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*
Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612; and
Laboratory of Molecular Immunoregulation, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1 nM). The mouse FPR (mFPR) is
a low-affinity receptor for fMLF (Kd
100 nM); therefore, other agonists for this receptor may exist.
Using mFPR-transfected rat basophilic leukemia cells, we found that a
recently identified synthetic peptide
Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a potent agonist for
mFPR. WKYMVm induced calcium mobilization with an EC50 of
1.2–1.5 nM. Optimal chemotaxis was achieved with 1 nM of WKYMVm, but
it required 100 nM of fMLF. WKYMVm stimulated rapid and potent
phosphorylation of the mitogen-activated protein kinases extracellular
signal-related kinases 1 and 2 when used at 50 nM. Pertussis toxin only
partially blocked calcium mobilization and production of inositol
1,4,5-trisphosphate in the stimulated mFPR cells, suggesting the
possibility that this receptor couples to G
proteins other than Gi
and Go. Competitive binding and desensitization data suggest that both
peptides interact with the same receptor but may use nonoverlapping
binding sites because WKYMVm was unable to effectively displace
[3H]fMLF bound to mFPR. These results provide evidence
for the presence of an alternative potent agonist for mFPR, and suggest
a potential usage of WKYMVm for probing the ligand-receptor
interactions with the murine formyl peptide receptor
homologs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The formyl peptide receptors (FPRs) of human and rabbit neutrophils
have been characterized biochemically as receptors that couple
to pertussis toxin (PTX)-sensitive G proteins (8, 9).
Molecular cloning of the human FPR provided the first direct evidence
that chemoattractant receptors share a seven-transmembrane domain
structure characteristic of the G protein-coupled receptor superfamily
(10, 11). Subsequent cloning efforts resulted in the
identification of a number of FPR orthologs and homologs in humans
(12, 13, 14), rabbits (15), and mice
(16). Although both human and rabbit FPRs are high
affinity receptors that bind fMLF with dissociation constants in
single-digit nanomolar range, the mouse FPR (mFPR) is a low affinity
receptor for fMLF (dissociation constant and EC50
100 nM) (16, 17). None of the other five mFPR homologs
bind fMLF with high affinity (17, 18); yet targeted
deletion of mFPR resulted in compromised host defense against
Listeria monocytogenes (19). These observations
led to speculations that there might be other agonists for mFPR. The
human FPR homolog formyl peptide receptor-like 1 (FPRL1; also termed
FPRH1 or FPR2), which shares 69% amino acid sequence identity, has
been identified as a receptor for both lipoxin A4
(20) and serum amyloid A (21). Peptide
sequences derived from the HIV-1 protein gp41 have also been shown to
bind human FPR and FPRL1 (22, 23), suggesting that these
receptors interact with a broad spectrum of agonists. This notion is
supported by the findings that the N-formyl group is not
necessary for high affinity interaction with FPR, as several nonformyl
peptides have been shown to possess potent agonist activities
(24, 25, 26).
Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a synthetic peptide that activates human neutrophils to generate superoxide anions (27, 28). The peptide was originally identified from a combinatorial library by functional screening based on stimulation of inositol phosphate production (29). Several hemopoietic cell lines have been shown to respond to the peptide with calcium mobilization and phospholipase D activation (27). Differentiation of HL-60 and U937 cells enhances the responsiveness to WKYMVm, indicating that the receptor may be up-regulated in granulocytes and monocytes when these cells mature (27). These original observations prompted a recent study of WKYMVm in cells expressing human FPR and FPRL1, and led to the discovery that WKYMVm is an agonist for both of these receptors (30). Because high affinity agonist(s) has not been identified for mFPR, we examined whether WKYMVm could act as such an agonist for this murine receptor.
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Materials and Methods |
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Indo-1/AM was obtained from Molecular Probe (Eugene, OR). The N-formyl peptide fMLF was purchased from Sigma (St. Louis, MO). WKYMVm (>90% purity) was synthesized by the Department of Biochemistry, Colorado State University (Fort Collins, CO) and by Sigma/Genosys (The Woodlands, TX), with equal potency. [3H]fMLF and myo-[3H]inositol were purchased from DuPont-NEN (Boston, MA). PTX was obtained from List Laboratories (Campbell, CA). The anti-phospho-p44/42 mitogen-activated protein (MAP) kinase (Thr 202/Tyr 204) monoclonal Ab (E10) was purchased from New England BioLabs (Beverly, MA).
DNA constructs and transfection
The mouse FPR1 gene was PCR amplified using genomic DNA from CD1 mice. The primers used are MFPR1 (5'-cagaattccagccatggacaccaacatgtctc-3'), and MFPR4 (5'-gcgaattctttacattgcatttaaagtg-3'). The 1.1-kb DNA fragment, containing the entire coding sequence of mFPR, was subcloned into the mammalian expression vector SFFV.neo (31) at the EcoRI site. DNA sequencing confirmed that the mouse FPR1 gene isolated here was identical with a previously published sequence (16). The human FPR cDNA expression construct was prepared in the same vector as described previously (32). Rat basophilic leukemia (RBL)-2H3 cells were transfected using Lipofectamine reagents (Life Technologies, Rockville, MD). Stable transfectants were selected with G418 (500 µg/ml) in DMEM with 20% FBS. Approximately 40 independent transfectants were pooled for assays.
Calcium mobilization assay
Calcium mobilization and desensitization were measured in Indo-1/AM-labeled cells. Briefly, cells were harvested with trypsin-free buffer (Life Technologies) and washed once with HBSS. Cells were adjusted to 5 x 106/ml in HBSS and incubated with 5 µM Indo-1/AM at 37°C for 45 min. After a brief wash with HBSS, the cells were resuspended to 1 x 106/ml in HBSS and stored on ice. Continuous fluorescent measurements of calcium-bound and free Indo-1/AM were made using a PTI (Photon Technology International, Monmouth Junction, NJ) spectrofluorometer, detecting at 405 and 485 nm, respectively, with an excitation wavelength of 340 nm. Intracellular Ca2+ level was expressed as relative fluorescence, calculated based on the ratio of Indo-1 fluorescence at 405 and 485 nm and standardized for Indo-1 loading and cell responsiveness. Dose response curves were generated based on intracellular free calcium concentrations, determined using the formula [Ca2+]i = 250 (F - Fmin)/(Fmax - F). Fmax is the ratio of fluorescence obtained with Triton X-100 (0.1%) and reflects the total available free calcium. Fmin is the ratio of fluorescence obtained with EDTA (2 mM) included in the assay buffer to remove free calcium released by Triton X-100 treatment. F is the ratio of fluorescence obtained after ligand stimulation. The maximal intracellular free calcium level was set as 100% response. Curve fitting was performed using Prism software (version 2.0; GraphPad, San Diego, CA).
Assay for inositol phosphates
Cells were seeded in 24-well plates at a density of 2 x 105 cells/well in 1 ml inositol-free medium with 10% dialyzed FBS and 1 µCi/ml of myo-[3H]inositol. Cells were incubated at 37°C for 24 h, detached with trypsin-free buffer, then washed twice with HBSS containing 100 mM LiCl and 1% BSA. After incubation in 200 µl of the same buffer for 10 min, cells were stimulated by peptides for the indicated time. The reaction was stopped by adding 600 µl chloroform/methanol (1:2), vortexed, and centrifuged; then the upper phase was transferred to a new 15-ml conical tube containing 0.5 g Dowex AG1-X8 resin. The resin-packed columns were washed three times with distilled water, and samples were eluted with a buffer containing 1 M ammonium formate and 0.1 M formic acid. Radioactivity of [3H]inositol phosphates was determined by scintillation counting.
Chemotaxis
Migration of cells induced by fMLP and WKYMVm was assessed using a 48-well microchemotaxis chamber (Neuroprobe, Cabin John, MD). Different concentrations of stimulants were placed in wells (50 µl) of the lower compartment. Cells (35 µl of a 1 x 106 cells/ml suspension) were seeded into wells of the upper compartment, which was separated from the lower compartment by a polycarbonate filter (10-µm pore size). The filter was precoated with 50 µg/ml collagen (type I; Sigma) for 60 min. After incubation at 37°C for 4 h, the filter was removed, fixed in methanol, and stained with hematoxylin. The cells that migrated across the filter were counted with light microscopy. Chemotaxis index was calculated as the ratio of the number of cells migrating toward stimuli over the number of cells migrating toward medium without stimuli. Checkerboard analysis was conducted with agonist ranging from 0.1 to 100 nM to verify that migration of the cells occurs only in the presence of higher concentrations of the stimulant in the lower wells of the chamber, i.e., positive concentration gradients.
Ligand binding assay
Cell membranes were prepared by nitrogen cavitation in 10 ml of bomb buffer (10 mM PIPES, pH 7.3, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2) containing 6 mg ATP, 0.2 µg chymostatin, 5 mg diisopropylfluorophosphate, and 0.5 mM PMSF. Cavitated cells were collected into 1 mM EGTA plus 1 mM EDTA. After centrifugation at 1500 rpm for 5 min to remove nuclei and cell debris, the supernatant was collected and subjected to high speed centrifugation (45,000 rpm for 60 min). Membranes were resuspended in 25 mM HEPES, pH 7.0, and 200 mM sucrose, and stored at -80°C. A membrane binding assay was conducted at room temperature (23°C) with 50 µg of membrane proteins and 3H-labeled fMLF in the presence or absence of unlabeled peptides for 60 min. The binding buffer contains 5 mM KCl, 147 mM NaCl, 1.9 mM KH2PO4, 1.1 mM Na2HPO4, 5.5 mM glucose, 0.15 mM CaCl2, 0.3 mM MgSO4, 1 mM MgCl2, and 10 mM HEPES, pH 7.4. The unbound ligand was removed by filtration through Whatman GF/C filters (Whatman, Maidstone, U.K.), which were then washed four times with ice-cold binding buffer. The filters were dried and radioactivity retained was counted with liquid scintillation spectrometry.
MAP kinase assay
Cells grown in six-well plates were stimulated with 50 nM fMLF and WKYMVm for indicated times. Some samples were treated with PTX (200 ng/ml for 16 h) before assays. The reaction was stopped by adding 1 ml ice-cold PBS, and the cells were scraped and harvested into tubes. After centrifugation, the cell pellet was resuspended in 1 ml ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1% Nonidet P-40, 150 mM NaCl, 1 mM Na3VO4, 5 mM NaF, and protease inhibitors). The contents were incubated on ice for 15 min, centrifuged, and the supernatant was transferred to Eppendorf tubes. Samples were analyzed by SDS-PAGE and Western blotting with antiphospho-extracellular signal-related kinase (ERK) 1/2 Ab (New England Biolabs) at 1:1000 dilution for 18 h. The membrane was washed and incubated with 1:5000 dilution of HRP-conjugated anti-mouse secondary Ab for 1 h. Excess Ab was removed by washing, and immunocomplexes were visualized using enhanced chemiluminescence detection (Pierce, Rockford, IL) according to the manufacturer’s instruction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify potentially novel agonists for the mFPR, a stable cell
line was established by transfection with cDNA coding for mFPR and G418
selection of the transfected RBL cells, a rat basophil leukemia cell
line used previously for characterization of chemoattractant receptors
(33, 34). Untransfected RBL cells did not respond to
WKYMVm or fMLF (data not shown) (29, 33). As a control,
human FPR (hFPR) cDNA was subcloned into the same expression vector and
transfected similarly into RBL cells. Both the hFPR and mFPR
transfected cells were characterized in calcium mobilization assays. As
shown in Fig. 1
A, the
prototype formyl peptide fMLF (50 nM) induced a potent calcium flux in
the hFPR cells. In contrast, fMLF at the same concentration only
induced a weak response in the mFPR cells (Fig. 1B). This
observation is in agreement with previous studies showing mFPR as a low
affinity receptor for fMLF (16, 19). We next examined the
responsiveness of these two cell lines to the peptide WKYMVm,
which activates human FPR and FPRL1 with high potency
(30). As demonstrated in Fig. 1, C and
D, both hFPR and mFPR cells responded equally well to
WKYMVm. Although fMLF and WKYMVm both induced calcium mobilization in a
dose-dependent manner in mFPR-expressing cells, WKYMVm stimulated the
cells with an EC50 of 
1.5 nM, whereas it
requires 200 nM of fMLF to achieve the same level of calcium
response (Fig. 2). Maximal calcium
response to fMLF could not be obtained due to a solubility problem with
fMLF above a 10-µM concentration. These results indicate that WKYMVm
is a potent agonist for both hFPR and mFPR, whereas fMLF is a potent
agonist for hFPR and a weak agonist for mFPR.
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WKYMVm induced a biphasic increase in intracellular calcium; a
rapid rise of [Ca2+]i is
followed by a weak but more sustained elevation of
[Ca2+]i (Fig. 3A). To verify that the
sustained increase in
[Ca2+]i is the result of
calcium influx, extracellular calcium was depleted before agonist
stimulation by the addition of EDTA. This treatment abolished the
sustained elevation of [Ca2+]i but had
only a small effect on the magnitude of the initial rise of
[Ca2+]i (Fig. 3B). We conclude that WKYMVm stimulates calcium influx
probably by activating a cell surface calcium channel
(35). Treatment of cells with
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid, which depletes intracellular calcium stores, completely abolished
the initial and sustained calcium responses (data not shown),
indicating that mobilization of intracellular calcium may be associated
with subsequent opening of the cell surface calcium channel. Calcium
influx was also observed in the mFPR and hFPR cells following fMLF
stimulation (Figs. 1
A and 3, C and
D).
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We asked whether WKYMVm could induce migration of cells, a
physiological function mediated by the chemoattractant receptors. As
shown in Fig. 4, WKYMVm induced
chemotaxis of mFPR cells with an optimal concentration of 1 nM (Fig. 4C). The chemotactic response of mFPR cells induced by
WKYMVm was bell-shaped, as typically seen with other
chemoattractants. The contribution of chemokinesis to peptide-induced
cell migration was excluded by checkerboard analyses in which cell
migration occurred only when higher concentrations of the stimulant
were present in the lower wells of the chemotaxis chamber (data not
shown). fMLF also stimulated chemotaxis of mFPR cells; however, the
response peaked at 100 nM (Fig. 4C). The weaker chemotactic
response to fMLF is similar to what was observed with mouse neutrophils
(17). Thus, WKYMVm is 100-fold more potent than fMLF in
the induction of chemotaxis through mFPR.
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fMLP has been shown to activate the MAP kinases ERK1 and ERK2 in
human neutrophils (36, 37). To investigate whether WKYMVm
has the ability to simulate this pathway through mFPR, cells
transfected to express mFPR and hFPR were treated with either WKYMVm or
fMLF. ERK1 and ERK2 activation was determined using an Ab against the
phosphorylated MAP kinases. Fig. 5 shows
that both mFPR and hFPR cells responded to WKYMVm with rapid tyrosine
phosphorylation of ERK1 and ERK2. Following agonist stimulation, the
response was observed at 1 min and peaked between 2 and 5 min. In
contrast, although fMLF at 50 nM stimulated a marked phosphorylation of
ERK1 and ERK2 in hFPR cells, its effect on the mFPR cells was minimal
at this concentration (Fig. 5, bottom).
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The human FPR is known to couple to Gi proteins (7, 9). PTX treatment of neutrophils abolishes chemotaxis and many
other cellular functions induced by fMLF (6). Several cell
lines transfected to express the hFPR displayed similar properties
(11, 32). Consistent with this finding, the hFPR cells
treated with PTX showed diminished calcium response to both WKYMVm and
fMLF (Fig. 6A, sample groups 3
and 4). In contrast, mFPR cells similarly treated with PTX retained a
significant portion (20–25%; p < 0.001) of the
calcium mobilization response induced by both WKYMVm and fMLF (Fig. 6A, sample groups 1 and 2). Therefore, the partial
resistance to PTX treatment is likely a property of the mouse receptor.
To further test the possibility that mFPR additionally couples to a
PTX-insensitive G protein, the cells were treated with PTX and assayed
for ERK activation. As shown in Fig. 6B, ERK phosphorylation
was effectively blocked by PTX in cells stimulated with fMLF (1 µM)
or with WKYMVm at a high concentration of 100 nM, suggesting that mFPR
couples to a PTX-sensitive G protein for activation of ERK. In an
inositol phosphate turnover assay, PTX treatment partially blocked
formation of phosphoinositol 1,4,5-trisphosphate
(IP3) in mFPR cells, but completely blocked
IP3 formation in hFPR cells. These results
combined suggest that mFPR couples to a PTX-insensitive G protein
subunit that contributes to agonist-induced IP3
formation and calcium mobilization.
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We showed in Fig. 1 that expression of mFPR in RBL cells rendered
the cells responsive to both WKYMVm and fMLF. Because WKYMVm induced
more potent responses than fMLF, we examined the binding properties of
these two peptides in mFPR. Membranes prepared from the mFPR cells were
incubated with [3H]fMLF in the absence or
presence of increasing amounts of unlabeled WKYMVm or fMLF. As shown in
Fig. 7, unlabeled fMLF reduced binding of
[3H]fMLF by 50 and 90% when used at 50 nM and
1 µM, respectively. In contrast, WKYMVm could not effectively
displace the bound radiolabeled fMLF despite its high potency in
stimulating chemotaxis and calcium mobilization through mFPR. This
property appears to be unique to mFPR because both WKYMVm and fMLF
reduced [3H]fMLF binding to the hFPR with
similar efficacy (30). Due to the lack of radiolabeled
WKYMVm, we were not able to directly determine whether fMLF binding to
mFPR could prevent subsequent receptor interaction with WKYMVm. To
examine this possibility further, desensitization experiments were
conducted in the mFPR cells, and the results were shown in Fig. 8. Homologous desensitization was
observed in cells stimulated with WKYMVm (Fig. 8A) and fMLF
(Fig. 8B). In contrast, stimulation of the cells with 1 µM
fMLF only minimally desensitized the cells for a subsequent response to
25 nM of WKYMVm (Fig. 8C). Therefore, WKYMVm was still able
to bind and activate mFPR in the presence of fMLF. The mFPR cells
treated with 25 nM of WKYMVm were completely desensitized to subsequent
stimulation with 1 µM of fMLF (Fig. 8D) but were able to
responded weakly to 4 µM of fMLF (Fig. 8E). These results
demonstrate cross-desensitization between fMLF and WKYMVm in the order
of the relative potency of these two agonists for the mFPR. Combined
with the binding data in Fig. 7, the above results indicate that both
peptides interact with and activate mFPR even in the presence of the
other peptide. One explanation for the lack of effective competition of
[3H]fMLF by WKYMVm is that the two peptides
interact with different and nonoverlapping binding sites on mFPR. A
direct examination of this possibility will be feasible when
[3H]WKYMVm becomes available.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 nM, the mFPR is
100-fold less effective in binding fMLF. In human and rabbit
neutrophils, chemotaxis occurs when the cells are stimulated with fMLF
(and most other chemoattractants) in the subnanomolar concentrations.
Higher concentrations of chemoattractants inhibit chemotaxis,
presumably due to receptor desensitization. Because mFPR is a low
affinity receptor for fMLF, accumulation of a higher concentration of
bacterially generated N-formyl peptides will be necessary
for the activation of most FPR-mediated phagocyte functions. These
observations suggest the possible presence of other agonists for
mFPR. WKYMVm is identified from a peptide library based on its ability to stimulate phosphoinositide hydrolysis in lymphocyte cell lines (29). WKYMVm is derived from WKYMVM-NH2 and is one of the most potent agonists among a group of peptides sharing a XKYX(P/V)M signature sequence. These peptides were suspected to bind and activate G protein-coupled receptors because the induced phosphoinositide hydrolysis and calcium mobilization could be blocked by PTX (29). In subsequent studies, Ryu and coworkers found that WKYMVm could induce superoxide generation in human neutrophils (27) through activation of phospholipase D (28), and these functions contributed to the killing of Staphylococcus aureus by WKYMVm-stimulated human monocytes. Despite extensive structural and functional characterization of these peptides, cellular receptors for WKYMVm were not identified until recently (30). An earlier observation that fMLF at up to 1 µM did not desensitize calcium response to WKYMVm led to the conclusion that this peptide binds to a unique receptor (27). It is now clear that the presence of FPRL1 in some of the hemopoietic cells tested, the high potency of WKYMVm, and the concentration of the peptide used (330 nM) in those experiments may have masked the effect of fMLF in these desensitization assays. Results shown in this and previous studies (30) indicate that WKYMVm is more potent than fMLF in the induction of calcium mobilization through the two receptors FPR and FPRL1 in human phagocytes, resulting in resistance to desensitization by fMLF.
WKYMVm contains no amino acid sequence similarity to fMLF or a formyl
group at the amino terminus, yet it is a potent agonist for FPR and
FPRL1. Exactly how this peptide interacts with its receptors is unclear
at present. It is intriguing that the human FPR interacts with both
fMLF and WKYMVm efficiently, whereas the mFPR favors WKYMVm over fMLF.
Furthermore, competitive binding assays with hFPR demonstrated that
WKYMVm, like fMLF, can displace [3H]fMLF bound
to hFPR, suggesting that these two peptides interact with the human
receptor using a similar mechanism or they may occupy the same or
overlapping binding pocket. A different type of interaction may exist
for mFPR, such that unlabeled WKYMVm cannot compete off the bound
[3H]fMLF. It appears that both mFPR and hFPR
contain structures necessary and sufficient for high affinity binding
of WKYMVm, but the ability to bind fMLF with high affinity was
developed only in hFPR, but not mFPR. The mFPR shares 77% sequence
identity with hFPR, and is a mouse ortholog of the hFPR
(16). Of the other five mFPR-like gene products, mFPRL1 is
an ortholog of hFPRL1 (74% sequence identity), which interacts with
fMLF with low affinity (13, 32) but binds lipoxin
A4 with high affinity (20). As
expected, mFPRL1 is also a receptor for lipoxin
A4 (38). The gene product of
Fpr-rs2, recently named mFPR2 (17), is
homologous to mFPRL1 (81% sequence identity) and hFPRL1 (76%
sequence identity) and responds poorly to fMLF
(EC50 5 µM) in calcium mobilization and
chemotaxis assays. It remains to be determined whether mFPRL1 and mFPR2
can bind WKYMVm. Thus, despite the presence of six FPR-like genes in
mice, none of them encode a high affinity receptor for fMLF.
The divergence of FPR and FPR-like receptors in ligand recognition is demonstrated by recent identification of additional agonists and antagonists for the hFPR and FPRL1. These agonists range from N-formyl and nonformyl peptides to lipids and proteins of larger sizes. Thus, the FPR family of receptors has evolved to be promiscuous with respect to ligand specificity, similar to many chemokine receptors. The biological functions of these agonist-receptor interactions are not all clear, but it is unusual for one receptor to bind both peptide and lipid agonists. It is possible that neutrophil recognition of and response to WKYMVm, or a ligand with similar structure, plays a more important role in lower organisms. This hypothesis needs to be tested in the future when FPR and FPR-like receptor genes from other mammals are cloned.
Our current study has revealed some of the properties of WKYMVm as well
as mFPR that were not appreciated previously. In general, WKYMVm
induces a calcium response that is equal to (in hFPR cells) or stronger
than (in mFPR cells) that induced by fMLF. In cells transfected to
express mFPR, both fMLF and WKYMVm induced calcium mobilization that
was not completely inhibited by PTX (Fig. 6). This compared with a
nearly complete inhibition by PTX of the same responses induced through
hFPR, suggesting that the mFPR may couple to a PTX-insensitive G
protein. Our results also indicate that the PTX-insensitive signaling
component of mFPR is responsible for IP3
formation and increased calcium mobilization, but not ERK activation.
These preliminary findings provide evidence that mFPR differs from hFPR
in certain signaling properties. The biological significance of such
differences remains to be determined.
The discovery by Schiffmann and coworkers that synthetic peptides with N-formyl group are potent leukocyte chemoattractants (1) led to the identification of fMLF as a primary bacterial chemoattractant several years later (2). fMLF has since been used widely as a prototypic chemotactic peptide to study a variety of leukocyte functions. Identification of WKYMVm as a potent synthetic chemotactic peptide specific for FPR and two FPR-related receptors may further advance our understanding of how small peptides activate G protein-coupled receptors. The observation that this peptide and fMLF appear to use nonoverlapping binding sites on mFPR, yet both are able to activate the same receptor, should provide a useful probe for detailed analysis of the relationship between structure, function, and evolution of the FPR family of receptors.
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Acknowledgments |
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Footnotes |
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2 R.H. and L.T. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Richard D. Ye, Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612.
4 Abbreviations used in this paper: fMLF, N-formyl-Met-Leu-Phe; FPR, formyl peptide receptor; mFPR, mouse FPR; FPRL1, formyl peptide receptor-like 1; PTX, pertussis toxin; RBL, rat basophilic leukemia; WKYMVm, Trp-Lys-Tyr-Met-Val-D-Met; MAP, mitogen-activated protein; hFPR, human FPR; IP3, phosphoinositol 1,4,5-trisphosphate; ERK, extracellular signal-related kinase.
Received for publication January 14, 2000. Accepted for publication July 19, 2000.
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) and fMLF (
). Med, medium
only. CI, chemotaxis index.
). After incubation at room temperature for 60 min,
the unbound radiolabeled fMLF was washed off, and bound radioactivity
was determined by liquid scintillation counting. Data presented are
means and SD from a representative experiment of three performed, with
samples in duplicate.