Fig. 5. EP2-G_s coupling and G protein selectivity.

(A) Superposition of the structures of EP2-G_s and β2AR-G_s (PDB ID: 3SN6 and 7BZ2) complex according to the transmembrane helices bundle. EP2 is in sky blue, BI 167107-β2AR is in green, and formoterol-β2AR is in light pink. Rotation of the α helices and the interaction of the Gα_s with H8 were highlighted, Gα_s in the EP2-G_s complex is in yellow orange, Gα_s in BI 167107-β2AR-G_s is in green, and Gα_s in formoterol-β2AR-G_s is in light pink. (B) Comparison of the G_s coupling interface in EP2 and known receptor-G_s complexes including β2AR-G_s (PDB ID: 3SN6 and 7BZ2), GPBAR-G_s (PDB ID: 7CFN), and A_2AR–mini-G_s (PDB ID: 6GDG). Residues involved in contacting with G_s of EP2 were illustrated as blue dots, and those of β2AR, GPBAR, and A_2AR were illustrated as green dots. (C) The detailed interactions of α5 helix of G_s with residues of TM2 and H8 of EP2 in the PGE₂-bound EP2-G_s complex structure. Polar interactions are depicted as blue dashed lines. (D) Effects of mutations in TM2 or H8 of EP2 on PGE₂-induced cAMP accumulation. Bars represent the differences of calculated potency (pEC₅₀) between WT EP2 and its mutant. Values are means ± SD from three independent experiments performed in triplicate. ***P < 0.001; comparison between WT EP2 and its mutant. All data were analyzed by two-sided one-way ANOVA with Tukey’s test. (E) The detailed interactions of ICL2 of EP2 with α5 helix of G_s in the PGE₂-bound EP2-G_s complex structure. H-bond is depicted as a dashed line. (F) Sequence alignment of residues involving G_s coupling in human, mouse, and rat EP2, which are compared with residues at corresponding positions in human EP3 and EP4.