Hydroxyeicosanoids Bind to and Activate the Low Affinity
Leukotriene B4 Receptor, BLT2*
Takehiko
Yokomizo
§¶,
Kazuhiko
Kato
,
Hiroshi
Hagiya
,
Takashi
Izumi§**, and
Takao
Shimizu
§
From the
Department of Biochemistry and Molecular
Biology, Faculty of Medicine, The University of Tokyo and the
§ Japan Science and Technology Corporation (CREST), Tokyo
113-0033, Japan, the
Pharmaceutical Research Center, Meiji Seika
Kaisha, Ltd., Yokohama 222-8567, Japan, and the ** Department of
Biochemistry, Faculty of Medicine, Gunma University, Maebashi 371-8511, Japan
Received for publication, December 18, 2000, and in revised form, January 17, 2001
 |
ABSTRACT |
Leukotriene B4, an arachidonate
metabolite, is a potent chemoattractant of leukocytes involved in
various inflammatory diseases. Two G-protein-coupled receptors
for leukotriene B4 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
[3H]LTB4 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.
 |
INTRODUCTION |
Leukotriene B4
((5S,12R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic
acid (LTB4))1 is
a metabolite of arachidonic acid and is one of the most potent activators of granulocytes and macrophages (1-3). BLT, the
LTB4-specific G-protein-coupled receptor (GPCR), is a
target for anti-inflammatory drugs, and many BLT antagonists have been
developed and are being evaluated. No BLT antagonists are yet approved
available for clinical use; their lack of efficacy may be due in part
to the presence of the other LTB4 receptors. We cloned
BLT1, a high affinity LTB4 receptor (4), and BLT2, a low
affinity LTB4 receptor (5), and showed that BLT1 is
expressed almost exclusively in peripheral leukocytes, whereas BLT2 is
expressed ubiquitously with the highest expression in spleen. The
structural similarities of these receptors (45% amino acid identity)
and the low homologies of BLTs to other known GPCRs suggest that these
BLTs comprise a novel receptor family. BLT1 and BLT2 form a gene
cluster both in human and mouse genomes. The human BLT2 open reading
frame overlaps the promoter region of BLT1 (6), suggesting the
expression of these two LTB4 receptors is tightly
intertwined. Human granulocytes, eosinophils, and mononuclear cells
express both BLT1 and BLT2 (7), so the precise pharmacological
characterization of these two receptors using native cells is
difficult. In this paper, we report the inhibitory effects of various
BLT antagonists and eicosanoids on LTB4 binding using CHO
cells stably expressing either human BLT1 or BLT2. We also show that
several hydroxyeicosanoids other than LTB4 bind to and
activate BLT2 but not BLT1. These findings provide insights into the
possible functions of BLT2 and information helpful for the isolation of
the other related GPCRs that recognize eicosanoids.
 |
EXPERIMENTAL PROCEDURES |
Materials--
U75302 (8) and all of the eicosanoids other than
LTB4 were purchased from Cayman Chemical Co.
LTB4 is a generous gift from Ono Pharmaceutical Co.
(Osaka, Japan). LY 255283 (9) and LY 223982 (10) were from Lilly
Research Laboratories. CP 105696 (11) and CP 195543 (12) are from
Pfizer Inc. ZK 158252 is from Schering AG (Berlin, Germany).
[3H]LTB4 (6956 GBq/mmol) was purchased from
PerkinElmer Life Sciences.
Cell Culture and Flow Cytometry--
CHO cells stably expressing
FLAG-tagged human BLT1 (CHO-FLAG-BLT1) or hemagglutinin (HA)-tagged
human BLT2 (CHO-HA-BLT2) were established as described previously (5,
13). The cells were cloned by limiting dilution and their expression of
the receptors confirmed by staining the cells with antibodies against
the epitope added to the amino terminus of the receptors. The cells
were fixed with PBS(
) containing 0.5% (w/v) paraformaldehyde for 5 min on ice and blocked with PBS(
) containing 2% goat serum (Life
Technologies, Inc.). The cells were incubated with 30 µg/ml anti-FLAG
antibody (clone M5, Eastman Kodak), 10 µg/ml anti-HA antibody (clone
CA12-5, Roche Molecular Biochemicals), or 30 µg/ml control mouse IgG
(Santa Cruz Biotechnology) in PBS(
) containing 2% oat serum for 30 min at room temperature, followed by staining with 500 × fluorescein isothiocyanate-labeled anti-mouse IgG (Beckman
Coulter, CA) for 30 min at room temperature. The cells were washed
twice with PBS(
), and analyzed with flow cytometry, Epics XL
(Beckman-Coulter). Cells expressing each receptor were maintained in
Ham's F12 medium supplemented with 10% fetal calf serum
(Sigma), 0.3 mg/ml G418 (Sigma), 100 IU/ml penicillin, and 100 µg/ml streptomycin.
Binding Assay--
Cells were harvested and sonicated in a
buffer containing 20 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 10 mM MgCl2, 2 mM EDTA-Na2, 2 mM
phenylmethylsulfonyl fluoride, and 1 µM pepstatin A. After centrifugation at 12,000 × g for 10 min at
4 °C, the remaining supernatants were further centrifuged at
105,000 × g for 60 min. The resulting pellets were
used for the binding assay as membrane fractions. The concentrations of
the protein were determined by the method of Bradford using a Bio-Rad
protein assay. The membrane fractions of CHO cells were examined for
[3H]LTB4 binding. The binding mixture (100 µl) contained membrane fractions (20 µg of protein) and 5 nM [3H]LTB4 (34.78 KBq/ml)
with/without various concentrations of antagonists or eicosanoids in
binding buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 10 mM NaCl, and 0.05% BSA). For
determination of the nonspecific binding, unlabeled LTB4
was added to the binding mixture to a final concentration of 10 µM. The mixtures were incubated at room temperature for
60 min with gentle agitation followed by rapid filtration through GF/C
filters (Packard) and washing with 3 ml of binding buffer. The
radioactivity levels of the filters were determined with a Top
Count scintillation counter (Packard).
Measurement of Intracellular Calcium Concentration--
CHO
cells were loaded with 3 µM Fura-2'AM (Dojin, Kumamoto,
Japan) in a modified Hepes-Tyrode's BSA buffer (25 mM
Hepes-NaOH, pH 7.4, 140 mM NaCl, 2.7 mM KCl,
1.0 mM CaCl2, 12 mM
NaHCO3, 5.6 mM D-glucose, 0.37 mM NaH2PO4, 0.49 mM
MgCl2, 0.01% (w/v) cremophore, and 0.1% (w/v) fatty
acid-free BSA (Fraction V, Bayer)) at 37 °C for 1 h. The cells
were washed twice and resuspended in Hepes-Tyrode's BSA buffer
(without cremophore) at a density of 1 × 106
cells/ml. 0.5 ml of the cell suspension was applied to a CAF-100 system (Jasco), and 5 µl of ligand solution was added. The ligands were dissolved in Hepes-Tyrode's BSA buffer after evaporation of the
stock solution in ethanol under nitrogen stream and used immediately.
Intracellular Ca2+ concentration was measured by the ratio
of emission fluorescence of 500 nm by excitation at 340 and 380 nm.
Chemotaxis Assay--
CHO cells expressing hBLT1 or hBLT2 were
examined for their chemotactic responses as described previously (4).
Briefly, 8 × 104 cells were left to migrate toward
the ligands for 4 h through fibronectin-coated filters in a
96-well Boyden chamber. After migration, the cells on the upper side of
the filter were wiped off and stained with Diff-Quik reagent, and the
migrated cells were quantified by measuring the
A595 nm using a microplate reader.
Presentation of the Data--
All figures shown contain
representative data from at least two independent experiments with
similar results.
 |
RESULTS |
Binding Profiles of Various BLT Antagonists to BLT1 and
BLT2--
In the light of our knowledge the actions of BLT antagonists
that have been developed as anti-inflammatory drugs has to be re-evaluated. Most were characterized using human peripheral leukocytes before the identification of two kinds of LTB4 receptors,
BLT1 and BLT2, and their coexpression in peripheral leukocytes (5), monocytes, and eosinophils (7). To distinguish the effects of BLT
antagonists on these two receptors, we examined their inhibitory effects on [3H]LTB4 binding to the membrane
fractions of CHO cells stably expressing either human BLT1 or BLT2. CHO
cells are useful for this purpose because they do not express any
intrinsic receptors for LTB4, as revealed by
[3H]LTB4 binding and calcium mobilization
assays (data not shown). Receptors tagged with FLAG or HA sequences at
their amino termini were introduced into the cells, and several clones
stably expressing each receptor were isolated. The proper expression of
the receptors on the cell surface was confirmed by staining the cells
with antibodies specific to the tag, which was followed by flow
cytometry (Fig. 1). The membrane
fractions of these cells (20 µg of membrane protein) were examined
for their binding with 5 nM
[3H]LTB4. Fig.
2 shows the competition of 5 nM [3H]LTB4 binding by various
BLT antagonists. LTB4 binding to BLT1 was inhibited by CP
105696, ZK 158252, CP 195543, and U75302 in a
dose-dependent manner but not by 10 µM LY
255283 or LY 223982. In contrast, LTB4 binding to BLT2 was
inhibited by ZK 158252, LY 255283, and CP 195543 but not by 10 µM U75302, CP105695 or LY 223982. We also examined their
agonistic activities on these receptors by calcium mobilization and
found that 1 and 10 µM U75302 acts as a weak agonist on
human BLT1, and 1 and 10 µM CP 195543 acts as a weak
agonist on human BLT2 (data not shown).

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Fig. 1.
Expression of FLAG-BLT1 or HA-BLT2 in CHO
cells. CHO cells were transfected with empty vector
(A), FLAG-BLT1 (B), or HA-BLT2 (C),
selected with 1 mg/ml G418, and cloned by limiting dilution. The cells
were stained with anti-FLAG, anti-HA, or control mouse IgG and
fluorescein isothiocyanate-labeled secondary antibody as described
under "Experimental Procedures." 10,000 cells were analyzed by flow
cytometry.
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Fig. 2.
Inhibition of
[3H]LTB4 binding to BLT1 or BLT2 by BLT
antagonists. 5 nM [3H]LTB4
binding to the membrane preparations (20 µg of membrane protein) from
CHO-FLAG-BLT1 (A) and CHO-HA-BLT2 (B) cells was
competed with various concentrations of BLT antagonists. Data are
represented as % inhibition compared with 10 µM
LTB4 as 100%. In CHO-FLAG-BLT1 cells, total binding
and nonspecific binding were 62424 ± 563 and 1818 ± 72 dpm,
respectively. In CHO-HA-BLT2 cells, total binding and nonspecific
binding were 5070 ± 236 and 1374 ± 123 dpm, respectively
(n = 3, average ± S.D.).
|
|
Binding Profiles of Various Eicosanoids to BLT1 and BLT2--
The
membrane fraction of HEK 293 cells transfected with human BLT2 cDNA
exhibited a specific and saturable binding for
[3H]LTB4 with a Kd value
of 23 nM, which is higher than that for human BLT1 by
20-fold (5). Thus, at that time we concluded that BLT2 is a low
affinity receptor for LTB4. In the present study, we
examined the inhibition by various eicosanoids of
[3H]LTB4 binding to the membrane fractions of
CHO cells stably expressing BLT1 or BLT2. Fig.
3 shows the inhibition of 5 nM [3H]LTB4 binding by various
eicosanoids at a concentration of 5 µM. LTB4
binding to BLT1 is very specific, because only LTB4, 20-hydroxy-LTB4, and 12-epi-LTB4
showed significant competitions at this concentration (Fig.
3A). These results are consistent with our previous results
using the membrane fractions of COS-7 cells transiently transfected
with BLT1 (4). On the other hand, the binding of LTB4 to
BLT2 was inhibited by various eicosanoids (Fig. 3B).
12(R)- and 12(S)-HETE, 12(S)-HPETE,
15(S)-HETE, and 15(S)-HPETE in addition to
LTB4, 20-hydroxy LTB4, and
12-epi-LTB4 showed significant inhibition
(>50%) of 5 nM [3H]LTB4
binding. Next, we tested various concentrations of LTB4, 20-hydroxy-LTB4, 12(S)-HPETE,
12(S)-HETE, 12(R)-HETE, and 15(S)-HETE in the competition with [3H]LTB4 binding to
BLT1 and BLT2. Only LTB4 and 20-hydroxy LTB4 showed dose-dependent inhibitions of LTB4
binding to BLT1 (Fig. 4A). In
the case of BLT2, however, all of the eicosanoids tested exhibited
dose-dependent inhibition of LTB4, with a rank
order of LTB4 > 12(S)-HETE > 12(S)-HPETE > 12(R)-HETE > 15(S)-HETE > 20-hydroxy LTB4 (Fig.
4B). These results clearly show that the recognition of the
ligands by BLT1 and BLT2 differs, with more promiscuity in BLT2.

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Fig. 3.
Inhibition of
[3H]LTB4 binding to BLT1 or BLT2 by
eicosanoids. 5 nM [3H]LTB4
binding to the membrane preparations (20 µg of membrane protein) from
CHO-FLAG-BLT1 (A) and CHO-HA-BLT2 (B)
cells was competed with 5 µM eicosanoids
(n = 3, average ± S.D.). ETE,
eicosatetraenoic acid; LX, lipoxin.
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Fig. 4.
Inhibition of
[3H]LTB4 binding to BLT1 or BLT2 by
eicosanoids. 5 nM [3H]LTB4
binding to the membrane preparations (20 µg of membrane protein) from
CHO-FLAG-BLT1 (A) and CHO-HA-BLT2 (B) cells was
competed with various concentrations of eicosanoids (n = 3, average ± S.D.).
|
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Calcium Mobilization by Various Eicosanoids through BLT1 and
BLT2--
We next examined whether these eicosanoids could activate
intracellular signaling through these two receptors. We used a calcium mobilization assay, because it is a very sensitive and quantitative method for detecting LTB4-BLT interaction. In CHO-FLAG-BLT1
cells, only LTB4 exhibited a robust increase in
intracellular calcium concentrations (Fig.
5A). The one exception was
seen in case of 12(S)-HPETE, where there was a very slight
signal seen at 10 µM (Fig. 5B). In contrast,
all of the ligands that were able to bind to BLT2 (Fig. 4B)
exhibited significant increases in intracellular calcium concentrations
in CHO-HA-BLT2 cells, showing that these eicosanoids are active on BLT2
(Fig. 5, A and B). Fig. 5, C and D, shows dose-response curves of increase in calcium
concentrations on CHO-FLAG-BLT1 and CHO-HA-BLT2 cells, respectively.
12(S)-HETE and 15(S)-HETE up to a concentration
of 10 µM did not induce measurable changes in
intracellular calcium through BLT1 (Fig. 5C). On the other
hand, these eicosanoids exhibited dose-dependent increases in calcium concentrations in CHO-HA-BLT2 cells (Fig. 5D).
12-epi-LTB4, a 12(S) epimer of
LTB4, acts as a full agonist for BLT1 and BLT2 (Fig. 5,
B and C), but a higher concentration of
12-epi-LTB4 is required. These results clearly
show that BLT2 is able to bind to various eicosanoids and to transduce
intracellular signaling.

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Fig. 5.
Calcium mobilization by various
eicosanoids through BLT1 or BLT2. A, typical results of
calcium mobilizations by various eicosanoids. B, calcium
increases in CHO cells by 10 µM eicosanoids
(n = 3, average ± S.D.). C and
D, dose responses of calcium mobilization by
LTB4, 12(S)-HETE, 15(S)-HETE, and
12-epi-LTB4 in CHO-FLAG-BLT1 (C) and
CHO-HA-BLT2 (D) cells (n = 3, average ± S.D.).
|
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Chemotactic Response of CHO-BLT2 Cells by Eicosanoids--
As
LTB4 is a potent chemoattractant for granulocytes,
eosinophils, and macrophages, and CHO cells expressing BLT1 or BLT2 migrate to LTB4 in vitro (4, 5, 7), we asked
whether other eicosanoids could evoke chemotactic responses through
BLT1 and BLT2. CHO cells expressing BLT1 showed clear chemotactic
activities toward very low concentration of LTB4 and high
concentration (>10 µM) of 12(R)-HETE and
12(S)-HETE, as shown in Fig.
6A. In the case of BLT2, the
optimum concentrations of LTB4 and 12(S)-HETE are close, and the 12(S)-HETE is more effective than
12(R)-HETE (Fig. 6B). The dose-response curves of
LTB4 and 12(S)-HETE in CHO-BLT2 cells are
bell-shaped as expected in in vitro chemotaxis assays.
12-epi-LTB4 also induced chemotaxis both in
CHO-BLT1 and CHO-BLT2 cells, with higher optimum concentrations than
LTB4. CHO cells transfected with an empty vector,
pcDNA3, as a control did not show any chemotaxis toward
LTB4, 12(S)-HETE, 12(R)-HETE, or
12-epi-LTB4 (Fig. 6C).

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Fig. 6.
Chemotactic responses of CHO cells toward
eicosanoids. CHO cells were left to migrate toward various
concentrations of eicosanoids for 4 h in 96-well Boyden chambers.
The cells that migrated to the lower side of the filters
were quantified by staining with Diff-Quik reagent followed by
measuring the A595 nm and represented as the
Chemotaxis Index (4). The absolute values of the OD of CHO-FLAG-BLT1,
CHO-HA-BLT2, and CHO-vector cells in the absence of the ligands are
0.059 ± 0.0023, 0.0705 ± 0.0005, and 0.0715 ± 0.005, respectively (n = 4, average ± S.E.).
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 |
DISCUSSION |
LTB4 is biologically important for the clearance of
microorganisms or foreign bodies by activating and recruiting
granulocytes to the inflamed lesions (14). Overproduction of
LTB4, however, is also involved in various inflammatory
diseases including psoriasis (15), bronchial asthma (16), rheumatoid
arthritis (17), ulcerative colitis (18), and ischemic reperfusion
injury in various tissues (13). Attempts to understand the biological functions of LTB4 and to develop potent and selective BLT
antagonists would be assisted by the molecular identification of
LTB4 receptors. Several groups including ours have cloned
and characterized two distinct LTB4 receptors, BLT1 and
BLT2. BLT1 (4, 19-24), a high affinity and leukocyte-restricted
LTB4 receptor, is presumably the classical BLT, and most of
the BLT antagonists developed thus far are targeted to this receptor.
Very recently, BLT2, with a structural similarity to BLT1, was
identified as a low affinity receptor for LTB4 (5, 7, 25).
One group reported that BLT2 is a high affinity receptor for
LTB4 when expressed in Cos-7 cells (26), but the clear
explanations for this discrepancy are not available. The
BLT2 gene is localized very close to the BLT1 gene both in human and mouse, suggesting that these two
receptors are linked genetically (6, 27). BLT2 shows a relatively
ubiquitous expression with the highest level in the spleen, followed by
the liver, ovary, and peripheral leukocytes (5, 7). Using
semiquantitative reverse transcriptase-polymerase chain reaction
analysis, BLT1 and BLT2 have been shown to be coexpressed in human
granulocytes, mononuclear cells, and eosinophils (7). A number of
papers have reported the inhibitory effects of various BLT antagonists on LTB4 binding and various LTB4-induced
phenomena, but most of the results were obtained using leukocytes that
intrinsically express both BLT1 and BLT2. To correctly understand the
pharmacological characters of BLT1 and BLT2, we introduced the
expression vectors for these receptors into CHO cells, which do not
express any intrinsic LTB4 receptors, and compared the
effects of various BLT antagonists and eicosanoids. These receptors are
properly expressed on the cell surface as revealed by flow cytometry
(Fig. 1). Binding assays using the fractionated cell membrane revealed
clear differences in pharmacological profiles of various BLT
antagonists on BLT1 and BLT2. CP 105696 and U75302 effectively
inhibited [3H]LTB4 binding to BLT1, but they
did not inhibit LTB4 binding to BLT2 (Fig. 2), showing that
these compounds are specific to BLT1. In addition, U75302 acts as a
weak agonist on BLT1, because it increased intracellular calcium in
CHO-BLT1 cells (data not shown). On the other hand, LY 255283 inhibited
[3H]LTB4 binding to BLT2 but not to BLT1. CP
195543 and ZK 158252 inhibited [3H]LTB4
binding to both receptors, but CP 195543 at 1 µM
increased intracellular calcium in CHO-BLT2 cells (data not shown).
Thus, CP 195543 acts as an antagonist for BLT1 but a weak agonist for BLT2. Various BLT antagonists have been developed and examined for
their effects on inflammatory animal models, but no compounds are
available for clinical use. If the expression/function of these two
receptors are intertwined, as would be predicted based on their
juxtaposition in the genome, a clinically important effect may lie in a
compound that has independent effects on each receptor as well as one
that blocks both. Our studies point to the need to re-evaluate past
studies in light of the independent actions of the compound on each
receptor and to find a way to screen future candidate compounds.
We next examined the ligand specificity for BLT1 and BLT2. From initial
screenings of various eicosanoids on ligand binding assays (Fig. 3), we
selected several hydroxy- or hydroperoxyeicosatetraenoic acids to
examine carefully the dose-dependent inhibition of
[3H]LTB4 binding to BLT1 and BLT2. To our
surprise, some of these eicosanoids effectively inhibited
LTB4 binding to BLT2 (Fig. 4). The rank order of potency in
binding to BLT2 is LTB4 > 12(S)-HETE > 12(S)-HPETE > 12(R)-HETE > 15(S)-HETE > 20-hydroxy LTB4 (Fig. 4B), which is different from the order of potency in binding
to BLT1 (LTB4 > 20-hydroxy-LTB4
12(R)-HETE, Fig. 4A (4)). We next examined
the agonistic activities of these eicosanoids using a calcium
mobilization assay and found that some of them act as agonists on BLT2
(Fig. 5). Although high concentrations of the ligands are required,
CHO-BLT2 cells exhibited clear calcium responses toward 1 µM 12(S)-HETE or 15(S)-HETE.
12(S)-HETE and 12(R)-HETE also induced
significant cell migration in CHO-BLT2 cells at lower concentrations
than for CHO-BLT1 cells (Fig. 6), suggesting that BLT2 recognizes these
eicosanoids and transduces intracellular signaling at physiological
concentrations. Despite the broad interests in leukotriene receptors,
limited information is available on receptors for HETE and HPETE. Among
these receptors, 12(S)-HETE binding sites have been
well characterized in human epidermal and carcinoma cells (28-30).
High affinity 12(S)-HETE binding sites in human skin were
reported, and Kd values for 12(S)-HETE were in the nM range. The rank order of potency in
inhibition of [3H]12(S)-HETE binding is
12(S)-HETE > 12(R)-HETE
LTB4, which is similar to that of BLT2. In murine
B16 melanoma cells, 12(S)-HETE-dependent activation of protein kinase C is mediated by a GPCR and
dependent on Gi-like G-protein (31).
12(S)-HETE-binding protein in the cytosolic and nuclear
fractions of Lewis lung carcinoma cells was characterized and shown to
interact as a heterodimer with steroid receptor coactivator-1 (SRC-1)
in the presence of 12(S)-HETE (32). The peroxisome
proliferator-activated receptor
(PPAR
) was also reported to
interact with 12(S)-HETE (33). These results suggest that
12(S)-HETE may possess a dual receptor system, cell surface
GPCRs, and intranuclear transcriptional factors. BLT2 can interact with
high concentrations of 12(S)-HETE, but the
Kd of BLT2 for 12(S)-HETE is higher than
the reported high affinity 12(S)-HETE receptor(s) in
keratinocytes and melanocytes. The selectivity of BLT2 in the
recognition of 12(S)-HETE and 15(S)-HETE and the structural information of BLT2 may be helpful for the identification of
the high affinity GPCR for HETEs.
We also showed by calcium mobilization and chemotaxis assays that
12-epi-LTB4 is active both on BLT1 and BLT2.
There are no published reports on the in vivo occurrence or
biological functions of 12-epi-LTB4, but it is
clear that it acts as an agonist for BLT1 and BLT2 in heterologously
expressed systems. Further study is required on the biosynthesis and
functions of 12-epi-LTB4.
In summary, we have revealed the specificity of various BLT antagonists
on two LTB4 receptors and identified the novel activation of BLT2 by various HETEs and HPETEs. These findings will lead to the
development of novel BLT antagonists that may be more specific and
therefore more potent as anti-inflammatory drugs, also raising the
possibility of the identification of other as yet unknown eicosanoid receptors.
 |
ACKNOWLEDGEMENTS |
We thank Dr. D. Wong (The University of
Tokyo) for critically reading this manuscript and S. Ishii, N. Uozumi,
and M. Taniguchi (The University of Tokyo) for discussions. We also
thank Ono Pharmaceutical Company for LTB4 and Pfizer Inc.,
Lilly Research Laboratories, and Schering AG for BLT antagonists.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid from the
Ministry of Education, Culture, Sports, Science and Technology
(Japan) and the Human Science Foundation.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Recipient of grants from the Yamanouchi Foundation for
Metabolic Disorders, the Uehara Memorial Foundation, and the Cell
Science Research Foundation. To whom correspondence should be
addressed: Dept. of Biochemistry and Molecular Biology, Faculty of
Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo
113-0033, Japan. Tel.: 81-3-5802-2925; Fax: 81-3-3813-8732; E-mail:
yokomizo-tky@umin.ac.jp.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M011361200
 |
ABBREVIATIONS |
The abbreviations used are:
LTB4, leukotriene B4
((5S,12R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid);
HETE, hydroxyeicosatetraenoic acid;
HPETE, hydroperoxyeicosatetraenoic acid;
BSA, bovine serum albumin;
GPCR, G-protein-coupled receptor;
CHO, Chinese hamster ovary;
HA, hemagglutinin;
PBS, phosphate-buffered saline.
 |
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