Modeling the 20‐HETE Binding Site on GPR75

Recently, the cytochrome P450‐derived eicosanoid 20‐Hydroxyeicosatetraenoic acid (20‐HETE) was shown to activate the orphan receptor GPR75. The pairing of 20‐HETE and GPR75 activates a signaling pathway that elicits increases in endothelial angiotensin converting enzyme (ACE) and promotes the development of hypertension. Little is known about the critical binding sites/pockets across GPR75 involved in interacting with 20‐HETE that ultimately leads to receptor activation. Computational docking analysis of GPR75 uncovered a putative ligand binding site located between transmembranes five and six on GPR75 (SiteScore: 1.182 and Dscore: 1.303). Independent analyses of ligand‐receptor docking using 20‐HETE placed the lipid in close proximity to the putative binding pocket (Docking Score: −3.229, Glide Emodel: −29.045). Additional modeling of GPR75 alongside another proposed ligand, the chemokine CCL5 (RANTES), revealed a potential protein‐protein interaction model occurring at the top (extracellular face) between the first and seventh transmembranes of GPR75. This distant site suggests the possibility of distinct ligand‐receptor interactions that promote unique signaling cascades. In order to establish a functional assay to further investigate the significance of the proposed 20‐HETE binding site/pocket, we have adapted the PRESTO‐Tango system. This system allows for the quantification of ligand‐dependent β‐arresstin2 recruitment, a common hallmark of G‐protein coupled receptor activation, biased agonism and receptor inactivation/internalization. Preliminary studies using the GPR75‐Tango construct demonstrated a robust signal wherein 20‐HETE stimulates the recruitment of β‐arresstin2 after GPR75 activation (EC50=3μM). Further studies will be conducted to assess the significance of residues located within the putative binding pocket with respect to 20‐HETE‐mediated activation of GPR75.Support or Funding InformationThis 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).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.