The ligand specificity of the G-protein-coupled receptor GPR34

Abstract

Lyso-PS (lyso-phosphatidylserine) has been shown to activate the G(i/o)-protein-coupled receptor GPR34. Since in vitro and in vivo studies provided controversial results in assigning lyso-PS as the endogenous agonist for GPR34, we investigated the evolutionary conservation of agonist specificity in more detail. Except for some fish GPR34 subtypes, lyso-PS has no or very weak agonistic activity at most vertebrate GPR34 orthologues investigated. Using chimaeras we identified single positions in the second extracellular loop and the transmembrane helix 5 of carp subtype 2a that, if transferred to the human orthologue, enabled lyso-PS to activate the human GPR34. Significant improvement of agonist efficacy by changing only a few positions strongly argues against the hypothesis that nature optimized GPR34 as the receptor for lyso-PS. Phylogenetic analysis revealed several positions in some fish GPR34 orthologues which are under positive selection. These structural changes may indicate functional specification of these orthologues which can explain the species- and subtype-specific pharmacology of lyso-PS. Furthermore, we identified aminoethyl-carbamoyl ATP as an antagonist of carp GPR34, indicating ligand promiscuity with non-lipid compounds. The results of the present study suggest that lyso-PS has only a random agonistic activity at some GPR34 orthologues and the search for the endogenous agonist should consider additional chemical entities.

Structure–Activity Relationships of Lysophosphatidylserine Analogs as Agonists of G-Protein-Coupled Receptors GPR34, P2Y10, and GPR174

“…The combined organic layer was washed with brine, dried over Na 2 SO 4 , and evaporated. The residue was column-chromatographed (hexane/AcOEt = 1:1) to yield acid 13 tert-Butyl O-(tert-Butoxy(diisopropylamino)phosphanyl)-N-(tert-butoxycarbonyl)-L-serinate (17). Bis(diisopropylamino)-tertbutylphoshine (643.1 mg, 2.112 mmol) was dissolved in a mixture of CH 2 Cl 2 (6 mL) and toluene (0.5 mL).…”

“…The residue was column-chromatographed (hexane/ AcOEt = 4:1) to yield 18 (322.7 mg, 0.852 mmol, 90%, colorless oil). 1 13 (19). In order to eliminate traces of water, phosphoamidite 17 (204.1 mg, 0.440 mmol) was dissolved in CH 2 Cl 2 (5 mL) and toluene (0.5 mL), and the solvent was evaporated under vacuum.…”

“…The residue was column-chromatographed (hexane/AcOEt = 4:1) to yield 19 (307.0 mg, 0.405 mmol, 92%, colorless oil). 1 13 tert-Butyl (tert-Butoxycarbonyl)-L-allothreoninate (22). L-alloThreonine 20 (997.3 mg, 8.372 mmol) was dissolved in 1 N aqueous NaOH (10 mL), H 2 O (10 mL) and dioxane (20 mL) at 0°C with stirring.…”

Identification of Determinants Required for Agonistic and Inverse Agonistic Ligand Properties at the ADP Receptor P2Y12

“…To measure label-free receptor activation, a dynamic mass redistribution (DMR) assay (Corning Epic Biosensor Measurements; Corning Life Sciences, Lowell, MA) with stably transfected human embryonic kidney (HEK) cells (HEK-FlpIn, P2Y 12 in pcDNA5/FRT) was performed as described previously (Schroder et al, 2010;Ritscher et al, 2012). Briefly, cells were seeded into fibronectin-coated Epic 384-well microplates (60,000 cells per well) and exposed to the various compounds.…”

Dendritic Cells Regulate GPR34 through Mitogenic Signals and Undergo Apoptosis in Its Absence

“…GPR34 was first described after mining GenBank for novel G protein-coupled receptor (GPCR) sequences and was assigned to the human X chromosome (4). Although lysophosphatidylserine was proposed as a potential endogenous ligand (5), subsequent studies found no support that this is the natural ligand but rather a surrogate agonist (6,7). Hence, GPR34 is still classified as an orphan in the International Union of Basic and Clinical Pharmacology database (8).…”