CDNA cloning and characterization of guinea-pig leukotriene B4 receptor.
Ontology highlight
ABSTRACT:
SUBMITTER: Masuda K
PROVIDER: S-EPMC1220439 | biostudies-other | 1999 Aug
REPOSITORIES: biostudies-other
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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
Project description:In this study, we introduced structure-based rational mutations in the guinea pig leukotriene B4 receptor (gpBLT1) in order to enhance the stabilization of the protein. Elements thought to be unfavorable for the stability of gpBLT1 were extracted based on the stabilization elements established in soluble proteins, determined crystal structures of G-protein-coupled receptors (GPCRs), and multiple sequence alignment. The two unfavorable residues His832.67 and Lys883.21, located at helix capping sites, were replaced with Gly (His83Gly2.67 and Lys88Gly3.21). The modified protein containing His83Gly2.67/Lys88Gly3.21 was highly expressed, solubilized, and purified and exhibited improved thermal stability by 4 °C in comparison with that of the original gpBLT1 construct. Owing to the double mutation, the expression level increased by 6-fold (Bmax=311 pmol/mg) in the membrane fraction of Pichia pastoris. The ligand binding affinity was similar to that of the original gpBLT1 without the mutations. Similar unfavorable residues have been observed at helix capping sites in many other GPCRs; therefore, the replacement of such residues with more favorable residues will improve stabilization of the GPCR structure for the crystallization.
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