Insights into divalent cation regulation and G13-coupling of orphan receptor GPR35

Abstract

Endogenous ions play important roles in the function and pharmacology of G protein-coupled receptors (GPCRs) with limited atomic evidence. In addition, compared with G protein subtypes Gs, Gi/o, and Gq/11, insufficient structural evidence is accessible to understand the coupling mechanism of G12/13 protein by GPCRs. Orphan receptor GPR35, which is predominantly expressed in the gastrointestinal tract and is closely related to inflammatory bowel diseases (IBDs), stands out as a prototypical receptor for investigating ionic modulation and G13 coupling. Here we report a cryo-electron microscopy structure of G13-coupled GPR35 bound to an anti-allergic drug, lodoxamide. This structure reveals a novel divalent cation coordination site and a unique ionic regulatory mode of GPR35 and also presents a highly positively charged binding pocket and the complementary electrostatic ligand recognition mode, which explain the promiscuity of acidic ligand binding by GPR35. Structural comparison of the GPR35–G13 complex with other G protein subtypes-coupled GPCRs reveals a notable movement of the C-terminus of α5 helix of the Gα13 subunit towards the receptor core and the least outward displacement of the cytoplasmic end of GPR35 TM6. A featured ‘methionine pocket’ contributes to the G13 coupling by GPR35. Together, our findings provide a structural basis for divalent cation modulation, ligand recognition, and subsequent G13 protein coupling of GPR35 and offer a new opportunity for designing GPR35-targeted drugs for the treatment of IBDs.

GPR35 and mediators from platelets and mast cells in neutrophil migration and inflammation

“…16,[27][28][29] Coupling to Gα13 has been validated with the recent determination of a GPR35-Gα 13 βγ structure. 25 Further studies are needed to determine if these G-proteins contribute independently or cooperatively to GPR35 dependent responses of myeloid cells.…”

“…The expression of GPR35 by a wide range of immune and nonimmune cell types makes it likely that its function will be context dependent. [10][11][12][13] In addition, positively charged residues in the ligand pocket allow for promiscuous binding with many acidic molecules, 12,13,21,25,30 suggesting that GPR35 may respond to different agonists depending on the cell type and tissue niche. In agreement with this view, DHICA analogs show strong cell line and assay dependent relative efficacy and potency against GPR35, a feature referred to as "biased agonism".…”

“…35 Additionally, increased extracellular concentrations of divalent cations (eg, Ca 2+ ) can augment the activity of some GPR35 agonists. 25 GPR35 can function as a pro-migratory and pro-adhesive receptor in response to several agonists, 9,26 and the absence of this receptor can result in 50% reduced neutrophil recruitment in vivo. 9 However, the overall migration activity obtained in vitro with GPR35 ligands is low compared to other pro-migratory GPCRs (eg, CXCR4).…”

“…The overall structure of human GPR35 with the agonist Lodoxamide is shown (PDB:8H8J). 25 "Endogenous ligands" indicates molecules that can be generated by physiological pathways; evidence for reduced in vivo GPR35 function under conditions of reduced metabolite production has so far only been reported for 5-HIAA. In addition to DHICA, several other tyrosine metabolites have activity in vitro on GPR35 in the μM to mM range.…”

“…Summary diagram of GPR35 ligands and functions. The overall structure of human GPR35 with the agonist Lodoxamide is shown (PDB:8H8J) 25 . “Endogenous ligands” indicates molecules that can be generated by physiological pathways; evidence for reduced in vivo GPR35 function under conditions of reduced metabolite production has so far only been reported for 5‐HIAA.…”