Abstract 13832: GPR39 is a High Affinity Vascular Smooth Muscle Receptor for P450 Eicosanoids
Epoxyeicosatrienoic (EETs) and hydroxyeicosatetraenoic acids (HETEs) are endogenous vasoactive P450 eicosanoids that play important roles in cardiovascular physiology and disease, but their mechanisms of action are not fully understood. We developed a clickable photo-crosslinking probe (EET-P) based on EETs regio-isomer 14,15-EET, which allowed us to purify and enrich 14,15-EET binding proteins in mouse heart vascular smooth muscle cells (VSMCs). Proteomic analysis generated a list of proteins bound by EET-P that included the orphan G-protein coupled receptor 39 (GPR39). Consistent with the molecular weight of GPR39, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis revealed a prominent ~50 kD protein crosslinked to EET-P, and pretreatment with either 14,15-EET or its antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) completely displaced EET-P binding. Heterologous expression of GPR39 but not a control plasmid in HEK293 cells conferred responsiveness to both 14,15-EET and 15-HETE, as assayed by extracellular-regulated kinase (ERK) activation. Immuno-histochemical analysis localized GPR39 expression in both mouse heart micro-vessels and primary cultured VSMCs. Live-cell imaging of VSMC intracellular calcium ([Ca2+]i) revealed a dual and antagonistic regulatory mechanism in which 15-HETE induces and 14,15-EET suppresses VSMC calcium transients. Short hairpin RNA (shRNA) knockdown of GPR39 in VSMCs was sufficient to abolish effects of both 15-HETE and 14,15-EET on VSMC calcium. Our results identify GPR39 as a high-affinity, lipid-sensing GPCR antagonistically regulated by two endogenous P450 eicosanoids with opposing actions on VSMC calcium, and offer a unique window into multi-ligand GPCRs and their role in integrating the multitude of hormonal signals to regulate local tissue function and response to disease state.
- Coronary microcirculation
- Cerebrovascular circulation
- Endothelium-derived relaxing factor
- Vasodilator agents
- Vasoconstrictor agents
Author Disclosures: N. Alkayed: None. Z. Cao: None. Z. Qian: None. F. Xie: None. B. Li: None. X. Xiao: None. A. Barnes: None. S. Kaul: None.
- © 2016 by American Heart Association, Inc.