From the ¶ Phagocyte Research Laboratory, Department of
Medical Microbiology and Immunology, University of Göteborg,
S-40530 Göteborg, Sweden, the
Département de Biologie Moléculaire et
Structurale/Biochimie et Biophysique des Systèmes Integrés
(UMR 5092, Commissariat à l'Energie Atomique
(CEA)/CNRS/Université Joseph Fourier), CEA/Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France, and
CEA/Saclay,
Departement d'Ingenierie et d'Etudes des Protéines,
Bâtiment 152, 91191 Gif-sur-Yvette, France
Received for publication, August 25, 2000, and in revised form, March 29, 2001
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ABSTRACT |
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Neutrophils express the G
protein-coupled N-formyl peptide receptor (FPR) and its
homologue FPRL1, whereas monocytes express FPR, FPRL1, and FPRL2, an
orphan receptor sharing 83% amino acid identity with FPRL1. FPRL1 is a
promiscuous receptor activated by serum amyloid A and by different
synthetic peptides, including the hexapeptide
Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm). By
measuring calcium flux in HL-60 cells transfected with FPR, FPRL1, or
FPRL2, we show that WKYMVm activated all three receptors, whereas the L-conformer WKYMVM activated exclusively FPRL1 and FPRL2.
The functionality of FPRL2 was further assessed by the ability of HL-60-FPRL2 cells to migrate toward nanomolar concentrations of hexapeptides. The half-maximal effective concentrations of WKYMVM for
calcium mobilization in HL-60-FPRL1 and HL-60-FPRL2 cells were 2 and 80 nM, respectively. Those of WKYMVm were 75 pM
and 3 nM. The tritiated peptide
WK[3,5-3H2]YMVM bound to FPRL1
(KD ~ 160 nM), but not to FPR. The
two conformers similarly inhibited binding of 125I-labeled
WKYMVm to FPRL2-expressing cells (IC50 ~ 2.5-3
µM). Metabolic labeling with orthophosphoric acid
revealed that FPRL1 was differentially phosphorylated upon addition of
the L- or D-conformer, indicating that it
induced different conformational changes. In contrast to FPRL1, FPRL2
was already phosphorylated in the absence of agonist and not evenly
distributed in the plasma membrane of unstimulated cells. However, both
receptors were internalized upon addition of either of the two
conformers. Taken together, the results indicate that neutrophils are
activated by WKYMVM through FPRL1 and that FPRL2 is a chemotactic
receptor transducing signals in myeloid cells.
The extravasation of leukocytes from the peripheral blood stream
to inflammatory sites is a key feature in the innate immune response to
infection (1). Different chemoattractants (e.g. N-formylated peptides, C5a, interleukin-8, leukotriene
B4, and platelet-activating factor) and chemokines
induce leukocyte infiltration and activation through binding to G
protein-coupled seven-transmembrane cell-surface receptors (2, 3). The
chemoattractant-mediated dissociation of G Two synthetic hexapeptides, Trp-Lys-Tyr-Met-Val-Met-NH2
(WKYMVM) and Trp-Lys-Tyr-Met-Val-D-Met-NH2
(WKYMVm),that stimulate phosphoinositide hydrolysis in myeloid
cells were identified by screening a peptide library (10, 11). The
D-methionine-containing hexapeptide (WKYMVm) was found to
be a very potent activator of several leukocyte effector functions such
as chemotaxis, mobilization of complement receptor-3, and activation of
the NADPH oxidase (11). The peptide WKYMVm activates neutrophils
through both the N-formyl peptide receptor
(FPR)1 and FPRL1
(N-formyl peptide
receptor-like-1) (12, 13). The latter was originally cloned from human phagocytes by low-stringency hybridization of a cDNA library with the FPR cDNA sequence, and it was initially defined as an orphan receptor (14-16). FPRL1 was later referred to as the LXA4 receptor since it was shown
to bind lipoxin A4 with high affinity (17). In addition,
several different peptides/proteins have been reported to stimulate
this receptor. These include a leucine zipper-like domain of the HIV-1
envelope gp41 (18); a peptide derived from the HIV-1 envelope gp120
(19); serum amyloid A, a protein secreted during the acute phase of inflammation (20); the mitochondrial necrotactic peptide MYFINILTL (21); LL-37, a cleaved antimicrobial peptide of a human antimicrobial protein of the cathelicidin family (22); and the neurotoxic prion
peptide fragment 106-126 (23). The binding of the peptide/protein agonists to FPRL1 results in a G protein-mediated signaling cascade leading to chemotaxis, mobilization of granules, and release of reactive oxygen species. Thus, in many ways, the FPR and
FPRL1/LXA4 receptors show great similarities in their
signaling abilities.
Based on the knowledge that the L-methionine-containing
peptide WKYMVM (but not N-formyl-Met-Leu-Phe) is able to
activate phosphoinositide hydrolysis in the human myeloma B-cell line
U266, it has been suggested that WKYMVM activates a receptor distinct from FPR (10). Here, we show that radioactive WKYMVM binds to FPRL1,
but not to FPR. The hexapeptides WKYMVM and WKYMVm induced a different
pattern of phosphorylation and internalization of FPRL1 in transfected
cells, suggesting that the two peptides bring about different
structural changes in the receptor. In addition, we show that the
L- and D-conformers elicit both calcium flux in
and chemotaxis of undifferentiated HL-60 cells transfected with the
FPRL2 (N-formyl peptide
receptor-like-2). Thus, this orphan
receptor, which shares 83% amino acid identity with FPRL1 and is
expressed on monocytes, but not on neutrophils or differentiated neutrophil-like HL-60 cells (24), is a functional chemotactic receptor.
Isolation of Human Neutrophils--
Blood neutrophils were
isolated from buffy coats from healthy blood donors using dextran
sedimentation and Ficoll-Paque gradient centrifugation (25). All cells
were washed and resuspended (1 × 107/ml) in KRG
buffer (Krebs-Ringer phosphate buffer containing 10 mM
glucose, 1 mM Ca2+, and 1.5 mM
Mg2+, pH 7.3).
Peptides and Peptide Receptor Antagonists--
The hexapeptide
Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) was synthesized and
HPLC-purified by Alta Bioscience (University of Birmingham, Birmingham,
United Kingdom). Diiodinated Trp-Lys-Tyr-Met-Val-Met-NH2 was synthesized and HPLC-purified by Neosystem (Strasbourg, France). The formylated peptides
N-formyl-methionyl-leucyl-phenylalanine (fMLF) and
N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK) were
from Sigma. Cyclosporin H was kindly provided by Novartis Pharma
(Basel, Switzerland). Recombinant serum amyloid A was from PeproTech
Inc. The peptide agonists and antagonists were dissolved in dimethyl
sulfoxide to 10 Stable Expression of FPR, FPRL1, and FPRL2 in Undifferentiated
HL-60 Cells--
The stable expression of FPR and FPRL1 in
undifferentiated cells has been previously described (13). The cDNA
encoding FPRL2 was cloned by polymerase chain reaction from genomic DNA
isolated from undifferentiated HL-60 cells using the sense primer
5'-cgggatccgcagacaagatggaaaccaacttctccattcc-3' and the reverse primer
5'-gctctagagctcacattgcttgtaactccgtctcctc-3'. After cleavage with
BamHI and XbaI, the polymerase chain reaction product was cloned in pcDNA3.1 and completely sequenced. The FPRL2 cDNA was further ligated into the pEFneo plasmid cleaved by
XbaI (26). Transfection of HL-60 cells was performed by
electroporation with a Bio-Rad Gene Pulser apparatus according to a
slightly modified version (27) of the technique described by Tonetti
et al. (28). Following electroporation, cells were allowed
to recover in 20 ml of culture medium for 48 h prior to selection
in a medium containing G418 (1 mg/ml). G418-resistant clones were
obtained by limited dilution into 24-well microtiter plates, and
FPRL2-expressing clones were identified for their ability to mobilize
intracellular calcium upon addition of WKYMVm (100 nM final concentration). Cells were cultured in RPMI 1640 medium/glutaMax I, and the maximal density was maintained below
2 × 106 cells/ml. Cells were centrifuged at each passage.
Stable Overexpression of FPR, FPRL1, and FPRL2 in RINm5F
Cells--
The stable expression of FPR in the insulin-secreting cell
line RINm5F has been previously described (29). The
FPR-expressing RINm5F cells expressed ~1.4 × 106 receptors/cell. FPRL1- and FPRL2-expressing RINm5F
cells were obtained by transfecting cells with 1 µg of pSV2neo DNA
and 20 µg of CDM8-FPRL1 or with 2 µg of pEFneo-FPRL2,
respectively. Cells were cultured in RPMI 1640 medium/glutaMax I with
400 µg/ml G418 and 10% heat-inactivated fetal calf serum. Resistant
clones were screened for FPRL1 or FPRL2 expression by
immunofluorescence using a rabbit polyclonal antibody directed against
the last 10 amino acid residues of FPRL1.
Preparation of Tritiated Hexapeptide
Trp-Lys-[3,5-3H2]Tyr-Met-Val-Met-NH2--
Diiodinated
Trp-Lys-Tyr-Met-Val-Met-NH2 (1.1 mg, 1 mmol) was treated
overnight with 99.5% tritium gas (4.2 hectopascal) over palladium oxide (5 mg) in freshly dried methanol (2 ml) and
triethylamine (30 ml). After elimination of the residual tritium gas
under reduced pressure, the catalyst was removed by filtration through
a Millipore FG filter. The filtrate was evaporated under vacuum and
rinsed three times with methanol. The resulting crude product (7.1 mCi) was dissolved in methanol (7.2 ml). Purification by analytical reverse-phased HPLC (Vydac C8 column, flow rate of 1 ml/min, linear gradient of 0.1% trifluoroacetic acid in water
to 0.01% trifluoroacetic acid in acetonitrile from 18:82 to 50:50 in
30 min) gave pure tritiated Trp-Lys-Tyr-Met-Val-Met-NH2
(0.308 mCi) with a specific activity of 8.5 Ci/mmol, which was compared
with an authentic sample of Trp-Lys-Tyr-Met-Val-Met-NH2.
Chemical and radiochemical purities were assessed by analytical HPLC
(same conditions as above). The retention time for
Trp-Lys-Tyr-Met-Val-Met-NH2 was 12.52 min; the purity was
99%; and the retention time for diiodinated Trp-Lys-Tyr-Met-Val-Met-NH2 was 19.68 min.
Peptide Iodination--
The highly potent FPRL1 agonist
Trp-Lys-Tyr-Met-Val-D-Met-NH2 was labeled with
125I using the solid-phase oxidizing agent IODO-GEN (30).
Briefly, 10 nmol of IODO-GEN were solubilized in 100 µl of
dichloromethane and coated as a film on the wall of a polypropylene
iodination vial by evaporation to dryness in a desiccator. Sodium
phosphate buffer (0.01 M, 75 µl, pH 8.0) was added to the
vial, followed by Na125I (1 mCi) and 4 nmol of
Trp-Lys-Tyr-Met-Val-D-Met-NH2 in 25 µl of
sodium phosphate buffer. The iodination was allowed to proceed for 10 min and terminated by the addition of 500 µl of water. After 5 min,
the diluted mixture was passed through a Waters Sep-Pak µC18 cartridge, followed by a wash with 10 ml of
potassium iodide (5 mM) and 10 ml of water. The cartridge
was then turned upside down, and the 125I-labeled peptide
was eluted with 2 ml of methanol. The iodinated peptide migrated with
RF = 0.35 on a Whatman Silica Gel K6 plate with a
solvent system of methanol/water/acetic acid (20:70:10, v/v/v). When
higher amounts of IODO-GEN were used, the peptide was oxidized during
the iodination procedure, and species with smaller
RF values were observed. The iodinated peptide could
be stored for several weeks at Binding Assays of Cells Stably Expressing FPR, FPRL1, or
FPRL2--
Equilibrium binding in RINm5F cells expressing either FPR
or FPRL1 was essentially performed on adherent cells as previously described (31). Briefly, cells were incubated with increasing concentrations of
Trp-Lys-[3,5-3H2]Tyr-Met-Val-Met-NH2
in Dulbecco's modified Eagle's medium and 0.5% bovine serum albumin
supplemented or not with an excess of nonradioactive peptide for 1 h at 4 °C. Plates were washed three times with chilled
phosphate-buffered saline; cells were solubilized in scintillation
mixture; and the radioactivity bound to the cells was quantified with a
Metabolic Labeling and Immunoprecipitation--
RINm5F cells
expressing either FPRL1 or FPRL2 were metabolically labeled with
[32P]orthophosphoric acid (0.3-0.5 mCi/ml) in 6-well
plates as previously described (32). Undifferentiated HL-60-FPRL2 cells
were metabolically labeled in suspension as previously described (33).
About 40 × 106 HL-60-FPRL2 cells were required for
each condition of immunoprecipitation. Receptor phosphorylation was
initiated by the addition of either WKYMVm or WKYMVM, and
phosphorylated receptors were immunoprecipitated with affinity-purified
rabbit IgGs directed against the last 10 residues of FPRL1 (34).
Neutrophil NADPH Oxidase Activity--
The NADPH oxidase
activity was determined using an isoluminol-enhanced chemiluminescence
system (35). The chemiluminescence activity was measured in a
six-channel Biolumat LB 9505 apparatus (Berthold Co., Wildbad, Germany)
using disposable 4-ml polypropylene tubes with a 900-µl reaction
mixture containing 1-2 × 105 neutrophils,
horseradish peroxidase (4 units), and isoluminol (2 × 10 Determination of Changes in Cytosolic Calcium in HL-60 Cells
Expressing FPR, FPRL1, or FPRL2--
HL-60 cells at a density of
1-3 × 106 cells/ml were washed with
phosphate-buffered saline. The cell pellets were resuspended at a
density of 2 × 107 cells/ml in RPMI 1640 medium
without phenol red and containing 0.1% BSA and loaded with 2 µM Fura-2/AM (Molecular Probes, Inc., Eugene, OR) for 30 min at 37 °C. Cells were then diluted with 2 volumes of the same
medium without BSA, washed once with KRG buffer, and resuspended in
RPMI 1640 medium without phenol red at a density of 2 × 107 cells/ml. Calcium measurements were carried out with a
Spex FluoroMAX fluorescence spectrophotometer with an excitation
wavelength of 340 nm; an emission wavelength of 505 nm; and slit widths
of 5 and 10 nm, respectively. Intracellular free calcium concentrations were calculated using the following formula:
[Ca2+]i = KD(F Chemotaxis Assay--
Undifferentiated HL-60 cells stably
transfected with pEFneo or pEFneo-FPRL2 were centrifuged, resuspended
in fresh complete RPMI 1640 medium (106 cells/ml), and
incubated overnight with 10 µg/ml
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Molecular Probes, Inc.). Cells were centrifuged and washed
once with complete medium and twice with chemotaxis buffer (RPMI 1640 medium supplemented with 1% BSA and 20 mM HEPES, pH 7.5).
2 × 105 cells in 100 µl of chemotaxis buffer were
loaded into the upper chamber of FluoroBlok inserts of 3-µm pore size
(Becton Dickinson, Le Pont-de-Claix, France). 500 µl of chemotaxis
buffer with or without the WKYMVm hexapeptide (at concentrations
indicated in the figures) were loaded in the lower chamber. The
FluoroBlok inserts were incubated at 37 °C for 5 h. Migrating
cells dropped from the filters to the bottom of the lower chamber. They
were counted in five fields (magnification × 100) using an
inverted fluorescence microscope equipped with an image analyzer
(SP-Eye, Photonic Science). The experiments were performed six
times, and the results are presented as chemotaxis indexes (number of
cells migrating in response to the chemoattractant divided by the
number of HL-60-FPRL2 cells spontaneously migrating toward control
medium) for different chemoattractant concentrations.
Microscopic Analysis of Internalization of FPRL1 and
FPRL2--
Ligand-mediated internalization of FPRL1 and FPRL2 was
followed by an indirect immunofluorescence microscopy staining
technique. Cells were seeded on polyornithine-coated coverslips 48 h prior to the experiments. To stop protein synthesis, cells were
incubated in RPMI 1640 medium containing 1% BSA and 100 µg/ml
cycloheximide for 3 h and maintained in the same medium throughout
the experiment. To study receptor internalization, RINm5F-FPRL1 cells
and RINm5F-FPRL2 cells were treated with either WKYMVM or WKYMVm at
37 °C for various time periods ranging from 0 to 30 min. Cells were
finally fixed and permeabilized with acetone at Peptide-induced NADPH Oxidase Activity in Neutrophils--
The
formylated peptide fMLF is a potent activator of the neutrophil NADPH
oxidase, and the generated reactive oxygen species are secreted from
the cells. When exposing neutrophils to WKYMVM, a similar superoxide
production was achieved (Fig. 1). The
time courses of the responses induced by the two peptides were very similar, but WKYMVM was a somewhat less potent activator, with an
EC50 of 75 nM compared with 50 nM
for fMLF (Fig. 1, inset).
The fMLF antagonist cyclosporin H is known to block FPR-evoked
responses (Refs. 37 and 38; for a review, see Ref. 3). Accordingly,
cyclosporin H (1 µM) blocked the NADPH oxidase activity induced by fMLF, whereas the inhibitor had no effect on the
response induced by WKYMVM (data not shown). To further determine the
specificity in the activation route for the WKYMVM and fMLF peptides,
we performed cross-desensitization experiments. Upon binding of fMLF to
its receptor, the occupied receptor is phosphorylated (33) and
interacts with the cytoskeleton (39-41). Cells are subsequently
desensitized and unable to generate oxidase-activating signals through
the same receptor. As illustrated in Fig.
2A, neutrophils stimulated with fMLF were unable to generate a second burst of superoxide when
challenged 10 min later with the same agonist (homologous desensitization). However, a second superoxide burst was observed when
cells were first stimulated with fMLF and then challenged with WKYMVM
(Fig. 2B). When the first stimulus was WKYMVM, no second
respiratory burst was observed in response to the same agonist
(homologous desensitization) (Fig. 2C), but a response was
obtained with fMLF (Fig. 2D). Thus, activation by one of the peptides did not induce desensitization to the other peptide (no heterologous or cross-desensitization), suggesting, as already proposed
by Baek et al. (10), that the two peptides act through different receptors.
Peptide-induced Activation of FPR or Its Homologues (FPRL1 and
FPRL2) in Transfected HL-60 Cells--
In addition to the
high-affinity FPR, human neutrophils express a structurally related
receptor originally known as FPRL1 (15). The second homologue (FPRL2)
is 83% identical to FPRL1, but its capacity to transduce signals has
not yet been assessed (24). FPRL2 is not expressed in neutrophils or
neutrophil-like HL-60 cells, but is present on monocytes (24). FPRL1 is
promiscuous and binds several different peptides, including the
D-methionine-containing chemoattractant WKYMVm (12, 13).
The hexapeptide WKYMVm is at least 6000-fold more potent than fMLF for
the activation of FPRL1. However, it can also activate phagocytes
through FPR (12, 13). To investigate whether WKYMVM is able to activate
receptors of the FPR family (FPR, FPRL1, and FPRL2), we stably
expressed these receptors in myeloid HL-60 cells. Although WKYMVM has
been shown to activate phosphoinositide hydrolysis in undifferentiated HL-60 cells (10), we did not observed a WKYMVM-mediated calcium mobilization in control cells, i.e. naive non-transfected
cells or cells transfected with CXCR2 (data not
shown).2 Likewise, no
calcium rise was observed when FPR-expressing cells were
stimulated with 100 nM WKYMVM, whereas an increase in
intracellular calcium concentration was observed when cells were
stimulated with fMLFK (Fig.
3A). In contrast, WKYMVM
induced a rise in intracellular calcium in FPRL1-expressing cells (Fig.
3B). The FPR antagonist cyclosporin H (1 µM)
had no inhibitory effect on the WKYMVM-induced calcium mobilization in
HL-60-FPRL1 cells (data not shown). In FPRL1-expressing cells, WKYMVM
mobilized calcium with an EC50 of ~2 nM,
whereas WKYMVm had an EC50 of ~75 pM (Fig.
4). We thus conclude that WKYMVM is
~25-fold less potent than WKYMVm; but, in contrast to the latter, it
appears to stimulate cells without activating FPR.
The three peptides fMLFK, WKYMVM, and WKYMVm were further tested on
HL-60 cells that stably expressed FPRL2. In a previous study, Durstin
et al. (24) have shown the lack of fMLF-induced calcium
mobilization in frog oocytes injected with FPRL2 cRNA. Consistently, no
fMLFK-induced response could be obtained in HL-60-FPRL2 cells. However,
both WKYMVM and WKYMVm were able to induce a sustained increase in
intracellular calcium (Fig. 3, C and D) with
EC50 values of ~80 and 3 nM, respectively
(Fig. 4). Interestingly, although 1 µM recombinant serum
amyloid A elicited a marked increase in intracellular calcium (~200
nM) in HL-60-FPRL1 cells, no calcium flux could be observed
in HL-60-FPRL2 cells (data not shown), indicating that HL-60-FPRL2
cells do not constitutively expressed FPRL1. Thus, the ability of the
hexapeptides to induce calcium mobilization demonstrates for the first
time that the orphan monocyte-expressed receptor FPRL2 transduces signals.
To further assess the functionality of FPRL2 in myeloid cells, we
examined the ability of WKYMVm to elicit the migration of HL-60-FPRL2
cells through 3-µm pores into a chamber containing various
concentrations of hexapeptide. Although naive HL-60 cells did not
migrate to the lower chambers, cells expressing FPRL2 migrated in a
bell-shaped dose-dependent manner in response to hexapeptide concentrations ranging from 1 nM to 1 µM. Maximal cell migration was observed at 10-50
nM WKYMVm (Fig. 5) and
500-1000 nM WKYMVM (data not shown). To make sure
that migration to the lower chamber was not due to chemokinesis, we
added increasing concentrations of hexapeptide to the upper chamber.
This did not increase spontaneous migration toward the control medium,
but prevented migration to lower chambers containing the hexapeptide (data not shown), suggesting that migration of HL-60-FPRL2 cells in a
concentration gradient is chemotaxis rather than chemokinesis.
Binding of Trp-Lys-Tyr-Met-Val-Met-NH2 and
Trp-Lys-Tyr-Met-Val-D-Met-NH2 to FPRL1 and
FPRL2--
The binding parameters of the hexapeptides were determined
with RINm5F cells that stably expressed either of the FPRs. The inability of WKYMVM to bind to FPR was assessed with the tritiated hexapeptide
Trp-Lys-[3,5-3H2]Tyr-Met-Val-Met-NH2.
The FPR-expressing cell line (RIN9815) has previously been shown to
expose 1.4 × 106 receptors/cell and to bind an
N-formyl peptide derivative (42) with a
KD of 21 nM (29). Despite a high level
of expression of FPR in this cell line, no specific binding of
[3H]WKYMVM could be measured for concentrations ranging
from 1 to 300 nM (Fig. 6).
Specific binding was easily measured with RINm5F-FPRL1 cells, where
>95% of the bound radioactivity could be displaced by an excess of
nonradioactive peptide (Fig. 6). Plotting the data from the equilibrium
binding study according to Scatchard revealed a KD
of 160 nM and a level of expression of ~1.8 × 106 receptors/cell (Fig. 6, inset). Due to the
low specific radioactivity of the tritiated ligand and to a low level
of surface receptor expression, we were unable to determine the binding
parameters of [3H]WKYMVM with HL-60-FPRL1 cells. However,
a competitive displacement assay showed that the half-maximal
concentration of WKYMVM to displace 125I-labeled
Trp-Lys-Tyr-Met-Val-D-Met-NH2 was ~120-180
nM (data not shown). This is consistent with the
KD value determined with RINm5F-FPRL1
cells.
The calcium mobilization assay suggests that the L- and
D-methionine-containing hexapeptides have a lower affinity
for FPRL2 than for FPRL1. We therefore determined the concentration
of each peptide that half-maximally inhibited the binding of
125I-labeled
Trp-Lys-Tyr-Met-Val-D-Met-NH2 in RINm5F-FPRL1
and RINm5F-FPRL2 cells. As shown in Fig.
7, WKYMVm was the best competitor for binding of 125I-labeled WKYMVm to FPRL1 (IC50 ~ 30-40 nM versus 130-150 nM for WKYMVM). This result is consistent with the observation that the half-maximal concentration of WKYMVm to mobilize calcium in HL-60-FPRL1 cells is lower than that of the L-methionine-containing
conformer. Although WKYMVm was more potent than WKYMVM for the
induction of a calcium increase in FPRL2-expressing HL-60 cells, the
two hexapeptides had a similar ability to compete for the binding of
125I-labeled WKYMVm to FPRL2 (IC50 ~ 2.5-3
µM) (Fig. 7). Thus, the data indicate altogether
that the hexapeptide WKYMVM is able to specifically bind to and
activate neutrophils through FPRL1 and that both L- and
D-methionine-containing hexapeptides are low-affinity agonists for the monocyte-restricted receptor, FPRL2.
Hexapeptide-induced Phosphorylation of FPRL1 and FPRL2--
We further examined whether FPRL1 and FPRL2 were phosphorylated
upon addition of either of the two hexapeptides. After metabolic labeling with [32P]orthophosphoric acid, cells were
incubated for various periods of time with no agonist, WKYMVM, or
WKYMVm. After cell lysis, receptors were immunoprecipitated with
affinity-purified rabbit immunoglobulins directed against the last 10 residues (PPAETELQAM) of FPRL1. These IgGs were found to
cross-react with the corresponding sequence in FPRL2 (PPEETELQAM), but
not with the C-terminal sequence of FPR (LPSAEVELQAK).
In the absence of ligand binding, there was no basal phosphorylation of
FPRL1. After the addition of the L- or
D-hexapeptide, FPRL1 was rapidly phosphorylated. As shown
in Fig. 8A, the time courses
of the responses induced by the two peptides were very similar
(t1/2 ~ 5-10 min). The immunoprecipitated materials migrated on SDS-polyacrylamide gel as two radioactive species
with relative molecular masses of 50 and 100 kDa (Fig. 8B).
The band at 100 kDa is specific of FPRL1 and may represent a dimeric
form of the receptor since it was absent from immunoprecipitates that
derived from stimulated RINm5F-FPRL2 cells or HL-60-FPRL2 cells (see
Fig. 9). Such a pattern of migration is very common with G
protein-coupled receptors (GPCRs) whether receptors are glycosylated or
not. Dimeric species have been documented for a number of GPCRs,
including the
Unexpectedly, increasing concentrations of either WKYMVM or WKYMVm had
a differential effect on the phosphorylation of FPRL1. Although FPRL1
phosphorylation steadily increased with the concentration of WKYMVM,
the level of receptor phosphorylation reached a maximum at 0.1 µM WKYMVm and then decreased at higher concentrations
(Fig. 8B). The same discrepancy in the pattern of
phosphorylation was observed when FPRL1 was expressed in Chinese
hamster ovary cells (data not shown).
Likewise, RINm5F-FPRL2 cells were stimulated with increasing
concentrations of either WKYMVM or WKYMVm. Although FPRL2 and FPRL1
have the same number of amino acids, FPRL2 had a slower electrophoretic
mobility, which is consistent with an additional glycosylation site in
the N-terminal region. In the absence of stimulation, FPRL2 was
immunoprecipitated as an already phosphorylated protein that migrated
on SDS-polyacrylamide gel with a relative molecular mass of 70-80 kDa
(Fig. 9A). This band was not
recovered from lysates of RINm5F-FPRL1 cells (see Fig. 8B).
As in the case of FPRL1, phosphorylated species with higher molecular
masses were detected that may represent a dimeric form or receptor
aggregates. After the addition of either of the two hexapeptides, the
level of receptor phosphorylation increased only slightly. A similar pattern of phosphorylation was observed when FPRL2 was
immunoprecipitated from lysates of undifferentiated HL-60-FPRL2 cells
(Fig. 9B). This suggests that the high level of FPRL2
phosphorylation in unstimulated cells is not linked to receptor
overexpression since HL-60-FPRL2 cells expressed physiologic amounts of
receptors (104 to 2 × 104
receptors/cells). There was no trace of phosphorylated FPRL1 present in
the immunoprecipitates from lysates of undifferentiated HL-60 and
HL-60-FPRL2 cells, confirming that FPRL1 is not constitutively expressed in undifferentiated HL-60 cells and undifferentiated HL-60-FPRL2 cells.
Hexapeptide-mediated Internalization of FPRL1 and FPRL2 in RINm5F
Cells--
Previous studies with chemoattractant and chemokine
receptors have indicated that the phosphorylation of the receptor
C-terminal domain is a key step for agonist-mediated receptor
internalization (32, 45-47). Therefore, we examined the subcellular
distribution of FPRL1 and FPRL2 in RINm5F cells by confocal
immunofluorescence microscopy. A complete absence of staining was
observed with non-transfected RINm5F cells or cells transfected with
FPR, indicating that there was no cross-reactivity of the antibody with
the C-terminal extremity of FPR (data not shown). In the absence of
agonist, FPRL1 remained evenly distributed at the cell surface, as
illustrated by the bright fluorescent staining of the plasma membrane
(Fig. 10, A and
B, upper left panels). The addition of WKYMVM
rapidly led to the formation of small receptor-containing vesicles that
moved to the subcortical area after 10 min. Larger vesicles that
accumulated in the perinuclear region were observable by 20 and 30 min.
When cells were stimulated with WKYMVm, a majority of the cells still presented a bright fluorescent staining of the plasma membrane after 10 min (Fig. 10B, upper right panel). By 20 min,
subcortical and cytoplasmic fluorescent vesicles were nevertheless
observable; and by 30 min, the perinuclear region was brightly stained
in a majority of the cells (Fig. 10B, lower
panels). Thus, despite an apparently poor ability to induce FPRL1
phosphorylation, receptors are internalized upon WKYMVm
application.
Likewise, the hexapeptide-mediated internalization of FPRL2 was
examined after stable expression in RINm5F cells (Fig.
11). Unlike FPRL1, FPRL2 was not evenly
distributed at the plasma membrane of unstimulated cells. A punctate
fluorescence at or underneath the plasma membrane was observed, which
may represent receptors already in endocytic vesicles. In ~10-20%
of the cells, fluorescent vesicles were seen farther down in the
cytoplasm. The addition of either L- or
D-methionine-containing peptide led to the accumulation of
fluorescent cytoplasmic vesicles that were best observed in a majority
of the cells after 20 min. Thus, like FPRL1, FPRL2 is internalized upon
binding of either of the two hexapeptides. This provides additional
support to the notion that FPRL2 is a functional receptor that can be
activated by the synthetic hexapeptides WKYMVM and WKYMVm and transduce
signals similar to those mediated through FPR and FPRL1.
The G protein-coupled seven-transmembrane receptors FPR, FPRL1,
and FPRL2 belong to the N-formyl peptide chemoattractant
receptor family (48). The first two of them have been identified in
both neutrophils and monocytes, whereas FPRL2 is expressed in
monocytes, but not in neutrophils and differentiated HL-60 cells (24). The prototypical chemoattractant receptor FPR binds to fMLF with high
affinity, whereas the homologue FPRL1 binds the same agonist with very
low affinity (16). Regarding FPRL2, no known natural or synthetic
ligand has thus far been documented. Here, we show that FPRL2 can
trigger calcium mobilization in myeloid cells following application of
nanomolar concentrations of the newly described neutrophil and monocyte
synthetic activators,
Trp-Lys-Tyr-Met-Val-D/L-Met-NH2 (WKYMVm/M) (11).
In the past few years, several studies have shown that FPRL1 is able to
bind a broad spectrum of ligands that do not bear any sequence
homologies to each other or to the classical FPR agonist fMLF.
LXA4, a lipid mediator originally reported to inhibit neutrophil activities mediated by other chemoattractants (for example,
fMLF-induced chemotaxis and granule secretion), was first described as
a high-affinity ligand for FPRL1. However, LXA4 has not
been documented to initiate chemotaxis of and intracellular calcium
increases in either neutrophils (17) or FPRL1-transfected HEK293 cells
(20). In contrast, all peptide and protein agonists of FPRL1 (see the
Introduction) have the capacity to induce chemotaxis of
FPRL1-expressing cells and to trigger intracellular calcium elevation
despite relatively modest affinities for FPRL1 (KD values in the high nanomolar to micromolar range).
The results of this study establish that the hexapeptide WKYMVM
activates neutrophils via FPRL1, but not via FPR. First, WKYMVM does
not induce intracellular calcium mobilization in FPR-expressing HL-60
cells. Second, WKYMVM does not desensitize the fMLF-mediated superoxide
release in neutrophils, indicating that it does not cross-react with
FPR. Third, [3H]WKYMVM does not bind to FPR-expressing
cells. In monocytes, WKYMVM is an agonist for FPRL1 and FPRL2. We show
here that FPRL2 is a functional receptor since it triggers calcium flux
in and migration of myeloid cells in response to WKYMVm/M binding.
In view of the high degree of amino acid identity between FPRL1 and
FPRL2 and judging from the ability of WKYMVm/M to mediate both calcium
mobilization in and migration of FPRL1-expressing cells (12, 13) and
undifferentiated FPRL2-expressing cells, it is likely that these two
receptors share many signal transduction steps and that they have
similar roles in host defense. It is worth noting here that, despite
previous studies showing that the WKYMVm/M hexapeptides mediate
phosphoinositide hydrolysis and calcium mobilization in HL-60 cells,
even undifferentiated HL-60 cells (10, 11), we exclude the possibility
that the hexapeptide-mediated activation of HL-60-FPRL2 cells could
result from constitutive expression of FPRL1. First, in contrast to Ryu and co-workers (10, 11), we did not observe calcium mobilization with
undifferentiated control cells,2 i.e. naive
non-transfected cells or cells transfected with either CXCR4 (data not
shown) or FPR (Fig. 3A). Second, if FPRL1 had been
constitutively expressed in HL-60-FPRL2 cells, an fMLFK-mediated calcium response would have been observed (Fig. 3C) since
fMLFK also activates FPRL1 (13). Third, HL-60-FPRL2 cells did not respond to serum amyloid A, an agonist that elicits calcium
mobilization in FPRL1-expressing HEK cells (20) and HL-60-FPRL1 cells
(data not shown). Finally, phosphorylated FPRL1 was not detected in lysates of WKYMVM-stimulated HL-60-FPRL2 cells.
Attenuation of the chemoattractant-evoked responses rapidly occurs
despite a persistent stimulation of the receptor. This attenuation of
responsiveness is thought to result from the desensitization of
receptors through their phosphorylation and rapid removal from the cell
surface by endocytosis (49, 50). Agonist binding to GPCRs is thought to
bring about a conformational change that allows interactions with
either the cognate G protein(s) or a specific kinase(s) that seems
critical for GPCR regulation (49, 51, 52).
Surprisingly, the stimulation of RINm5F-FPRL1 cells with increasing
concentrations of either WKYMVM or WKYMVm leads to distinct patterns of
FPRL1 phosphorylation. The higher efficacy of WKYMVm for ligand
displacement and intracellular calcium mobilization is not correlated
with a higher ability to induce FPRL1 phosphorylation and
internalization. At first glance, this departs from the general model
for GPCR regulation since there is, in general, a good correlation between the efficacy of ligands in GPCR activation and phosphorylation (53, 54). This paradox could be explained if one considers the
possibility that the binding of WKYMVm (but not WKYMVM) induces a
conformational change that impairs the recognition of FPRL1 by the
antibody we used. However, it has not been possible to directly test
this hypothesis by immunoblot analysis of FPRL1 in immunoprecipitates
since FPRL1 migrates at the same level as IgG heavy chains. An impaired
interaction between FPRL1 and the antibody could result from the
WKYMVm-mediated phosphorylation of the threonine residue present in the
antigenic epitope (PPAETELQAM). Such a scenario could also explain why
FPRL1 appears to be distributed in the plasma membrane and not in
fluorescent vesicles by 10 min after the addition of WKYMVm. Judging
from the time course of phosphorylation, FPRL1 is not fully
phosphorylated after 10 min. Consequently, the antibody would stain the
plasma membrane, i.e. non-phosphorylated receptors, but not
the endocytic vesicles that should exclusively contain phosphorylated
receptors. Endocytic vesicles would remain undetectable until receptors
become dephosphorylated at later time points.
Thus, it appears that closely related agonists can induce distinct
activated conformations of FPRL1 that result in a differential phosphorylation. Conformationally distinct forms of FPRL1 may be
involved in coupling with the cognate G protein(s) and in the interactions with a specific kinase(s). This could explain the disproportion between the affinities for FPRL1 and the relative efficacy of the two peptides for calcium mobilization. Although the
affinity of WKYMVm is only 3-4-fold higher than that of WKYMVM, WKYMVm
is a 25-fold more potent agonist for calcium mobilization. This
suggests that WKYMVm induces a conformation allowing a better coupling with the G protein.
Surprisingly, despite a high level of amino acid identity to FPRL1,
FPRL2 has a distinct behavior regarding the phosphorylation process and
its distribution in unstimulated cells. In contrast to FPRL1, FPRL2
displays a strong basal phosphorylation in the absence of agonist,
suggesting that it may undergo constitutive phosphorylation on a number
of residues without any visible interference in the coupling with the G
protein. This hypothesis is further supported by the observation that
the level of receptor phosphorylation is only slightly increased upon
agonist binding, as if only a few additional serine/threonine residues
were unmasked and able to incorporate radioactive phosphate.
Constitutive phosphorylation would be consistent with the punctate
pattern of receptor distribution at/near the plasma membrane of
unstimulated RINm5F-FPRL2 cells. In addition, fluorescent vesicles with
an intracellular localization are observed in ~10-20% of cells.
Such a pattern suggests that FPRL2 may be internalized at a low rate in
the absence of stimulation. An agonist-mediated internalization
nevertheless occurs since FPRL2 is rapidly concentrated inside the
cells following stimulation with either of the two hexapeptides.
In summary, the hexapeptide WKYMVM appears to be a more specific
agonist that the D-methionine-containing peptide. WKYMVM provides a means to investigate more thoroughly how the binding of
LXA4 to FPRL1 can inhibit neutrophil functions otherwise
activated through the same receptor by other agonists. The orphan
receptor FPRL2 is temporarily adopted by the synthetic hexapeptides
WKYMVm/M. It is clear that FPRL2 is a functional receptor with regard
to chemotaxis of myeloid cells and agonist-mediated calcium flux and
internalization. The next challenge will be the isolation of a natural
ligand able to activate this receptor. This may take time, but the
availability of radioactive synthetic agonists will undoubtedly be of
great use in the screening of antagonists.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i2 from the
G
subunit complex results in the activation of several downstream
signaling effector enzymes that promote intracellular calcium
mobilization, modifications in the metabolism of phosphoinositides, and
activation of mitogen-activated protein kinases (4). The integration by
the cell of the different chemoattractant-activated signaling pathways
results in directed cell migration, recruitment of new receptors from
the granules to the cell surface, release of proteolytic enzymes,
production of large amounts of superoxide by the neutrophil NADPH
oxidase, and increased gene transcription (5-8). The extent of the
cellular response is dependent on the identity of the agonist and on
the level of expression and desensitization of the receptors involved in the activation process (9).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 M and stored at
70 °C until used. Further dilutions were made in
H2O.
20 °C in methanol containing 0.5%
-mercaptoethanol. A specific activity of 100-130 Ci/mmol was
routinely obtained.
-counter. Competitive binding assays were performed on adherent
cells at 4 °C for 1 h using a single concentration of
125I-labeled
Trp-Lys-Tyr-Met-Val-D-Met-NH2 in Dulbecco's
modified Eagle's medium and 0.5% BSA containing increasing
concentrations of unlabeled ligands.
5 M) (36). The tubes were
equilibrated in the Biolumat for 5 min at 37 °C, after which the
stimulus (0.1 ml) was added. By a direct comparison of the superoxide
dismutase-inhibitable reduction of cytochrome c and
superoxide dismutase-inhibitable chemiluminescence, 7.2 × 107 cpm were found to correspond to production of 1 nmol of
superoxide (a millimolar extinction coefficient for cytochrome
c of 21.1 was used). Details about the chemiluminescence
technique is given in Ref. 35.
Fmin)/(Fmax
F) with a KD for Fura-2 of 224 nM, where Fmax is the fluorescence
in the presence of 0.04% Triton X-100 and Fmin
is the fluorescence obtained after the addition of 5 mM
EGTA plus 30 mM Tris-HCl, pH 7.4.
20 °C for 30 s. Permeabilized cells were treated for immunofluorescent staining with
a 1:400 dilution of affinity-purified rabbit anti-FPRL1 polyclonal
antibody and a 1:500 dilution of BODIPY-labeled goat anti-rabbit
antibody as previously described (32). For detailed microscopic
analysis, we used a Leica TCS-SP2 confocal scanning microscope.
BODIPY-labeled antibody was excited at 488 nm with an argon laser, and
its fluorescence was collected between 500 and 600 nm.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Neutrophil NADPH oxidase activity induced by
WKYMVM and fMLF. Shown are the time courses of the
neutrophil chemiluminescence responses induced by WKYMVM (100 nM; ------) and fMLF (100 nM; - - -).
Inset, dose-response curves for WKYMVM ( ) and fMLF
(
).
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Fig. 2.
Desensitization of neutrophil
superoxide production. In A and B, cells
were first activated with 100 nM fMLF and 10 min later
restimulated with the same concentration of fMLF (A;
homologous desensitization) or 100 nM WKYMVM (B;
no heterologous desensitization). In C and D, the
cells were first activated with 100 nM WKYMVM and 10 min
later restimulated with the same concentration of WKYMVM (C;
homologous desensitization) or 100 nM fMLF (D;
no heterologous desensitization). The arrows indicate the
times for the addition of agonist, and the curves are from a
representative experiment. CL, chemiluminescence.
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Fig. 3.
Calcium mobilization in undifferentiated
HL-60 cells transfected with FPR, FPRL1, or FPRL2. Cells were
loaded with 2 µM Fura-2 and analyzed with respect to the
rise in intracellular calcium mediated by peptide application.
A, after stimulation with WKYMVM (100 nM), the
[Ca2+]i of HL-60-FPR cells remained at a resting
level (~ 90 nM), whereas it peaked at 180 nM
after application of fMLFK (100 nM). B,
[Ca2+]i peaked at 400 nM after
stimulation of HL-60-FPRL1 cells with WKYMVM (100 nM).
C, no [Ca2+]i rise was induced by the
addition of fMLFK (1 µM) to HL-60-FPRL2 cells, whereas
[Ca2+]i peaked at 580 nM after the
addition of WKYMVm (100 nM). D, the addition of
WKYMVM (200 nM) induced a [Ca2+]i
increase that peaked at 540 nM in HL-60-FPRL2 cells.
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Fig. 4.
Dose-response curves of intracellular calcium
mobilization. The [Ca2+]i increases (peak
value in percent of the maximal value) induced by different
concentrations of either WKYMVm ( ) or WKYMVM (
) are shown for
HL-60-FPRL1 cells (------) and HL-60-FPRL2 cells (- - -). The
curves are from a representative experiment.
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Fig. 5.
FPRL2 is a functional chemotactic
receptor. Undifferentiated myeloid HL-60 and HL-60-FPRL2 cells
migrated toward various concentrations of peptide through polyethylene
terephthalate filters as described under "Experimental Procedures."
Chemotaxis indexes are the means ± S.D. of six independent
experiments.
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Fig. 6.
Specific binding of [3H]WKYMVM
to FPRL1 expressed in RINm5F cells. A, RINm5F cells
(4 × 106 cells/plate) that stably expressed either
FPR ( ) or FPRL1 (
) were incubated with increasing concentrations
of
Trp-Lys-[3,5-3H2]Tyr-Met-Val-Met-NH2
(1-300 nM) in the absence or presence of 10 µM fMLFK or 10 µM WKYMVM, respectively. The
specifically bound radioactivity is expressed as counts/min in the
absence of unlabeled agonist minus counts/min in the presence of a
100-fold excess of unlabeled agonist. No specific binding could be
observed with RINm5F-FPR cells. Inset, Scatchard plot of
[3H]WKYMVM binding to RINm5F-FPRL1 cells. Data are
representative of two independent experiments. R,
receptors.
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Fig. 7.
Relative affinities of L- and
D-methionine-containing hexapeptides for FPRL1 and
FPRL2. RINm5F-FPRL1 cells (------) and RINm5F-FPRL2 cells
(- - -) (3 × 106 cells/plate) were incubated
with 5 and 40 nM 125I-labeled WKYMVm,
respectively, in the absence or presence of either WKYMVm ( ) or
WKYMVM (
) at 4 °C for 1 h. About 95 and 60% of bound
radioactivity were displaced by a 1000-fold excess of unlabeled peptide
in the case of FPRL1 and FPRL2, respectively. Results are expressed as
the percentage of inhibition of the specific binding. Data are
representative of two experiments.
2-adrenergic receptor (43) and the C5a
anaphylatoxin receptor (32, 34) (for a review, see Ref. 44). Whether
these higher molecular mass species have a functional role in signal
transduction is still a matter of debate.
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Fig. 8.
Phosphorylation of FPRL1 expressed in RINm5F
cells. FPRL1 was immunoprecipitated from cells that were
metabolically labeled with [32P]orthophosphoric acid.
Cells were stimulated with either 5 µM WKYMVM or 0.5 µM WKYMVm for various time periods at 37 °C
(A) or were not stimulated (NS) or stimulated
with increasing concentrations of either WKYMVM or WKYMVm for 20 min at
37 °C (B). Immunoprecipitates were treated with 2-fold
Laemmli sample buffer under reducing conditions for 10 min at 37 °C
and then subjected to SDS-polyacrylamide gel electrophoresis analysis
and autoradiography. Data are representative of three independent
experiments.
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Fig. 9.
Phosphorylation of FPRL2 expressed in RINm5F
and HL-60 cells. A, after metabolic labeling with
[32P]orthophosphoric acid, FPRL2 was immunoprecipitated
from RINm5F-FPRL2 cells that were not stimulated (NS) or
stimulated with either WKYMVM or WKYMVm for 20 min at 37 °C.
B, phosphorylated FPRL2 was immunoprecipitated from
HL-60-FPRL2 cells that were not stimulated or stimulated with 5 µM hexapeptide for 20 min at 37 °C. Immunoprecipitates
were treated and analyzed as described in the legend to Fig. 8. Data
are representative of at least two independent experiments.
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Fig. 10.
Internalization time courses of FPRL1 in
RINm5F cells. Shown are confocal immunofluorescence
microscopic images of the medial plane of cells that were not
stimulated or stimulated with 10 µM WKYMVM (A)
or WKYMVm (B) for various time periods at 37 °C. In
unstimulated cells, FPRL1 localized in the plasma membrane. By 20-30
min after stimulation with either of the two peptides, FPRL1
accumulated in intracellular vesicles.
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Fig. 11.
Internalization of FPRL2 in RINm5F
cells. Shown are confocal immunofluorescence microscopic images of
the medial plane of cells that were not stimulated or stimulated with
10 µM WKYMVM or WKYMVm for 20 min at 37 °C. FPRL2 was
unevenly distributed at/near the plasma membrane of unstimulated cells.
After stimulation with either of the two peptides, FPRL2 accumulated in
intracellular vesicles.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Ina Attree-Delic for help in confocal microscopy and Michèle Keramidas for help in the chemotaxis assay.
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FOOTNOTES |
---|
* Work performed in France was supported in part by grants from the Commissariat à l'Energie Atomique, CNRS, and the University Joseph Fourier. Work performed in Sweden was supported by the Swedish Medical Research Council, the King Gustaf V 80-Year Foundation, the Fredrik and Ingrid Thuring Foundation, the Anna-Greta Crafoord Foundation for Rheumatological Research, the Vårdal Foundation, and the Swedish Society of Medicine.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 a fellowship from the "Direction Générale de l'Armement."
** Supported by grants from the Commissariat à l'Energie Atomique.
To whom correspondence should be addressed. Tel.:
33-438-78-31-38; Fax: 33-438-78-51-85; E-mail: fboulay@cea.fr.
Published, JBC Papers in Press, April 2, 2001, DOI 10.1074/jbc.M007769200
2 No calcium response was observed when cells were centrifuged (and not simply diluted) at each passage. When this step was omitted, a low level of constitutive expression of FPRL1 occurred since WKYMVM induced a small calcium rise in control or FPR-expressing HL-60 cells. This is probably due to the fact that a small population of cells becomes differentiated under these conditions. However, the amplitude of the response remained small (between 25 and 75 nM) compared with that obtained with FPRL2-expressing cells ([Ca2+]i > 550 nM).
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ABBREVIATIONS |
---|
The abbreviations used are: FPR, N-formyl peptide receptor; LXA4, lipoxin A4; HIV-1, human immunodeficiency virus-1; HPLC, high pressure liquid chromatography; fMLF, N-formyl-methionyl-leucyl-phenylalanine; fMLFK, N-formyl-methionyl-leucyl-phenylalanyl-lysine; BSA, bovine serum albumin; GPCR, G protein-coupled receptor, BODIPY, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene.
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