20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension.
BACKGROUND
20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle contractility, migration, and proliferation, as well as endothelial cell dysfunction and inflammation. Increased levels of 20-HETE in experimental animals and in humans are associated with hypertension, stroke, myocardial infarction, and vascular diseases.


OBJECTIVE
To date, a receptor/binding site for 20-HETE has been implicated based on the use of specific agonists and antagonists. The present study was undertaken to identify a receptor to which 20-HETE binds and through which it activates a signaling cascade that culminates in many of the functional outcomes attributed to 20-HETE in vitro and in vivo.


RESULTS
Using crosslinking analogs, click chemistry, binding assays, and functional assays, we identified G-protein receptor 75 (GPR75), currently an orphan G-protein-coupled receptor (GPCR), as a specific target of 20-HETE. In cultured human endothelial cells, 20-HETE binding to GPR75 stimulated Gαq/11protein dissociation and increased inositol phosphate accumulation and GPCR-kinase interacting protein-1-GPR75 binding, which further facilitated the c-Src-mediated transactivation of epidermal growth factor receptor. This results in downstream signaling pathways that induce angiotensin-converting enzyme expression and endothelial dysfunction. Knockdown of GPR75 or GPCR-kinase interacting protein-1 prevented 20-HETE-mediated endothelial growth factor receptor phosphorylation and angiotensin-converting enzyme induction. In vascular smooth muscle cells, GPR75-20-HETE pairing is associated with Gαq/11- and GPCR-kinase interacting protein-1-mediated protein kinase C-stimulated phosphorylation of MaxiKβ, linking GPR75 activation to 20-HETE-mediated vasoconstriction. GPR75 knockdown in a mouse model of 20-HETE-dependent hypertension prevented blood pressure elevation and 20-HETE-mediated increases in angiotensin-converting enzyme expression, endothelial dysfunction, smooth muscle contractility, and vascular remodeling.


CONCLUSIONS
This is the first report to identify a GPCR target for an eicosanoid of this class. The discovery of 20-HETE-GPR75 pairing presented here provides the molecular basis for the signaling and pathophysiological functions mediated by 20-HETE in hypertension and cardiovascular diseases.
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Victor
V
Garcia
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Ankit
A
Gilani
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Brian
B
Shkolnik
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Varunkumar
V
Pandey
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Frank Fan
FF
Zhang
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Rambabu
R
Dakarapu
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Shyam K
SK
Gandham
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
N Rami
NR
Reddy
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Joan P
JP
Graves
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Artiom
A
Gruzdev
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Darryl C
DC
Zeldin
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
Jorge H
JH
Capdevila
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.).
John R
JR
Falck
From the Department of Pharmacology, New York Medical College School of Medicine, Valhalla (V.G., A.G., B.S., V.P., F.F.Z., M.L.S.); Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas (R.D., S.K.G., N.R.R., J.R.F.); Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC (J.P.G., A.G., D.C.Z.); and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN (J.H.C.). Michal_schwartzman@nymc.edu.
Michal Laniado
ML
Schwartzman
D000818:Animals; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism* / Q000494:pharmacology / Q000633:toxicity; D006973:Hypertension / Q000139:chemically induced / Q000378:metabolism*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D066253:Vascular Remodeling / Q000187:drug effects / Q000502:physiology*
C120151:GPR75 protein, human; D006893:Hydroxyeicosatetraenoic Acids; D043562:Receptors, G-Protein-Coupled; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid
cardiovascular diseases; cytochrome P-450 enzyme system; hypertension; receptor, epidermal growth factor; vascular remodeling