Fig. 4: Ternary complex formation of β1AR isoprenaline-bound with non-canonical G proteins (secondary coupling).
From: Binding kinetics drive G protein subtype selectivity at the β1-adrenergic receptor
a In cell measurement of the dissociation of the Gβγ G protein subunit from Gα from different families using the BRET2-based TRUPATH assay for G protein activation. Signal was measured for varying isoprenaline concentrations to obtain concentration-response curves for β1AR-E (black) and β1AR-W (grey). b pEC50 values for isoprenaline bound β1AR measured using the TRUPATH assay. Values were obtained for both β1AR-E and β1AR-W constructs signalling via Gs, Gi1, Go1 and Gq. For both (a) and (b), data are mean ± SEM of n = 3 independent biological experiments each performed in duplicate. c In vitro BLI binding kinetics and affinity data for different mini-G proteins binding to detergent solubilised avi-β1AR-E (black) and avi-β1AR-W (grey). The mini-Gs shows a dramatically increased kon rate compared to the other proteins. Whilst the koff rates are more comparable, the significant differences in kon are the major contributor to the higher affinity of the mini-Gs ternary complex. See Supplementary Table 6 for exact p values. In brief, all kon values for β1AR-E (log scale) are significantly different from each other (p < 0.05) except those for mini-Go1 and mini-Gs/i. None of the koff values for β1AR-E are significantly different from each other (p < 0.05) except those for mini-Gs/q and mini-Gs/i. All KD values (log scale) for β1AR-E are significantly different from each other (p < 0.05) except between mini-Go1 and mini-Gs/i / mini-Gs/q. Statistical significance was calculated with unpaired two-tailed t-tests, with a Bonferroni correction to the significance level for multiple comparisons. All parameters between β1AR-E and β1AR-W for each binding partner are significantly different (p < 0.05) aside from koff rates for mini-Gs. See Supplementary Table 7 for exact p values comparing β1AR-E and β1AR-W kinetic parameters. Statistical significance was calculated with unpaired two-tailed t-tests. For convenience, the individual values for kon, koff and KD are indicated above the bars in the charts. Values are the averages of n = 3 individual repeats, and error bars indicate the SD of these replicates (Supplementary Table 5). See Methods and Supplementary Methods for details.
