Residues from transmembrane helices 3 and 5 participate in leukotriene B4 binding to BLT1

Leukotrienes are inflammatory mediators that bind to seven transmembrane, G-protein-coupled receptors (GPCRs). Here we examine residues from transmembrane helices 3 and 5 of the leukotriene B4 (LTB4) receptor BLT1 to elucidate how these residues are involved in ligand binding. We have selected these residues on the basis of (1) amino acid sequence analysis, (2) receptor binding and activation studies with a variety of leukotriene-like ligands and recombinant BLT1 receptors, (3) previously published recombinant BLT1 mutants, and (4) a computed model of the active structure of the BLT1 receptor. We propose that LTB4 binds with the polar carboxylate group of LTB4 near the extracellular surface of BLT1 and with the hydrophobic LTB4 tail pointing into the transmembrane regions of the receptor protein. The carboxylate group and the two hydroxyls of LTB4 interact with Arg178 and Glu185 in transmembrane helix 5. Residues from transmembrane helix 3, Val105 and Ile108, also line the pocket deeper inside the receptor. LTB4 is becoming increasingly important as an immunomodulator during a number of pathologies, including atherosclerosis. Detailed information about the LTB4 binding mechanism, and the receptor residues involved, will hopefully aid in the design of new immunomodulatory drugs.     

The intracellular trafficking of opioid receptors directed by carboxyl tail and a di-leucine motif in Neuro2A cells

The mu- and delta-opioid receptors (MOR and DOR) differ significantly in their intracellular trafficking. MORs recycle back to the cell surface upon agonist treatment, whereas most internalized DORs are targeted to lysosomes for degradation. By exchanging the carboxyl tail domains of MOR and DOR and expressing the receptor chimeras in mouse neuroblastoma Neuro2A cells, it could be demonstrated that the carboxyl tail domain is not the sole determinant in directing the intracellular trafficking in these Neuro2A cells. Deletion of the dileucine motif (Leu245-Leu246) within the third intracellular loop of DOR or the mutation of Leu245 to Met slowed the lysosomal targeting of these delta-opioid receptors. Meanwhile the mutation of Met264 to Leu increased the rate of agonist-induced receptor internalization and the lysosomal targeting of the wild type and the delta-opioid receptor carboxyl tail chimera of the mu-opioid receptor. These studies suggest interplay between a di-leucine motif and the carboxyl tail in the lysosomal targeting of the receptor.     

Phosphorylation-independent beta-arrestin translocation and internalization of leukotriene B4 receptors

Leukotriene B4 (LTB4) activates the G-protein-coupled receptor leukotriene B4 receptor 1 (BLT1) to mediate a diverse array of cellular responses in leukocytes including chemotaxis, calcium mobilization, degranulation, and gene expression. To determine the role of phosphorylation in BLT1 regulation, we generated mutants of BLT1 in which all of the serine/threonine residues in the C-tail are converted to alanine or to aspartate/glutamate. These mutants expressed in rat basophilic leukemia RBL-2H3 cells bound LTB4 with similar affinity and activated all of the known functional activities of BLT1, albeit at different levels. The conversion of phosphorylation sites to alanine resulted in enhanced G-protein-mediated activities, whereas conversion to aspartate/glutamate resulted in reduced responses and a right shift in dose response, indicating that receptor phosphorylation is a critical regulator of G-protein-mediated pathways. Surprisingly, translocation of beta-arrestin and receptor internalization was completely independent of BLT1 phosphorylation. Real-time analysis of beta-arrestin translocation and receptor internalization using digital fluorescence video microscopy in cells expressing a red fluorescent protein labeled BLT1 and a green fluorescent protein-tagged beta-arrestin confirmed phosphorylation-independent beta-arrestin translocation and internalization of BLT1. In beta-arrestin-deficient mouse embryo fibroblasts, the BLT1 receptors failed to display endosomal localization upon stimulation. In these cells, co-expression of beta-arrestin-green fluorescent protein with BLT1-red fluorescent protein resulted in co-localization of BLT1 and beta-arrestin upon activation. Thus, receptor phosphorylation-dependent mechanisms regulate G-protein-mediated pathways; however, phosphorylation-independent mechanisms regulate beta-arrestin association and internalization of BLT1.     

Critical role for polar residues in coupling leukotriene B4 binding to signal transduction in BLT1

Leukotriene B(4) (LTB(4)) mediates a variety of inflammatory diseases such as asthma, arthritis, atherosclerosis, and cancer through activation of the G-protein-coupled receptor, BLT1. Using in silico molecular dynamics simulations combined with site-directed mutagenesis we characterized the ligand binding site and activation mechanism for BLT1. Mutation of residues predicted as potential ligand contact points in transmembrane domains (TMs) III (H94A and Y102A), V (E185A), and VI (N241A) resulted in reduced binding affinity. Analysis of arginines in extracellular loop 2 revealed that mutating arginine 156 but not arginine 171 or 178 to alanine resulted in complete loss of LTB(4) binding to BLT1. Structural models for the ligand-free and ligand-bound states of BLT1 revealed an activation core formed around Asp-64, displaying multiple dynamic interactions with Asn-36, Ser-100, and Asn-281 and a triad of serines, Ser-276, Ser-277, and Ser-278. Mutagenesis of many of these residues in BLT1 resulted in loss of signaling capacity while retaining normal LTB(4) binding function. Thus, polar residues within TMs III, V, and VI and extracellular loop 2 are critical for ligand binding, whereas polar residues in TMs II, III, and VII play a central role in transducing the ligand-induced conformational change to activation. The delineation of a validated binding site and activation mechanism should facilitate structure-based design of inhibitors targeting BLT1.     

Palmitoylation of Protease-activated Receptor-1 Regulates Adaptor Protein Complex-2 and -3 Interaction with Tyrosine-based Motifs and Endocytic Sorting

Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface.     

Helix 8 of the leukotriene B4 receptor is required for the conformational change to the low affinity state after G-protein activation

Recent studies have revealed that G-protein-coupled receptors contain a putative cytoplasmic helical domain, helix 8. Leukotriene B4 (LTB4) receptor 1 derivatives with truncated or mutated helix 8 showed much higher LTB4 binding than wild-type (WT) receptors. Similar to the WT receptor, LTB4 promoted guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding in these mutants. Unlike the WT receptor, however, the addition of GTPgammaS did not inhibit LTB4 binding to the mutant receptors. Scatchard analyses revealed that mutants maintained high affinity for LTB4, even in the presence of excess GTPgammaS. Consistently, mutant receptors showed a more prolonged Ca2+ mobilization and cellular metabolic activation than the WT receptor. From mutational studies and three-dimensional modeling based on the structure of bovine rhodopsin, we conclude that the helix 8 of LTB4 receptor 1 plays an important role in the conformational change of the receptor to the low affinity state after G-protein activation, possibly by sensing the status of coupling Galpha subunits as GTP-bound.     

Fluorescent leukotriene B4: potential applications

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a Kd of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.     

Mechanisms of homomeric alpha1 glycine receptor endocytosis

Little is known regarding the mechanism(s) by which glycine receptors are endocytosed. Here we examined the endocytosis of homomeric alpha1 glycine receptors expressed in HEK 293 cells using immunofluorescence/confocal microscopy and whole-cell patch-clamp recordings. Our studies demonstrate that constitutive endocytosis of glycine receptors is blocked by the dominant negative dynamin construct K44A and that intracellular dialysis with peptide P4, a dynamin/amphiphysin-disrupting peptide, increased whole-cell glycine-gated chloride currents. To examine whether receptor endocytosis could be regulated by PKC, experiments with the PKC activator PMA (phorbol 12-myristate 13-acetate) were performed. PMA, but not its inactive analogue PMM (phorbol 12-monomyristate), stimulated receptor endocytosis and inhibited glycine-gated chloride currents. Similar to constitutive endocytosis, PKC-stimulated endocytosis was blocked by dynamin K44A. Mutation of a putative AP2 adaptin dileucine motif (L314A, L315A) present in the receptor cytoplasmic loop blocked PMA-stimulated receptor endocytosis and also prevented PMA inhibition of glycine receptor currents. In patch-clamp experiments, intracellular dialysis of a 12-amino acid peptide corresponding to the region of the receptor containing the dileucine motif prevented PKC modulation of wild-type glycine receptors. Unlike PKC modulation of the receptor, constitutive endocytosis was not affected by mutation of this dileucine motif. These results demonstrate that PKC activation stimulates glycine receptor endocytosis, that both constitutive endocytosis and PKC-stimulated endocytosis are dynamin-dependent, and that PKC-stimulated endocytosis, but not constitutive endocytosis, occurs via the dileucine motif (L314A, L315A) within the cytoplasmic loop of the receptor.     

Resolvin E1 selectively interacts with leukotriene B4 receptor BLT1 and ChemR23 to regulate inflammation

Resolvin E1 (RvE1) is a potent anti-inflammatory and proresolving mediator derived from omega-3 eicosapentaenoic acid generated during the resolution phase of inflammation. RvE1 possesses a unique structure and counterregulatory actions that stop human polymorphonuclear leukocyte (PMN) transendothelial migration and PMN infiltration in several murine inflammatory models. To examine the mechanism(s) underlying anti-inflammatory actions on PMNs, we prepared [(3)H]RvE1 and characterized its interactions with human PMN. Results with membrane fractions of human PMN demonstrated specific binding with a K(d) of 48.3 nM. [(3)H]RvE1 specific binding to human PMN was displaced by leukotriene B(4) (LTB(4)) and LTB(4) receptor 1 (BLT1) antagonist U-75302, but not by chemerin peptide, a ligand specific for another RvE1 receptor ChemR23. Recombinant human BLT1 gave specific binding with [(3)H]RvE1 with a K(d) of 45 nM. RvE1 selectively inhibited adenylate cyclase with BLT1, but not with BLT2. In human PBMC, RvE1 partially induced calcium mobilization, and blocked subsequent stimulation by LTB(4). RvE1 also attenuated LTB(4)-induced NF-kappaB activation in BLT1-transfected cells. In vivo anti-inflammatory actions of RvE1 were sharply reduced in BLT1 knockout mice when given at low doses (100 ng i.v.) in peritonitis. In contrast, RvE1 at higher doses (1.0 mug i.v.) significantly reduced PMN infiltration in a BLT1-independent manner. These results indicate that RvE1 binds to BLT1 as a partial agonist, potentially serving as a local damper of BLT1 signals on leukocytes along with other receptors (e.g., ChemR23-mediated counterregulatory actions) to mediate the resolution of inflammation.     

Attenuated Th1 induction by dendritic cells from mice deficient in the leukotriene B4 receptor 1

Dendritic cells (DCs) are important antigen-presenting cells that control Th1- and Th2-type immunological reactions by releasing cytokines and interacting directly with T cells. Leukotriene B4 (LTB4), a classical proinflammatory lipid mediator for phagocytes, was recently identified as an important attractant for effector CD4⁺ and CD8⁺ T cells. However, little information is available on the roles of LTB4 and its receptor BLT1 in DCs. Here we show that functional BLT1 expressed in mouse bone marrow-derived DCs (BMDCs) plays important role in initiating Th1-type immune response. Detailed analyses using BMDCs revealed that BLT1-deficient DCs produced less IL-12p70 than WT DCs, leading to attenuated IFN-γ production in an allogeneic mixed lymphocyte reaction. Adoptive transfer of antigen-loaded BLT1-deficient DCs into naïve WT mice induced a weakened Th1- and enhanced Th2-response in vivo compared to WT DCs. BLT1-deficient mice consistently showed much attenuated delayed-type hypersensitivity (DTH), in which Th1-type cellular responses play a key role, and popliteal lymph node cells of BLT1-deficient mice showed reduced production of Th1 cytokines after DTH induction compared to cells from WT mice. Thus, in addition to its role in inflammation, the LTB4–BLT1 axis is important in initiating Th1-type immunological reactions mediated by DCs.     

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.     

A conserved motif for the transport of G protein-coupled receptors from the endoplasmic reticulum to the cell surface

The structural determinants for the export trafficking of G protein-coupled receptors are poorly defined. In this report, we determined the role of carboxyl termini (CTs) of alpha2B-adrenergic receptor (AR) and angiotensin II type 1A receptor (AT1R) in their transport from the endoplasmic reticulum (ER) to the cell surface. The alpha2B-AR and AT1R mutants lacking the CTs were completely unable to transport to the cell surface and were trapped in the ER. Alanine-scanning mutagenesis revealed that residues Phe436 and Ile433-Leu444 in the CT were required for alpha2B-AR export. Insertion or deletion between Phe436 and Ile443-Leu444 as well as Ile443-Leu444 mutation to FF severely disrupted alpha2B-AR transport, indicating there is a defined spatial requirement, which is essential for their function as a single motif regulating receptor transport from the ER. Furthermore, the carboxyl-terminally truncated as well as Phe436 and Ile443-Leu444 mutants were unable to bind ligand and the alpha2B-AR CT conferred its transport properties to the AT1R mutant without the CT in a Phe436-Ile443-Leu444-dependent manner. These data suggest that the Phe436 and Ile443-Leu444 may be involved in both proper folding and export from the ER of the receptor. Similarly, residues Phe309 and Leu316-Leu317 in the CT were identified as essential for AT1R export. The sequence F(X)6LL (where X can be any residue, and L is leucine or isoleucine) is highly conserved in the membrane-proximal CTs of many G protein-coupled receptors and may function as a common motif mediating receptor transport from the ER to the cell surface.     

Involvement of BLT1 endocytosis and Yes kinase activation in leukotriene B4-induced neutrophil degranulation

One of the important biological activities of human neutrophils is degranulation, which can be induced by leukotriene B4 (LTB4). Here we investigated the intracellular signaling events involved in neutrophil degranulation mediated by the high affinity LTB4 receptor, BLT1. Peripheral blood neutrophils as well as the promyeloid PLB-985 cell line, stably transfected with BLT1 cDNA and differentiated into a neutrophil-like cell phenotype, were used throughout this study. LTB4-induced enzyme release was inhibited by 50-80% when cells were pretreated with the pharmacological inhibitors of endocytosis sucrose, Con A and NH4Cl. In addition, transient transfection with a dominant negative form of dynamin (K44A) resulted in approximately 70% inhibition of ligand-induced degranulation. Pretreating neutrophils or BLT1-expressing PLB-985 cells with the Src family kinase inhibitor PP1 resulted in a 30-60% inhibition in BLT1-mediated degranulation. Yes kinase, but not c-Src, Fgr, Hck, or Lyn, was found to exhibit up-regulated kinase activity after LTB4 stimulation. Moreover, BLT1 endocytosis was found to be necessary for Yes kinase activation in neutrophils. LTB4-induced degranulation was also sensitive to inhibition of PI3K. In contrast, it was not affected by inhibition of the mitogen-activated protein kinase MEK kinase, the Janus kinases, or the receptor tyrosine kinase epidermal growth factor receptor or platelet-derived growth factor receptor. Taken together, our results suggest an essential role for BLT1 endocytosis and Yes kinase activation in LTB4-mediated degranulation of human neutrophils.     

Role of an acidic cluster/dileucine motif in cation-independent mannose 6-phosphate receptor traffic

The endocytic trafficking of the cation-independent mannose 6-phosphate receptor (CI-MPR) involves multiple sorting steps. A cluster of acidic amino acids followed by a dileucine motif in the cytoplasmic tail has been proposed to mediate receptor sorting from the trans Golgi network (TGN) to late endosomes. Mutations in this motif impair lysosomal enzyme sorting by preventing association of CI-MPR with coat proteins. The role of the acidic cluster/dileucine motif in the post-endocytic transport of the receptor was examined using the CI-MPR mutants, AC01 and D160E (Chen HJ, Yuan J, Lobel P. J Biol Chem 1997;272:7003-7012). Following internalization, wild type (WT) CI-MPR is transported through sorting endosomes into the endocytic recycling compartment (ERC), after which it traffics to the TGN and other organelles. However, the mutants localize mostly to the ERC and only a small portion reaches the TGN, suggesting that the sorting of the CI-MPR mutants from the ERC into the TGN is severely impaired. We observed no defect in receptor internalization or in the rate of tail mutant recycling to the cell surface compared to the WT. These results demonstrate that the acidic cluster/dileucine motif of CI-MPR is critical for receptor sorting at early stages of intracellular transport following endocytosis.     

Co-expression of two LTB4 receptors in human mononuclear cells

Leukotriene B4 (LTB4) is one of the most potent chemoattractants and activators of leukocytes, and is involved in inflammatory diseases. Two G-protein-coupled-receptors for LTB4, BLT1 and BLT2, have been isolated, and shown to be a high- and low-affinity receptor, respectively. The tissue distributions of these receptors are different, and distinct roles of each receptor remain elusive. We compared the expression of these two receptors using semi-quantitative PCR analyses, and show that these two receptors are expressed in various subsets of human lymphocytes in different quantities. BLT1 expression is highest in CD14+ monocytes, while BLT2 expression is high in CD8+ cytotoxic T-, CD4+ helper T-, and CD19+ B-cells. Moreover, BLT2 expression in these lymphocytes decreased upon activation of the cells. We also established CHO cells stably expressing both receptors, and found that these cells could migrate toward LTB4 with a broad range of LTB4. These findings suggest novel roles of LTB4 in immune system, and the biological significance of high- and low- affinity LTB4 receptors in chemotaxis.     

Leukotriene B4 is an indirectly acting vasoconstrictor in guinea pig aorta via an inducible type of BLT receptor

Leukotriene B(4) (LTB(4)) is a potent leukocyte chemoattractant recently implicated in the pathogenesis of atherosclerosis. The aim of this study was to assess the effects of LTB(4) on isolated aortic preparations. Rings of guinea pig aorta were challenged with LTB(4) for recording mechanical responses and measurements of mediator release, and LTB(4) receptor (BLT(1)) expression was assessed by RT-PCR. Single concentrations of LTB(4) induced concentration-dependent contractions that were inhibited by treatment with antihistamines, indomethacin, or the thromboxane receptor antagonist BAYu3405 as well as by denudation of endothelium. In addition, LTB(4) increased the release of histamine and thromboxane in the bath. The contractions induced by LTB(4) were inhibited by either the unselective BLT receptor antagonist ONO-4057 or the selective BLT(1) receptor antagonist U-75302. Pretreatment with all-trans-retinoic acid enhanced the contractions and the release of histamine induced by LTB(4), without affecting either the contractions induced by histamine or the histamine release evoked by calcium ionophore A23187. Analysis by RT-PCR indicated the expression of a BLT(1) receptor in the guinea pig aorta and that BLT(1) receptor mRNA was upregulated after treatment with retinoic acid. These results suggest that LTB(4) contracts the guinea pig aorta via an indirect mechanism involving the release of histamine and thromboxane and that this BLT(1) receptor-mediated response can be upregulated by all-trans-retinoic acid.     

BLT1 and BLT2: the leukotriene B(4) receptors

Two receptors for leukotriene B(4) (LTB(4)) have been molecularly identified: BLT1 and BLT2. Both receptors are G protein-coupled seven transmembrane domain receptors, whose genes are located in very close proximity to each other in the human and mouse genomes. The two receptors differ in their affinity and specificity for LTB(4): BLT1 is a high-affinity receptor specific for LTB(4), whereas BLT2 is a low-affinity receptor that also binds other eicosanoids. The two receptors also differ in their pattern of expression with BLT1 being expressed primarily in leukocytes, whereas BLT2 is expressed more ubiquitously. By mediating the activities of LTB(4), these receptors participate both in host immune responses and in the pathogenesis of inflammatory diseases. Reduced disease severity in animal inflammatory models seen with LTB(4) receptor antagonists and in mice with targeted deletion of BLT1 have revealed important roles for LTB(4) and its receptors in regulating pathologic inflammation.     

Leukotriene B4 augments and restores Fc gammaRs-dependent phagocytosis in macrophages

Phagocytosis by macrophages is essential for host defense, i.e. preventing invasion of pathogens and foreign materials. Macrophages engulf immunoglobulin G (IgG)-opsonized particles through the action of the receptors for the Fc of IgG (FcγRs). Leukotriene B(4) (LTB(4)) is a classical lipid chemoattractant derived from arachidonic acid. Leukotriene B(4) receptor 1 (BLT1), a high affinity LTB(4) receptor, is expressed in a variety of immune cells such as neutrophils, macrophages, and dendritic cells. Although LTB(4) has been shown to enhance macrophage phagocytosis, few studies have investigated the intracellular mechanisms involved in this in detail. Furthermore, there have been no reports of the direct cross-talk between LTB(4)-BLT1 and IgG-FcγRs signaling. Here, we show that FcγRs-dependent phagocytosis was attenuated in BLT1-deficient macrophages as compared with wild-type (WT) cells. Moreover, cross-talk between LTB(4)-BLT1 and IgG-FcγRs signaling was identified at the level of phosphatidylinositol 3-OH kinase (PI3K) and Rac, downstream of Syk. In addition, the trimeric G(i) protein (G(i)) was found to be essential for BLT1-dependent phagocytosis. Surprisingly, we found that LTB(4)-BLT1 signaling restores phagocytosis in the absence of FcγRs signaling. These data indicate that LTB(4)-BLT1 signaling plays a pivotal role in macrophage phagocytosis and innate immunity.     

Leukotriene B4 receptor and the function of its helix 8

More than 30 lipid ligands, which express their biological activities through cognate G-protein-coupled receptors (GPCRs), have been reported. Among them, leukotriene B(4) (LTB(4)) is a potent lipid mediator involved in host defense, inflammation, and the immune responses. Two GPCRs for LTB(4) (BLT1 and BLT2) have been cloned and analyzed. Recent studies using genetically engineered mice suggest that BLT1 plays an important role in several inflammatory diseases including ischemic reperfusion tissue injury, atherosclerosis, and bronchial asthma. BLT1 is also a good tool to study the molecular mechanism of GPCR activation and inactivation in vitro. In this brief review, we focus on the biological and biochemical properties of BLT1 with special attention to the putative helix 8 of the receptor.