[Comparative Computational Modeling of Agonist Binding to the Leukotriene Receptors BLT1 and BLT2].

The leukotriene B4 receptors BLT1 and BLT2 are promising targets for the treatment of allergic and inflammatory diseases. However, no working model of ligand binding to either of these receptors has been developed so far. Under the assumption that homologous receptors bind their ligands in a similar way, computational modeling of agonist binding to BLT1 and BLT2 was performed using fully flexible docking in Galaxy 7TM. For both receptors, the carboxyl group of the ligand forms a salt bridge with an arginine residue, while the tail hydroxyl groups form hydrogen bonds with three amino acid residues. The differential specificity of ligands to BLT1 and BLT2 is explained by the replacement of histidine with tyrosine. In BLT1, the histidine residue binds the 5-OH group of the ligand, while the tyrosine residue in BLT2 repels it. The presented models are in agreement with experimental data and may be useful for developing new BLT1- and BLT2-targeted drugs.

The leukotriene B4 receptor BLT2 protects barrier function via actin polymerization with phosphorylation of myosin phosphatase target subunit 1 in human keratinocytes.

Leukotriene B4 (LTB4 ) receptor type 2 (BLT2) is a novel G-protein-coupled receptor, which selectively binds to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) with stronger affinity than to LTB4 . Recently, 12-HHT has been shown to have a protective effect on the epidermal barrier in human keratinocytes or transfectant cells overexpressing BLT2. Because the protective activity of BLT2 in high-calcium conditions, which occurs in well-differentiated cells, is exerted through increasing the integrity of tight junctions, we investigated the effects of 12-HHT on the barrier function of human keratinocytes in low-calcium conditions that mimic the basal layer; to our knowledge, this has not been reported previously. After stimulation with or without 12-HHT, barrier function was measured using transepithelial electrical resistance (TER) and dextran permeability assay. Expression levels of adhesion molecules and actin polymerization were also evaluated. Treatment with 12-HHT increased TER, along with decreased epidermal permeability of dextran in human keratinocytes. Furthermore, 12-HHT induced actin polymerization with phosphorylation of myosin phosphatase target subunit 1. These results suggest that the ligation of BLT2 protects permeability barrier function by enhancing cell-cell contact, even under low-calcium conditions, and indicate that a BLT2 agonist could be a novel therapeutic target for barrier-disrupted skin diseases.

Leukotriene production profiles and actions in the bovine endometrium during the oestrous cycle.

We have previously shown the influence of leukotrienes (LTs) on reproductive functions in vivo: LTB4 is luteotrophic and supports corpus luteum function inducing PGE2 and progesterone (P4) secretion, whereas LTC4 is luteolytic and stimulates PGF2α secretion in cattle. The aim of this study was to examine expression and production profiles of LTs and their actions in the endometrium. LT receptors (LTB4R for LTB4 and CysLTR2 for LTC4), 5-lipoxygenase (LO), 12-LO synthase (LTCS) and LTA4 hydrolase (LTAH) mRNA and protein expression, as well as LT production were measured in bovine endometrial tissue during the luteal phases of the oestrous cycle. The action of LTs on uterine function was studied by measuring the level of PGs after stimulating uterine slices with LTs on Days 8-10 of the cycle. Expression of 5-LO and LTB4R mRNA and protein were highest on Days 2-4 of the cycle, while CysLTR2 and LTCS were highest on Days 16-18 (P<0.05). LTB4 concentration was highest on Days 2-4 of the cycle, whereas the greatest LTC4 level was on Days 16-18 (P<0.05). Both LTB4 and C4 increased the content of PGE2 and F2α in endometrial slices at a dose of 10(-7)M (P<0.05). In summary, mRNA expression and activation of receptors for LTB4 and production occur in the first part of the cycle, whereas LTC4 and its receptors predominate at the end of the cycle. The 12-LO and 5-LO pathways are complementary routes of LT production in the bovine uterus.

Dual role of leukotriene B4 receptor type 1 in experimental sepsis.

BACKGROUND: The controversial results from different studies suggested that leukocyte recruitment mediated by leukotriene B4 (LTB4) and its receptor might improve pathogen clearance, but might also aggravate organ injury during sepsis. The present study was performed to compare the effect of BLT1 ligand LTB4 and its antagonist U-75302 on the development of sepsis. METHODS: Sepsis in mice was induced by cecal ligation and puncture (CLP). The mice were allocated into sham group, CLP group, U-75302 group, and LTB4 group. In the latter three groups, CLP mice were treated by intraperitoneal saline, U-75302, and LTB4, respectively. Their effect on the progression of sepsis were compared by histopathologic tests, level of systemic cytokines, counts of immune cells and bacterial clearance, and survival rate. RESULTS: The histopathologic tests showed that U-75302 attenuated lung injury, whereas LTB4 aggravated liver injury. LTB4 increased the plasma levels of interleukin-6, tumor necrosis factor-α, and U-75302 increased the level of plasma interleukin-10. LTB4 increased whereas U-75302 reduced the neutrophil numbers in the peritoneal lavage fluid. LTB4 also increased the number of peritoneal and splenic CD4(+) and CD8(+) T cells. Bacterial clearance in blood and peritoneal lavage fluid was significantly enhanced in the LTB4 group. Both U-75302 and LTB4 did not change the survival rate significantly compared with vehicle, but mortality in the LTB4 group was significantly higher than in the U-75302 group. Dose response analyses were also performed to compare the effect of U-75302 and LTB4 at different doses. Different doses of both agents did not influence the survival rate of CLP mice. CONCLUSIONS: U-75302 attenuates sepsis-induced organ injury, whereas LTB4 increases the leukocyte recruitment toward infection site, but LTB4 showed a more lethal effect than U-75302 during polymicrobial sepsis.

Development and Characterization of a Fluorescent Ligand for Leukotriene B4 Receptor 2 in Cells and Tissues.

The leukotriene B4 receptor 2 (BLT2) is a G-protein coupled receptor activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), which has been proposed as a promising therapeutic target for diabetic wound healing and gastrointestinal lesions. In this study, the rational design of a fluorescent probe based on the synthetic BLT2 agonist CAY10583 is described. The synthesis of several derivatives of CAY10583 coupled to fluorescein resulted in a traceable ligand suitable for different fluorescence-based techniques. An HTRF-based displacement assay (Tag-lite) on stably transfected CHO-K1 cells was developed to characterize binding properties of diverse BLT2 ligands. Highly specific binding to the BLT2 receptor was demonstrated in staining experiments on mouse skin tissue, and specific modulation of BLT2-induced cAMP signaling provided further evidence for receptor binding and ligand functionality. In conclusion, the fluorescent ligands developed in this study are suitable to investigate the pharmacology of BLT2 receptor ligands in a variety of assay systems.

Electrostatically-driven fast association and perdeuteration allow detection of transferred cross-relaxation for G protein-coupled receptor ligands with equilibrium dissociation constants in the high-to-low nanomolar range.

The mechanism of signal transduction mediated by G protein-coupled receptors is a subject of intense research in pharmacological and structural biology. Ligand association to the receptor constitutes a critical event in the activation process. Solution-state NMR can be amenable to high-resolution structure determination of agonist molecules in their receptor-bound state by detecting dipolar interactions in a transferred mode, even with equilibrium dissociation constants below the micromolar range. This is possible in the case of an inherent ultra-fast diffusive association of charged ligands onto a highly charged extracellular surface, and by slowing down the (1)H-(1)H cross-relaxation by perdeuterating the receptor. Here, we demonstrate this for two fatty acid molecules in interaction with the leukotriene BLT2 receptor, for which both ligands display a submicromolar affinity.

Structural insights on ligand recognition at the human leukotriene B4 receptor 1.

The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design.

Intracerebroventricular injection of leukotriene B4 attenuates antigen-induced asthmatic response via BLT1 receptor stimulating HPA-axis in sensitized rats.

BACKGROUND: Basic and clinical studies suggest that hypothalamic-pituitary-adrenal (HPA) axis is the neuroendocrine-immune pathway that functionally regulates the chronic inflammatory disease including asthma. Our previous studies showed corresponding changes of cytokines and leukotriene B4 (LTB4) between brain and lung tissues in antigen-challenged asthmatic rats. Here, we investigated how the increased LTB4 level in brain interacts with HPA axis in regulating antigen-induced asthmatic response in sensitized rats. METHODS: Ovalbumin-sensitized rats were challenged by inhalation of antigen. Rats received vehicle, LTB4 or U75302 (a selective LTB4 BLT1 receptor inhibitor) was given via intracerebroventricular injection (i.c.v) 30 min before challenge. Lung resistance (RL) and dynamic lung compliance (Cdyn) were measured before and after antigen challenge. Inflammatory response in lung tissue was assessed 24 h after challenge. Expression of CRH mRNA and protein in hypothalamus were evaluated by RT-PCR and Western Blot, and plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were measured using the ELISA kits. RESULTS: Antigen challenge decreased pulmonary function and induced airway inflammation, evoked HPA axis response in sensitized rats. Administration of LTB4 via i.c.v markedly attenuated airway contraction and inflammation. Meanwhile, LTB4 via i.c.v markedly increased CORT and ACTH level in plasma before antigen challenge, and followed by further increases in CORT and ACTH levels in plasma after antigen challenge in sensitized rats. Expression of CRH mRNA and protein in hypothalamus were also significantly increased by LTB4 via i.c.v in sensitized rats after antigen challenge. These effect were completely blocked by pre-treatment with BLT1 receptor antagonist U75302 (10 ng), but not by BLT2 antagonist LY255283. CONCLUSIONS: LTB4 administered via i.c.v down-regulates the airway contraction response and inflammation through activation of the HPA axis via its BLT1 receptor. This study expands our concept of the regulatory role of intracranial inflammatory mediators in inflammatory diseases including asthma. The favourable effects of LTB4 on the HPA axis may help to explain the phenomenon of self-relief after an asthmatic attack.

Helix 8 of leukotriene B4 type-2 receptor is required for the folding to pass the quality control in the endoplasmic reticulum.

Many G protein-coupled receptors (GPCRs) possess a putative cytoplasmic helical domain, termed helix 8 (H8), at the proximal region of the C-terminal tail. However, the significance of this domain is not fully understood. Here, we demonstrate the requirement of H8 for the proper folding of GPCRs for passage through the quality control in the endoplasmic reticulum (ER). In the human leukotriene B(4) type-2 receptor (hBLT2), lack of H8 led to an accumulation of the receptor (hBLT2/DeltaH8) in the ER. Similar results were obtained in two representative human GPCRs, dopamine type-1 and lysophosphatidic acid type-2 receptors, which were engineered to lack H8. Treatment with the several ligands, which act as pharmacological chaperones, facilitated the surface expression of hBLT2/DeltaH8. The surface-trafficked hBLT2/DeltaH8 exhibited an agonist-evoked increase in Ca(2+), demonstrating that H8 is not critical for ligand binding and activation of coupled G proteins. Thus, these results suggest that the H8 region of hBLT2 plays an important role in transport-competent receptor folding.

Leukotriene B4-induced changes in vascular permeability are mediated by neutrophil release of heparin-binding protein (HBP/CAP37/azurocidin).

In humans, heparin-binding protein (HBP) and the potent chemotactic lipid leukotriene B(4) (LTB(4)) are important mediators of innate immune responses. Here we show that human neutrophils (PMNs) challenged with LTB(4) (30 s to 5 min) release HBP as determined by Western blot analysis. This response peaks at 100 nM of agonist and is mediated by the BLT1 receptor. Protein phosphatase-1 (30 microM) and wortmannin (0.5 microM) block the LTB(4)-mediated HBP release from PMNs, which suggests involvement of the 1-phosphatidylinositol 3-kinase intracellular pathway during degranulation. Furthermore, postsecretory supernatants from LTB(4)-stimulated PMNs induce intracellular calcium mobilization in endothelial cells in vitro and increase in vascular permeability in vivo, as assessed in a mouse model of pleurisy. Selective removal of HBP from the supernatant significantly reduces these activities attributing a key role to HBP in the LTB(4)-induced change in vascular permeability. This lipid-protein axis could offer novel opportunities for pharmacological intervention in key steps of the vascular response to inflammation.

Structure of the agonist 12-HHT in its BLT2 receptor-bound state.

G Protein-Coupled receptors represent the main communicating pathway for signals from the outside to the inside of most of eukaryotic cells. They define the largest family of integral membrane receptors at the surface of the cells and constitute the main target of the current drugs on the market. The low affinity leukotriene receptor BLT2 is a receptor involved in pro- and anti-inflammatory pathways and can be activated by various unsaturated fatty acid compounds. We present here the NMR structure of the agonist 12-HHT in its BLT2-bound state and a model of interaction of the ligand with the receptor based on a conformational homology modeling associated with docking simulations. Put into perspective with the data obtained with leukotriene B4, our results illuminate the ligand selectivity of BLT2 and may help define new molecules to modulate the activity of this receptor.

First Structure-Activity Relationship Study of Potent BLT2 Agonists as Potential Wound-Healing Promoters.

The first potent leukotriene B4 (LTB4) receptor type 2 (BLT2) agonists, endogenous 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), and synthetic CAY10583 (CAY) have been recently described to accelerate wound healing by enhanced keratinocyte migration and indirect stimulation of fibroblast activity in diabetic rats. CAY represents a very valuable starting point for the development of novel wound-healing promoters. In this work, the first structure-activity relationship study for CAY scaffold-based BLT2 agonists is presented. The newly prepared derivatives showed promising in vitro wound-healing activity.

A synthetic leukotriene B4 receptor type 2 agonist accelerates the cutaneous wound healing process in diabetic rats by indirect stimulation of fibroblasts and direct stimulation of keratinocytes.

AIMS: The synthetic leukotriene B4 receptor type 2 (BLT2) agonist CAY10583 (CAY) accelerates wound healing in diabetic mice by promoting keratinocyte migration. However, its effects on fibroblast activity and granulation are unknown. We investigated the mechanisms by which CAY promotes wound healing. METHODS: CAY was applied to wounds on streptozotocin-induced diabetic rats, and wound closure, granulation thickness, and epithelialization gaps were analyzed. BLT2 expression was examined by RT-PCR. Migration and proliferation were studied by scratch assays and MTS assays. Keratinocyte supernatants with CAY were applied to fibroblasts, and cytokines were measured by enzyme-linked immunosorbent assays. RESULTS: CAY significantly accelerated wound healing in diabetic rats (CAY, 78.05±12.22% vs. control, 59.84±11.09%; p=0.0222), with increased re-epithelialization and granulation compared to controls. BLT2 was expressed in keratinocytes, but not in fibroblasts. Keratinocyte treatment with the CAY supernatant enhanced fibroblast proliferation and migration (fibroblast scratch closure: CAY, 75.95±4.09% vs. control, 49.69±4.49%; p<0.0001). CAY-treated keratinocytes exhibited increased TGF-ß1 and bFGF expression. CONCLUSIONS: CAY directly promotes keratinocyte migration and indirectly enhances fibroblast proliferation by increasing keratinocyte production of TGF-ß1 and bFGF, accelerating wound closure. CAY is a promising pharmaceutical agent for diabetic wounds.

In vivo modulation of LPS induced leukotrienes generation and oxidative stress by sesame lignans.

The role of inflammation and oxidative stress is critical during onset of metabolic disorders and this has been sufficiently established in literature. In the present study, we evaluated the effects of sesamol and sesamin, two important bioactive molecules present in sesame oil, on the generation of inflammatory and oxidative stress factors in LPS injected rats. Sesamol and sesamin lowered LPS induced expression of cPLA2 (61 and 56%), 5-LOX (44 and 51%), BLT-1(32 and 35%) and LTC4 synthase (49 and 50%), respectively, in liver homogenate. The diminished serum LTB4 (53 and 64%) and LTC4 (67 and 44%) levels in sesamol and sesamin administered groups, respectively, were found to be concurrent with the observed decrease in the expression of cPLA2 and 5-LOX. The serum levels of TNF-α (29 and 19%), MCP-1 (44 and 57%) and IL-1ß (43 and 42%) were found to be reduced in sesamol and sesamin group, respectively, as given in parentheses, compared to LPS group. Sesamol and sesamin offered protection against LPS induced lipid peroxidation in both serum and liver. Sesamol, but not sesamin, significantly restored the loss of catalase and glutathione reductase activity due to LPS (P<.05). However, both sesamol and sesamin reverted SOD activities by 92 and 98%, respectively. Thus, oral supplementation of sesamol and sesamin beneficially modulated the inflammatory and oxidative stress markers, as observed in the present study, in LPS injected rats. Our report further advocates the potential use of sesamol and sesamin as an adjunct therapy wherein, inflammatory and oxidative stress is of major concern.

Structure-based analysis of GPCR function: conformational adaptation of both agonist and receptor upon leukotriene B4 binding to recombinant BLT1.

We produced the human leukotriene B(4) (LTB(4)) receptor BLT1, a G-protein-coupled receptor, in Escherichia coli with yields that are sufficient for the first structural characterization of this receptor in solution. Overexpression was achieved through codon optimization and the search for optimal refolding conditions of BLT1 recovered from inclusion bodies. The detergent-solubilized receptor displays a 3D-fold compatible with a seven transmembrane (TM) domain with ca 50% alpha-helix and an essential disulfide bridge (circular dichroism evidence); it binds LTB(4) with K(a)=7.8(+/-0.2)x10(8)M(-1) and a stoichiometric ratio of 0.98(+/-0.02). Antagonistic effects were investigated using a synthetic molecule that shares common structural features with LTB(4). We report evidence that both partners, LTB(4) and BLT1, undergo a rearrangement of their respective conformations upon complex formation: (i) a departure from planarity of the LTB(4) conjugated triene moiety; (ii) a change in the environment of Trp234 (TM-VI helix) and in the exposure of the cytoplasmic region of this transmembrane helix.

Structure-based analysis of GPCR function: evidence for a novel pentameric assembly between the dimeric leukotriene B4 receptor BLT1 and the G-protein.

We produced human leukotriene B(4) (LTB(4)) receptor BLT1 as a recombinant protein in Escherichia coli. This detergent-solubilized receptor displays two states with regard to its affinity for LTB(4): (i) a low-affinity state (K(a)=7.8x10(8)M(-1)) that involves a receptor homodimer (BLT1.LTB(4))(2); we report evidence for a central role of the sixth transmembrane helix in regulating the stability of this homodimer; (ii) a high-affinity state (K(a)=1.3x10(10)M(-1)) upon interaction of the receptor with the heterotrimeric GDP-loaded G-protein, Galpha(i2)beta(1)gamma(2). Association of the G-protein with recombinant BLT1 induces GDP-GTP exchange by the Galpha subunit. These results indicate that isolated BLT1 is fully representative of the in vivo receptor with regard to high-affinity recognition of LTB(4), association with a G-protein and activation of Galpha. Using a combination of mass spectrometry after chemical cross-linking and neutron-scattering in solution with the native complex, we establish unambiguously that only one G-protein trimer binds to a receptor dimer to form the stoichiometrically defined (BLT1.LTB(4))(2):Galpha(i2)beta(1)gamma(2) pentameric assembly. This suggests that receptor dimerization could be crucial to transduction of the LTB(4)-induced signal.

12-Hydroxyheptadecatrienoic acid promotes epidermal wound healing by accelerating keratinocyte migration via the BLT2 receptor.

Leukotriene B4 (LTB4) receptor type 2 (BLT2) is a G protein-coupled receptor (GPCR) for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4. Despite the well-defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive. As mouse BLT2 is highly expressed in epidermal keratinocytes, we investigated the role of the 12-HHT/BLT2 axis in skin wound healing processes. 12-HHT accumulated in the wound fluid in mice, and BLT2-deficient mice exhibited impaired re-epithelialization and delayed wound closure after skin punching. Aspirin administration reduced 12-HHT production and resulted in delayed wound closure in wild-type mice, which was abrogated in BLT2-deficient mice. In vitro scratch assay using primary keratinocytes and a keratinocyte cell line also showed that the 12-HHT/BLT2 axis accelerated wound closure through the production of tumor necrosis factor α (TNF) and matrix metalloproteinases (MMPs). A synthetic BLT2 agonist accelerated wound closure in cultured cells as well as in C57BL/6J and diabetic mice. These results identify a novel mechanism underlying the action of the 12-HHT/BLT2 axis in epidermal keratinocytes and accordingly suggest the use of BLT2 agonists as therapeutic agents to accelerate wound healing, particularly for intractable wounds, such as diabetic ulcers.

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. Recent studies have demonstrated that ER-accumulated receptors are effectively exported from ER using membrane permeable ligands as pharmacological chaperones. Here, we identified several residues of the platelet-activating factor receptor and leukotriene B(4) type-II receptor that are crucial for export from ER. Moreover, we used their specific ligands as pharmacological chaperones to traffic ER-accumulated GPCRs to the cell surface in order to examine the functional deficiencies of each mutant receptor. Here, we introduce the novel technique of site-specific N-terminal labeling of cell surface proteins in living cells with Sortase-A, a transpeptidase isolated from Staphylococcus aureus, to evaluate the trafficking of receptors after agonist stimulation.

Ras-induced invasion and metastasis are regulated by a leukotriene B4 receptor BLT2-linked pathway.

Ras signaling pathways are well-recognized for their involvement in cancer cell proliferation; however, considerably less is known regarding their contribution to invasion and metastasis. Here, we demonstrate that a novel BLT2, a low-affinity leukotriene B(4) receptor-linked signaling cascade involving the generation of reactive oxygen species (ROS) via Nox1, NF-kappaB stimulation and subsequent upregulation of matrix metalloproteinase-9 (MMP-9) is a potential mechanism by which Ras promotes invasion and metastasis. We found that inhibition of BLT2 signaling markedly suppressed Ras-evoked metastasis and reduced the associated mortality in mice. Consistent with the proposed role of BLT2 as a key downstream mediator of Ras signaling to metastasis, BLT2 expression alone resulted in the formation of numerous metastatic lung nodules and the nodules formation was significantly attenuated by the inhibition of MMP-9, a downstream component of BLT2. Together, our results reveal the previously unsuspected function of BLT2-linked cascade in driving oncogenic Ras-induced metastasis and would provide a valuable insight into invasion and metastasis.

Engineering a G protein-coupled receptor for structural studies: stabilization of the BLT1 receptor ground state.

Structural characterization of membrane proteins is hampered by their instability in detergent solutions. We modified here a G protein-coupled receptor, the BLT1 receptor of leukotriene B(4), to stabilize it in vitro. For this, we introduced a metal-binding site connecting the third and sixth transmembrane domains of the receptor. This modification was intended to restrain the activation-associated relative movement of these helices that results in a less stable packing in the isolated receptor. The modified receptor binds its agonist with low-affinity and can no longer trigger G protein activation, indicating that it is stabilized in its ground state conformation. Of importance, the modified BLT1 receptor displays an increased temperature-, detergent-, and time-dependent stability compared with the wild-type receptor. These data indicate that stabilizing the ground state of this GPCR by limiting the activation-associated movements of the transmembrane helices is a way to increase its stability in detergent solutions; this could represent a forward step on the way of its crystallization.