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) 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.

Efficient, long-term transgene expression in Xenopus laevis dermal melanophores.

Xenopus laevis dermal melanophores provide an excellent model system for the investigation of complex cellular processes. Specifically, the expression of exogenous genes in Xenopus melanophores is the basis of recombinant bioassays for the study of receptor-ligand interactions. However, due to their slow rate of cell division and to the relatively low efficiency of current transfection protocols, long-term expression of exogenous genes and the generation of stable melanophore cell lines remains problematic. In this report we demonstrate the efficient, long-term expression of two exogenous proteins, the enhanced green fluorescent protein (EGFP) and the human CD4 (hCD4) cell surface receptor, following stable introduction into Xenopus melanophores via an HIV-1 based vector. Transduction of melanophores with the EGFP expression vector resulted in up to 80% EGFP+ cells. After 1 year in continuous culture in the absence of antibiotic selection, more than 60% of the cells remained EGFP+. Furthermore, we demonstrate the expression of hCD4 melanophores for over 9 months in continuous culture in the absence of antibiotic selection. Our results indicate that lentivirus vectors provide an efficient means of introducing genetic information into Xenopus melanophores, resulting in sustained levels of gene expression. The significance of this gene transfer system for the study of cellular signal transduction pathways is discussed.

Melatonin-induced organelle movement in melanophores is coupled to tyrosine phosphorylation of a high molecular weight protein.

Melanophores, brown to black pigment cells from, for example, Xenopus laevis, contain mobile melanin filled organelles, and are well suited for studies on organelle movement. The intracellular regulation of the movement seems to be controlled by serine and threonine phosphorylations and dephosphorylations. Melatonin induces aggregation of the melanosomes to the cell centre through a G(i/o)-protein-coupled receptor, Mel1c, which leads to an inhibition of PKA and a stimulation of PP2A. However, this study shows that the melatonin-induced aggregation of melanosomes is also accompanied by tyrosine phosphorylation of a protein with a molecular weight of approximately 280 kDa. Cells pre-incubated with genistein, an inhibitor of tyrosine phosphorylations, showed inhibited melanosome movement after melatonin stimulation, and a lower degree of tyrosine phosphorylation of the approximately 280 kDa protein. The adenylyl cyclase activator forskolin, and the G(i/o) protein inhibitor pertussis toxin, also inhibited tyrosine phosphorylation of the approximately 280 kDa protein. The results indicate that melatonin stimulation generates tyrosine phosphorylation of a high molecular weight protein, an event that seems to be essential for melanosome aggregation.

Leukotriene binding, signaling, and analysis of HIV coreceptor function in mouse and human leukotriene B4 receptor-transfected cells.

The mouse leukotriene B4 receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4 (LTB4, Kd = 0.24 +/- 0.03 nM). In competition binding experiments, LTB4 was the most potent competitor (Ki = 0.23 +/- 0.05 nM) followed by 20-hydroxy-LTB4 (Ki = 1.1 +/- 0.2 nM) and by 6-trans-12-epi-LTB4 and LTD4 (Ki > 1 microM). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation.