Characterization of the soluble leukotriene B4 receptor from sheep lung membranes.
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ABSTRACT:
SUBMITTER: Votta B
PROVIDER: S-EPMC1131700 | biostudies-other | 1990 Aug
REPOSITORIES: biostudies-other
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Project description:Leukotriene B4 (LTB4) is a pro-inflammatory arachidonate metabolite. We have characterized the LTB4 receptors in sheep lung membranes and have assessed the contribution of the guanine-nucleotide-binding (G) protein in the regulation of receptor affinity states. Saturation isotherms have demonstrated a single class of LTB4 receptor with a Kd of 0.18 +/- 0.03 nM and a density (Bmax.) of 410 +/- 84 fmol/mg of protein in sheep lung membranes. The effect of the G-protein on receptor affinity was assessed in the presence of non-hydrolysable GTP analogues (e.g. GTP[S]) and in membranes following alkali treatment (pH 12.1) to remove the G-protein. Saturation isotherms produced either in the presence of GTP[S] (Kd.GTP[S] = 0.51 +/- 0.02 nM) or with alkali-treated membranes (Kd.alk. = 0.52 +/- 0.02 nM) demonstrated a 3-fold shift in receptor affinity for [3H]LTB4 binding. In competition experiments, the rank order of affinity of LTB4 analogues was LTB4 greater than 20-OH-LTB4 greater than trans-homo-LTB4 greater than 6-trans-LTB4 greater than 20-COOH-LTB4, using either untreated or alkali-treated membranes, both in the presence and absence of GTP[S]. These findings demonstrate that, in sheep lung membranes, there is only one class of LTB4 receptor. Removal of the G-protein or uncoupling of the receptor from the G-protein shifted the agonist-binding affinity of the receptor by 3-4-fold, without affecting the specificity of the LTB4 receptor in either the high- or the low-affinity state.
| S-EPMC1133708 | biostudies-other
Project description:The movement of leukocytes into tissues is regulated by the local production of chemical mediators collectively referred to as chemoattractants. Although chemoattractants constitute a diverse array of molecules, including proteins, peptides, and lipids, they all appear to signal leukocytes through a related family of seven transmembrane-spanning G protein-coupled receptors. The eosinophil is a potent proinflammatory cell that is attracted into tissues during allergic inflammation, parasitic infection, and certain malignancies. Since the molecular mechanisms controlling eosinophil recruitment are incompletely understood, we performed a degenerate polymerase chain reaction on cDNA isolated from murine eosinophils to identify novel chemoattractant receptors. We report the isolation of a cDNA that encodes a 351-amino acid glycoprotein that is 78% identical to a human gene that has been reported to be a purinoceptor (P2Y7) and a leukotriene B4 (LTB4) receptor (BLTR). Chinese hamster ovary (CHO) cells transfected with this cDNA specifically bound [3H]LTB4 with a dissociation constant of 0.6 +/- 0.1 nM. Furthermore, LTB4 induced a dose-dependent intracellular calcium flux in transfected CHO cells. In contrast, [35S]dATP did not specifically bind to these transfectants. This mRNA was expressed at high levels in interleukin 5-exposed eosinophils, elicited peritoneal macrophages and neutrophils, and to a lesser extent interferon gamma stimulated macrophages. Low levels of expression were detected in the lung, lymph node, and spleen of unchallenged mice. Western blot analysis detected the mBLTR protein in murine eosinophils and alveolar macrophages as well as human eosinophils. In addition, elevated levels of mBLTR mRNA were found in the lungs of mice in a murine model of allergic pulmonary inflammation in a time course consistent with the influx of eosinophils. Our findings indicate that this murine receptor is an LTB4 receptor that is highly expressed on activated leukocytes, including eosinophils, and may play an important role in mediating eosinophil recruitment into inflammatory foci.
| S-EPMC2212531 | biostudies-literature
Project description:Leukotriene B4 receptor 1 (BLT1) plays crucial roles in the acute inflammatory responses and is a valuable target for anti-inflammation treatment, however, the mechanism by which leukotriene B4 (LTB4) activates receptor remains unclear. Here, we report the cryo-electron microscopy (cryo-EM) structure of the LTB4 -bound human BLT1 in complex with a Gi protein in an active conformation at resolution of 2.91 Å. In combination of molecule dynamics (MD) simulation, docking and site-directed mutagenesis, our structure reveals that a hydrogen-bond network of water molecules and key polar residues is the key molecular determinant for LTB4 binding. We also find that the displacement of residues M1013.36 and I2717.39 to the center of receptor, which unlock the ion lock of the lower part of pocket, is the key mechanism of receptor activation. In addition, we reveal a binding site of phosphatidylinositol (PI) and discover that the widely open ligand binding pocket may contribute the lack of specificity and efficacy for current BLT1-targeting drug design. Taken together, our structural analysis provides a scaffold for understanding BLT1 activation and a rational basis for designing anti-leukotriene drugs.
| S-EPMC8894450 | biostudies-literature
Project description:The cDNA for leukotriene B(4) (LTB(4)) receptor (BLT) was cloned from a guinea-pig leucocyte cDNA library. The cloned receptor cDNA encodes 348 amino acid residues and shares 73% identity with the amino acid sequence of human BLT. Northern blot analysis showed the highest expression of the receptor mRNA in leucocytes, followed by lung and spleen. The membrane fractions of HEK-293 and Cos-7 cells transfected with the cDNA showed specific LTB(4)-binding activities, with K(d) values of 0.27 and 0.17 nM respectively. Xenopus laevis oocytes injected with the cRNA of guinea-pig BLT showed LTB(4)-induced Cl(-) currents, indicating that the cloned receptor is functional. LTB(4) is metabolized to 20-hydroxy-LTB(4) and then to 20-carboxy-LTB(4), a transformation considered as a major inactivation pathway of the compound. Using the cloned receptor, we analysed the agonistic effects of LTB(4) and these two metabolites. 20-Carboxy-LTB(4) is a much weaker agonist, with a K(d) value higher than that of LTB(4) by three orders of magnitude, corresponding to a much weaker chemotactic activity. Although 20-hydroxy-LTB(4) is as potent as LTB(4) in inhibiting [(3)H]LTB(4) binding and cAMP formation, it is less potent than LTB(4) in the mobilization of intracellular Ca(2+) and the chemotaxis of Chinese hamster ovary cells expressing the guinea-pig BLT. The present study demonstrated that although LTB(4) and 20-hydroxy-LTB(4) bind to the receptor with similar affinities, they do differ in activating intracellular signalling.
| S-EPMC1220439 | biostudies-other
Project description:Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo.
| S-EPMC5602668 | biostudies-literature
Project description:Chronic inflammation is the underlying cause of many diseases, including type 1 diabetes, obesity, and non-alcoholic fatty liver disease. Macrophages are continuously recruited to tissues during chronic inflammation where they exacerbate or resolve the pro-inflammatory environment. Although leukotriene B4 receptor 2 (BLT2) has been characterized as a low affinity receptor to several key eicosanoids and chemoattractants, its precise roles in the setting of inflammation and macrophage function remain incompletely understood. Here we used zebrafish and mouse models to probe the role of BLT2 in macrophage function during inflammation. We detected BLT2 expression in bone marrow derived and peritoneal macrophages of mouse models. Transcriptomic analysis of Ltb4r2-/- and WT macrophages suggested a role for BLT2 in macrophage migration, and studies in vitro confirmed that whereas BLT2 does not mediate macrophage polarization, it is required for chemotactic function, possibly mediated by downstream genes Ccl5 and Lgals3. Using a zebrafish model of tailfin injury, we demonstrated that antisense morpholino-mediated knockdown of blt2a or chemical inhibition of BLT2 signaling impairs macrophage migration. We further replicated these findings in zebrafish models of islet injury and liver inflammation. Moreover, we established the applicability of our zebrafish findings to mammals by showing that macrophages of Ltb4r2-/- mice have defective migration during lipopolysaccharide stimulation in vivo. Collectively, our results demonstrate that BLT2 mediates macrophage migration during inflammation, which implicates it as a potential therapeutic target for inflammatory pathologies.
| S-EPMC10790086 | biostudies-literature
Project description:Chronic exposure to crystalline silica (CS) causes silicosis, an irreversible lung inflammatory disease that may eventually lead to lung cancer. In this study, we demonstrate that in K-ras(LA1) mice, CS exposure markedly enhances the lung tumour burden and genetic deletion of leukotriene B4 receptor-1 (BLT1(-/-)) attenuates this increase. Pulmonary neutrophilic inflammation induced by CS is significantly reduced in BLT1(-/-)K-ras(LA1) mice. CS exposure induces LTB4 production by mast cells and macrophages independent of inflammasome activation. In an air-pouch model, CS-induced neutrophil recruitment is dependent on LTB4 production by mast cells and BLT1 expression on neutrophils. In an implantable lung tumour model, CS exposure results in rapid tumour growth and decreased survival that is attenuated in the absence of BLT1. These results suggest that the LTB4/BLT1 axis sets the pace of CS-induced sterile inflammation that promotes lung cancer progression. This knowledge may facilitate development of immunotherapeutic strategies to fight silicosis and lung cancer.
| S-EPMC4418220 | biostudies-literature
Project description:Although lung cancer is the leading cause of cancer-related deaths worldwide and KRAS is the most frequently mutated oncogene in lung cancer cases, the mechanism by which KRAS mutation drives lung cancer has not been fully elucidated. Here, we report that the expression levels of leukotriene B4 receptor-2 (BLT2) and its ligand-producing enzymes (5-LOX, 12-LOX) were highly increased by mutant KRAS and that BLT2 or 5-/12-LOX blockade attenuated KRAS-driven lung cell proliferation and production of interleukin-6 (IL-6), a principal proinflammatory mediator of lung cancer development. Next, we explored the roles of BLT2 and 5-/12-LOX in transgenic mice with lung-specific expression of mutant KRAS (KrasG12D) and observed that BLT2 or 5-/12-LOX inhibition decreased IL-6 production and tumor formation. To further determine whether BLT2 is involved in KRAS-driven lung tumor formation, we established a KrasG12D/BLT2-KO double-mutant mouse model. In the double-mutant mice, we observed significantly suppressed IL-6 production and lung tumor formation. Additionally, we observed high BLT2 expression in tissue samples from patients with KrasG12D-expressing lung adenocarcinoma, supporting the contributory role of BLT2 in KRAS-driven human lung cancer. Collectively, our results suggest that BLT2 is a potential contributor to KRAS-driven lung cancer and identify an attractive therapeutic target for KRAS-driven lung cancer.
| S-EPMC8569214 | biostudies-literature
Project description:BackgroundPolymorphisms spanning genes involved in the production of leukotriene B4 (LTB4) e.g. ALOX5AP and LTA4H are associated with asthma susceptibility, suggesting a role for LTB4 in disease. The contribution of LTB4receptor polymorphism is currently unknown. The aim of this study was to characterise the genes for the two pivotal LTB4 receptors, LTB4R1 and LTB4R2 in lung tissue and determine if polymorphisms spanning these genes are associated with asthma and disease severity.MethodsRapid amplification of cDNA ends (RACE) was used to characterise the LTB4R1 and LTB4R2 gene structure in lung. The LTB4R1/2 locus on chromosome 14q11.2 was screened for polymorphic variation. Six LTB4R single nucleotide polymorphisms (SNPs) were genotyped in 370 Caucasian asthma families and 299 Adult Asthma Individuals (n=1877 total) and were evaluated for association with asthma and severity (BTS) outcome measures using Family Based Association Test, linear regression and chi square.ResultsLTB4R1 has complex mRNA arrangement including multiple 5'-untranslated exons, suggesting additional levels of regulation. Three potential promoter regions across the LTB4R1/2 locus were identified with some airway cell specificity. 22 SNPs (MAF>0.01) were validated across the LTB4R locus in the Caucasian population. LTB4R1 and LTB4R2 SNPs were not associated with asthma susceptibility, FEV1 or severity.ConclusionsLTB4R1 and LTB4R2 shows splice variation in the 5'-untranslated region and multiple promoter regions. The functional significance of this is yet to be determined. Both receptor genes were shown to be polymorphic. LTB4R polymorphisms do not appear to be susceptibility markers for the development of asthma in Caucasian subjects.
| S-EPMC3607986 | biostudies-literature
Project description:Chronic inflammation is the underlying cause of many diseases, including type 1 diabetes, obesity, and non-alcoholic fatty liver disease. Macrophages are continuously recruited to tissues during chronic inflammation where they exacerbate or resolve the pro-inflammatory environment. Although leukotriene B4 receptor 2 (BLT2) has been characterized as a low affinity receptor to several key eicosanoids and chemoattractants, its precise roles in the setting of inflammation and macrophage function remain incompletely understood. Here we used zebrafish and mouse models to probe the role of BLT2 in macrophage function during inflammation. We detected BLT2 expression in bone marrow derived and peritoneal macrophages of mouse models. Transcriptomic analysis of Ltb4r2-/- and WT macrophages suggested a role for BLT2 in macrophage migration, and studies in vitro confirmed that whereas BLT2 does not mediate macrophage polarization, it is required for chemotactic function, possibly mediated by downstream genes Ccl5 and Lgals3. Using a zebrafish model of tailfin injury, we demonstrated that antisense morpholino-mediated knockdown of blt2a or chemical inhibition of BLT2 signaling impairs macrophage migration. We further replicated these findings in zebrafish models of islet injury and liver inflammation. Moreover, we established the applicability of our zebrafish findings to mammals by showing that macrophages of Ltb4r2-/- mice have defective migration during lipopolysaccharide stimulation in vivo. Collectively, our results demonstrate that BLT2 mediates macrophage migration during inflammation, which implicates it as a potential therapeutic target for inflammatory pathologies.
2023-12-01 | GSE242723 | GEO