Uncovering the signalling, structure and function of the 20‐HETE‐GPR75 pairing: Identifying the chemokine CCL5 as a negative regulator of GPR75

The G-protein-coupled receptor GPR75 (Gq) and its ligand, the cytochrome P450-derived vasoactive eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), are involved in the activation of pro-inflammatory and hypertensive signalling cascades contributing to diabetes, obesity, vascular dysfunction/remodelling, hypertension and cardiovascular disease. Little is known as to how, where and with what affinity 20-HETE interacts with GPR75.To better understand the pairing of 20-HETE and its receptor (GPR75), we used surface plasmon resonance (SPR) to determine binding affinity/kinetics. The PRESTO-Tango receptor-ome methodology for GPR75 overexpression was coupled with FLIPR Calcium 6 assays, homogeneous time-resolved fluorescence (HTRF) IP-1 and β-arrestin recruitment assays to determine receptor activation and downstream signalling events.SPR confirmed 20-HETE binding to GPR75 with an estimated KD of 1.56 × 10-10 M. In GPR75-transfected HTLA cells, 20-HETE stimulated intracellular Ca2+ levels, IP-1 accumulation and β-arrestin recruitment, all of which were negated by known 20-HETE functional antagonists. Computational modelling of the putative ligand-binding pocket and mutation of Thr212 within the putative 20-HETE binding site abolished 20-HETE's ability to stimulate GPR75 activation. Knockdown of GPR75 in human endothelial cells nullified 20-HETE-stimulated intracellular Ca2+ . The chemokine CCL5, a suggested GPR75 ligand, binds to GPR75 (KD of 5.85 × 10-10 M) yet fails to activate GPR75; however, it inhibited 20-HETE's ability to activate GPR75 signalling.We have identified 20-HETE as a high-affinity ligand for GPR75 and CCL5 as a low-affinity negative regulator of GPR75, providing additional evidence for the deorphanization of GPR75 as a 20-HETE receptor.