Fig. 5. Engagement and activation of G_s by GCGR and β₂AR.

(A) Conformation of TM6 in the GCGR-G_s (cyan) and β₂AR-G_s (pink, PDB 3SN6) (50) complex structures. (B) The GTP-turnover assay shows that β₂AR (blue) activates G_s much faster than GCGR (red), with GCGR inducing a maximal GTP-turnover rate of 0.11 GTP min⁻¹ G_s⁻¹ compared with (inset) 8 GTP min⁻¹G_s⁻¹ for β₂AR. (C) Monitoring of GPCR-G_s association by means of FRET between Cy3B-labeled GCGR (red) or Cy3B-labeled β₂AR (blue) with Sulfo-Cy5-labeled G_s. The decrease in donor fluorescence shows comparable rates of association between the receptors and G_s. (D) The rate of receptor-induced [³H]-GDP dissociation from G_s shows faster release for β₂AR (blue; k_off = 0.042 s⁻¹) compared with GCGR (red; k_off = 0.0022 s⁻¹). (E) Bodipy-GTPγS binding to nucleotide-free complex is slower for the GCGR-G_s complex (k_on = 0.001 s⁻¹) compared with the β₂AR-G_s complex (k_on = 0.003 s⁻¹). (F) Concentration-response curves for G_s dissociation rates in HEK293 cells show slower G_s activation by GCGR compared with β₂AR. In (B) and (D), data represent mean ± SEM from at least three independent experiments performed in triplicates. In (C) and (E), data represent mean ± SEM of triplicate measurements. In (F), data represent mean ± SEM of three to seven independent experiments.