Molecular cloning and functional. characterization of murine cysteinyl-leukotriene 1 (CysLT(1)) receptors

We sought to clone and characterize the murine cysteinyl-leukotriene D-4 re ceptor (mCysLT(1)R) to complement our studies with leukotriene-deficient mi ce. A cDNA, cloned from trachea mRNA by reverse transcriptase-polymerase ch ain reaction, has two potential initiator ATG codons that would encode for polypeptides of 352 and 339 amino acids, respectively. These two potential forms, predicted to be seven transmembrane-spanning domain proteins, have 8 7% amino acid identity with the human CysLT(1) receptor (KysLT(1)R). Membra ne fractions of Cos-7 cells transiently expressing the short mCysLT(1)R dem onstrated high affinity and specific binding for leukotriene D-4 (LTD4, K-d = 0.25 +/- 0.04 nM). In competition binding experiments, LTD4 was the most potent competitor (K-i = 0.8 +/- 0.2 nM) followed by LTE4 and LTC4 (K-i = 86.6 +/- 24.5 and 100.1 +/- 17.1 nM, respectively) and LTB4 (K-i > 1.5 muM) . Binding of LTD4 was competitively inhibited by the specific CysLT(1) rece ptor antagonists MK-571 [(+)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)pheny l) ((3-(dimethylamino)-3-oxopropyl)thio)methyl)thio)propanoic acid], pranlu kast (Onon (TM)), and zafirlukast (Accolate (TM)), while the CysLT(1)/CysLT (2) receptor antagonist BAY-u9773 [6(R)-(4'-carboxyphenylthio)-5(S)-hydroxy -7(E),9(E), I I(Z), 14(Z)-eicosatetrenoic acid] was 1000 times less potent than LTD4. In transiently transfected HEK293-T cells expressing either the long or short form of mCysLT(1)R, LTD4 induced an increase of intracellular calcium. In Xenopus laevis melanophores transiently expressing either isof orm, LTD4 induced the dispersion of pigment granules, consistent with the a ctivation by LTD4 of a G(alphaq) (calcium) pathway. Functional elucidation of mCysLT(1)R properties as described here will enable further experiments to clarify the selective role of LTD4 in murine models of inflammation and asthma. (C) 2001 Elsevier Science Inc. All rights reserved.