Identifying the 20‐HETE Binding Site on the 20‐HETE Receptor (GPR75)

The G-protein coupled receptor GPR75 and its ligand the cytochrome P450 (CYP)-derived vasoactive eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), are implicated in the activation of pro-inflammatory and hypertensive signaling cascades including decreased nitric oxide (NO) production and increased angiotensin converting enzyme (ACE) protein levels. Little is known as to how and where 20-HETE interacts with GPR75. Computational modeling of the putative ligand binding pocket identified a site located across the fifth and sixth transmembrane on GPR75 (SiteScore: 1.182 and Dscore: 1.303). An independent analysis modeling the ligand-receptor interaction between 20-HETE and GPR75 places 20-HETE in close proximity to the putative binding site location (Docking Score: −3.229, Glide Emodel: −29.045). Using the PRESTO-TANGO methodology as a measure of receptor activation through the quantification of a ligand-dependent β-arrestin recruitment, 20-HETE (1 nM) increased β-arrestin recruitment by 3.48-fold (± 0.63; p<0.05) compared to vehicle treated HTLA cells transfected with the WT-GPR75-TANGO construct. Recruitment of β-arrestin was not observed with other structurally similar lipids including arachidonic acid, arachidonoyl ethanolamide and 12(S)-HETE. Moreover, co-treatment of WT GPR75-TANGO cells with equimolar concentrations of 20-HETE and the 20-HETE antagonist N-disodium succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA) prevented the 20-HETE-mediated increase in GPR75 receptor activation, i.e., β-arrestin recruitment [3.45 ± 0.19 vs. 0.89 ± 0.15 fold, 20-HETE (1 nM) vs. 20-HETE (1 nM) + AAA (1 nM), respectively; p<0.05]. Analysis of the 20-HETE-GPR75 interaction revealed several potentially key amino acids involved in hydrogen bond interactions with 20-HETE including Ser205, Thr212 and Ser209. To assess the significance of these amino acids with respect to 20-HETE-mediated activation of GPR75, we generated site-directed mutants of each residue. Cells transfected with the Ser205Ala and Ser219Ala mutant GPR75-TANGO constructs exhibited similar increases in 20-HETE receptor activation when treated with 1nM 20-HETE (3.73 ± 0.79-fold and 3.01 ± 0.57-fold increases in luminescence, respectively, compared to vehicle treated controls; p<0.05). Interestingly, Thr212Asn GPR75-TANGO transfected cells showed no significant changes to receptor activation when exposed to 20-HETE (1nM) (1.00 ± 0.29 vs. 0.84 ± 0.18-fold, vehicle vs. 20-HETE). Taken together, this study identifies Thr212 as a critical residue of the 20-HETE receptor binding site located across GPR75’s 5th and 6th transmembranes. Further studies are necessary to uncover other essential residues and their role in 20-HETE binding and receptor activation. Support or Funding Information This study was supported by National Institute of Health grants HL39793 (M.L. Schwartzman), HL39793-01S1 (V. Garcia) and the Robert A. Welch Foundation (I-0011, J.R. Falck).