Intracellular TAS2Rs act as a gatekeeper for the excretion of harmful substances via ABCB1 in keratinocytes.

Bitter taste receptors (TAS2Rs) are not only expressed in the oral cavity but also in skin. Extraoral TAS2Rs are thought to be involved in non-taste perception and tissue-specific functions. Keratinocytes that express TAS2Rs in the skin provide a first-line defense against external threats.

Stepwise phosphorylation of BLT1 defines complex assemblies with β-arrestin serving distinct functions.

G protein-coupled receptors (GPCRs) utilize complex cellular systems to respond to diverse ligand concentrations. By taking BLT1, a GPCR for leukotriene B (LTB ), as a model, our previous work elucidated that this system functions through the modulation of phosphorylation status on two specific residues: Thr and Ser . Ser phosphorylation occurs at a lower LTB concentration than Thr , leading to a shift in ligand affinity from a high-to-low state.

Therapeutic target of leukotriene B receptors, BLT1 and BLT2: Insights from basic research.

Leukotriene B (LTB) is a lipid mediator rapidly generated from arachidonic acid in response to various stimuli. This lipid mediator exerts its biological activities by binding to cognate receptors. Two LTB receptors have been cloned; BLT1 and BLT2 as a high- and a low-affinity receptors, respectively.

Constitutively active GPR43 is crucial for proper leukocyte differentiation.

The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation.

Role of leukotriene B4 (LTB4)-LTB4 receptor 1 signaling in post-incisional nociceptive sensitization and local inflammation in mice.

Leukotriene B4 (LTB4) is a potent lipid mediator involved in the recruitment and activation of neutrophils, which is an important feature of tissue injury and inflammation. The biological effects of LTB4 are primarily mediated through the high-affinity LTB4 receptor, BLT1. Postoperative incisional pain is characterized by persistent acute pain at the site of tissue injury and is associated with local inflammation.

Recent advances in function and structure of two leukotriene B receptors: BLT1 and BLT2.

Leukotriene B (LTB) is generated by the enzymatic oxidation of arachidonic acid, which is then released from the cell membrane and acts as a potent activator of leukocytes and other inflammatory cells. Numerous studies have demonstrated the physiological and pathophysiological significance of this lipid in various diseases. LTB exerts its activities by binding to its specific G protein-coupled receptors (GPCRs): BLT1 and BLT2.

Bitter taste receptor T2R38 is expressed on skin-infiltrating lymphocytes and regulates lymphocyte migration.

Bitter taste receptors (T2Rs) are G protein-coupled receptors involved in the perception of bitter taste on the tongue. In humans, T2Rs have been found in several sites outside the oral cavity. Although T2R38 has been reported to be expressed on peripheral lymphocytes, it is poorly understood whether T2R38 plays immunological roles in inflammatory skin diseases such as atopic dermatitis (AD).

Stepwise phosphorylation of leukotriene B receptor 1 defines cellular responses to leukotriene B.

Leukotriene B (LTB) receptor type 1 (BLT1) is abundant in phagocytic and immune cells and plays crucial roles in various inflammatory diseases. BLT1 is phosphorylated at several serine and threonine residues upon stimulation with the inflammatory lipid LTB Using Phos-tag gel electrophoresis to separate differentially phosphorylated forms of BLT1, we identified two distinct types of phosphorylation, basal and ligand-induced, in the carboxyl terminus of human BLT1. In the absence of LTB, the basal phosphorylation sites were modified to various degrees, giving rise to many different phosphorylated forms of BLT1.

Leukotriene receptors as potential therapeutic targets.

Leukotrienes, a class of arachidonic acid-derived bioactive molecules, are known as mediators of allergic and inflammatory reactions and considered to be important drug targets. Although an inhibitor of leukotriene biosynthesis and antagonists of the cysteinyl leukotriene receptor are clinically used for bronchial asthma and allergic rhinitis, these medications were developed before the molecular identification of leukotriene receptors. Numerous studies using cloned leukotriene receptors and genetically engineered mice have unveiled new pathophysiological roles for leukotrienes.

Real-time monitoring of pH-dependent intracellular trafficking of ovarian cancer G protein-coupled receptor 1 in living leukocytes.

G-protein coupled receptors (GPCRs) are involved in many diseases and important biological phenomena; elucidating the mechanisms underlying regulation of their signal transduction potentially provides both novel targets for drug discovery and insight into living systems. A proton-sensing GPCR, ovarian cancer G protein-coupled receptor 1 (OGR1), has been reported to be related to acidosis and diseases that cause tissue acidification, but the mechanism of proton-induced activation of OGR1-mediated signal transduction in acidic conditions remains unclear. Here, pH-dependent intracellular trafficking of OGR1 was visualized in living leukocytes by a real-time fluorescence microscopic method based on sortase A-mediated pulse labeling of OGR1.

Na-mimicking ligands stabilize the inactive state of leukotriene B receptor BLT1.

Most G-protein-coupled receptors (GPCRs) are stabilized in common in the inactive state by the formation of the sodium ion-centered water cluster with the conserved Asp inside the seven-transmembrane domain. We determined the crystal structure of the leukotriene B (LTB) receptor BLT1 bound with BIIL260, a chemical bearing a benzamidine moiety. Surprisingly, the amidine group occupies the sodium ion and water locations, interacts with D66, and mimics the entire sodium ion-centered water cluster.

Microfluidic preparation of anchored cell membrane sheets for in vitro analyses and manipulation of the cytoplasmic face.

Molecular networks on the cytoplasmic faces of cellular plasma membranes are critical research topics in biological sciences and medicinal chemistry. However, the selective permeability of the cell membrane restricts the researchers from accessing to the intact intracellular factors on the membrane from the outside. Here, a microfluidic method to prepare cell membrane sheets was developed as a promising tool for direct examination of the cytoplasmic faces of cell membranes.

Quantitative image cytometry for analyzing intracellular trafficking of G protein-coupled receptors on a chemical-trapping single cell array.

G protein-coupled receptors (GPCRs) are important targets in medical and pharmaceutical research fields, because they play key roles in a variety of biological processes. Recently, intracellular trafficking of GPCRs involving endosomal internalization and recycling to the plasma membrane has been studied as a regulation mechanism for GPCR activities. However, the absence of a quantitative single-cell analysis method has hampered conditional GPCR trafficking studies and the possibility of gaining significant insights into the mechanism of regulation of GPCR signaling.

The leukotriene B receptor BLT1 is stabilized by transmembrane helix capping mutations.

In this study, we introduced structure-based rational mutations in the guinea pig leukotriene B 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 His83 and Lys88, located at helix capping sites, were replaced with Gly (His83Gly and Lys88Gly).

The absence of the leukotriene B4 receptor BLT1 attenuates peripheral inflammation and spinal nociceptive processing following intraplantar formalin injury.

Background: Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation, and its biological effects are mediated primarily through the high affinity LTB4 receptor BLT1. Although numerous studies have reported that LTB4-BLT1 signaling is involved in inflammatory diseases, the role of BLT1 signaling in pain remains undefined. To clarify the role of LTB4-BLT1 signaling in acute inflammatory pain induced by tissue injury, we performed pain behavioral analysis and assessment of local inflammation induced by peripheral formalin injections in BLT1 knockout mice.

The atypical N-glycosylation motif, Asn-Cys-Cys, in human GPR109A is required for normal cell surface expression and intracellular signaling.

Asparagine-linked glycosylation (N-glycosylation) is necessary for the proper folding of secreted and membrane proteins, including GPCRs. Thus, many GPCRs possess the N-glycosylation motif Asn-X-Ser/Thr at their N-termini and/or extracellular loops. We found that human GPR109A (hGPR109A) has an N-glycosylation site at Asn(17) in the N-terminal atypical motif, Asn(17)-Cys(18)-Cys(19).

Visualization of the pH-dependent dynamic distribution of G2A in living cells.

G2A (from G2 accumulation) receptor is a member of the proton-sensing G-protein coupled receptor (GPCR) family and induces signal transduction events that regulate the cell cycle, proliferation, oncogenesis, and immunity. The mechanism by which G2A-mediated signal transduction is regulated by the extracellular pH remains unresolved. Here, we first visualize the pH-dependent G2A distribution change in living cells by a sortase A-mediated pulse labeling technology: the short-peptide tag-fused human G2A on human embryo kidney HEK293T cell surfaces was labeled with a small fluorescent dye in the presence of lysophosphatidylcholine, and the labeled G2A was chased at acidic and neutral pHs in real time by microscope time course observations.

Interplay between CXCR2 and BLT1 facilitates neutrophil infiltration and resultant keratinocyte activation in a murine model of imiquimod-induced psoriasis.

Psoriasis is an inflammatory skin disease with accelerated epidermal cell turnover. Neutrophil accumulation in the skin is one of the histological characteristics of psoriasis. However, the precise mechanism and role of neutrophil infiltration remain largely unknown.

Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis.

Microenvironment-based alterations in phenotypes of mast cells influence the susceptibility to anaphylaxis, yet the mechanisms underlying proper maturation of mast cells toward an anaphylaxis-sensitive phenotype are incompletely understood. Here we report that PLA2G3, a mammalian homolog of anaphylactic bee venom phospholipase A2, regulates this process. PLA2G3 secreted from mast cells is coupled with fibroblastic lipocalin-type PGD2 synthase (L-PGDS) to provide PGD2, which facilitates mast-cell maturation via PGD2 receptor DP1.

Amino acid residues of G-protein-coupled receptors critical for endoplasmic reticulum export and trafficking.

Analysis of the structural features of rhodopsin-type G-protein-coupled receptors (GPCRs) revealed the existence of an additional α-helix, termed helix 8, in the C-terminal tail. Furthermore, these GPCRs were determined to possess several conserved residues in their transmembrane domains. The functional deficiencies of receptors in which these domains or residues have been mutated have not been examined in living cells due to their accumulation in the endoplasmic reticulum (ER), although the ligand affinities of these receptors have been tested in membrane preparations.

Discovery of INT131: a selective PPARγ modulator that enhances insulin sensitivity.

PPARγ is a member of the nuclear hormone receptor family and plays a key role in the regulation of glucose homeostasis. This Letter describes the discovery of a novel chemical class of diarylsulfonamide partial agonists that act as selective PPARγ modulators (SPPARγMs) and display a unique pharmacological profile compared to the thiazolidinedione (TZD) class of PPARγ full agonists. Herein we report the initial discovery of partial agonist 4 and the structure-activity relationship studies that led to the selection of clinical compound INT131 (3), a potent PPARγ partial agonist that displays robust glucose-lowering activity in rodent models of diabetes while exhibiting a reduced side-effects profile compared to marketed TZDs.

Identification and characterization of a novel chemotype MEK inhibitor able to alter the phosphorylation state of MEK1/2.

A small molecule compound, JTP-74057/GSK1120212/trametinib, had been discovered as a very potent antiproliferative agent able to induce the accumulation of CDK inhibitor p15INK4b. To conduct its drug development rationally as an anticancer agent, molecular targets of this compound were identified as MEK1/2 using compound-affinity chromatography. It was shown that JTP-74057 directly bound to MEK1 and MEK2 and allosterically inhibited their kinase activities, and that its inhibitory characteristics were similar to those of the known and different chemotype of MEK inhibitors PD0325901 and U0126.

Trehalose treatment suppresses inflammation, oxidative stress, and vasospasm induced by experimental subarachnoid hemorrhage.

Background: Subarachnoid hemorrhage (SAH) frequently results in several complications, including cerebral vasospasm, associated with high mortality. Although cerebral vasospasm is a major cause of brain damages after SAH, other factors such as inflammatory responses and oxidative stress also contribute to high mortality after SAH. Trehalose is a non-reducing disaccharide in which two glucose units are linked by α,α-1,1-glycosidic bond, and has been shown to induce tolerance to a variety of stressors in numerous organisms.