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INTRODUCTION |
Leukotriene B4
(LTB4)1 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
(1). 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 (2). LTB4 mediates its effects
through a membrane G-protein-coupled receptor termed BLTR (3).
Additionally, LTB4 was shown to bind to the intracellular
transcription factor peroxisome proliferator-activated receptor (4).
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 (5).
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 (6, 7).
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 (8).
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 (9, 10), endotoxic shock (11), and induced asthma
(12-14). 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 (15).
There has been considerable controversy about the molecular
identification of the BLTR. In 1996 two independent research groups (16, 17) 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 (16) but later retracted this finding to indicate specific binding (18). 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
(19). However, Yokomizo et al. (3) 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 (20, 21). 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 (22). 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.
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EXPERIMENTAL PROCEDURES |
Materials--
[
-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.
Screening of Genomic Library--
A 1.1-kb fragment
(NheI-PstI) from the cDNA, described in Ref.
19 and labeled with [
-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.
Plasmid Constructs--
A 2.2- kb XbaI fragment that
hybridized to the probe was gel extracted and inserted into the
XbaI 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.
Cell Culture and Transfection--
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 × 106
cells/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).
Northern Blot and Reverse Transcriptase-PCR Analysis--
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 [
-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.
Binding Experiments--
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 mM
HEPES, 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 mM HEPES.
Intracellular Calcium Measurements--
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 (23). 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 (24).
Functional Bioassay--
Xenopus laevis melanophores
were maintained in culture and used as described previously (25-28).
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 (25), 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 (A0)
reading obtained at 620 nm using a Molecular Devices
Vmax 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 (Af). Data were
plotted with y = 1
(Af/A0). Data are presented as mean ± S.E.
cAMP Assay--
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.
BLT Receptor Binding Assays to Glycogen-elicited Neutrophil
Membranes--
5-Lipoxygenase-deficient mice (29) 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 (29). Binding was carried out on
membranes as mentioned above.
Cell-Cell Fusion and Virus Infection Assays--
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. 19), 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 (30, 31). 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
) (32, 33). 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.
Data Analysis--
Multiple analysis of variance tests followed
by Bonferroni analysis were performed on cAMP data. *p < 0.05; ***p < 0.001.
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RESULTS |
Isolation of m-BLT Receptor Gene--
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 (19).
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 number
AF077673). This open reading frame revealed a deduced amino acid
sequence with 77% identity to the h-BLTR (3). The third intracellular
loop of the m-BLTR showed 100% identity to the human receptor, with
two protein kinase C phosphorylation sites. The
N-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. (34)
published a m-BLTR sequence cloned from a murine eosinophil cDNA
library that was identical to the m-BLTR gene cloned here.
LTB4 Binding--
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.
1A), 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 a
Kd = 0.24 ± 0.03 nM and
Bmax = 743 ± 168 fmol/mg of protein (Fig.
1B). Displacement curves of
[3H]LTB4 binding indicated that the binding
site was specific for LTB4 with a Ki of
0.23 ± 0.05 nM (n = 4), followed by 20-hydroxy-LTB4, a metabolite of LTB4
(Ki = 1.1 ± 0.2 nM,
n = 4), and by the nonenzymatic breakdown product of
LTA4, 6-trans-12-epi-LTB4, and
LTD4 (Ki > 1 µM) (Fig.
1C).

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Fig. 1.
[3H]LTB4
binding to membrane fractions of HEK 293 cells transiently or stably
(HEK-m-BLTR) transfected with pCR3.1-m-BLTR. Panel A,
total binding of 0.2 nM
[3H]LTB4 to membrane fractions (100 µg) of nontransfected cells (Ct) or transiently
transfected HEK 293 cells. Total binding (black column) and
nonspecific binding (gray column) are shown (mean ± S.E., n = 4). Cells transfected with a control vector
(pCR3.1 Uni) have the same total and nonspecific binding as membrane
fractions from parental cells (not shown). Panel B,
Scatchard analysis and saturation isotherms of
[3H]LTB4 binding to membrane fractions of
HEK-m-BLTR cells stably transfected with pCR3.1-m-BLTR. Data from a
representative experiment of four giving similar results are shown. The
linear Scatchard plot gives a correlation coefficient of 0.99. In this
particular experiment the Bmax and
Kd values were 474 fmol/mg of protein and 0.19 nM, respectively. The specific ( ), nonspecific ( ),
and total binding ( ) isotherms are drawn (inset).
Panel C, displacement curves of specific
[3H]LTB4 binding using several compounds with
structures similar to that of LTB4. Inhibition of 0.1 nM [3H]LTB4 binding to the
membrane fractions of HEK-m-BLTR cells, by LTB4 ( ),
20-hydroxy-LTB4 ( ), 6-trans-12-epi
LTB4 ( ), and LTD4 ( ) is shown. Each point
reflects the mean ± S.E. of four experiments.
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Functional Expression of the m-BLT Receptor--
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 (27). 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.

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Fig. 2.
m-BLTR-induced pigment aggregation in
melanophores. The cloned m-BLTR was transfected into
Xenopus melanophores via electroporation. LTB4
(0.03 pM-10 nM) was added to cells exposed to
room light for 1 h. The ordinate scale is explained
under "Experimental Procedures." Each data point is mean ± S.E. of triplicates (n = 3).
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In CHO-m-BLTR cells, LTB4 inhibited in a
concentration-dependent manner the cAMP production induced
by 25 µM forskolin (Fig. 3A). Maximum inhibition (58%)
was obtained at 100 nM.

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Fig. 3.
LTB4 bound to the m-BLTR induces
activation of signal transduction pathways in CHO-m-BLTR.
Panel A, LTB4 induced a
concentration-dependent inhibition of forskolin (25 µM)-stimulated cAMP accumulation. Different
concentrations of LTB4 (0.01-100 nM) were
added to cells stimulated by forskolin (F, 100%). The
stimulated level of cAMP by forskolin was 436 ± 83 pmol/106 cells. Data presented are mean ± S.E.
(n = 5). Panel B,
concentration-dependent increase in intracellular calcium
induced by LTB4. The addition of 1 µM
thapsigargin induced the depletion of ER calcium (inset).
LTB4 (100 nM)-induced intracellular calcium
increase was inhibited subsequently (n = 4).
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With the same cell line, LTB4 induced a
concentration-dependent increase in the intracellular
calcium levels (EC50 = 4.4 nM; Fig.
3B). 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).
BLT Receptor Binding in Leukotriene-deficient
Mice--
Previously, we (29) and another group (35) 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).

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Fig. 4.
Absence of leukotrienes in
5-lipoxygenase-deficient mice does not appreciably alter BLTR binding
to neutrophil membranes. Wild type (WT) and
5-lipoxygenase-deficient ( / ) male mice (five each) were injected
intraperitoneally with 1% sterile glycogen. Neutrophils were obtained
by peritoneal lavage 5 h later. Crude membranes were prepared and
binding of [3H]LTB4 examined in
duplicate.
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Is the BLT Receptor a Coreceptor for HIV/SIV Entry into
CD4-expressing Cells?--
Primate lentiviruses utilize CD4 and a
coreceptor (most often the chemokine receptors CCR5 and CXCR4) to enter
target cells (20). 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
(36). 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 (37). 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 (22). 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-clone 3, and sm62B
were unable to use h-BLTR to infect cells. In separate experiments using transiently transfected HEK 293 cells the number of specific binding sites was found to be approximately 90 fmol/mg of protein.

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Fig. 5.
Infection assay using luciferase reporter
viruses to test for h-BLT coreceptor function. 293T or feline CCCS
cells were transfected with the indicated coreceptors and CD4 and
infected with luciferase reporter viruses pseudotyped with the SIV and
HIV-1 Env proteins listed. Data are presented from a representative
experiment. Infections were performed two or three times depending on
the Env protein.
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The results described above indicate that the h-BLTR does not serve as
a coreceptor for the virus strains tested or does so only
inefficiently. We therefore employed a more sensitive cell-cell fusion
assay (Fig. 6) in an attempt to detect
h-BLT coreceptor activity. We have found that coreceptors that support
virus infection also support Env-mediated cell-cell fusion. However,
there are cases when a given Env-coreceptor combination supports
cell-cell fusion but does not support virus infection (38, 39). Cells expressing the indicated Env proteins (including four primary HIV-1
strains that utilize CXCR4: 89.6, UG024.2, 93ZR001.3, and DH12) were
mixed with cells expressing CD4 and either CCR5, CXCR4, or h-BLTR.
Cell-cell fusion was quantified 7 h later using a gene-reporter system (30, 31). Although fusion was readily observed with the major
HIV-1 coreceptors, fusion using this sensitive assay was not observed
when CD4 and h-BLTRs were coexpressed. Thus, the h-BLTR did not
function as a coreceptor for either virus infection or Env-mediated
cell-cell fusion.

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Fig. 6.
h-BLTR does not function as an HIV coreceptor
in cell-cell fusion assays. Quail QT6 target cells were
transfected with plasmids expressing CD4, a "coreceptor" (CXCR4,
CCR5 or h-BLTR), and luciferase under control of the T7 promoter. 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. Cell-cell fusion results in
cytoplasmic mixing and luciferase production, measured as relative
light units (RLU). Results depict one representative
experiment (n = 2) performed in triplicate.
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DISCUSSION |
The high level of sequence identity for the m-BLTR gene cloned
here with the human sequences R2 (17), CMKRL1 (16), BLTR (3), and the
clone classified as a P2Y7 purinergic receptor (19)
combined with the binding studies confirm the fact that these are mouse
and human orthologs of the BLTR. Additional confirmation comes from a
recent independent cloning of m-BLTR cDNA from murine eosinophils
(34). LTB4 affinity for m-BLTR was similar to that for
h-BLTR (0.24 versus 0.154 nM, respectively). The
NH2 terminus as well as the three putative extracellular
loops of the receptor show the lowest percentage identity between
species (between 25 and 75% identity), in contrast with the high
percent identity for the transmembrane domains (between 86 and 95%
identity) and the intracellular loops (between 61 and 100%). The third
intracellular loop of the m-BLTR, which shows two putative protein
kinase C phosphorylation sites, is identical to the human sequence.
These phosphorylation sites could be critical in the process of
homologous desensitization of the BLTR that has been established in
neutrophils (40), eosinophils (41), and granulocytes (42). The
potential protein kinase C as well the casein kinase 2 (a Ser/Thr
protein kinase) phosphorylation sites of the COOH-teminal loop could
also be involved in the regulation of the activation/desensitization of
the receptor or in the uncoupling of the receptor to G protein.
The rank order of LTB4 binding affinity and of related
compounds for the m-BLTR is similar to the h-BLTR (3) and consistent with previous experiments using guinea pig lung membranes (43) and
porcine spleen membranes (44). 20-Hydroxy-LTB4, a
metabolite of LTB4, was the most potent competitor after
LTB4. There was a discrepancy in the efficiency of
LTB4 to induce an increase in Cai2+
and to inhibit cAMP production in CHO cells. There was also a distinction in the ability of LTB4 to inhibit cAMP
production in CHO cells versus melanophores. These
differences could be caused by different affinities of the receptor for
Go- and Gi-like proteins or by a difference in
the type of G protein coupled to the m-BLTR in these two cell types.
The 5'-upstream sequence of the m-BLTR gene did not contain a TATA box
but revealed the presence of putative consensus sequences for binding
various transcription factors, including myeloid-specific factors,
which could be implicated in the regulation of transcription of the
m-BLTR. There was also a repetitive poly(A) sequence whose role remains
to be defined.
The alteration of LTB4 synthesis in vivo could
modify m-BLTR expression. However, we found that LTB4
binding to neutrophil membranes from 5-lipoxygenase knockout mice, who
do not synthesize any leukotrienes, and from wild type mice were
similar. In some pathological situations (45, 46), a modification in
the level of BLTR binding has been demonstrated. The functional
characterization of the promoter region, combined with mutation and
deletion studies of this region, would help to elucidate transcription
factors involved in the regulation of transcription of m-BLTR, and
potentially link the results to inflammatory pathologies where
LTB4 is involved. The recent discovery of high expression
of m-BLTR in eosinophils (34) will be important to explore in the
context of airway inflammation models.
In addition to CCR5 and CXCR4, a host of other chemokine and orphan
seven-transmembrane domain receptors have been shown to support
infection by smaller numbers of HIV or SIV strains (for review, see
Ref. 37). Recently, Owman et al. (22) reported that the
h-BLTR functions as an efficient coreceptor for a number of virus
strains, especially primary virus isolates that utilize CXCR4. However,
we were unable to detect coreceptor activity for any of the 16 SIV and
HIV-1 Env proteins examined using either virus infection or a sensitive
cell-cell fusion assay with h-BLTR. There are a number of possible
explanations for these discrepancies. First, it is simply possible that
h-BLTR may function as a coreceptor for the strains tested by Owman
et al. (22) but does not function as a coreceptor for the
strains tested here. Second, h-BLT coreceptor function may be dependent
upon the cell type or assay used. Owman et al. (22) used a
PCR-based entry assay, which may be overly sensitive, whereas we used a
reporter virus infection and cell-cell fusion assays. It will be
important to demonstrate h-BLT coreceptor function in stable cell lines
using more standard methods of viral quantification, such as p24
measurements. Third, coreceptor function can be highly dependent upon
surface expression levels (47, 48). Reagents are not yet available
which make it possible to measure BLTR surface expression accurately.
It is possible that we expressed suboptimal amounts of plasma membrane
BLTR in our system, although this receptor did express well for binding
studies in transiently transfected HEK 293 cells. Regardless of the
explanation, the importance of h-BLTR as a viral coreceptor will rest
upon the number and types of virus strains that can use it for
infection, the levels at which it is expressed in vivo, and
the CD4-positive cell types in which it is expressed. Only additional
work will clarify these points.