Structural basis for ligand recognition and signaling of the lysophosphatidylserine receptors GPR34 and GPR174

Abstract

Lysophosphatidylserine (LysoPS) is a naturally occurring lipid mediator involved in various physiological and pathological processes especially those related to the immune system. GPR34, GPR174, and P2Y10 have been identified as the receptors for LysoPS, and its analogues have been developed as agonists or antagonists for these receptors. However, the lack of structural information hinders the drug development with novel characteristics, such as nonlipid ligands and allosteric modulators. Here, we determined the structures of human GPR34 and GPR174 in complex with LysoPS and G protein by cryo-EM. Combined with structural analysis and functional studies, we elucidated the lipid-binding modes of these receptors. By structural comparison, we identified the structural features of GPR34 and GPR174 in active state. Taken together, our findings provide insights into ligand recognition and signaling of LysoPS receptors and will facilitate the development of novel therapeutics for related inflammatory diseases and autoimmune diseases.

Fig 1. Overall structures of GPR34-Gi complex and GPR174-Gs complex.

(A) Cryo-EM density map and model of GPR34-Gi complex. Density of LysoPS is shown as mesh in the middle and colored plum. (B) The map and model of ECL2 of GPR34-Gi complex (extracellular view). (C) Cryo-EM density map and model of GPR174-Gs complex. Density of LysoPS is shown as mesh in the middle and colored salmon. (D) The map and model of ECL2 of GPR174-Gs complex (extracellular view). GPR34 is colored marine green. GPR174 is colored blue. ECL2 is colored orange. Gα_i, Gα_s, Gβ, and Gγ subunits are colored gold, cyan, pink, and light green, respectively. ScFv16 and Nb35 are colored grey.

Fig 2. Recognition of LysoPS by GPR34 and GPR174.

(A) Interactions between LysoPS and GPR34. Polar interactions are depicted as black dashed lines. (B) Normalized ΔBRET of GPR34 mutants in response to 10 μM LysoPS relative to wild-type GPR34 in Gi-dissociation assays. (C) Interactions between LysoPS and GPR174. Polar interactions are depicted as black dashed lines. The π–π interaction is depicted as a red dashed line. (D) Net cAMP accumulation of CHO cells expressing GPR174 mutants in response to 10 μM LysoPS. WT-basal represents net cAMP accumulation of cells expressing wild-type GPR174 in the absence of exogenous LysoPS. Data represent mean ± SEM from 3 independent experiments. Mutants with expression less than half that of the wild-type receptor are labeled with red stars. The data used to generate graphs in Fig 2B and 2D are available in S1 Data.

Fig 3. Comparison of LysoPS recognition by GPR34 and GPR174.

(A) Interactions between GPR174 and the polar head of LysoPS (18:0) or LysoPS (18:1). (B) Interactions between GPR174 and the acyl tail of LysoPS (18:0) or LysoPS (18:1). The double bond of LysoPS (18:1) is colored red; the corresponding single bond in LysoPS (18:0) is colored orange. (C) Charged interactions between LysoPS and GPR34 in the positively charged pocket. (D) Charged interactions between LysoPS and GPR174 in the positively charged pocket. (E) Structural superposition of LysoPS bound GPR34 and GPR174 (extracellular view). (F) Comparison between acyl tails of LysoPS binding in GPR34 and GPR174. Polar or charged interactions are depicted as black dashed lines.

Fig 4. Comparison of lysophospholipid-binding modes.

The ligand-binding pockets of GPR34 (A), GPR174 (B), S1P₁ (C, PDB: 7td3), LPA₁ (D, PDB: 7dt0), GPR119 (E, PDB: 7xz5), and LPA₆ (F, PDB: 5xsz). GPR34, GPR174, S1P₁, LPA₁, GPR119, and LPA₆ are colored marine green, blue, dark gold, orange, plum, and light blue, respectively. The ligands are shown as sticks. The blobs represent the surface of the atomic models.

Fig 5. G protein coupling of GPR34 and GPR174.

(A) Structural superposition of GPR34-Gα_i and rhodopsin-Gα_i (PDB: 6cmo). Rhodopsin is colored pink, and Gi coupled to it is colored cyan. (B) Structural superposition of GPR34-Gα_i and CB2-Gα_i (PDB: 6pt0). CB2 is colored brown, and Gα_i coupled to it is colored cyan. The displacements are indicated by arrows. (C) Interactions between the C terminus of α5 and GPR34. (D) Interactions between α5/β6 of Gα_i and ICL3 of GPR34. (E) Relative intrinsic activity (RAi) plot of GPR34 mutants of the G protein–coupling sites in Gi-dissociation assays. RAi was calculated as [E_max(test)/EC₅₀(test)]/[E_max(WT)/EC₅₀(WT)]. E_max and EC₅₀ are from the averaged concentration-response curves of 3 independent experiments. (F) Superposition of GPR174-Gα_s, β₂AR-Gα_s (PDB: 3sn6), and CCK_AR-Gα_s (PDB: 7ezk). GPR174, β₂AR, and CCRK_AR are colored blue, light yellow, and magenta, respectively. Gα_s subunits in these structures are colored light blue, yellow, and plum, respectively. (G) Interactions between the C terminus of α5 helix and GPR174. (H) Interactions between Gα_s and ICL3 of GPR174. (I) Interactions between Gα_s and ICL2 of GPR174. Polar interactions are depicted as black dashed lines. (J) Basal activity and maximal LysoPS-induced activity of GPR174 mutants in cAMP accumulation assays. Data are from the averaged concentration-response curves of 3 independent experiments. The data used to generate graphs in Fig 5E and 5J are available in S1 Data.