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.     

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.     

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.     

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.     

Structural determinants regulating expression of the high affinity leukotriene B4 receptor: involvement of dileucine motifs and alpha-helix VIII

Mutational analysis of determinants located in the C-terminal (C) tail of the high affinity leukotriene (LT) B(4) receptor, BLT1, was performed to assess their significance in BLT1 trafficking. When expressed in COS-7 cells, a BLT1 deletion mutant lacking the C-tail (G291stop) displayed higher numbers of binding sites and increased signal transduction compared with wild-type (WT) BLT1. Addition of the C-tail from either the platelet-activating factor receptor or the LTD(4) receptor, CysLT1, did not restore WT phenotype. Moreover, the number of LTB(4) binding sites was higher in the chimeras than in the WT BLT1, suggesting the requirement for specific structural determinants within the BLT1 C-tail. Elimination of a distal C-tail dileucine motif (Leu(304)-Leu(305)), but not the proximal (Leu(292)-Leu(293)) motif, altered BLT1 pharmacological characteristics and caused a moderate constitutive receptor activation. Surprisingly, all mutant receptors were efficiently delivered to the plasma membrane, but not to a greater extent than WT BLT1, as assessed by flow cytometry. Furthermore, substitution of Leu(304)-Leu(305) prevented LTB(4)-induced BLT1 internalization. Molecular modeling of BLT1 on the bovine rhodopsin receptor scaffold strongly suggested the involvement of the distal dileucine motif (Leu(304)-Leu(305)) in a hydrophobic core, including intrahelical interactions within alpha-helix VIII and interhelical interactions with residues of helix I. Disruption of this hydrophobic core is proposed to increase the population of receptors in the active form, to restrain their trafficking and to facilitate the activation of BLT1 as indicated by the increased maximal level of binding of the ligand and constitutive activation of the receptor.     

Leukotriene B4 receptors BLT1 and BLT2: expression and function in human and murine mast cells

Leukotriene B4 (LTB4) is a potent activator and chemoattractant for leukocytes and is implicated in several inflammatory diseases. The actions of LTB4 are mediated by two cell surface receptors, BLT1, which is predominantly expressed in peripheral blood leukocytes, and BLT2, which is expressed more ubiquitously. Recently, BLT1 expression and LTB4-dependent chemotaxis have been reported in immature mast cells (MCs). We now show the first evidence for BLT2 mRNA expression, in addition to BLT1, in murine bone marrow-derived MCs (mBMMCs) and in a human MC line (HMC-1). Protein expression of BLT1 was confirmed by mAb staining in HMC-1 cells and shown to be predominantly intracellular. Both HMC-1 cells and mBMMCs migrated to LTB4 in a dose-dependent manner in chemotaxis assays. Migration to LTB4 could be inhibited by either a BLT1- or BLT2-selective antagonist. Significant dose-dependent migration of mBMMCs also was observed to 12-(S)-hydroxyeicosotetraenoic acid, a BLT2-selective agonist, demonstrating functional BLT2 activity in these cells. Stimulation of mBMMCs with LTB4 induced transient, dose-dependent, ERK phosphorylation and changes in Akt phosphorylation. Dose-dependent ERK phosphorylation also was observed in response to 12-(S)-hydroxyeicosotetraenoic acid, indicating signaling downstream of BLT2. Pretreatment of mBMMCs with stem cell factor significantly down-regulated expression of BLT1 and BLT2 mRNA and inhibited their migration to LTB4. This study demonstrates expression of functional LTB4 receptors, both BLT1 and BLT2, in murine and human MCs and a regulatory role for stem cell factor in their expression. These receptors may mediate recruitment and accumulation of MCs in response to LTB4 production in areas of inflammation.     

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.     

Leukotriene B4 receptor 1 expression on dendritic cells is required for the development of Th2 responses and allergen-induced airway hyperresponsiveness

Dendritic cells (DC) are important APCs that control allergen-induced airway responses by interacting directly with T cells. Leukotriene B(4) (LTB(4)), interacting with its high-affinity receptor, LTB(4) receptor 1 (BLT1), is known to attract and activate leukocytes during inflammation. We have previously shown that BLT1 expression on Ag-primed T cells is required for the development of airway hyperresponsiveness (AHR; Miyahara et al. 2005. Am. J. Respir. Crit. Care Med. 172: 161-167). However, the role for the LTB(4)-BLT1 pathway in DC function in allergen-induced airway responses has not been defined. Bone marrow-derived DCs (BMDC) were generated. Naive BALB/c mice received OVA-pulsed BLT1-deficient (BLT1(-/-)) BMDCs or wild-type BMDCs intratracheally and were then challenged with OVA for 3 days. Airway responses were monitored 48 h after the last allergen challenge. BLT1(-/-) BMDCs showed normal maturation judged from surface expression of CD markers. Compared with recipients of wild-type BMDCs, mice that received BLT1(-/-) BMDCs developed significantly lower AHR to inhaled methacholine, lower goblet cell metaplasia, and eosinophilic infiltration in the airways and decreased levels of Th2 type cytokines in the bronchoalveolar lavage fluid. Migration of BLT1(-/-) BMDCs into peribronchial lymph nodes was significantly impaired compared with BLT1(+/+) BMDCs after intratracheal instillation. These data suggest that BLT1 expression on DCs is required for migration of DCs to regional lymph nodes as well as in the development of AHR and airway inflammation.     

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.     

Identification of the intracellular region of the leukotriene B4 receptor type 1 that is specifically involved in Gi activation

Many G-protein-coupled receptors can activate more than one G-protein subfamily member. Leukotriene B4 receptor type 1 (BLT1) is a high affinity G-protein-coupled receptors for leukotriene B4 functioning in host defense, inflammation, and immunity. Previous studies have shown that BLT1 utilizes different G-proteins (the Gi family and G16 G-proteins) in mediating diverse cellular events and that truncation of the cytoplasmic tail of BLT1 does not impair activation of Gi and G16 proteins. To determine responsive regions of BLT1 for G-protein coupling, we performed an extensive mutagenesis study of its intracellular loops. Three intracellular loops (i1, i2, and i3) of BLT1 were found to be important for both Gi and G16 coupling, as judged by Gi-dependent guanosine 5'-(gamma-thio) triphosphate (GTPgammaS) binding and G16-dependent inositol phosphate accumulation assays. The i3-1 mutant, with a mutation at the i3 amino terminus, exhibited greatly reduced GTPgammaS binding but intact inositol phosphate accumulation triggered by leukotriene B4 stimulation. These results suggest that the i3-1 region is required only for Gi activation. Moreover, in the i3-1 mutant, the deficiency in Gi activation was accompanied by a loss of the high affinity leukotriene B4 binding state seen with the wild type receptor. A three-dimensional model of BLT1 constructed based on the structure of bovine rhodopsin suggests that the i3-1 region may consist of the cytoplasmic end of the transmembrane helix V, which protrudes the helix into the cytoplasm. From mutational studies and three-dimensional modeling, we propose that the extended cytoplasmic helix connected to the transmembrane helix V of BLT1 might be a key region for selective activation of Gi proteins.     

Increased acute inflammation, leukotriene B4-induced chemotaxis, and signaling in mice deficient for G protein-coupled receptor kinase 6

Directed migration of polymorphonuclear neutrophils (PMN) is required for adequate host defense against invading organisms and leukotriene B(4) (LTB(4)) is one of the most potent PMN chemoattractants. LTB(4) exerts its action via binding to BLT1, a G protein-coupled receptor. G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases (GRK) in an agonist-dependent manner, resulting in receptor desensitization. Recently, it has been shown that the human BLT1 is a substrate for GRK6. To investigate the physiological importance of GRK6 for inflammation and LTB(4) signaling in PMN, we used GRK6-deficient mice. The acute inflammatory response (ear swelling and influx of PMN into the ear) after topical application of arachidonic acid was significantly increased in GRK6(-/-) mice. In vitro, GRK6(-/-) PMN showed increased chemokinetic and chemotactic responses to LTB(4). GRK6(-/-) PMN respond to LTB(4) with a prolonged increase in intracellular calcium and prolonged actin polymerization, suggesting impaired LTB(4) receptor desensitization in the absence of GRK6. However, pre-exposure to LTB(4) renders both GRK6(-/-) as well as wild-type PMN refractory to restimulation with LTB(4), indicating that the presence of GRK6 is not required for this process to occur. In conclusion, GRK6 deficiency leads to prolonged BLT1 signaling and increased neutrophil migration.     

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.     

Endothelium-dependent vascular responses induced by leukotriene B4

Leukotriene B(4) (LTB(4)) is an inflammatory mediator derived from the 5-lipoxygenase pathway of arachidonic acid metabolism and has recently implicated in the pathogenesis of atherosclerosis. There are two membrane bound receptors for LTB(4): BLT(1) and BLT(2), which represent the high and low affinity receptors, respectively. BLT receptors are expressed on leukocytes, and LTB(4) is a potent chemoattractant for neutrophils, eosinophils, and T lymphocytes. Recent studies have in addition shown that LTB(4) is an indirectly acting vasoconstrictor of isolated vascular preparations. In the guinea pig aorta, the LTB(4)-induced contractions were inhibited by endothelium-denudation. In addition, pre-treatment with the NO synthase inhibitor, L-NOARG, significantly enhanced the contractions induced by LTB(4). The contractile response induced by LTB(4) in the guinea pig aorta was abolished by the selective BLT(1) receptor antagonist U75302 and the expression of BLT(1) receptor mRNA in the guinea pig aorta was established by RT-PCR. Taken together, these results suggest that LTB(4) activates BLT(1) receptors on the endothelium of the guinea pig aorta, associated with the release of both contractile factors and NO.     

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.     

Involvement of leukotriene B4 receptor 1 signaling in platelet-activating factor-mediated neutrophil degranulation and chemotaxis

Platelet-activating factor (PAF) is a potent lipid mediator of inflammation that can act on human neutrophils. When neutrophils are stimulated with PAF at concentrations greater than 10 nM, a double peak of intracellular calcium mobilization is observed. The second calcium peak observed in PAF-treated neutrophils has already been suggested to come from the production of endogenous leukotriene B4 (LTB4). Here we demonstrate the involvement of endogenous LTB4 production and subsequent activation of the high affinity LTB4 receptor (BLT1) in this second calcium mobilization peak observed after stimulation with PAF. We also show that the second, but not the first peak, could be desensitized by prior exposure to LTB4. Moreover, when neutrophils were pre-treated with pharmacological inhibitors of LTB4 production or with the specific BLT1 antagonist, U75302, PAF-mediated neutrophil degranulation was inhibited by more than 50%. On the other hand, pre-treating neutrophils with the PAF receptor specific antagonist (WEB2086) did not prevent any LTB4-induced degranulation. Also, when human neutrophils were pre-treated with U75302, PAF-mediated chemotaxis was reduced by more than 60%. These data indicate the involvement of BLT1 signaling in PAF-mediated neutrophil activities.     

Threonine 308 within a putative casein kinase 2 site of the cytoplasmic tail of leukotriene B(4) receptor (BLT1) is crucial for ligand-induced,G-protein-coupled receptor-specific kinase 6-mediated desensitization

Desensitization of G-protein-coupled receptors may involve phosphorylation of serine and threonine residues. The leukotriene B(4) (LTB(4)) receptor (BLT1) contains 14 intracellular serines and threonines, 8 of which are part of consensus target sequences for protein kinase C (PKC) or casein kinase 2. In this study, we investigated the importance of PKC and GPCR-specific kinase (GRK) phosphorylation in BLT1 desensitization. Pretreatment of BLT1-transfected COS-7 cells with PKC activators caused a decrease of LTB(4)-induced inositol phosphate (IP) accumulation. This reduction was prevented with the PKC inhibitor, staurosporine, and not observed in cells expressing a BLT1 deletion mutant (G291stop) lacking the cytoplasmic tail. Moreover LTB(4)-induced IP accumulation was significantly inhibited by overexpression of GRK2, GRK5, and especially GRK6, in cells expressing wild type BLT1 but not in those expressing G291stop. GRK6-mediated desensitization correlated with increased phosphorylation of BLT1. The G319stop truncated BLT1 mutant displayed functional characteristics comparable with wild type BLT1 in terms of desensitization by GRK6, but not by PKC. Substitution of Thr(308) within a putative casein kinase 2 site to proline or alanine in the full-length BLT1 receptor prevented most of GRK6-mediated inhibition of LTB(4)-induced IP production but only partially affected LTB(4)-induced BLT1 phosphorylation. Our findings thus suggest that Thr(308) is a major residue involved in GRK6-mediated desensitization of BLT1 signaling.