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

Abstract

Background and Purpose: 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. Experimental Approach: 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. Key Results: SPR confirmed 20-HETE binding to GPR75 with an estimated K D of 1.56 Â 10 À10 M. In GPR75-transfected HTLA cells, 20-HETE stimulated intracellular Ca 2+ 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 Ca 2+ .The chemokine CCL5, a suggested GPR75 ligand, binds to GPR75 (K D of 5.85 Â 10 À10 M) yet fails to activate GPR75; however, it inhibited 20-HETE's ability to activate GPR75 signalling. Conclusions and Implications: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.

Blockade of 20-hydroxyeicosatetraenoic acid receptor lowers blood pressure and alters vascular function in mice with smooth muscle-specific overexpression of CYP4A12-20-HETE synthase

“…That this sensitization to constrictor stimuli was 20-HETE-dependent is evident by the ability of its receptor blocker, AAA, to prevent the increased contraction to phenylephrine. We have shown that 20-HETE-GPR75 pairing results in a rapid increase in intracellular calcium and that AAA blocks the binding/pairing and the ensuing signaling cascade [3,4]. Hence, it is reasonable to suggest that in these mice, the vascular smooth muscle 20-HETE-GPR75 -signaling pathway is amplified leading to increasing intracellular calcium via mechanisms that may include inhibition of BK Ca 2þ channels and activation of L-type Ca þ2 channels and/or activation of the ROCK leading to increased phosphorylation of MLC [12,13,15].…”

“…Both male and female mice were used in this study as they exhibited similar phenotypic changes with respect to Cyp4a12 overexpression, blood pressure and vascular function and remodeling. A water-soluble 20-HETE receptor antagonist, N-disodium succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA), was used to assess the contribution of 20-HETE to the phenotype of these mice [4,38,39]. AAA was administered to the mice, via the drinking water (vehicle), at a dose of 10 mg/kg/day.…”

“…20-HETE is a vasoactive eicosanoid that exerts its actions in conduit and resistant vessels and contributes to the regulation of blood pressure [1,2]. Recent studies by Garcia and colleagues identified the G-protein-coupled receptor GPR75 as a 20-HETE receptor (20HR) demonstrating that 20-HETE binds to and triggers a signaling cascade that culminates with changes in endothelial and smooth muscle functions [3,4].…”

The Functions of Cytochrome P450 ω-hydroxylases and the Associated Eicosanoids in Inflammation-Related Diseases

“…20-HETE can activate nuclear factor-kappa B (NF-κB) and stimulate the production of inflammatory cytokines in human endothelial cells (Ishizuka et al, 2008). Recent studies have proved that 20-HETE could bind to the G-protein coupled receptor 75 (GPR75) to promote c-Src-mediated-EGFR and trigger the downstream MAPK pathway to induce ACE expression and endothelial dysfunction in human endothelial cells (Garcia et al, 2017;Pascale et al, 2021). 20-HETE/GPR75 also triggered PI3K/ AKT pathway to promote vascular smooth muscle cells migration, hypertrophy.…”

Activation of GPR75 Signaling Pathway Contributes to the Effect of a 20-HETE Mimetic, 5,14-HEDGE, to Prevent Hypotensive and Tachycardic Responses to Lipopolysaccharide in a Rat Model of Septic Shock

“…Thus, it has been proposed that 20-HETE-GPR75 pairing may be a therapeutic target to treat cardiovascular and renal diseases in which changes in the 20-HETE formation play a role in pathogenesis. Recently, Pascale et al 30 reported that 20-HETE is a high-affinity ligand for GPR75, whereas RANTES/CCL5 is a low-affinity negative regulator of GPR75. Hence, these data raise the possibility that GPR75 plays a crucial role in the pathogenesis of septic shock.…”