1 The family includes the endocannabinoid N-arachidonoyl ethanolamide (AEA, also known as anandamide), and the cannabinoid system is the best understood signalling pathway in the class, with cognate G-protein-coupled receptors (GPCRs) CB 1 and CB 2 and well-elucidated pathways for synthesis and degradation of AEA. Recent lipidomic studies confirm the widespread occurrence of N-acylamides in mammalian tissue, including brain. N-acylamides, also known as lipoamines, are a family of diverse naturally occurring lipids derived from the conjugation of saturated, unsaturated, or hydroxylated fatty acids to amines such as dopamine, ethanolamine, or amino acids (or simply to amide, as in linoleamide). Antagonists, such as those described here, will be vital to understand the physiological role of this long-studied target. Our data suggest distinct modes of binding for small-molecule and lipid agonists to the GPR132 receptor. Structure-directed mutagenesis indicates a critical role for arginine at position 203 in transmembrane domain 5 to mediate GPR132 activation by N-acylamides. Small-molecule ligands are envisaged to occupy a “classical” site encapsulated in the 7TM bundle. Molecular docking to a homology model suggested a site for lipid binding, predicting the acyl side-chain to extend into the membrane bilayer between TM4 and TM5 of GPR132. The synthetic cannabinoid CP-55 940 also activates GPR132. A telmisartan analog, GSK1820795A, antagonizes the actions of N-acylamides at GPR132. SB-583831 and SB-583355 are peptidomimetic molecules containing core amino acids (glycine and phenylalanine, respectively), and structurally related to previously described ligands. We describe pharmacological tools for GPR132, identified through drug screening. N-linoleoylglycine and 9-HODE also activate rat and mouse GPR132, despite limited sequence conservation to human. Physiological concentrations of N-acylglycines in tissue are sufficient to activate GPR132. The order-of-potency is N-palmitoylglycine > 9-HODE ≈ N-linoleoylglycine > linoleamide > N-oleoylglycine ≈ N-stereoylglycine > N-arachidonoylglycine > N-docosehexanoylglycine. Here, we identify N-acylamides in particular N-acylglycines, as lipid activators of GPR132 with comparable activity to 9-HODE. Other suggested GPR132 agonists including lysophosphatidylcholine (LPC) have not been readily reproduced. The G-protein-coupled receptor GPR132, also known as G2A, is activated by 9-hydroxyoctadecadienoic acid (9-HODE) and other oxidized fatty acids.
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