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

Abstract

We sought to clone and characterize the murine cysteinyl-leukotriene D(4) receptor (mCysLT(1)R) to complement our studies with leukotriene-deficient mice. A cDNA, cloned from trachea mRNA by reverse transcriptase-polymerase chain 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 87% amino acid identity with the human CysLT(1) receptor (hCysLT(1)R). Membrane fractions of Cos-7 cells transiently expressing the short mCysLT(1)R demonstrated high affinity and specific binding for leukotriene D(4) (LTD(4), K(d) = 0.25 +/- 0.04 nM). In competition binding experiments, LTD(4) was the most potent competitor (K(i) = 0.8 +/- 0.2 nM) followed by LTE(4) and LTC(4) (K(i) = 86.6 +/- 24.5 and 100.1 +/- 17.1 nM, respectively) and LTB(4) (K(i) > 1.5 microM). Binding of LTD(4) was competitively inhibited by the specific CysLT(1) receptor antagonists MK-571 [(+)-3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-(dimethylamino)-3-oxopropyl)thio)methyl)thio)propanoic acid], pranlukast (Onon), and zafirlukast (Accolate), while the CysLT(1)/CysLT(2) receptor antagonist BAY-u9773 [6(R)-(4'-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetrenoic acid] was 1000 times less potent than LTD(4). In transiently transfected HEK293-T cells expressing either the long or short form of mCysLT(1)R, LTD(4) induced an increase of intracellular calcium. In Xenopus laevis melanophores transiently expressing either isoform, LTD(4) induced the dispersion of pigment granules, consistent with the activation by LTD(4) 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 LTD(4) in murine models of inflammation and asthma.