Molecular cloning and characterization of another leukotriene B4 receptor

Leukotriene B(4) is a potent lipid mediator known to be implicated mainly in inflammatory actions. Previous pharmacological studies indicated the existence of only one class of G protein-coupled receptor for leukotriene B(4), for which a candidate gene, namely BLT, had been identified. Here we report the isolation of another gene encoding a functional G protein-coupled receptor for leukotriene B(4), named JULF2. JULF2 is a novel G protein-coupled receptor of 358 amino acids that shares 36.6% amino acid identity with human BLT. According to genomic information, the JULF2 gene is located on the chromosome 14, about 4 kilobases upstream of the BLT gene. During screening of endogenous ligands for JULF2, we found that leukotriene B(4) induced inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells, stably expressing JULF2. Additionally, Chinese hamster ovary cells expressing exogenous JULF2 showed chemotactic responses with leukotriene B(4) in a pertussis toxin-sensitive manner. A large amount of JULF2 mRNA was detected in the human spleen and the peripheral blood leukocytes. Furthermore, JULF2 mRNA was expressed in mononuclear lymphocytes, in which BLT mRNA was barely detected. The discovery of this second leukotriene B(4) receptor will eventually lead to a better understanding of the classification of leukotriene B(4) receptors and reconsideration of the pathophysiological role of leukotriene B(4).     

ONO-4057, a novel, orally active leukotriene B4 antagonist: effects on LTB4-induced neutrophil functions.

ONO-4057(5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E- hexenyl]oxyphenoxy]valeric acid), an orally active leukotriene B4(LTB4) antagonist, displaced the binding of [3H] LTB4 to the LTB4 receptor in human neutrophil (Ki = 3.7 +/- 0.9 nM). ONO-4057 inhibited the LTB4-induced rise in cytosolic free calcium (the concentration causing 50% inhibition (IC50) = 0.7 +/- 0.3 microM) and inhibited human neutrophil aggregation, chemotaxis or degranulation induced by LTB4 (IC50 = 3.0 +/- 0.1, 0.9 +/- 0.1 and 1.6 +/- 0.1 microM) without showing any agonist activity at concentration up to 30 microM. ONO-4057 did not inhibit fMLP or C5a-induced neutrophil activation at concentrations up to 30 microM. In the in vivo study, ONO-4057 given orally, prevented LTB4-induced transient neutropenia or intradermal neutrophil migration in guinea pig (the dose causing 50% efficacy (ED50) = 25.6mg/kg or 5.3mg/kg). Furthermore, ONO-4057 given topically, suppressed phorbol-12-myristate-13-acetate (PMA)-induced neutrophil infiltration in guinea pig ear (the effective dose = 1 mg/ear). These results indicate that ONO-4057 is a selective and orally active LTB4 antagonist and may be a potential candidate for the treatment of various inflammatory diseases.     

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.     

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.     

Hydroxyeicosanoids bind to and activate the low affinity leukotriene B4 receptor, BLT2

Leukotriene B(4), an arachidonate metabolite, is a potent chemoattractant of leukocytes involved in various inflammatory diseases. Two G-protein-coupled receptors for leukotriene B(4) have been cloned and characterized. BLT1 (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624) is a high affinity receptor exclusively expressed in leukocytes, and BLT2 (Yokomizo, T., Kato, K., Terawaki, K., Izumi, T., and Shimizu, T. (2000) J. Exp. Med. 192, 421-432) is a low affinity receptor expressed more ubiquitously. Here we report the binding profiles of various BLT antagonists and eicosanoids to either BLT1 or BLT2 using the membrane fractions of Chinese hamster ovary cells stably expressing the receptor. BLT antagonists are grouped into three classes: BLT1-specific U-75302, BLT2-specific LY255283, and BLT1/BLT2 dual-specific ZK 158252 and CP 195543. We also show that 12(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroperxyeicosatetraenoic acid, and 15(S)-hydroxyeicosatetraenoic acid competed with [(3)H]LTB(4) binding to BLT2, but not BLT1, dose dependently. These eicosanoids also cause calcium mobilization and chemotaxis through BLT2, again in contrast to BLT1. These findings suggest that BLT2 functions as a low affinity receptor, with broader ligand specificity for various eicosanoids, and mediates distinct biological and pathophysiological roles from BLT1.     

Lipopolysaccharide modulates receptors for leukotriene B4, C5a, and formyl-methionyl-leucyl-phenylalanine on rabbit polymorphonuclear leukocytes

Peripheral blood polymorphonuclear leukocytes (PMNL) isolated from rabbits after an i.v. injection of endotoxin exhibited decreased chemotactic migration in response to leukotriene B4 (LTB4) and C5a, but not N-formyl-methionyl-leucyl-phenylalanine (fMLP), after endotoxin treatment. The binding of radiolabeled LTB4, fMLP, and C5a to isolated PMNL was assessed in order to determine whether altered receptor expression could account for the observed functional changes. Control PMNL expressed binding sites for fMLP, LTB4, and C5a similar to those previously characterized from human PMNL. Control PMNL expressed a single class of 14,600 +/- 2700 receptors for fMLP with a mean dissociation constant (Kd) of 2.0 +/- 0.6 nM at 0 degrees C, whereas two subclasses of binding sites were expressed for LTB4: 10,300 +/- 6800 high-affinity and 85,600 +/- 53,000 low-affinity binding sites per PMNL with mean Kd for LTB4 of 0.75 +/- 0.43 nM and 70 +/- 58 nM (mean +/- SD, n = 5), respectively. Control PMNL bound [125I]-C5a in a dose-dependent and saturable manner at 24 degrees C. At saturating concentrations of C5a, PMNL obtained from control rabbits bound 270,000 +/- 50,000 molecules of [125I]-C5a with half-maximal binding occurring at [125I]-C5a concentrations of 5.5 +/- 1.9 nM. The binding of LTB4 and C5a to PMNL obtained 24 hr after an i.v. injection of endotoxin was markedly decreased compared with control PMNL. PMNL from endotoxin-treated rabbits exhibited 68% fewer high-affinity binding sites per PMNL for LTB4 and a 51% decrease in the amount of [125I]-C5a bound at saturating concentrations compared with control PMNL. There was no significant change in the Kd of the high-affinity binding sites for LTB4, no change in the Kd and number of the low-affinity binding sites for LTB4, and a small decrease in the apparent Kd for C5a to 3.3 +/- 1.1 nM. Even though the pretreatment with i.v. endotoxin did not alter chemotactic or degranulation responses elicited by fMLP, the endotoxin pretreatment induced an eightfold increase in the receptor density without altering the Kd for fMLP. Decreased receptor expression could account in large part for the decreased chemotactic responsiveness towards C5a and LTB4 induced by LPS. The finding that a substantial increase in receptors for fMLP need not be accompanied by a comparable functional change suggests that decreased efficiency in receptor coupling to intracellular biochemical events may also result from i.v. endotoxin.     

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.     

Solubilization and characterization of leukotriene B4 receptor-GTP binding protein complex from porcine spleen

A high amount of leukotriene B4 (LTB4) binding protein was observed in the porcine spleen. It was solubilized and partially purified from spleen membrane with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Scatchard analysis indicated the presence of a single class of receptor with Kd and Bmax values of 0.26 nM and 120 fmol/mg protein, respectively. The receptor was specific for LTB4, and Ki values for 20-hydroxy- and 20-carboxy-LTB4, both inactive metabolites of LTB4, were 1.7 nM and over 1,000 nM, respectively. By the addition of 10 microM GTP gamma S, a low affinity binding site appeared with a Kd value of 390 nM. A pretreatment of the receptor-GTP binding protein complex with islet-activating protein (IAP) increased the inhibitory effect of GTP gamma S on LTB4 binding, indicating that the LTB4 receptor is coupled with an IAP-sensitive GTP-binding protein in the porcine spleen.     

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.     

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.     

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.     

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.     

In vitro and in vivo pharmacological characterization of SB 201993, an eicosanoid-like LTB4 receptor antagonist with anti-inflammatory activity.

Leukotriene B4 (LTB4) and 12-(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-[R]-HETE) have been postulated to contribute to the pathophysiology of inflammatory diseases. SB 201993, (E)-3-[[[[6-(2-carboxyethenyl)-5-[[8-(4-methoxyphenyl)octyl] oxy]-2-pyridinyl] methyl] thio] methyl] benzoic acid, identified from a chemical series designed as ring-fused analogs of LTB4, was evaluated as an antagonist of LTB4- and 12-(R)-HETE-induced responses in vitro and for anti-inflammatory activity in vivo. SB 201993 competitively antagonized [3-H]-LTB4 binding to intact human neutrophils (Ki = 7.6 nM) and to membranes of RBL 2H3 cells expressing the LTB4 receptor (RBL 2H3-LTB4R; IC50 = 154 nM). This compound demonstrated competitive antagonism of LTB4- and 12-(R)-HETE-induced Ca2+ mobilization responses in human neutrophils (IC50s of 131 nM and 105 nM, respectively) and inhibited LTB4-induced Ca2+ mobilization in human cultured keratinocytes (IC50 = 61 nM), RBL 2H3-LTB4R cells (IC50 = 255 nM) and mouse neutrophils (IC50 = 410 nM). SB 201993 showed weak LTD4-receptor binding affinity (Ki = 1.9 microM) and inhibited 5-lipoxygenase (IC50 of 3.6 microM), both in vitro and ex vivo. In vivo, SB 201993 inhibited LTB4-induced neutrophil infiltration in mouse skin and produced dose-related, long lasting topical anti-inflammatory activity against the fluid and cellular phases of arachidonic acid-induced mouse ear inflammation (ED50 of 580 microg/ear and 390 microg/ear, respectively). Similarly, anti-inflammatory activity was also observed in the murine phorbol ester-induced cutaneous inflammation model (ED50 of 770 and 730 microg/ear, respectively, against the fluid and cellular phases). These results indicate that SB 201993 blocks the actions of LTB4 and 12-(R)-HETE and inhibits a variety of inflammatory responses; and thus may be a useful compound to evaluate the role of these mediators in disease models.     

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.     

Development of receptors for leukotriene B4 on HL-60 cells induced to differentiate by 1 alpha,25-dihydroxyvitamin D3

The incubation of HL-60 human promyelocytic leukemia cells for 7 days with 100 nM 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced differentiation into monocyte-like cells, as assessed by morphologic and biochemical characteristics. Stereospecific receptors for leukotriene B4 (LTB4) developed on the surface of the HL-60 cell-derived monocytes that had the capacity to transduce LTB4 stimulation of a transient increase in the cytosolic concentration of calcium ([Ca+2]in). HL-60 cell-derived monocytes, but not undifferentiated HL-60 cells, expressed a high affinity subset of 6400 +/- 3700 receptors per cell with a dissociation constant (Kd) of 2.3 +/- 1 nM (mean +/- SD, n = 3) and a low affinity subset of approximately 2.2 X 10(6) receptors per cell with an apparent Kd of 680 +/- 410 nM. Derivatives of LTB4 inhibited the binding of [3H]LTB4 to HL-60 cell-derived monocytes with a rank order of potency of LTB4 greater than 20-OH-LTB4 greater than 3-aminopropyl amide-LTB4, which is similar to the order for LTB4 receptors of human blood PMNL. In contrast, leukotrienes C4 and D4 and formyl-methionyl chemotactic peptides did not inhibit the binding of [3H] LTB4, which demonstrates the specificity of these receptors for isomers of 5,12-dihydroxy-eicosatetraenoic acid. LTB4 stimulated an increase in [Ca+2]in in HL-60 cell-derived monocytes which reached 50% of the maximal level at an LTB4 concentration of 0.5 nM (EC50). Preincubation of HL-60 cell-derived monocytes with 10 nM LTB4 resulted in a selective loss of high affinity receptors, as assessed by binding of [3H]LTB4, and a 200-fold increase in the EC50 for stimulation by LTB4 of increases in [Ca+2]in, without alterations in either the low affinity receptors for LTB4 or the responsiveness of [Ca+2]in to formyl-methionyl chemotactic peptides. HL-60 cells that are induced to differentiate into monocytes thus develop stereospecific receptors for LTB4 with binding and transductional characteristics similar to those of human blood PMNL.     

Cloning and functional expression of the hNPY Y5 receptor in human endometrial cancer (HEC-1B) cells

Aiming to develop a functional assay for the human NPY Y5 receptor based on adenylyl cyclase activity, HEC-1B cells, in which cAMP synthesis can be efficiently stimulated with forskolin, were selected for the transfection with the pcDNA3-Y5-FLAG and the pcDEF3-Y5 vectors. After optimization of the transfection procedure, the binding of [3H]propionyl-NPY to transiently and stably expressed Y5 receptors was determined. The affinities of NPY, NPY derivatives, and rPP (pNPY > or = p(Leu31Pro34)NPY = p(2-36)NPY > or = p(D-Trp32)NPY > p(13-36)NPY > rPP) were in accordance with the NPY Y5 receptor subtype. For [3H]propionyl-pNPY approximately 1.7 x 10(5) and 1 x 10(6) binding sites per transiently and stably transfected cell, respectively, were determined. The KD values were 2.4 +/- 0.4 and 1.7 +/-0.2 nM, respectively. Due to the high expression of the receptor protein, both stably and transiently transfected cells can be conveniently used in routine radioligand binding studies. By contrast, functional assays were only feasible with HEC-1B cells stably expressing the Y5 receptor. In these cells, 10 nM pNPY inhibited the forskolin-stimulated cAMP synthesis by 75%. This effect was partially antagonized by the Y5 antagonist N-?trans-[4-(2-naphthylmethylamino)-methyl]cyclohexylmethyl) naphthalene-2-sulfonamide. Although the genetic variability of cancer cells is in principle incompatible with a stable phenotype, both ligand binding characteristics and functionality of the Y5 receptor remained unchanged for more than 30 passages.