Ligand recognition, unconventional activation, and G protein coupling of the prostaglandin E ₂ receptor EP2 subtype

Abstract

Selective modulation of the heterotrimeric G protein α S subunit–coupled prostaglandin E2 (PGE2) receptor EP2 subtype is a promising therapeutic strategy for osteoporosis, ocular hypertension, neurodegenerative diseases, and cardiovascular disorders. Here, we report the cryo–electron microscopy structure of the EP2-Gs complex with its endogenous agonist PGE2 and two synthesized agonists, taprenepag and evatanepag (CP-533536). These structures revealed distinct features of EP2 within the EP receptor family in terms of its unconventional receptor activation and G protein coupling mechanisms, including activation in the absence of a typical W6.48 “toggle switch” and coupling to Gs via helix 8. Moreover, inspection of the agonist-bound EP2 structures uncovered key motifs governing ligand selectivity. Our study provides important knowledge for agonist recognition and activation mechanisms of EP2 and will facilitate the rational design of drugs targeting the PGE2 signaling system.

Development of fluorescent peptide G Protein Coupled Receptor activation biosensors for NanoBRET characterisation of intracellular allosteric modulators

“…NanoBRET was monitored for 30 min every 1.16 minutes on the PHERAstar, using the BRET ratiometric measurements described above. In experiments to understand TMR-GαS19cha18 selectivity, agonist binding assays were repeated using the same protocol employing Hek-EP 2 -tsNluc or Hek-ssCXCR2-tsNluc cell membranes, stimulated with PGE2 9 or CXCL8 (Stratech Scientific, Cambridge, UK), or vehicle. In these experiments the extent of recruitment was assessed 30 min after membrane addition at 37°C.…”

Molecular basis for lipid recognition by the prostaglandin D2receptor CRTH2

“…2E). Supporting distinct lipid recognition mechanisms for CRTH2 and other prostanoid receptors, EP2 residues R 7.40 , Y 2.65 , and T ECL2 , that interact with the carboxyl group of PGE 2 are highly conserved in all prostanoid receptors except for CRTH2 (33), while CRTH2 residues, R170 ECL2 , Y184 ECL2 , and K210 5.42 , that form a polar interaction network with the carboxyl group of PGD 2 are not conserved in most of other prostanoid receptors (Fig. 2F).…”

“…Distinctive Lipid Recognition by CRTH2. Recently, structures of several prostaglandin receptors have been reported, including crystal structures of EP3 bound to two prostaglandin agonists, misoprostol and PGE 2 , antagonist-bound EP4 and TP, and cryo-EM structures of the EP2-G s and EP4-G i complexes with PGE 2 (28)(29)(30)(31)(32)(33). The structural comparison of 15mPGD 2 -bound CRTH2 to those structures of other prostanoid receptors reveals significant differences in lipid recognition and receptor activation.…”

“…Indeed, previous studies on other GPCRs suggested that W 6.48 functions as a "toggle switch" or a part of a "transmission switch" with F 6.44 that links the ligand binding at the extracellular region to the activation of the receptor at the cytoplasmic region (43,44). For EP2 and EP4, the conserved W 6.48 is replaced by S 6.48 (31)(32)(33). Structural studies on the EP2 signaling complex with PGE 2 suggested that a direct interaction between the ω-chain of PGE 2 and M124 3.40 instead of W 6.48 , which packs against F273 6.44 , may stabilize the active conformation of EP2 (33) (Fig.…”

“…The structural analysis of antagonistbound CRTH2 suggested that PGD 2 may enter the ligand-binding pocket through the ligand entry port by opposite charge attraction (27). Recently, crystal structures of several other prostanoid receptors, EP3, EP4, and TP, and cryogenic electron microscopy (cryo-EM) structures of the EP2 and EP4 signaling complexes with PGE 2 have also been reported (28)(29)(30)(31)(32)(33). To further study the binding of lipid agonists to CRTH2 and investigate if CRTH2 differs from other prostanoid receptors in lipid recognition and receptor activation, we determined a 2.6-Å resolution, room temperature crystal Significance Many amphipathic lipids, including cannabinoids, lysophospholipids, prostanoids, and leukotrienes, bind to G proteincoupled receptors (GPCRs) through the lipid bilayer.…”