GPR75 Identified as the First 20-HETE Receptor

Abstract

Editorial T he discovery by Garcia et al 1 that 20-hydroxyeicosatet-raenoic acid (20-HETE) activates GPR75 and signals via Gα q/11 /phospholipase C/protein kinase C (PKC) and Proto-Oncogene Tyrosine-Protein Kinase (c-Src)/epidermal growth factor receptor pathways to elicit vascular effects represents a transformative milestone in the field of cytochrome P450 (CYP) eicosanoids. It is the first demonstration that a member of this class of eicosanoids acts via a G-protein-coupled receptor (GPR). The GPR75 was previously deorphanized, and the chemokine, RANTES (regulated on activation, normal T cell expressed and secreted)/CCL5 (chemokine (C-C motif) ligand 5), was identified as its endogenous ligand. Activation of this receptor was reported to protect hippocampus from β-amyloid toxicity and to stimulate insulin secretion in pancre-atic islet cells. 2,3 Article, see p 1776 Studies over the past 35 years have revealed that 20-HETE is a major metabolite of arachidonic acid produced by enzymes of CYP 4A and 4F families in the blood vessels, kidney , heart, lung, and other tissues. It plays a critical role in the regulation of vascular reactivity, sodium transport, endothe-lial dysfunction, oxidative stress, cell proliferation, vascular hypertrophy, inflammation, angiogenesis, and the control of blood pressure. 4,5 Increased levels of 20-HETE are associated with hypertension, stroke, myocardial infarction, vasospasm, and vascular restenosis. 4,5 The existence of 20-HETE receptors was first foreseen by the finding that inactive analogues of 20-HETE are competitive antagonists of its vasoconstrictor actions. 6 Subsequent studies that indicate the vasoconstrictor and natriuretic actions of 20-HETE are phospholipase C/PKC dependent, and its effects on cell migration and proliferation, endothelial dysfunction, and inflammation are associated with the activation of the c-Src and mitogen-activated protein kinases pathways, further suggest that 20-HETE acts via GPRs. 4,5,7,8 However, the identification of this elusive receptor using binding studies has been fraught with difficulties because 20-HETE is rapidly esterified into membrane phospho-lipids, avidly binds to proteins, and distributes intracellularly. 4 Garcia et al 1 overcome these limitations using a novel multi-step strategy by cross-linking a relatively polar and photoac-tive 20-HETE antagonist to the cell surface, then using click chemistry to attach a fluorescent tag, followed by the isolation of the labeled proteins, proteomics, and bioinformatics to identify binding partners and ultimately the receptor. Their successful approach indicates that this strategy is a viable template for identification of receptors for other CYP eicosanoids and lipid mediators. Garcia et al 1 went on to develop an antibody to GPR75 for immunoprecipitation studies to determine the mechanisms of the G-protein signaling. They demonstrated that activation of GPR75 by 20-HETE in human endothelial cells promotes dis-sociation of the Gα q/11 subunit and release of c-Src from G1T1 (GPCR-kinase interacting protein 1) which is bound to the receptor (Figure [A]). Ga q/11 activates phospholipase C that in turn hydrolyzes phosphatidylinositol 4, 5-bisphosphate (PIP 2) to inositol trisphosphate (IP 3) and diacylglycerol, which promotes phosphorylation, activation, and translocation of PKC. 9 c-Src released from GPR75 binds to and phosphorylates the epidermal growth factor receptor, which activates the mito-gen-activated protein kinases/inhibitor of nuclear factor κ-B kinase subunit β/nuclear factor κ-light-chain-enhancer of activated B cells pathway. This leads to uncoupling of endothelial nitric oxide synthase, endothelial dysfunction, and increased expression of angiotensin-converting enzyme. 8 Similarly, the authors found that 20-HETE activation of GPR75 in rat aor-tic vascular smooth muscle cells promoted disassociation of Gα q/11 which is known to activate the phospholipase C/diac-ylglycerol/IP 3 /PKC pathway to increase intracellular calcium and the vasoconstrictor response to Gq receptor agonists. They also demonstrated increased association of PKCα and c-Src and enhanced tyrosine phosphorylation of the calcium-activated potassium β channel subunit in vascular smooth muscle cells. This is consistent with previous reports that the vasoconstrictor response to 20-HETE is associated with increased TRPC6 and decreased calcium-activated potassium channel activities that depolarize the membrane and promote calcium entry through voltage-sensitive calcium channels. 5,10 One limitation of this study, however, is that the authors did not directly show that knockdown of GPR75 blocks the va-soconstrictor effect of 20-HETE or its inhibitory effect on calcium-activated potassium channel activity. The most exciting aspect of the study of Garcia et al 1 is that they established a role for GPR75 in a 20-HETE-dependent mouse model of hypertension. Mutations in CYP4A11 and CYP4F2 are associated with the development of hypertension in man. 11,12 Studies in CYP4A14 knockout inducible CYP4A12 transgenic, and dihydrotestosterone-treated mouse models indicate that increased vascular 20-HETE production contributes to