM), oxyntomodulin (10 μM) and exendin-P5 (10 μM). Data shown are mean ± s.e.m. of three independent experiments performed in triplicate. Right; Quantification of the rate of ligand-induced conformational change for each agonist was calculated by applying a one-phase association curve to the kinetic data with values from each individual experiment shown in circles with the mean ± s.e.m. of the four individual experiments. * Represents statistically different to GLP-1 (P < 0.05) when assessed using a one-way ANOVA of variance with a Dunnett’s post hoc test. Exact P values are shown on the relevant figures. Source data are provided in the Source Data file.
