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

• Published in: Biochemical pharmacology Vol. 62; no. 9; pp. 1193 - 1200
• Main Authors: Martin, Viviane, Sawyer, Nicole, Stocco, Rino, Unett, David, Lerner, Michael R, Abramovitz, Mark, Funk, Colin D
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.11.2001; Elsevier Science
• Subjects: Aequorin - metabolism; Amino Acid Sequence; Animals; Antagonist; Asthma; Base Sequence; Biological and medical sciences; Biotechnology; Cells, Cultured; Cloning, Molecular; DNA, Complementary - analysis; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; GPCR; Humans; Leukotriene; Luminescent Measurements; Melanophores - metabolism; Membrane Proteins; Methods. Procedures. Technologies; Mice; Molecular cloning; Molecular Sequence Data; Murine; Radioligand Assay; Receptor; Receptors, Leukotriene - genetics; Receptors, Leukotriene - metabolism; Sequence Analysis, Protein; Sequence Homology, Amino Acid; Xenopus laevis - metabolism; Leukotriene; GPCR; Antagonist; Receptor; Murine; Asthma; CysLT1 leukotriene receptor; Respiratory disease; Ligand; Arachidonic acid derivatives; Rodentia; Inflammation; Leukotriene D4; Gene expression; Vertebrata; Mammalia; Complementary DNA; Mouse; Genetics; Obstructive pulmonary disease; Molecular biology; Molecular cloning; Aminoacid sequence
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/S0006-2952(01)00774-2

We sought to clone and characterize the murine cysteinyl-leukotriene D 4 receptor (mCysLT 1R) 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 1R). Membrane fractions of Cos-7 cells transiently expressing the short mCysLT 1R 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 μM). 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 1R, 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 αq (calcium) pathway. Functional elucidation of mCysLT 1R properties as described here will enable further experiments to clarify the selective role of LTD 4 in murine models of inflammation and asthma.