FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1989046199> ?p ?o ?g. }

• W1989046199 endingPage "8603" @default.
• W1989046199 startingPage "8597" @default.
• W1989046199 abstract "The mouse leukotriene B4receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4(LTB4, K d = 0.24 ± 0.03 nm). In competition binding experiments, LTB4was the most potent competitor (K i = 0.23 ± 0.05 nm) followed by 20-hydroxy-LTB4(K i = 1.1 ± 0.2 nm) and by 6-trans-12-epi-LTB4 and LTD4(K i > 1 μm). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation. The mouse leukotriene B4receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4(LTB4, K d = 0.24 ± 0.03 nm). In competition binding experiments, LTB4was the most potent competitor (K i = 0.23 ± 0.05 nm) followed by 20-hydroxy-LTB4(K i = 1.1 ± 0.2 nm) and by 6-trans-12-epi-LTB4 and LTD4(K i > 1 μm). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation. Leukotriene B4(LTB4) 1The abbreviations used are:LTB4, leukotriene B4, 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid; BLTR, leukotriene B4 receptor; h-BLTR, human leukotriene B4 receptor; HIV, human immunodeficiency virus; m-BLTR, mouse leukotriene B4 receptor; 6-trans-12-epi-LTB4, 5(S),12(S)-dihydroxy-14-cis-6,8,10-trans-eicosatetraenoic acid; kb, kilobase(s); PCR, polymerase chain reaction; HEK, human embryonic kidney; CHO, Chinese hamster ovary; HEK-m-BLTR, stable transfected HEK cells with pCR3.1-m-BLTR; CHO-m-BLTR, stable transfected CHO cells with pCR3.1-m-BLTR; SIV, simian immunodeficiency virus.1The abbreviations used are:LTB4, leukotriene B4, 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid; BLTR, leukotriene B4 receptor; h-BLTR, human leukotriene B4 receptor; HIV, human immunodeficiency virus; m-BLTR, mouse leukotriene B4 receptor; 6-trans-12-epi-LTB4, 5(S),12(S)-dihydroxy-14-cis-6,8,10-trans-eicosatetraenoic acid; kb, kilobase(s); PCR, polymerase chain reaction; HEK, human embryonic kidney; CHO, Chinese hamster ovary; HEK-m-BLTR, stable transfected HEK cells with pCR3.1-m-BLTR; CHO-m-BLTR, stable transfected CHO cells with pCR3.1-m-BLTR; SIV, simian immunodeficiency virus. and the cysteinyl-leukotrienes (LTC4, LTD4, and LTE4), derived from arachidonic acid metabolism, are synthesized sequentially by 5-lipoxygenase and then by either LTA4 hydrolase or LTC4 synthase, respectively (1Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2320) Google Scholar). The biological actions of the cysteinyl-leukotrienes are mediated through at least two G protein-coupled receptors referred to as CysLT1 and CysLT2 whose molecular identities remain uncharacterized (2Metters K.M. J. Lipid Mediators Cell Signal. 1995; 12: 413-427Crossref PubMed Scopus (69) Google Scholar). LTB4 mediates its effects through a membrane G-protein-coupled receptor termed BLTR (3Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar). Additionally, LTB4 was shown to bind to the intracellular transcription factor peroxisome proliferator-activated receptor (4Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1205) Google Scholar). This facet of binding has been proposed to be part of a negative feedback mechanism to limit the inflammatory actions of LTB4. However the binding to peroxisome proliferator-activated receptor has been questioned in recent experiments (5Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1861) Google Scholar). LTB4, a dihydroxy fatty acid, is one of the most potent known chemoattractant mediators, acting mainly on neutrophils but also on related myeloid cells, mast cells, and endothelial cells (6Ford-Hutchinson A.W. Bray M.A. Doig M.V. Shipley M.E. Smith M.J. Nature. 1980; 286: 264-265Crossref PubMed Scopus (1584) Google Scholar, 7Nohgawa M. Sasada M. Maeda A. Asagoe K. Harakawa N. Takano K. Yamamoto K. Okuma M. J. Leukocyte Biol. 1997; 62: 203-209Crossref PubMed Scopus (42) Google Scholar). LTB4 induces chemotaxis, chemokinesis, and aggregation, causing the migration of neutrophils to sites of inflammation where the cells degranulate, resulting in the release of lysosomal enzymes in addition to other antibacterial and anti-microbicidal agents (8Henderson Jr., W.R. Ann. Intern. Med. 1994; 121: 684-697Crossref PubMed Scopus (588) Google Scholar). LTB4 also promotes the adherence of neutrophils to vascular endothelial cells and their transmigration, which amplifies the inflammatory response. LTB4 has been implicated in the pathophysiology of various diseases like arthritis, inflammatory bowel disease, and psoriasis. The exact role of LTB4 in the etiology of these disorders has been debated vigorously. Inhibitors of 5-lipoxygenase and the 5-lipoxygenase-activating protein have been used efficiently in models of ulcerative colitis (9Kjeldsen J. Laursen L.S. Hillingso J. Mertz-Nielsen A. Bukhave K. Rask-Madsen J. Lauritsen K. Pharmacol. Toxicol. 1995; 77: 371-376Crossref PubMed Scopus (7) Google Scholar, 10Hillingso J. Kjeldsen J. Laursen L.S. Lauritsen K. von Spreckelsen S. Depre M. Friedman B.S. Malmstrom K. Shingo S. Bukhave K. Raskmadsen J. Clin. Pharmacol. Ther. 1995; 57: 335-341Crossref PubMed Scopus (19) Google Scholar), endotoxic shock (11Yoshikawa D. Goto F. Circul. Shock. 1992; 38: 29-33PubMed Google Scholar), and induced asthma (12Shindo K. Koide K. Fukumura M. Thorax. 1997; 52: 1024-1029Crossref PubMed Scopus (27) Google Scholar, 13Diamant Z. Timmers M.C. van der Veen H. Friedman B.S. De Smet M. Depre M. Hilliard D. Bel E.H. Sterk P.J. J. Allergy Clin. Immunol. 1995; 95: 42-51Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 14Fisher A.R. Rosenberg M.A. Roth M. Loper M. Jungerwirth S. Israel E. Thorax. 1997; 52: 1074-1077Crossref PubMed Scopus (19) Google Scholar). The development of specific and highly potent BLTR antagonists has lagged behind cysteinyl receptor antagonists, which are currently available in the clinic for treatment of asthmatic inflammatory symptoms. One BLTR antagonist has shown encouraging results in a murine model of collagen-induced arthritis (15Griffiths R.J. Pettipher E.R. Koch K. Farrell C.A. Breslow R. Conklyn M.J. Smith M.A. Hackman B.C. Wimberly D.J. Milici A.J. Scampoli D.N. Cheng J.B. Pillor J.S. Pazoles C.J. Doherty N.S. Melvin L.S. Reiter L.A. Biggars M.S. Falkner F.C. Mitchell D.Y. Liston T.E. Showell H.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 517-521Crossref PubMed Scopus (197) Google Scholar). There has been considerable controversy about the molecular identification of the BLTR. In 1996 two independent research groups (16Owman C. Nilsson C. Lolait S.J. Genomics. 1996; 37: 187-194Crossref PubMed Scopus (50) Google Scholar, 17Raport C.J. Schweickart V.L. Chantry D. Eddy Jr., R.L. Shows T.B. Godiska R. Gray P.W. J. Leukocyte Biol. 1996; 59: 18-23Crossref PubMed Scopus (82) Google Scholar) cloned identical orphan receptor genes believed to encode members of the chemotactic factor subfamily of G protein-coupled seven transmembrane receptors. Initially, one group indicated that the receptor was unable to bind LTB4 (16Owman C. Nilsson C. Lolait S.J. Genomics. 1996; 37: 187-194Crossref PubMed Scopus (50) Google Scholar) but later retracted this finding to indicate specific binding (18Owman C. Lolait S.J. Santen S. Olde B. Biochem. Biophys. Res. Commun. 1997; 241: 390-394Crossref PubMed Scopus (7) Google Scholar). A third group cloned the identical receptor sequence, which was classified as a purinergic P2Y7 receptor on the basis of its affinity for binding ATP (19Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). However, Yokomizo et al. (3Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar) challenged this identification and provided convincing proof that the human BLTR (h-BLTR) had been cloned. Intense interest in the role of chemokine receptors for facilitation of HIV entry into CD4-positive cells is evident from recent surveys of the literature (20Doms R.W. Peiper S.C. Virology. 1997; 235: 179-190Crossref PubMed Scopus (236) Google Scholar, 21Baggiolini M. Dewald B. Moser B. Annu. Rev. Immunol. 1997; 15: 675-705Crossref PubMed Scopus (1984) Google Scholar). The h-BLTR is structurally related to chemokine receptors (e.g. CCR5) and is expressed in various immune cells. Recent evidence has suggested that the h-BLTR may function as a coreceptor for entry of primary HIV-1 isolates into CD4-positive cells (22Owman C. Garzino-Demo A. Cocchi F. Popovic F. Sabirsh A. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9530-9534Crossref PubMed Scopus (53) Google Scholar). If true, this finding would add a significant new dimension to the interplay of leukotrienes, inflammation, and AIDS pathogenesis. We report here the cloning and characterization of the m-BLTR, the signaling pathways for this G protein-coupled receptor, and a detailed analysis as to whether the h-BLTR can function as an HIV coreceptor. [α-32P]dCTP was purchased from NEN Life Science Products and [3H]LTB4 from Amersham Pharmacia Biotech. The mouse strain 129 Sv genomic library in Lambda Fix II and the cloning vector pBluescript II KS were from Stratagene (La Jolla, CA), and the mammalian expression vector pCR3.1 Uni was from Invitrogen (Carlsbad, CA). LTB4, 20-hydroxy-LTB4, 6-trans-12-epi LTB4, and LTD4 were purchased from Cayman Chemical Co. (Ann Arbor, MI). Dulbecco's modified Eagle's medium, Ham's F-12, Opti-MEM, L-15, conditioned frog medium, phosphate-buffered saline, fetal bovine serum, and LipofectAMINE were from Life Technologies, Inc., and restriction enzymes were from New England Biolabs (Beverly, MA). Ampli-Taq DNA polymerase was obtained from Perkin-Elmer. A 1.1-kb fragment (NheI-PstI) from the cDNA, described in Ref.19Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar and labeled with [α-32P]dCTP, was used as probe to screen the genomic mouse library by standard procedures. Putative positive clones were taken through two additional rounds of screening until plaque-purified. Phage DNA was purified and subjected to restriction enzyme digestion and Southern blot analysis. A 2.2- kb XbaI fragment that hybridized to the probe was gel extracted and inserted into theXbaI site of pBluescript II KS. The insert was sequenced entirely on both strands using automated sequencing (Applied Biosystems Big Dye Terminator, Ready Reaction Kit Reagents; ABI 373 sequencer) at the Department of Genetics, University of Pennsylvania. The open reading frame was amplified by PCR using primers CDF367 (5′-GCCATGGCTGCAAACACTACATCTC-3′) and CDF370 (5′-AGTTCACTTCGAAGACTCAGG-3′). The sequence of the amplified product was verified and cloned into pCR3.1 Uni. Human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells from the American Type Culture Collection (ATCC) were maintained in Dulbecco's modified Eagle's medium and Ham's F-12, respectively, containing 100 units/ml of penicillin and 100 μg/ml streptomycin, supplemented with 10% (v/v) fetal bovine serum in a 5% CO2 incubator at 37 °C. Cells were seeded at a density of 1–1.5 × 106cells/100-mm dish. Plasmid DNA and LipofectAMINE were mixed with Opti-MEM and added for 12 h on cells, at 70–80% confluence for transient transfection or at 40–50% confluence to select stable transfected cell lines. Cells were selected 24 h after transfection with G418. Isolation of stable clones was achieved by serial dilution or pipette lifting colonies followed by trypsinization and replating colonies in medium containing 2 mg/ml G418. When single clones where isolated and passed several times, the G418 concentration was reduced gradually to 0.5 mg/ml. Experiments were performed on two cell lines transfected with pCR3.1-m-BLTR (HEK-m-BLTR and CHO-m-BLTR). Total RNA was prepared from different murine tissues using TRIzol reagent (Life Technologies, Inc.). RNA blot analysis was carried out with 15 μg of total RNA from different tissues. The full-length m-BLTR DNA was used as probe, labeled with [α-32P]dCTP. Blots were prehybridized 20 min at 68 °C in QuickHyb solution (Stratagene) and hybridized at 68 °C for 1 h. The final washing conditions were 0.1 × SSC, 0.1% SDS at 60 °C. cDNA was synthesized from 5 μg of total RNA with random primers (for reverse transcriptase-PCR). PCR primers CDF370 and CDF367 were used to amplify m-BLTR cDNA. Cells were washed twice with cold phosphate-buffered saline without calcium and magnesium, harvested, and homogenized with a hand-held Polytron on ice in 5 ml of buffer A (10 mm HEPES, 2 mm EDTA, pH 7.4, and a mixture of protease inhibitors (Boehringer Mannheim)). The homogenate was centrifuged 5 min at 140 × g, and the supernatant was then centrifuged at 100,000 g for 1 h. The pellet was resuspended in buffer A to 2 mg/ml of protein for [3H]LTB4 binding experiments performed within several hours. To determine the specific binding on membrane fractions isolated from transiently transfected cells, cells were isolated 36–40 h after transfection. For cold competition experiments, HEK-m-BLTR cell membrane fractions (200 μg/ml of protein) were incubated with 0.1 nm[3H]LTB4 in 0.5 ml of 10 mmHEPES, pH 7.4, containing 20 mm MgCl2 and various concentrations of either LTB4 (0.01–100 nm), 20-hydroxy-LTB4, 6-trans-12-epi LTB4, or LTD4 (0.1 nm-1 μm). For saturation experiments, stable transfected cell membranes (200 μg/ml of protein) were incubated with various concentrations of [3H]LTB4 (0.01–2.5 nm) in 0.25 ml of 10 mm HEPES, pH 7.4, containing 20 mm MgCl2 in the presence or absence of 2.5 μm LTB4. All samples, in duplicate, were incubated at room temperature for 1 h. The total and nonspecific binding were determined as the amount of [3H]LTB4 bound to the membrane fractions in the presence or absence of 2.5 μm LTB4. Bound and free radioligand were separated by filtration through Whatman GF/C filters presoaked with 0.1% bovine serum albumin in 10 mmHEPES. Confluent CHO-m-BLTR cells were harvested and washed with HEPES-buffered saline. Cells were then loaded with the fluorescent dye FURA-2/AM (Molecular Probes, Eugene, OR) at 10 μm, washed and resuspended in HEPES-buffered saline containing: 142 mm NaCl, 2.4 mm KCl, 1.2 mm K2HPO4, 1.3 mm Ca2+, 10 mm d-glucose, 10 mm HEPES, pH 7.4, and 250 μm sulfinpyrazone, the latter being added in order to reduce excretion of the dye (23Di Virgilio F. Fasolato C. Steinberg T.H. Biochem. J. 1988; 256: 959-963Crossref PubMed Scopus (123) Google Scholar). Measurements of change in Ca2+ levels in stirred cell suspensions were made using a Perkin-Elmer model LS50B luminescence spectrometer and were expressed as ratios of fluorescence emitted at 510 nm in response to excitation at 340 and 380 nm (data sampling interval, 0.5 s). Calcium concentrations were calculated from these ratios after determining the maximum and minimum ratios of fluorescence in the presence and absence of saturating levels of Ca2+, respectively, according to the ratiometric method described previously (24Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Xenopus laevis melanophores were maintained in culture and used as described previously (25Graminski G.F. Jayawickreme C.K. Potenza M.N. Lerner M.R. J. Biol. Chem. 1993; 268: 5957-5964Abstract Full Text PDF PubMed Google Scholar, 26Lerner M.R. Trends Neurosci. 1994; 17: 142-146Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 27Potenza M.N. Graminski G.F. Schmauss C. Lerner M.R. J. Neurosci. 1994; 14: 1463-1476Crossref PubMed Google Scholar, 28McClintock T.S. Graminski G.F. Potenza M.N. Jayawickreme C.K. Roby-Shemkovitz A. Lerner M.R. Anal. Biochem. 1993; 209: 298-305Crossref PubMed Scopus (40) Google Scholar). Briefly, transient expression of pCR3.1-m-BLTR in melanophores was achieved by electroporation. Melanophores were plated (15,000/well) in 96-well tissue culture plates and cultured for 2 days. Before the addition of agonist, cells were washed, incubated with 0.7 × L-15, 0.1% bovine serum albumin as described (25Graminski G.F. Jayawickreme C.K. Potenza M.N. Lerner M.R. J. Biol. Chem. 1993; 268: 5957-5964Abstract Full Text PDF PubMed Google Scholar), and then exposed to room light for 1 h. This exposure causes the cells to disperse their pigment granules and darken. The plates were incubated for 1 h in room light and base-line absorbance (A 0) reading obtained at 620 nm using a Molecular DevicesV max kinetic microtiter plate reader. The agonist LTB4 was added to the microtiter wells in 20-μl aliquots at 10 × their final concentration. Dose-response data were obtained 1 h later (A f). Data were plotted with y = 1 − (A f/A 0). Data are presented as mean ± S.E. CHO-m-BLTR were plated in 12-well plates at a density of 200,000 cells/well. 2 days later, the cultured medium was removed and replaced with 0.5 ml of culture medium with 25 μm forskolin. After 15 min, different concentrations of LTB4 were added. The medium was removed 10 min after the addition of agonist, and the cAMP produced was extracted by adding 0.5 ml of ethanol to each well. The supernatant was evaporated to dryness, and the pellet was dissolved in Tris (0.05 m) EDTA (4 mm) buffer, pH = 7.5. The cAMP content was measured using a [3H]cAMP radioimmunoassay kit from Amersham Pharmacia Biotech. 5-Lipoxygenase-deficient mice (29Chen X.S. Sheller J.R. Johnson E.N. Funk C.D. Nature. 1994; 372: 179-182Crossref PubMed Scopus (354) Google Scholar) and wild-type controls (five mice each) from our colony were injected intraperitoneally with glycogen and the neutrophils harvested 5 h later as described previously (29Chen X.S. Sheller J.R. Johnson E.N. Funk C.D. Nature. 1994; 372: 179-182Crossref PubMed Scopus (354) Google Scholar). Binding was carried out on membranes as mentioned above. For cell-cell fusion assays, quail QT6 target cells were transfected with plasmids expressing CD4, the desired coreceptor, and luciferase under control of the T7 promoter. The h-BLTR, previously characterized as the P2Y7 receptor (gift from S. Kunapuli; Ref. 19Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), was used for these studies (see “Results”). The next day, QT6 effector cells expressing the desired Env protein by transfection and T7 polymerase as a consequence of infection by recombinant vaccinia viruses were added. In this assay, cell-cell fusion results in cytoplasmic mixing and luciferase production, which can be easily quantified. Additional details can be found in previous papers (30Nussbaum O. Broder C.C. Berger E.A. J. Virol. 1994; 68: 5411-5422Crossref PubMed Google Scholar, 31Rucker J. Doranz B.J. Edinger A.E. Long D. Berson J.F. Doms R.W. Methods Enzymol. 1997; 288: 118-133PubMed Google Scholar). For infection assays, we used luciferase reporter viruses. Human 293T cells were transfected with plasmids expressing the desired Env (in a pcDNA3 or psv7d background) and with the NL4–3 luciferase virus backbone (pNL-Luc-E−R−) (32Chen B.K. Saksela K. Andino R. Baltimore D. J. Virol. 1994; 68: 654-660Crossref PubMed Google Scholar, 33Connor R.I. Chen B.K. Choe S. Landau N.R. Virology. 1995; 206: 935-944Crossref PubMed Scopus (1088) Google Scholar). Virus was harvested from the media the next day and used to infect feline CCCS cells (for HXBch) or 293T cells (for all other Evs) expressing the indicated CD4/coreceptor combinations. Infection was quantified by measuring luciferase activity 3-days postinfection. Multiple analysis of variance tests followed by Bonferroni analysis were performed on cAMP data. *p< 0.05; ***p < 0.001. To clarify the confusion surrounding the molecular identity of the h-BLTR and to advance the study of the role of LTB4 in murine inflammation models, we sought to clone and characterize the m-BLTR. A mouse genomic library was screened using a 1.1-kb fragment from the cDNA identified previously as encoding the purinergic P2Y7 receptor (19Akbar G.K.M. Dasari V.R. Webb T.E. Ayyanathan K. Pillarisetti K. Sandhu A.K. Athwal R.S. Daniel J.L. Ashby B. Barnard E.A. Kunapuli S.P. J. Biol. Chem. 1996; 271: 18363-18367Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Among the positive clones that hybridized to the probe, one genomic clone with a 2.2-kb XbaI fragment was found to display an open reading frame of 1.056 kb, encoding a 351-amino acid receptor with seven putative transmembrane domains, two potential glycosylation sites, and several phosphorylation sites (GenBank accession numberAF077673). This open reading frame revealed a deduced amino acid sequence with 77% identity to the h-BLTR (3Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (850) Google Scholar). The third intracellular loop of the m-BLTR showed 100% identity to the human receptor, with two protein kinase C phosphorylation sites. TheN-glycosylation sites and several of the phosphorylation sites are conserved between both species. The putative promoter region (−1 to −618) contained a CAAT-like box, a 36-nucleotide poly(A) tract, and several other conserved sequences for the putative binding of GATA-1, PEA-3, c-Myc, c-Myb, Sp-1, and NF-IL6 transcription factors. While this manuscript was under review, Huang et al. (34Huang W.-W. Garcia-Zepeda E.A. Sauty A. Oettgen H.C. Rothenberg M.E. Luster A.D. J. Exp. Med. 1998; 188: 1063-1074Crossref PubMed Scopus (133) Google Scholar) published a m-BLTR sequence cloned from a murine eosinophil cDNA library that was identical to the m-BLTR gene cloned here. Because the genomic clone appeared to be intronless, we proceeded directly to expression studies. m-BLTR was subcloned in the expression vector pCR3.1 and used to transfect HEK 293 cells. Transient transfected HEK cells displayed specific binding for LTB4 (Fig.1 A), as did cells transfected with the original human P2Y7 receptor clone (not shown), whereas nontransfected (Ct) and mock transfected cells did not. Membrane fractions of HEK-m-BLTR cells showed a reversible, saturable and high affinity binding for LTB4 with aK d = 0.24 ± 0.03 nm andB max = 743 ± 168 fmol/mg of protein (Fig.1 B). Displacement curves of [3H]LTB4 binding indicated that the binding site was specific for LTB4 with a K i of 0.23 ± 0.05 nm (n = 4), followed by 20-hydroxy-LTB4, a metabolite of LTB4(K i = 1.1 ± 0.2 nm,n = 4), and by the nonenzymatic breakdown product of LTA4, 6-trans-12-epi-LTB4, and LTD4 (K i > 1 μm) (Fig.1 C). The tissue distribution of the m-BLTR was investigated by Northern blot analysis of total RNA using two different probes. No signal was detected in the tissues tested (spleen, lung, kidney, liver, pancreas, uterus, testis, heart, aorta, brain). However, using reverse transcriptase-PCR, the m-BLTR mRNA was detected in all tissues, but not aorta (data not shown). The mouse heart and lung cDNAs were sequenced and found to be identical with the positive clone identified by mouse genomic library screening. X. laevis melanophores provide a rapid, functional and visual readout of receptor activation. These cells disperse their pigment granules upon stimulation of receptors that are coupled to Gs, Go, and Gq and lead to accumulation of second messengers and thus appear dark. In contrast, stimulation of receptors that are coupled to Gi cause a decrease in second messenger levels and result in pigment granule aggregation, and the cells appear light (27Potenza M.N. Graminski G.F. Schmauss C. Lerner M.R. J. Neurosci. 1994; 14: 1463-1476Crossref PubMed Google Scholar). A long term culture of melanophores was used to evaluate the functional activation of the m-BLTR. Stimulation of the receptor caused pigment aggregation in a concentration-dependent fashion with an EC50 = 0.13 nm (Fig. 2) consistent with coupling to Gi. In CHO-m-BLTR cells, LTB4 inhibited in a concentration-dependent manner the cAMP production induced by 25 μm forskolin (Fig.3 A). Maximum inhibition (58%) was obtained at 100 nm. With the same cell line, LTB4 induced a concentration-dependent increase in the intracellular calcium levels (EC50 = 4.4 nm; Fig.3 B). With 1 μm thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase pump, the intracellular increase of calcium induced by 100 nm LTB4 was inhibited by 89.5 ± 1.4% (n = 4). Previously, we (29Chen X.S. Sheller J.R. Johnson E.N. Funk C.D. Nature. 1994; 372: 179-182Crossref PubMed Scopus (354) Google Scholar) and another group (35Goulet J.L. Snouwaert J.N. Latour A.M. Coffman T.M. Koller B.H. Proc. Natl. Acad. Sci U. S. A. 1994; 91: 12852-12856Crossref PubMed Scopus (188) Google Scholar) developed mice with disruptions in the 5-lipoxygenase gene. These mice were unable to synthesize cysteinyl-leukotrienes or LTB4 in various inflammatory cell types. To test whether the absence of ligand influences receptor expression, we tested 5-lipoxygenase-deficient and control mice for alterations in LTB4 binding using membranes from glycogen-elicited neutrophils. Although we obtained specific and competitive binding in these membranes, the lack of ability to synthesize LTB4 in 5-lipoxygenase deficient mice did not substantially alter BLTR binding (Fig.4). Primate lentiviruses utilize CD4 and a coreceptor (most often the chemokine receptors CCR5 and CXCR4) to enter target cells (20Doms R.W. Peiper S.C. Virology. 1997; 235: 179-190Crossref PubMed Scopus (236) Google Scholar). The importance of CCR5 as a HIV-1 coreceptor was demonstrated by the finding that individuals who lack CCR5 because of a naturally occurring polymorphism are highly resistant to HIV infection (36Samson M. Libert F. Doranz B.J. Rucker J. Liesnard C. Farber C.M. Saragosti S. Lapoumeroulie C. Cognaux J. Forceille C. Muyldermans G. Verhofstede C. Burtonboy G. Georges M. Imai T. Rana S. Yi Y. Smyth R.J. Collman R.G. Doms R.W. Vassart G. Parmentier M. Nature. 1996; 382: 722-725Crossref PubMed Scopus (2447) Google Scholar). In addition to CCR5 and CXCR4, approximately one dozen other chemokine and related orphan receptors have been shown to function as coreceptors for more limited numbers of virus strains in vitro (37Hoffman T.L. Doms R.W. AIDS. 1998; 12: S17-S26PubMed Google Scholar). However, the in vivo relevance of these alternative coreceptors is uncertain. Recently, using a PCR-based entry assay, h-BLTR was shown to serve as a coreceptor for some X4 HIV-1 stains (22Owman C. Garzino-Demo A. Cocchi F. Popovic F. Sabirsh A. Gallo R.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9530-9534Crossref PubMed Scopus (53) Google Scholar). To investigate the ability of the BLTR to serve as a coreceptor for SIV and additional HIV-1 strains using a virus infection assay, we expressed CD4 and h-BLTR in human 293T or CCCS cells. The cells were then infected with luciferase reporter viruses bearing various HIV-1 and SIV Env proteins (Fig.5). None of the viral Env proteins tested could mediate infection of cells expressing CD4 and h-BLTR, although infection was readily observed when cells expressed either CCR5 or CXCR4 (depending on the virus strain). In addition to the results depicted in Fig. 5, SIVmac1A11, smPBj6.6, macB670" @default.
• W1989046199 created "2016-06-24" @default.
• W1989046199 creator A5003988177 @default.
• W1989046199 creator A5009388371 @default.
• W1989046199 creator A5010176130 @default.
• W1989046199 creator A5027181810 @default.
• W1989046199 creator A5032705392 @default.
• W1989046199 creator A5033489214 @default.
• W1989046199 creator A5048283029 @default.
• W1989046199 creator A5082912190 @default.
• W1989046199 date "1999-03-01" @default.
• W1989046199 modified "2023-09-30" @default.
• W1989046199 title "Leukotriene Binding, Signaling, and Analysis of HIV Coreceptor Function in Mouse and Human Leukotriene B4Receptor-transfected Cells" @default.
• W1989046199 cites W1494346716 @default.
• W1989046199 cites W1571692670 @default.
• W1989046199 cites W1595120220 @default.
• W1989046199 cites W1597767121 @default.
• W1989046199 cites W1671834350 @default.
• W1989046199 cites W1672565987 @default.
• W1989046199 cites W1731073298 @default.
• W1989046199 cites W1895644457 @default.
• W1989046199 cites W1973735123 @default.
• W1989046199 cites W1979299780 @default.
• W1989046199 cites W1981101682 @default.
• W1989046199 cites W1987090231 @default.
• W1989046199 cites W2004351423 @default.
• W1989046199 cites W2008235942 @default.
• W1989046199 cites W2008599526 @default.
• W1989046199 cites W2011103103 @default.
• W1989046199 cites W2012241540 @default.
• W1989046199 cites W2018982364 @default.
• W1989046199 cites W2023213942 @default.
• W1989046199 cites W2026444780 @default.
• W1989046199 cites W2036323747 @default.
• W1989046199 cites W2037507488 @default.
• W1989046199 cites W2047146671 @default.
• W1989046199 cites W2047569480 @default.
• W1989046199 cites W2057529727 @default.
• W1989046199 cites W2060106009 @default.
• W1989046199 cites W2072901265 @default.
• W1989046199 cites W2087019177 @default.
• W1989046199 cites W2087339573 @default.
• W1989046199 cites W2095942761 @default.
• W1989046199 cites W2098532478 @default.
• W1989046199 cites W2106234499 @default.
• W1989046199 cites W2114817502 @default.
• W1989046199 cites W2116296362 @default.
• W1989046199 cites W2126255146 @default.
• W1989046199 cites W2132940813 @default.
• W1989046199 cites W2141260882 @default.
• W1989046199 cites W2160272188 @default.
• W1989046199 cites W2171826251 @default.
• W1989046199 cites W2271095628 @default.
• W1989046199 cites W243604420 @default.
• W1989046199 cites W4211232555 @default.
• W1989046199 cites W1987305904 @default.
• W1989046199 doi "https://doi.org/10.1074/jbc.274.13.8597" @default.
• W1989046199 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10085095" @default.
• W1989046199 hasPublicationYear "1999" @default.
• W1989046199 type Work @default.
• W1989046199 sameAs 1989046199 @default.
• W1989046199 citedByCount "37" @default.
• W1989046199 countsByYear W19890461992012 @default.
• W1989046199 countsByYear W19890461992013 @default.
• W1989046199 countsByYear W19890461992014 @default.
• W1989046199 crossrefType "journal-article" @default.
• W1989046199 hasAuthorship W1989046199A5003988177 @default.
• W1989046199 hasAuthorship W1989046199A5009388371 @default.
• W1989046199 hasAuthorship W1989046199A5010176130 @default.
• W1989046199 hasAuthorship W1989046199A5027181810 @default.
• W1989046199 hasAuthorship W1989046199A5032705392 @default.
• W1989046199 hasAuthorship W1989046199A5033489214 @default.
• W1989046199 hasAuthorship W1989046199A5048283029 @default.
• W1989046199 hasAuthorship W1989046199A5082912190 @default.
• W1989046199 hasBestOaLocation W19890461991 @default.
• W1989046199 hasConcept C14036430 @default.
• W1989046199 hasConcept C159047783 @default.
• W1989046199 hasConcept C185592680 @default.
• W1989046199 hasConcept C203014093 @default.
• W1989046199 hasConcept C2776042228 @default.
• W1989046199 hasConcept C2776452501 @default.
• W1989046199 hasConcept C2776685179 @default.
• W1989046199 hasConcept C2776914184 @default.
• W1989046199 hasConcept C2779543280 @default.
• W1989046199 hasConcept C3013748606 @default.
• W1989046199 hasConcept C54009773 @default.
• W1989046199 hasConcept C54355233 @default.
• W1989046199 hasConcept C81885089 @default.
• W1989046199 hasConcept C86803240 @default.
• W1989046199 hasConcept C95444343 @default.
• W1989046199 hasConceptScore W1989046199C14036430 @default.
• W1989046199 hasConceptScore W1989046199C159047783 @default.
• W1989046199 hasConceptScore W1989046199C185592680 @default.
• W1989046199 hasConceptScore W1989046199C203014093 @default.
• W1989046199 hasConceptScore W1989046199C2776042228 @default.
• W1989046199 hasConceptScore W1989046199C2776452501 @default.
• W1989046199 hasConceptScore W1989046199C2776685179 @default.
• W1989046199 hasConceptScore W1989046199C2776914184 @default.

• next