Single hormone or synthetic agonist induces G_s/G_i coupling selectivity of EP receptors via distinct binding modes and propagating paths

To accomplish concerted physiological reactions, nature has diversified functions of a single hormone at at least two primary levels: 1) Different receptors recognize the same hormone, and 2) different cellular effectors couple to the same hormone-receptor pair [R.P. Xiao, Sci STKE 2001, re15 (2001); L. Hein, J. D. Altman, B.K. Kobilka, Nature 402, 181-184 (1999); Y. Daaka, L. M. Luttrell, R. J. Lefkowitz, Nature 390, 88-91 (1997)]. Not only these questions lie in the heart of hormone actions and receptor sign-aling but also dissecting mechanisms underlying these questions could offer therapeutic routes for refractory diseases, such as kidney injury (KI) or X-linked nephrogenic diabetes insipidus (NDI). Here, we identified that G(s)-biased signaling, but not G(i) activation downstream of EP4, showed beneficial effects for both KI and NDI treatments. Notably, by solving Cryo-electron microscope (cryo-EM) structures of EP3-G(i), EP4-G(s), and EP4-G(i) in complex with endogenous prostaglandin E2 (PGE2)or two synthetic agonists and comparing with PGE2-EP2-G(s) structures, we found that unique primary sequences of prostaglandin E2 receptor (EP) receptors and distinct conformational states of the EP4 ligand pocket govern the G(s)/G(i) transducer coupling selectivity through different structural propagation paths, especially via TM6 and TM7, to generate selective cyto-plasmic structural features. In particular, the orientation of the PGE2 omega-chain and two distinct pockets encompassing agonist L902688 of EP4 were differentiated by their G(s)/G(i) coupling ability. Further, we identified common and distinct features of cytoplasmic side of EP receptors for G(s)/G(i) coupling and provide a structural basis for selective and biased agonist design of EP4 with therapeutic potential.