Extended Data Fig. 9: WT β2AR and mutants of GsGDP coupling interface in the nano-luciferase BRET assay for Gα subunit and Gβγ dissociation.
From: Mechanistic insights into G-protein coupling with an agonist-bound G-protein-coupled receptor
A) Concentration response curves (CRC) of wild-type (WT) β2AR and the mutants in the Nano-luciferase BRET assay for Gα subunit and Gβγ dissociation. Single mutant K273Q6.35, the double mutant K270Q6.32/K273Q6.35, the triple mutant K267Q6.29/K270Q6.32/K273Q6.35 and the wild-type were titrated with isoproterenol (ISO) and used to determine the EC50. The CRC curves were normalized on the WT response. 6 experiments in triplicates per concentration were performed for WT, double and triple lysine mutants and 7 experiments for the single lysine K273Q6.35. Error bars indicate the standard error of the mean. B) Mean maximal delta BRET values of wild-type and mutant β2AR in the Nano-luciferase BRET assay for Gα subunit and Gβγ dissociation. Error bars indicate the standard error of the mean, number of experiments (N) indicated at the bottom of the bars, N is 6 for K267Q6.29/K270Q6.32/K273Q6.35. Statistical significance was calculated using t-test with Welch´s correction, two-tailed p values < 0.05 = * (p value = 0.0167), < 0.001 = *** (p value = 0.0002), < 0.0001 = **** C) EC50 values of agonist-induced Gs dissociation using Nano-luciferase BRET assay. Number of independent experiments (N) performed in triplicates indicated in columns; error bars indicate the SEM – Standard Error of the Mean. P values were calculated using unpaired t-test with Welch´s correction. Two-tailed P value < 0.05 = * (p value of 0.0486 for wt vs. K273Q6.35, p value of 0.0119 for wt vs. K267Q6.29/K273Q6.35. EC50 values of the triple mutant could not be determined due to low delta BRET% values.
