Fig. 6. Conformational changes in GCGR and β₂AR upon ligand binding, G protein coupling, and G protein dissociation.

(A and B) DEER data showing no change in the distance distribution, between Apo (gray shading) and ZP3780 binding (red), of the TM4-TM6 and TM4-TM5 pairs. The upper limit of reliable distance (r) and width (σ) determination are shown as gray and black bars, respectively. (C) The apo (gray) spectrum of bimane-labeled GCGR (TM6) does not change upon ZP3780 addition (red). Further addition of G_s (purple) results in a decrease in fluorescence intensity and a 4-nm λ_max shift owing to outward movement of TM6. (D) β₂AR labeled with bimane in TM6 shows a decrease in fluorescence and a redshift in λ_max when agonist, epinephrine (Epi) (blue), is added to apo (black), which changes further upon G_s (green) addition. (E) The addition of GDP (orange) or GTPγS (cyan) to NBD-labeled GCGR (TM6) bound to ZP3780 and coupled to G_s (purple) does not result in an increase of the fluorescence intensity to the level of ZP3780-bound GCGR in the absence of G protein (red). (F) The addition of GDP (brown) or GTPγS (light purple) to bimane-labeled β₂AR bound to Epi and coupled to G_s (green) leads to an increase in fluorescence intensity and a blueshift in λ_max to the same values as that for for Epi-bound β₂AR in the absence of G protein (blue). In (C) to (F), data represent mean ± SEM of triplicate measurements.