N‐Palmitoylglycine and Other N‐acylamides Activate the Lipid Receptor G2A/GPR132

The G-protein-coupled receptor GPR132, also known as G2A, is activated by 9-hydroxyoctadecadienoic acid (9-HODE) and other oxidized fatty acids. Other suggested GPR132 agonists including lysophosphatidylcholine (LPC) have not been readily reproduced. Here, we identify N-acylamides in particular N-acylglycines, as lipid activators of GPR132 with comparable activity to 9-HODE. The order-of-potency is N-palmitoylglycine > 9-HODE approximate to N-linoleoylglycine > linoleamide > N-oleoylglycine approximate to N-stereoylglycine > N-arachidonoylglycine > N-docosehexanoylglycine. Physiological concentrations of N-acylglycines in tissue are sufficient to activate GPR132. N-linoleoylglycine and 9-HODE also activate rat and mouse GPR132, despite limited sequence conservation to human. We describe pharmacological tools for GPR132, identified through drug screening. SKF-95667 is a novel GPR132 agonist. SB-583831 and SB-583355 are peptidomimetic molecules containing core amino acids (glycine and phenylalanine, respectively), and structurally related to previously described ligands. A telmisartan analog, GSK1820795A, antagonizes the actions of N-acylamides at GPR132. The synthetic cannabinoid CP-55 940 also activates GPR132. 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. Small-molecule ligands are envisaged to occupy a "classical" site encapsulated in the 7TM bundle. Structure-directed mutagenesis indicates a critical role for arginine at position 203 in transmembrane domain 5 to mediate GPR132 activation by N-acylamides. Our data suggest distinct modes of binding for small-molecule and lipid agonists to the GPR132 receptor. Antagonists, such as those described here, will be vital to understand the physiological role of this long-studied target.