GPR75 identified as the first 20-HETE receptor - A chemokine receptor adopted by a new family

The discovery by Garcia et al. ¹ that 20-HETE activates GPR75 and signals via Gα_q/11/PLC/PKC and c-Src/EGFR pathways to elicit vascular effects represents a transformative milestone in the field of cytochrome P450 eicosanoids. It is the first demonstration that a member of this class of eicosanoids acts via a GPR. The GPR75 was previously deorphanized and the chemokine, RANTES/CCL5 was identified as its endogenous ligand. Activation of this receptor was reported to protect hippocampus from amyloid β toxicity and to stimulate insulin secretion in pancreatic islet cells.^2,3

Studies over the past 35 years have revealed that 20-HETE is a major metabolite of AA 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, endothelial 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 competive antagonists of its vasoconstrictor actions. ⁶ Subsequent studies indicating that the vasoconstrictor, and natriuretic actions of 20-HETE are PLC/PKC-dependent, while its effects on cell migration and proliferation, endothelial dysfunction, inflammation are associated with activation of the c-Src and MAPK pathways, futher suggest that 20-HETE act 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 phospholipids, avidly binds to proteins, and distributes intracellularly. ⁴ Garcia et al. ¹ overcome these limitations utilizing a novel multi-step strategy by cross-linking a relatively polar and photoactive 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 meditators.

Garcia et al. ¹ went on to developed 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 dissociation of the Gα_q/11 subunit and release of c-Src from G1T1 which is bound to the receptor (Figure 1A). Ga_q/11 activates PLC that in turn hydrolyzes PIP2 to IP3 and DAG, which promotes phosphorylation, activation and translocation of PKC. ⁹ c-Src released from GPR75 binds to and phosphorylates the EGFR, which activates the MAPK/IKK-β/NF-κB pathway. This leads to uncoupling of eNOS, endothelial dysfunction, and increased expression of ACE. ⁸ Similarly, the authors found that 20-HETE activation of GPR75 in rat aortic VSMCs promoted disassociation of Gα_q/11 which is known to activate the PLC/DAG/IP3/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 BK β channel subunit in VSMCs. This is consistent with previous reports that the vasoconstrictor response to 20-HETE is associated with increased TRPC6 and decreased BK channel activities that depolarize the membrane and promote calcium entry through voltage-sensitive calcium channels. ^5,10 One limitation of the present study, however, is that the authors did not directly show that knockdown of GPR75 blocks the vasoconstrictor effect of 20-HETE or its inhibitory effect on BK channel activity.

Figure 1.

Hypothetical incorporation of the GPR75 signaling pathways in attempt to explain the established vascular and renal tubular effects of 20-HETE. The effects of 20-HETE to uncouple eNOS, promote endothelial dysfunction and increase the expression of ACE in the endothelium are presented in Panel A. Panel B presents the vasoconstrictor action of 20-HETE in VSMCs. The natriuretic effects of 20-HETE in the proximal tubule and thick ascending loop of Henle are summarized in Panels C and D. Arrows indicate increased activity, while crosses indicate inhibitory actions. Question marks indicate actions that are not fully defined.

The most exciting aspect of the study of Garcia et al ¹ is 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 KO, inducible CYP4A12 transgenic and DHT-treated mouse models indicate that increased vascular 20-HETE production contributes to the development of hypertension. These models are associated with uncoupling of eNOS, endothelial dysfunction, increased vascular reactivity and hypertrophy, and activation of the local RAS. ¹² Inhibition of 20-HETE also lowers pressure in a variety of angiotensin II-dependent models of hypertension. ¹¹ Garcia et al. ¹ has now demonstrated that knockdown of the expression of GPR75 mimics the effects of 20-HETE inhibitors to prevent the development of hypertension and vascular hypertrophy in a CYP4A12 transgenic mouse model. These findings imply that GPR75 may be a viable target for the treatment of hypertension.

The identification of GPR75 as the first 20-HETE receptor raises many questions and opportunities for followup investigation. First, is GPR75 the only receptor for 20-HETE? Additional bands were labeled by the 20-HETE antagonist. Does this suggest the existence of other receptors or binding partners? For example, there is some evidence that 20-HETE, like DAG, may serve as an intracellular second messenger and directly activate PKC and/or small G-proteins. ⁴ It is vitally important to determine whether GPR75 is expressed and 20-HETE is produced in all the tissues where it acts. GPR75 is highly expressed in the brain. CYP4A enzymes have been identified in pial vessels, astrocytes and neonatal hippocampal neurons, ^4,5 but it is difficult to detect nonesterfied 20-HETE in brain tissue. ¹³ 20-HETE inhibitors reduce infarct size after stroke and protect neonatal hippocampal neurons from hypoxic injury. ^4,5 However, it is not known whether the neurodegenerative effects of 20-HETE are mediated by activation GPR75. In contrast, activation of GPR75 with its endogenous ligand, RANTES/CCL5, is neuroprotective.² Moreover, administration of CCL5 did not activate GPR75 signaling in endothelial cells, and the size of GPR75 protein isolated was smaller than expected.¹ This suggests that multiple isoforms or splice variants of GPR75 might be expressed different tissues.

The study of Garcia et al. ¹ has established that the signaling pathways activated by 20-HETE via GPR75 are consistent with its known actions in the endothelium and VSMCs (Figures 1A, B). 20-HETE also inhibits sodium reabsorption in the PT and TALH in part by activating PKC which phosphorylates serine 23 to inhibit Na⁺/K⁺-ATPase. ¹⁴ Moreover, 20-HETE inhibits the NHE3 exchanger in the PT via a signaling pathway that has not been fully determined, and blocks a 70 pS ROMK potassium channel possibly via c-Src ¹⁵ which recycles potassium essential for reabsorption of sodium by the NKCC transporter in the TALH (Figures 1C, D). ^4,5 Data presented in the study of Garcia et al. ¹ and the GeneAtlas database indicates that the expression of GPR75 at the mRNA and protein levels is relatively low in the kidney. Therefore, it will be important to establish that GPR75 is expressed in the PT and TALH, and mediates the natriuretic actions of 20-HETE. Similarly, GPR75 is highly expressed in the lung which produces 20-HETE. However, 20-HETE dilates, rather than constricts, bronchiole smooth muscle and pulmonary arteries ^4,5 and it will be interesting to determine if GPR75 plays a role in the vasodilatory responses.

Overall, the discovery that GPR75 serves as a 20-HETE receptor is a scientific breakthrough in understanding the role of CYP450 eicosanoids in the regulation of cardiovascular function. Since 20-HETE plays a critical role in the pathogenesis of hypertension, stroke, myocardial infarction, vascular hypertrophy, renal ischemia/reperfusion injury, angiogenesis, cell proliferation and cancer, ^4,5 it is likely that the identification of this first 20-HETE receptor will herald the discovery of additional receptors for CYP metabolites of AA and other fatty acids, and lead to the development of drugs targeting these pathways for the treatment of cardiovascular disease.

Non-standard Abbreviations and Acronyms

20-HETE

20-Hydroxyeicosatetraenoic acid

AA

Arachidonic acid

ADP

Adenosine diphosphate

Akt

Protein Kinase B

ATP

Adenosine Tri-Phosphate

BK

Calcium-activated potassium channel

CaM

Calmodulin

CCL5

Chemokine (C-C motif) ligand 5

c-Src

Proto-oncogene tyrosine-protein kinase

CYP

Cytochrome P450

DAG

Diacylglycerol

DHT

Dihydrotestosterone

EGFR

Epidermal growth factor receptor

eNOS

Endothelial nitric oxide synthase

G1T1

GPCR-kinase interacting protein 1

GPR

G protein-coupled receptor

HIC-5

Hydrogen peroxide-inducible clone 5 protein

Hsp90

Heat shock protein 90

IKK-β

inhibitor of nuclear factor kappa-B kinase subunit beta

IP3

Inositol triphosphate

IP3R

Inositol triphosphatereceptor

MAPK

Mitogen-activated protein kinases

MEK

Mitogen-activated protein kinase kinase

MLCK

Myosin light-chain kinase

NADPH

Nicotinamide adenine dinucleotide phosphate

NF-κB

Nuclear factor kappa-light-chain-enhancer of activated B cells

NHE3

Sodium–hydrogen antiporter 3

NKCC2

Na-K-2Cl cotransporter

PI3K

Phosphatidylinositol 3-kinase

PIP2

Phosphatidylinositol 4, 5-bisphosphate

PKC

Protein kinase C

PLC

Phospholipase C

PT

Proximal tubule

RANTES

Regulated on activation, normal T cell expressed and secreted

ROMK

Renal outer medullary potassium channel

ROS

Reactive oxygen species

SK

Small conductance calcium-activated potassium channels

TALH

Thick ascending loop of Henle

TRP

Transient receptor potential channels

VGCC

Voltage gated calcium channel

VSMC

Vascular smooth muscle cell