Identification, Molecular Cloning, Expression, and Characterization of a Cysteinyl Leukotriene Receptor

Abstract

The cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory disorders, in particular asthma, for which the CysLT receptor antagonists pranlukast, zafirlukast, and montelukast, have been introduced recently as novel therapeutics. Here we report on the molecular cloning, expression, localization, and pharmacological characterization of a CysLT receptor (CysLTR), which was identified by ligand fishing of orphan seven-transmembrane-spanning, G protein-coupled receptors. This receptor, expressed in human embryonic kidney (HEK)-293 cells responded selectively to the individual CysLTs, LTC(4), LTD(4), or LTE(4), with a calcium mobilization response; the rank order potency was LTD(4) (EC(50) = 2.5 nM) > LTC(4) (EC(50) = 24 nM) > LTE(4) (EC(50) = 240 nM). Evidence was provided that LTE(4) is a partial agonist at this receptor. [(3)H]LTD(4) binding and LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor were potently inhibited by the structurally distinct CysLTR antagonists pranlukast, montelukast, zafirlukast, and pobilukast; the rank order potency was pranlukast = zafirlukast > montelukast > pobilukast. LTD(4)-induced calcium mobilization in HEK-293 cells expressing the CysLT receptor was not affected by pertussis toxin, and the signal appears to be the result of the release from intracellular stores. Localization studies indicate the expression of this receptor in several tissues, including human lung, human bronchus, and human peripheral blood leukocytes. The discovery of this receptor, which has characteristics of the purported CysLT(1) receptor subtype, should assist in the elucidation of the pathophysiological roles of the CysLTs and in the identification of additional receptor subtypes.

CysLT1 leukotriene receptor antagonists inhibit the effects of nucleotides acting at P2Y receptors

“…Expression of P2Y 2,4,6,11,13,14 in dU937 cells. Total RNA was subjected to RT and the resulting cDNA was amplified with primers specifically designed for human P2Y 1,2,4,6,11,12,13,14 receptors.…”

“…Total RNA was subjected to RT and the resulting cDNA was amplified with primers specifically designed for human P2Y 1,2,4,6,11,12,13,14 receptors. PCR products were separated on 2% ethidium bromidestained agarose gel and visualized under UV light.…”

“…Conversely, the hP2Y 1,2,4,6,11,12,13,14 receptors represent a large family of GPCRs responding to either adenine or uracil nucleotides, or to sugar-nucleotides. Montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937).…”

“…In particular, when the CysLT 1 receptor is expressed in recombinant systems it shows a preferential coupling to G q/11 , whereas when constitutively expressed it has been reported to activate both pertussis toxin (PTX)-sensitive and -insensitive G-proteins [8,9]. This was demonstrated in dimethyl sulfoxide-differentiated U937 (dU937) cells, an immortalized cell line known to constitutively express a high density of CysLT 1 receptors upon differentiation to monocytes/macrophages [10,11], CysLT 1 receptors respond to LTD 4 with a strong increase in cytosolic Ca 2+ concentration ([Ca 2+ ] i ) partially sensitive to PTX, and with the activation of the Ras-MAPK cascade totally dependent upon G i/o [12]. These signaling effects were totally inhibited by various specific CysLT 1 -receptor antagonists, and no CysLT 2 receptor mRNA was detected [13,14], thus indicating that in monocytemacrophage like U937 cells LTD 4 -induced responses can be totally ascribed to a CysLT 1 receptor.…”

Mecanismos intracelulares induzidos por leucotrienos durante a diferenciação osteogênica

“…Estas proteínas G são classificadas em quatro famílias com base em suas subunidades α: Gαs, que ativa adenilato ciclase e tirosina quinase Src; Gαi, que inibe classicamente a adenilato ciclase, mas também pode ativar tirosina quinase Src; Gαq, no qual estimula a fosfolipase C e a tirosina quinase de Bruton; e Gα12, que ativa a tirosina quinase de Bruton, proteínas ativadoras de GTPase e os fatores de troca de nucleotídeos de guanina, também chamados GEFs (do inglês p115 Rho guanine exchange factor Lsc (LANDRY et al, 2006) (PERES et al, 2007. Com relação aos LTs, seus receptores podem acoplar-se às proteínas Gαi e Gαq, resultando em reduções sensíveis à toxina pertussis (PTX) do cAMP e aumento do Ca 2+ intracelular, respectivamente (LYNCH et al, 1999;SARAU et al, 1999;PERES et al, 2007).…”