Extended Data Fig. 7: Experimental validation of the β2AR–GsGDP coupling interface by functional and binding studies.
From: Mechanistic insights into G-protein coupling with an agonist-bound G-protein-coupled receptor
A) Kinetic traces capturing the initial onset of Gs activation at β2AR WT and mutants upon stimulation (t = 0 s) with 10 µM isoproterenol (ISO) by quantifying Gβγ association with a membrane-associated reporter GRK3-C-tail following after dissociating from Gα (Gβγ-release-BRET). Data represent mean ± SEM from (n) independent experiments: WT (n = 14), R63Q/N69M (n = 7), Y141V/Q142L (n = 7), K273Q (n = 7), K270Q/K273Q (n = 7), K267Q/K270Q/K273Q (n = 7), G276A/G280A (n = 7) and G276V/G280V (n = 6). B) Surface expression levels of β2AR WT and mutants for the Gs activation assay (Gβγ-release- BRET). Data represent mean ± SEM from (n) independent experiments: WT (n = 5), R63Q/N69M (n = 4), Y141V/Q142L (n = 5), K273Q (n = 5), K270Q/K273Q (n = 3); K267Q/K270Q/K273Q (n = 5), G276A/G280A (n = 5) and G276V/G280V (n = 5). C) Surface expression levels of β2AR WT and mutants for the cAMP accumulation assay. Data represent mean ± SEM from 7 independent experiments. D) Concentration response curves of β2AR WT and mutants and derived pEC50 values for Gs-mediated cAMP accumulation. Data represent mean ± SEM from 7 independent experiments. Statistical significance (P) was determined by one-way ANOVA, followed by Dunnett’s multiple comparisons test. P values: WT high (P = 0.5183), R63Q/N69M (P = 0.0051), Y141V/Q142L (P = < 0.0001), K273Q (P = 0.801), K270Q/K273Q (P = 0.0008), K267Q/K270Q/K273Q (P = < 0.0001), G276A/G280A (P = 0.9956) and G276V/G280V (P = 0.202). ns; not significant, P < 0.05 = *, P < 0.01 = **, P < 0.001 = ***, P < 0.0001 = ****. E) A representative graph of an SDS-PAGE gel showing purified β2AR WT, K273Q, K267Q/K270Q/K273Q (3 M) fractions used for the Glo assay (for gel source data, see Supplementary Fig 1). F) Saturation binding experiments of [3H]-DHA to purified β2AR WT, K273Q and K267Q/K270Q/K273Q showing similar affinities (KD). Data indicate similar sub-micromolar affinities (Ki) of purified β2AR WT, K273Q and K267Q/K270Q/K273Q. Data represent mean ± SEM from 3 independent experiments. H) [3H]-DHA binding affinities (KD) and estimated isoproterenol affinities (pKi) for WT and mutant receptors expressed in HEK293 membranes. KD and pKi values indicate similar estimated binding affinities of isoproterenol for WT and mutants. KD values were determined from similar [3H]-DHA saturation as in F). pKi values were determined from similar competition curves as in G) to first determine isoproterenol IC50 followed by application of the Cheng-Prussoff equation to factor in radioligand affinity and concentration (see Methods). Data from [3H]-DHA saturation binding experiments (KD) represent mean ± SEM from 3 independent experiments. Data from [3H]-DHA competition binding experiments (pKi) represent mean ± SEM from 5 independent experiments.
