Large-scale human exome sequencing can identify rare protein-coding variants with a large impact on complex traits such as body adiposity. We sequenced the exomes of 645,626 individuals from the United Kingdom, the United States, and Mexico and estimated associations of rare coding variants with body mass index (BMI). We identified 16 genes with an exome-wide significant association with BMI, including those encoding five brain-expressed G protein–coupled receptors (CALCR, MC4R, GIPR, GPR151, and GPR75). Protein-truncating variants in GPR75 were observed in ~4/10,000 sequenced individuals and were associated with 1.8 kilograms per square meter lower BMI and 54% lower odds of obesity in the heterozygous state. Knock out of Gpr75 in mice resulted in resistance to weight gain and improved glycemic control in a high-fat diet model. Inhibition of GPR75 may provide a therapeutic strategy for obesity.

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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.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical mediator of vascular function. eNOS is tightly regulated at various levels, including transcription, co- and posttranslational modifications, and by various protein–protein interactions. Using stable isotope labeling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we identified several eNOS interactors, including the protein plasminogen activator inhibitor-1 (PAI-1). In cultured human umbilical vein endothelial cells (HUVECs), PAI-1 and eNOS colocalize and proximity ligation assays demonstrate a protein–protein interaction between PAI-1 and eNOS. Knockdown of PAI-1 or eNOS eliminates the proximity ligation assay (PLA) signal in endothelial cells. Overexpression of eNOS and HA-tagged PAI-1 in COS7 cells confirmed the colocalization observations in HUVECs. Furthermore, the source of intracellular PAI-1 interacting with eNOS was shown to be endocytosis derived. The interaction between PAI-1 and eNOS is a direct interaction as supported in experiments with purified proteins. Moreover, PAI-1 directly inhibits eNOS activity, reducing NO synthesis, and the knockdown or antagonism of PAI-1 increases NO bioavailability. Taken together, these findings place PAI-1 as a negative regulator of eNOS and disruptions in eNOS–PAI-1 binding promote increases in NO production and enhance vasodilation in vivo.

:Arachidonic acid–derived lipid mediators play crucial roles in the development and progression of cardiovascular diseases. Eicosanoid metabolites generated by lipoxygenases and cytochrome P450 enzymes produce several classes of molecules, including the epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids (HETE) family of bioactive lipids. In general, the cardioprotective effects of EETs have been documented across a number of cardiac diseases. In contrast, members of the HETE family have been shown to contribute to the pathogenesis of ischemic cardiac disease, maladaptive cardiac hypertrophy, and heart failure. The net effect of 12(S)- and 20-HETE depends upon the relative amounts generated, ratio of HETEs:EETs produced, timing of synthesis, as well as cellular and subcellular mechanisms activated by each respective metabolite. HETEs are synthesized by and affect multiple cell types within the myocardium. Moreover, cytochrome P450–derived and lipoxygenase- derived metabolites have been shown to directly influence cardiac myocyte growth and the regulation of cardiac fibroblasts. The mechanistic data uncovered thus far have employed the use of enzyme inhibitors, HETE antagonists, and the genetic manipulation of lipid-producing enzymes and their respective receptors, all of which influence a complex network of outcomes that complicate data interpretation. This review will summarize and integrate recent findings on the role of 12(S)-/20-HETE in cardiac diseases.