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. 2023 Sep 22;14(1):5901.
doi: 10.1038/s41467-023-41654-3.

Specific binding of GPR174 by endogenous lysophosphatidylserine leads to high constitutive Gs signaling

Yingying Nie #  1   2 , Zeming Qiu #  2   3 , Sijia Chen  2 , Zhao Chen  2   3 , Xiaocui Song  2 , Yan Ma  2   3 , Niu Huang  2   3 , Jason G Cyster  4   5 , Sanduo Zheng  6   7   8
Affiliations

Specific binding of GPR174 by endogenous lysophosphatidylserine leads to high constitutive Gs signaling

Yingying Nie et al. Nat Commun. .

Abstract

Many orphan G protein-coupled receptors (GPCRs) remain understudied because their endogenous ligands are unknown. Here, we show that a group of class A/rhodopsin-like orphan GPCRs including GPR61, GPR161 and GPR174 increase the cAMP level similarly to fully activated D1 dopamine receptor (D1R). We report cryo-electron microscopy structures of the GPR61‒Gs, GPR161‒Gs and GPR174‒Gs complexes without any exogenous ligands. The GPR174 structure reveals that endogenous lysophosphatidylserine (lysoPS) is copurified. While GPR174 fails to respond to exogenous lysoPS, likely owing to its maximal activation by the endogenous ligand, GPR174 mutants with lower ligand binding affinities can be specifically activated by lysoPS but not other lipids, in a dose-dependent manner. Moreover, GPR174 adopts a non-canonical Gs coupling mode. The structures of GPR161 and GPR61 reveal that the second extracellular loop (ECL2) penetrates into the orthosteric pocket, possibly contributing to constitutive activity. Our work definitively confirms lysoPS as an endogenous GPR174 ligand and suggests that high constitutive activity of some orphan GPCRs could be accounted for by their having naturally abundant ligands.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. A group of class A oGPCRs dramatically increase cAMP levels via the Gs pathway.
a GloSensor cAMP accumulation assay and NanoBiT mini-Gs recruitment assay performed in Expi293F cells transiently expressing any of 81 oGPCRs or D1R with relative luminescence unit (RLU) value shown. Receptors that show high cAMP levels compared with the other oGPCRs are colored in red. b Sequence alignment of ICL2 in caoGPCRs, D1R and β2AR. Residues are highlighted based on the Clustal color scheme. c Concentration–response curves in the cAMP accumulation assay at Expi293F cells transiently transfected with GPR174 or D1R treated with lysoPS or dopamine. Each data point represents mean ± SEM from three independent experiments. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. The endogenous lysoPS is copurified with GPR174.
a Cryo-EM map of the GPR174 and mini-Gs complex. b Ribbon representation of the GPR174 and mini-Gs complex. LysoPS and cholesterol are shown as sticks. c Cryo-EM density map of lysoPS and cholesterol in the GPR174‒Gs complex at a contour level of 3.5σ. d Lipidomics analysis of the GPR174‒Gs and D1R‒Gs complexes by MS. The relative abundance of representative LPs with the acyl chain length from 16:0 to 20:1 determined by MS is shown. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Specific recognition of lysoPS by GPR174.
a Electrostatic potential of the lysoPS binding surface colored from red to blue for negatively and positively charged regions. b GPR174 shows perfect shape complementarity with lysoPS. The surface of interior pocket of GPR174 is shown in gray. c Detailed interactions between GPR174 and lysoPS. d and e Concentration–response curves of GPR174 mutants treated with increasing concentrations of lysoPS in the cAMP accumulation assay (d) and the NanoBiT Gαs recruitment assay (e). All measurements were performed in three independent times, shown as mean ± SEM. Source data are provided as a Source Data file. f Concentration–response curves of GPR174/Y993.33A mutants treated with different LPs in the cAMP accumulation assay. Each data point represents mean ± SEM from three independent experiments. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. ECL2 is penetrated into the orthosteric binding pocket of GPR161 and GPR61.
a and d Ribbon representation of the GPR161‒Gs (a) and GPR61‒Gs (d) complexes with ECL2 colored in orange and gray respectively. b and e Detailed interactions between ECL2 and other regions of GPR161 (b) or GPR61 (e). c and f The basal activity of GPR161 (c) or GPR61 (f) mutants determined by the cAMP assay. Bar graphs represent mean ± SEM from three independent experiments. Expression levels of mutants were determined by western blot using an anti-Flag antibody. Statistical analysis was performed using one-way ANOVA method (ns, not significant; *P < 0.1; **P < 0.01; ***P < 0.001; ****P < 0.0001). Source data are provided as a Source Data file. P values for GPR161 mutants versus WT are <0.0001, <0.0001, <0.0001, 0.5954, 0.0098, 0.0067, <0.0001, 0.9996, <0.0001, 0.0003, <0.0001, 0.3316, <0.0001 (from left to right). P values for GPR61 mutants versus WT are 0.001, <0.0001, 0.0001, <0.0001, <0.0001, 0.2203, 0.0230, <0.0001, <0.0001 (from left to right).
Fig. 5
Fig. 5. In contrast to GPR61, GPR161, and β2AR, GPR174 adopts a non-canonical Gs coupling mode.
a Structural overlay of β2AR‒Gs and GPR174‒Gs complexes from the side view. b Comparison of structures of inactive β2AR (PDB: 2RH1), active β2AR (PDB: 3SN6), active μOR (PDB: 6DDE) and active GPR174 in two orthogonal views. c Structural overlay of GPR174‒Gs, CCK1R‒Gs (PDB: 7MBX), GPR52‒Gs (PDB: 6LI3), and NK1R‒Gs (PBD: 7RMI) complexes from the cytoplasmic view. All these complexes adopt a non-canonical Gs coupling mode where the hook of Gαs is distorted and the outward movement of TM6 is less pronounced compared to β2AR. α5 of Gαs is shown with other regions hidden.
Fig. 6
Fig. 6. The non-canonical Gs coupling mode is determined by a larger hydrophobic residue at position 5.65 of TM5.
a Receptors that adopt canonical Gs coupling mode have a small hydrophobic residue A/V at position 5.65, while receptors that adopt non-canonical Gs coupling prefer a large hydrophobic residue (L/C5.65). b Comparison of the binding interface between β2AR–Gαs and GPR174–Gαs. The hook of Gαs is distorted when bound to GPR174 due to a potential steric clash between L2125.65 and the tri-leucine pocket formed by L394(-1), L393(−2), and L388(−7). c Mutation of L2125.65 in GPR174 remarkably reduces its basal activity. Each data point represents mean ± SEM from three independent experiments. Source data are provided as a Source Data file. d The effects of mutations in the GPR174‒Gs interface on the potency and maximum effect of lysoPS in the context of the Y993.33A mutant evaluated by the cAMP assay. Each data point represents mean ± SEM from three independent experiments. Source data are provided as a Source Data file. e Detailed interactions between GPR174 and Gαs. f and g The presence of a larger hydrophobic residue (L or C) at 5.65 in GPR52 (f) and CCK1R (g) leads to a non-canonical Gs coupling mode.
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