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

Abstract

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 signaling 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 cytoplasmic structural features. In particular, the orientation of the PGE₂ ω-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.

Keywords: Gs/Gi coupling selectivity; cryo-EM; propagating paths; prostaglandin E2 receptor.

Fig. 1.

Functional characterization and biased properties of different EP4 agonists. (A) Schematic diagram showing that EP4 mediates membrane trafficking of AQP2, and restrains the KIM-1, inflammatory factor responses in AKI via the EP4-G_s pathway in renal. (B) Chemical structures of PGE₂, Rivenprost, L902688, and KMN-80. The carboxylic acid ester of Rivenprost’s α chain can be hydrolyzed into carboxylic acid (35). (C) Schematic diagram of the effects of Rivenprost and L902688 on AKI mice. The Cdh16Cre⁺/Gnas^flox/flox (Cko) mice represented the renal tubule-specific Gα_s knockout mice, while the Gnas^flox/flox (flox) mice were used as control. “√”, beneficial to treat AKI; “X”, not conducive to treat AKI; “XX”, accentuated the AKI. (D and E) The effects of Rivenprost on SCR (D) and BUN (E) levels of AKI mice (n = 8). Data are mean ± SEM. ***P < 0.001. Data were analyzed by Student’s t test. ^###P < 0.001, ^&&&P < 0.001. Data were analyzed by one-way ANOVA with Tukey’s test. (F) The Emax of ΔBRET values of AQP2 trafficking stimulated with Rivenprost alone, Rivenprost and PTX (100 ng/mL), Rivenprost and NF449 (100 μM), or Rivenprost and MF498 (1 μM). Data are mean ± SEM, (n = 4).^###P < 0.001. Data were analyzed by Student’s t test. ns, no significant difference. ***P < 0.001; **P < 0.01. Data were analyzed by one-way ANOVA with Tukey’s test. (G) Dextran permeability in response to L902688 (5 μM), Rivenprost (5 μM), Rivenprost (5 μM) and PTX (100 ng/mL), Rivenprost (5 μM) and NF449(100 μM), Rivenprost (5 μM) and MF498 (1 μM) in HUVECs. Data are mean ± SEM, (n = 4). ^###P < 0.001; ns, no significant difference. Data were analyzed by Student’s t test. ***P < 0.001, **P < 0.01. Data were analyzed by one-way ANOVA with Tukey’s test.

Fig. 2.

Cryo-EM structure of agonist-bound EP4-G_s, EP4-G_i and EP3-G_i complexes. (A) Cryo-EM density of the Rivenprost-EP4-G_s complex (Left), the L902688-EP4-G_i complex (Middle) and the PGE₂-EP3-G_i complex (Right). (B) Vertical positions comparison of the agonists in EP4-G_s complexes, EP4-G_i complexes, and the EP3-G_i complex. The brown dashed line indicates the insertion depth of the E-ring of L902688 in the EP4-G_s complex (lower line) or EP4-G_i complex (upper line). (C) Schematic diagram showing that the nature diversifies hormone function at three primary levels: 1) the yellow box: different receptors can recognize the same ligands, probably via variable amino acid composition at the receptor level; and 2) the green box: the same receptor–ligand pair can couple to different cellular effectors, in certain cases potentially dependent on different structural states with the same primary amino acid sequence for the particular receptor at the conformational level (additionally regulated by the presence of the effector or local effector concentrations); 3) the blue dotted box: different alternative splicing or posttranslational modifications of GPCR may serve as a putative layer for the generations of diverse signals downstream of a single hormone–receptor pair engagement.

Fig. 3.

Different binding modes of endogenous PGE₂ in distinct EP receptor subtypes. (A) Comparison of PGE₂ binding sites between EP4, EP2, and EP3. The L/M^3.32-L^7.36 hydrophobic interactions and the Y^2.65-T^ECL2-R^7.40 motif are shown as blue- and red-filled circles, respectively. The F102^3.35 engaged with PGE₂ in the EP4-G_s complex is highlighted by orange-filled circles. Residues without interaction are shown as blank circles. Residues circled by the red dashed line constituted the different interaction modes with PGE₂ in EP2 or EP3 compared to those in EP4, and the combined mutation of the allergic positions in EP4 that mutated to those of EP2 (M^3.32T^3.36L^7.42S^7.46) or of EP3 (Q^2.54G^3.36S^3.39Q^7.46) are shown. The F102, S103, and P322 in EP4 are surrounded by a blue dashed box. Ballesteros–Weinstein residue numbers are provided for reference. (B) The ω-chain of PGE₂ showed differences in the ligand interaction pattern in EP receptor structures with magenta in EP2-G_s, blue in EP4-G_s, orange in EP4-G_i, and green in EP3-G_i. The red dashed line represents the same horizontal plane. (C) Mutagenesis studies showing the effects of residues on ligand-binding pocket. Heatmap of the ΔpEC₅₀ (pEC₅₀ of mutant - pEC₅₀ of wild type) and Emax (normalized to 100% wild type) showed differences in the Gα_s-Gγ/Gα_i-Gγ dissociation assay. Data are mean ± SEM, (n = 3). (D) Comparison of the biased properties of the combined mutations of EP4. The bias factor (β value) of EP4-MTLS or EP4-QGSQ was calculated using the EP4-WT as the reference by the method in the Materials and Methods of Gα_s-Gγ/Gα_i–Gγ dissociation assay. Data are mean ± SEM, (n = 3).

Fig. 4.

The selective binding pockets of synthetic agonists in EP4. (A) Cutaway view of Rivenprost in EP4 receptors. The residues T76^2.61 and S103^3.36 form a hydrogen bond with Rivenprost. (B) Sequence alignment of EP family members in ligand pocket. Note that T76^2.61 was only found in EP4; L304^7.42 was only found in EP2; S^3.36 is conserved in EP2 and EP4, but replaced by G^3.36 in EP1 and EP3. (C) The decreasing folds of the equivalent mutations of T/V^2.61, S/G^3.36 and A/L^7.42 between EP subfamily receptors in ligand binding. Data are mean ± sem.

Fig. 5.

A G-protein-subtype-dependent agonist binding mode in EP4. (A) The distinct configurations and positions of L902688 in the EP4-G_s and EP4-G_i complex. The red arrows indicate the movement of corresponding L902688 regions. Hydrogen bonds and polar interactions are shown by red dashed lines. (B) Interactional differences of EP4 in L902688 pocket between G_s and G_i signaling complex. (C) Schematic diagram of the ternary complex model (–54). The model involves the interaction of the hormone (ligand), the receptor (EP4), and the downstream effector (G protein). The K_Lo represents the ligand interacting with the free receptor to constitute a low-affinity state, which then coupled with G protein, representing as K_C. The K_G represents that the free receptor coupled with G protein, and the K_Hi represents the ligand binding to such a precoupled receptor-G protein complex. The scheme also shows the formation of the high-affinity ternary complex for effective signaling. (D and E) The competition binding curves of L902688 in response to wild-type EP4 receptor, EP4-Gα_i/EP4-Gα_s fusion protein (EP4-G_i/EP4-G_s) and EP4-Gα_i/EP4-Gα_s with A318L mutation (EP4-A318L-G_i/EP4-A318L-G_s). L902688 showed two different binding states to EP4-G_i/EP4-G_s, containing a high-affinity state (K_Hi) and a low-affinity state (K_Lo). Gα fusion induced a fraction of EP4 into the high-affinity state, at 34% and 47% in EP4-G_s and EP4-G_i, and a large shift of K_Hi. In contrast, the K_Lo of both EP4-G_s and EP4-G_i are similar to wild-type EP4. A318L mutant weakened L902688 K_Hi and K_Lo, both in EP4-G_s and EP4-G_i. Importantly, the high-affinity fraction reduced from 34% in EP4-G_s to 9% in EP4-A318L-G_s chimeric mutation, while EP4-A318L-G_i chimeric mutation showed no reduction. Data are mean ± SEM, (n = 3).

Fig. 6.

Distinct configurations and propagating paths of G_s and G_i-bias activation mechanisms of EP4. (A) Schematic diagram representing the propagating paths that contribute to the G_s/G_i bias decision mechanism. The ligand pocket in L902688-EP4-G_s (magenta) was deeper than that in L902688-EP4-G_i (blue). The propagating paths started with residues constituting two different ligand-binding pockets and extended to the G_s/G_i protein interface region. The residues in TM2, TM3, TM6, and TM7 are shown as brown, blue, magenta, and green circles, respectively. (B and C) The different structural rearrangement of the ω-chain of L902688 in the EP4-G_i and EP4-G_s complex. (D–F) Key residues along the propagating paths connected the ligand-binding pocket to the cytoplasmic side by comparison of L902688-EP4-G_s (green); L902688-EP4-G_i (blue); ONO-AE3-208-EP4 complexes (gray). (G) Effects of the residues’ mutations along the propagating path in response to L902688. The heat map is filled according to the ΔpEC₅₀ and Emax (100% wild type). “X”, no detectable signal. Data are mean ± SEM, (n = 3). (H and I) The competition binding curves of EP4-Gα_s (H) and EP4-Gα_i fusion protein (I) with V320A, N321A, and D325R mutants in response to L902688. Data are mean ± SEM, (n = 3). (J) The FlAsH-BRET responses of EP4 intracellular region between the ICL2 (S2) or TM7 terminus (S4) and the C terminus in response to the G_s/G_i signal. Data are mean ± SEM, (n = 3 or 4). The concentration (M) of Rivenprost and L902688, and the mutants along the propagating path are shown left. The G_s and G_i signal’s effects were surrounded by red and green dotted lines, respectively. **P < 0.01; ns, no significant difference. Data were analyzed by one-way ANOVA with Tukey’s test.