ACCELERATED COMMUNICATION Molecular Cloning and Characterization of a Second Human Cysteinyl Leukotriene Receptor: Discovery of a Subtype Selective Agonist

The cysteinyl leukotrienes (CysLTs) are potent biological mediators in the pathophysiology of inflammatory diseases, in particular of airway obstruction in asthma. Pharmacological studies have suggested the existence of at least two types of CysLT receptors, designated CysLT1 and CysLT2. The CysLT1 receptor has been cloned recently. Here we report the molecular cloning, expression, localization, and functional characterization of a human G protein-coupled receptor that has the expected characteristics of a CysLT2 receptor. This new receptor is selectively activated by nanomolar concentrations of CysLTs with a rank order potency of LTC4 5 LTD4 .. LTE4. The leukotriene analog BAY u9773, reported to be a dual CysLT1/ CysLT2 antagonist, was found to be an antagonist at CysLT1 sites but acted as a partial agonist at this new receptor. The structurally different CysLT1 receptor-selective antagonists zafirlukast, montelukast, and MK-571 did not inhibit the agonist-mediated calcium mobilization of CysLT2 receptors at physiological concentrations. Localization studies indicate highest expression of CysLT2 receptors in adrenal glands, heart, and placenta; moderate levels in spleen, peripheral blood leukocytes, and lymph nodes; and low levels in the central nervous system and pituitary. The human CysLT2 receptor gene is located on chromosome 13q14.12–21.1. The new receptor exhibits all characteristics of the thus far poorly defined CysLT2 receptor. Moreover, we have identified BAY u9773 as a CysLT2 selective agonist, which could prove to be of immediate use in understanding the functional roles of the CysLT2 receptor. Cysteinyl leukotrienes (CysLTs) are the products of the 5-lipoxygenase pathway in arachidonic acid metabolism. They are predominantly produced by myeloid cells associated with the inflammatory responses (Samuelsson et al., 1987) and are potent constrictors of pulmonary smooth muscles (Dahlén et al., 1980), trachea, and parenchyma in human airways, where they induce microvascular permeability (Dahlén et al., 1981) and mucus secretion (Marom et al., 1982). Leukotrienes have been implicated in a number of pathological inflammatory diseases including asthma, allergic rhinitis, inflammatory bowel disease, and psoriasis (Busse and Gaddy, 1991). The effects of CysLTs are mediated via specific plasma membrane receptors belonging to the superfamily of G protein-coupled receptors. There is evidence for the existence of two CysLT receptor subtypes (Fleisch et al., 1982; Labat et al., 1992; Coleman et al., 1995; Metters, 1995): CysLT1 and the CysLT2 receptors, the latter of which encompasses all receptors that cannot be inhibited by CysLT1-specific antagonists (Coleman et al., 1995). The CysLT1 receptor has been studied intensively because of the availability of CysLT1-specific antagonists and of the existence of cell lines expressing it endogenously (Saussy et al., 1989). Recently, a cDNA encoding a CysLT1 receptor was cloned (Lynch et al., 1999; Sarau et al., 1999). The pharmacological profile of the cloned CysLT1 showed a rank order potency of LTD4.LTC4.LTE4 and was potently inhibited by the CysLT1 antagonists pranlukast, montelukast, zafirlukast, and pobilukast. The CysLT2 receptor, on the other hand, is pharmacologically less defined, mainly beThis work was supported in part by grants from Neotherapeutics, the Eric and Lila Nelson Chair in Neuropharmacology, and from BioStar (S97–107). ABBREVIATIONS: CysLT, cysteinyl leukotrienes; PCR, polymerase chain reaction; GPCR, G protein-coupled receptor; HEK 293T, human embryonic kidney cells stably expressing the simian virus 40 large T antigen. 0026-895X/00/061601-08$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 6 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 463/866282 Mol Pharmacol 58:1601–1608, 2000 Printed in U.S.A. 1601 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from cause of the lack of selective agonists and antagonists. In man, CysLT2 receptors have been indirectly shown to be responsible for contracting pulmonary veins, contractions that were resistant to a number of CysLT1-selective antagonists (Labat et al., 1992). In our quest to identify the natural ligands of orphan Fig. 1. Nucleotideand deduced amino acid sequence of the human HPN321(CysLT2) receptor as derived from clone RP11–108P5 (AL137118). Putative poly-adenylation signals are underlined. Position 2123 to 2430 corresponds to an expressed sequence tag (AW235714) found in the dBEST database from a subtracted kidney library. The sequence of HPN321 has been deposited in GenBank (accession no. AF279611). 1602 Nothacker et al. at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from GPCRs, we cloned in silico a receptor, HPN321, that exhibited moderate similarity to the CysLT1 receptor. We describe here the pharmacological characterization of this novel receptor and conclude that we cloned a receptor exhibiting the expected characteristics of the CysLT2 receptor. Experimental Procedures Materials. LTB4, LTC4, LTD4, and LTE4 were from Cayman Chemical (Ann Arbor, MI). BAY u9773 and MK-571 were from BIOMOL Research Laboratories (Plymouth Meeting, PA). LY-17883 was from Sigma (St. Louis, MO). Zafirlukast (ICI 204,219; Accolate) and montelukast (MK-476; Singulair) were purchased from the local pharmacy. All other standard chemicals used were either from