Recently, single-nucleotide polymorphisms (SNPs) in G-protein-coupled receptors (GPCRs) have been suggested to contribute to physiopathology and therapeutic effects. Leukotriene B4 receptor 2 (BLT2), a member of the GPCR family, plays a critical role in the pathogenesis of several inflammatory diseases, including cancer and asthma. However, no studies on BLT2 SNP effects have been reported to date. In this study, we demonstrate that the BLT2 SNP (rs1950504, Asp196Gly), a Gly-196 variant of BLT2 (BLT2 D196G), causes enhanced cell motility under low-dose stimulation of its ligands. In addition, we demonstrated that Akt activation and subsequent production of reactive oxygen species (ROS), both of which act downstream of BLT2, are also increased by BLT2 D196G in response to low-dose ligand stimulation. Furthermore, we observed that the ligand binding affinity of BLT2 D196G was enhanced compared with that of BLT2. Through homology modeling analysis, it was predicted that BLT2 D196G loses ionic interaction with R197, potentially resulting in increased agonist-receptor interaction. To the best of our knowledge, this report is the first to describe a SNP study on BLT2 and shows that BLT2 D196G enhances ligand sensitivity, thereby increasing cell motility in response to low-dose ligand stimulation.
Asthma ; Cell Movement* ; Humans ; Ligands ; Reactive Oxygen Species ; Receptors, G-Protein-Coupled ; Receptors, Leukotriene B4 ; Therapeutic Uses

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.
Calcium ; Carbachol ; Humans ; Hydrogen-Ion Concentration ; Ion Channels ; Kidney ; Mass Screening ; Mutagenesis, Site-Directed ; Mutant Proteins ; Protein Kinase C ; Receptors, Muscarinic