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 5 th and 6 th 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).