title: 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.
abstract: The cysteinyl leukotrienes (cysLTs) LTC₄, LTD₄, and LTE₄ are lipid mediators with physiological and pathophysiological functions. They exert their effects through G protein-coupled receptors (GPCRs), most notably via CysLT₁ and CysLT₂ receptor. The roles of the CysLT₂ receptor are beginning to emerge. Both LTC₄ and LTD₄ are potent agonists for the CysLT₂ receptor; however, LTC₄ is rapidly converted to LTD₄, which is also the main endogenous ligand for the CysLT₁ receptor. A selective and potent agonist at the CysLT₂ receptor would facilitate studies to discern between receptor subtypes. We show here that N-methyl LTC₄ (NMLTC₄), a metabolically stable LTC₄ mimetic, is a potent and selective CysLT₂ receptor agonist. Two expression systems were used to evaluate the functional activity of NMLTC₄ at human and/or mouse CysLT₁ and CysLT₂ receptors. Through the aequorin cell-based assay for calcium-coupled GPCRs, NMLTC₄ was almost equipotent to LTC₄ at CysLT₂ receptors but was the least efficacious at CysLT₂ receptors. In a β-galactosidase-β-arrestin complementation assay, the human (h) CysLT₂ receptor can couple with β-arrestin-2, and NMLTC₄ is slightly more potent for eliciting β-arrestin-2 binding compared with cysLTs. Furthermore, LTE₄ is nearly inactive in this assay compared with its weak partial agonist activity in the aequorin system. In a vascular leakage assay, NMLTC₄ is potent and active in mice overexpressing hCysLT₂ receptor in endothelium, whereas the response is abrogated in CysLT₂ receptor knockout mice. Therefore, NMLTC₄ is a potent subtype selective agonist for the CysLT₂ receptor in vitro and in vivo, and it will be useful to elucidate its biological roles.
authors.affiliation: Department of Biochemistry, Queen's University, 18 Stuart Street, 433 Botterell Hall, Kingston, ON K7L 3N6 Canada.
authors.forename: Dong
authors.initials: D
authors.lastname: Yan
authors.forename: Rino
authors.initials: R
authors.lastname: Stocco
authors.forename: Nicole
authors.initials: N
authors.lastname: Sawyer
authors.forename: Michael E
authors.initials: ME
authors.lastname: Nesheim
authors.forename: Mark
authors.initials: M
authors.lastname: Abramovitz
authors.forename: Colin D
authors.initials: CD
authors.lastname: Funk
mesh_terms: D000331:Aequorin / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D017997:Leukotriene C4 / Q000031:analogs & derivatives* / Q000494:pharmacology; D015289:Leukotrienes / Q000378:metabolism*; D051379:Mice; D018077:Receptors, Leukotriene / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; C000605258:Arrb2 protein, mouse; D019390:Arrestins; D017931:DNA Primers; D015289:Leukotrienes; D018077:Receptors, Leukotriene; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; C112381:cysteinyl-leukotriene; D017997:Leukotriene C4; D000331:Aequorin; C043074:leukotriene C4 monomethyl ester; C412375:cysteinyl leukotriene receptor 2; D003545:Cysteine; D002118:Calcium