Differential Signaling of Cysteinyl Leukotrienes and a Novel Cysteinyl Leukotriene Receptor 2 (CysLT2) Agonist, N-Methyl-Leukotriene C4, in Calcium Reporter and β Arrestin Assays

The cysteinyl leukotrienes (cysLTs) LTC 4 , LTD 4 , and LTE 4 are lipid mediators with physiological and pathophysiological functions. They exert their effects through G protein-coupled receptors (GPCRs), most notably via CysLT 1 and CysLT 2 receptor. The roles of the CysLT 2 receptor are beginning to emerge. Both LTC 4 and LTD 4 are potent agonists for the CysLT 2 receptor; however, LTC 4 is rapidly converted to LTD 4 , which is also the main endogenous ligand for the CysLT 1 receptor. A selective and potent agonist at the CysLT 2 receptor would facilitate studies to discern between receptor subtypes. We show here that N -methyl LTC 4 (NMLTC 4 ), a metabolically stable LTC 4 mimetic, is a potent and selective CysLT 2 receptor agonist. Two expression systems were used to evaluate the functional activity of NMLTC 4 at human and/or mouse CysLT 1 and CysLT 2 receptors. Through the aequorin cell-based assay for calcium-coupled GPCRs, NMLTC 4 was almost equipotent to LTC 4 at CysLT 2 receptors but was the least efficacious at CysLT 1 receptors. In a β-galactosidase–β-arrestin complementation assay, the human (h) CysLT 2 receptor can couple with β-arrestin-2, and NMLTC 4 is slightly more potent for eliciting β-arrestin-2 binding compared with cysLTs. Furthermore, LTE 4 is nearly inactive in this assay compared with its weak partial agonist activity in the aequorin system. In a vascular leakage assay, NMLTC 4 is potent and active in mice overexpressing hCysLT 2 receptor in endothelium, whereas the response is abrogated in CysLT 2 receptor knockout mice. Therefore, NMLTC 4 is a potent subtype selective agonist for the CysLT 2 receptor in vitro and in vivo, and it will be useful to elucidate its biological roles.