Formyl peptides are potent neutrophil chemoattractants. In humans and rabbits, the formyl peptide receptor (FPR) binds N-formyl-Met-Leu-Phe (fMLF) with high affinity (Kd ≈ 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 Gα 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.

Bacterially derived and synthetic peptides that contain N-formyl-methionine are chemoattractants for phagocytic leukocytes (1, 2). In mammalian cells, cleavage products of mitochondrial proteins bearing N-formyl-methionine have also been shown to possess neutrophil chemotactic activity (3). The prototype formyl peptide, N-formyl-Met-Leu-Phe (fMLF),4 binds to human and rabbit neutrophil receptors with high affinity (4). fMLF stimulates neutrophil chemotaxis with an EC50 in the subnanomolar range. At higher concentrations, formyl peptides induce release of granule contents and production of superoxide anions (reviewed in Refs. 5, 6). fMLF has also been shown to trigger phagocytosis and to regulate integrin activation. One of the very few externally generated chemoattractants, fMLF is believed to play an important role in host defense against bacterial infection (6, 7).

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.

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).

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 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 × 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 × 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)/(FmaxF). 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).

Cells were seeded in 24-well plates at a density of 2 × 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.

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 × 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 Ι; 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.

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.

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.

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. 1,B). 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.

FIGURE 1.

WKYMVm and fMLF induce calcium mobilization in transfected cells. Stable RBL cell lines expressing the hFPR and mFPR were labeled with Indo-1/AM and subjected to stimulation with fMLF and WKYMVm as marked by arrowheads. Real-time intracellular free calcium levels were determined as described in Materials and Methods. The results were expressed as relative fluorescence (FL) based on ratio of fluorescence detected at 405 and 485 nm. Data shown are representative of three independent experiments.

FIGURE 1.

WKYMVm and fMLF induce calcium mobilization in transfected cells. Stable RBL cell lines expressing the hFPR and mFPR were labeled with Indo-1/AM and subjected to stimulation with fMLF and WKYMVm as marked by arrowheads. Real-time intracellular free calcium levels were determined as described in Materials and Methods. The results were expressed as relative fluorescence (FL) based on ratio of fluorescence detected at 405 and 485 nm. Data shown are representative of three independent experiments.

Close modal
FIGURE 2.

Dose-dependent calcium mobilization in mFPR cells. The cells were stimulated with WKYMVm and fMLF at various concentrations, and real-time calcium mobilization was measured as described in Materials and Methods. The magnitude of calcium mobilization was determined by integration of [Ca2+]i above base level for 80 s after agonist stimulation, and the values are expressed as percent calcium response. Maximal calcium response (100%) was that produced by WKYMVm at 10 μM.

FIGURE 2.

Dose-dependent calcium mobilization in mFPR cells. The cells were stimulated with WKYMVm and fMLF at various concentrations, and real-time calcium mobilization was measured as described in Materials and Methods. The magnitude of calcium mobilization was determined by integration of [Ca2+]i above base level for 80 s after agonist stimulation, and the values are expressed as percent calcium response. Maximal calcium response (100%) was that produced by WKYMVm at 10 μM.

Close modal

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. 3,A). 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. 3,B). 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).

FIGURE 3.

Influx of extracellular calcium contributes to prolonged elevation of [Ca2+]i. Indo-1-labeled mFPR cells were resuspended in HBSS (containing 0.15 mM CaCl2) and then stimulated with WKYMVm (50 nM; A). In a parallel experiment, EDTA was added to 2 mM before stimulation of the cells with WKYMVm (50 nM; B). The same experiments were then repeated using fMLF (4 μM) as agonist. Data shown were from one representative experiment of a total of three experiments, with similar results.

FIGURE 3.

Influx of extracellular calcium contributes to prolonged elevation of [Ca2+]i. Indo-1-labeled mFPR cells were resuspended in HBSS (containing 0.15 mM CaCl2) and then stimulated with WKYMVm (50 nM; A). In a parallel experiment, EDTA was added to 2 mM before stimulation of the cells with WKYMVm (50 nM; B). The same experiments were then repeated using fMLF (4 μM) as agonist. Data shown were from one representative experiment of a total of three experiments, with similar results.

Close modal

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. 4,C). 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. 4 C). 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.

FIGURE 4.

WKYMVm induces chemotaxis of mFPR cells. Approximately 35,000 cells were loaded into the top well of a 48-well microchemotaxis chamber. Various concentrations of WKYMVm and fMLF were placed in bottom wells. Chemotaxis assay was conducted at 37°C for 4 h. Migrated cells were stained and counted as described in Materials and Methods. A, Chemotaxis of mFPR cells with medium placed in bottom wells. B, Chemotaxis of mFPR cells with 1 nM of WKYMVm. C, Chemotaxis of mFPR cells in response to different concentrations of WKYMVm (▪) and fMLF (▨). Med, medium only. CI, chemotaxis index.

FIGURE 4.

WKYMVm induces chemotaxis of mFPR cells. Approximately 35,000 cells were loaded into the top well of a 48-well microchemotaxis chamber. Various concentrations of WKYMVm and fMLF were placed in bottom wells. Chemotaxis assay was conducted at 37°C for 4 h. Migrated cells were stained and counted as described in Materials and Methods. A, Chemotaxis of mFPR cells with medium placed in bottom wells. B, Chemotaxis of mFPR cells with 1 nM of WKYMVm. C, Chemotaxis of mFPR cells in response to different concentrations of WKYMVm (▪) and fMLF (▨). Med, medium only. CI, chemotaxis index.

Close modal

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).

FIGURE 5.

Activation of ERKs by peptide agonists in FPR-transfected RBL cells. RBL cells stably transfected to express mFPR (left) or hFPR (right) were stimulated with WKYMVm peptide (50 nM, upper panels) or fMLF (50 nM, lower panels). After various time intervals the cells were harvested and the phosphorylated ERKs were determined by Western blotting with anti-phospho-ERK Abs as detailed in Materials and Methods. Brackets mark the positions of the 44- and 42-kDa forms of phosphorylated ERK. Equal loading of protein on the blots was determined by probing with total ERK (data not shown). Data shown are representative of two independent experiments.

FIGURE 5.

Activation of ERKs by peptide agonists in FPR-transfected RBL cells. RBL cells stably transfected to express mFPR (left) or hFPR (right) were stimulated with WKYMVm peptide (50 nM, upper panels) or fMLF (50 nM, lower panels). After various time intervals the cells were harvested and the phosphorylated ERKs were determined by Western blotting with anti-phospho-ERK Abs as detailed in Materials and Methods. Brackets mark the positions of the 44- and 42-kDa forms of phosphorylated ERK. Equal loading of protein on the blots was determined by probing with total ERK (data not shown). Data shown are representative of two independent experiments.

Close modal

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. 6,A, 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. 6,A, 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. 6 B, 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 α subunit that contributes to agonist-induced IP3 formation and calcium mobilization.

FIGURE 6.

Differential inhibition by PTX. A, mFPR and hFPR cells were treated with PTX (200 ng/ml, 16 h; ▪) before calcium mobilization assays. The control cells were incubated with medium only (▨). Calcium mobilization to WKYMVm (W-pep) and fMLF were determined as described in Materials and Methods. Maximal calcium response for each sample group, determined in the absence of PTX treatment, was set as 100%. Data presented are means and SD combined from three independent experiments. B, Effect of PTX on ERK phosphorylation. The mFPR cells were treated with or without PTX similar to (A), and then stimulated with fMLF (1 μM) or WKYMVm (W-pep, 100 nM) for 2 min. Phosphorylated ERK species (ERK-P) was detected using an anti-phospho-ERK Ab as in Fig. 5. Equal loading of protein samples was shown by Western blot probing total ERK (bottom). The control cells were without agonist stimulation. C, Effect of PTX on IP3 production. Cell labeling and assay procedures were described in Materials and Methods. The mFPR and hFPR cells were stimulated without (Ctrl) and with fMLF or WKYMVm (W-pep). Agonist concentrations were 100 nM except in sample group 2, where fMLF was used at 400 nM. Data presented are combined means and SD from two separate experiments, each with duplicate samples.

FIGURE 6.

Differential inhibition by PTX. A, mFPR and hFPR cells were treated with PTX (200 ng/ml, 16 h; ▪) before calcium mobilization assays. The control cells were incubated with medium only (▨). Calcium mobilization to WKYMVm (W-pep) and fMLF were determined as described in Materials and Methods. Maximal calcium response for each sample group, determined in the absence of PTX treatment, was set as 100%. Data presented are means and SD combined from three independent experiments. B, Effect of PTX on ERK phosphorylation. The mFPR cells were treated with or without PTX similar to (A), and then stimulated with fMLF (1 μM) or WKYMVm (W-pep, 100 nM) for 2 min. Phosphorylated ERK species (ERK-P) was detected using an anti-phospho-ERK Ab as in Fig. 5. Equal loading of protein samples was shown by Western blot probing total ERK (bottom). The control cells were without agonist stimulation. C, Effect of PTX on IP3 production. Cell labeling and assay procedures were described in Materials and Methods. The mFPR and hFPR cells were stimulated without (Ctrl) and with fMLF or WKYMVm (W-pep). Agonist concentrations were 100 nM except in sample group 2, where fMLF was used at 400 nM. Data presented are combined means and SD from two separate experiments, each with duplicate samples.

Close modal

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. 8,A) and fMLF (Fig. 8,B). 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. 8,C). 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. 8,D) but were able to responded weakly to 4 μM of fMLF (Fig. 8,E). 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.

FIGURE 7.

Competitive binding of 3H-labeled fMLF with unlabeled fMLF and WKYMVm. Membranes were prepared from mFPR cells and subject to direct binding assays as described in Materials and Methods. A fixed amount (30 nM) of 3H-labeled fMLF was added to each sample, along with variable amounts of unlabeled fMLF (▪) or WKYMVm (▴). 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.

FIGURE 7.

Competitive binding of 3H-labeled fMLF with unlabeled fMLF and WKYMVm. Membranes were prepared from mFPR cells and subject to direct binding assays as described in Materials and Methods. A fixed amount (30 nM) of 3H-labeled fMLF was added to each sample, along with variable amounts of unlabeled fMLF (▪) or WKYMVm (▴). 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.

Close modal
FIGURE 8.

Desensitization of calcium mobilization induced by WKYMVm and fMLF. Stable RBL cells expressing mFPR were labeled with Indo-1/AM and stimulated with fMLF and WKYMVm at the indicated concentrations. The experiments were conducted in the presence of EDTA (2 mM) in assay buffer and without removal of the first agonist. Relative fluorescence (FL) was determined as described in Materials and Methods. Data shown are representative of three independent experiments that produced similar results.

FIGURE 8.

Desensitization of calcium mobilization induced by WKYMVm and fMLF. Stable RBL cells expressing mFPR were labeled with Indo-1/AM and stimulated with fMLF and WKYMVm at the indicated concentrations. The experiments were conducted in the presence of EDTA (2 mM) in assay buffer and without removal of the first agonist. Relative fluorescence (FL) was determined as described in Materials and Methods. Data shown are representative of three independent experiments that produced similar results.

Close modal

The signaling properties of the human and rabbit FPRs have been studied extensively in neutrophils. Less is known about the mFPR. Targeted deletion of mFPR resulted in mice that were developmentally normal, but displayed increased susceptibility to challenge with L. monocytogenes (19). Although this study clearly illustrated a role of mFPR in antibacterial host defense, several questions remain unanswered. Deficiency of mFPR does not seem to affect neutrophil migration to L. monocytogenes-infected liver and spleen, as neutrophilic abscesses were found in these organs (19). Also, no histological differences between mFPR−/− and mFPR+/+ mice were observed in liver and spleen after infection with L. monocytogenes. It is noted that although the human and rabbit FPR bind fMLF with dissociation constants at ∼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.

We thank Meiying Wang for generating the transfected cells, Larry Shepard for help in inositol phosphate assays, Dick Green for helpful discussions, and Heather Gray for proofreading the manuscript.

1

This work was supported by National Institutes of Health Grant AI33503 (to R.D.Y.) and was conducted during the tenure of an Established Investigatorship (to R.D.Y.) from the American Heart Association. D.D.B. was supported in part by a fellowship from Arthritis Foundation.

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.

1
Schiffmann, E., B. A. Corcoran, S. M. Wahl.
1975
. N-formylmethionyl peptides as chemoattractants for leucocytes.
Proc. Natl. Acad. Sci. USA
72
:
1059
2
Marasco, W. A., S. H. Phan, H. Krutzsch, H. J. Showell, D. E. Feltner, R. Nairn, E. L. Becker, P. A. Ward.
1984
. Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli.
J. Biol. Chem.
259
:
5430
3
Carp, H..
1982
. Mitochondrial N-formylmethionyl proteins as chemoattractants for neutrophils.
J. Exp. Med.
155
:
264
4
Niedel, J., J. Davis, P. Cuatrecasas.
1980
. Covalent affinity labeling of the formyl peptide chemotactic receptor.
J. Biol. Chem.
255
:
7063
5
Becker, E. L..
1987
. The formylpeptide receptor of the neutrophil: a search and conserve operation.
Am. J. Pathol.
129
:
16
6
Snyderman, R., R. J. Uhing.
1992
. Phagocytic cells: stimulus-response coupling mechanisms. J. I. Gallin, and I. M. Goldstein, and R. Snyderman, eds.
Inflammation: Basic Principles and Clinical Correlates
421
Raven Press, New York.
7
Prossnitz, E. R., R. D. Ye.
1997
. The N-formyl peptide receptor: a model for the study of chemoattractant receptor structure and function.
Pharmacol. Ther.
74
:
73
8
Snyderman, R., M. C. Pike, S. Edge, B. Lane.
1984
. A chemoattractant receptor on macrophages exists in two affinity states regulated by guanine nucleotides.
J. Cell Biol.
98
:
444
9
Bokoch, G. M., A. G. Gilman.
1984
. Inhibition of receptor mediated release of arachidonic acid by pertussis toxin.
Cell
39
:
301
10
Boulay, F., M. Tardif, L. Brouchon, P. Vignais.
1990
. Synthesis and use of a novel N-formyl peptide derivative to isolate a human N-formyl peptide receptor cDNA.
Biochem. Biophys. Res. Commun.
168
:
1103
11
Boulay, F., M. Tardif, L. Brouchon, P. Vignais.
1990
. The human N-formylpeptide receptor: characterization of two cDNA isolates and evidence for a new subfamily of G protein-coupled receptors.
Biochemistry
29
:
11123
12
Murphy, P. M., T. Ozcelik, R. T. Kenney, H. L. Tiffany, D. McDermott, U. Francke.
1992
. A structural homologue of the N-formyl peptide receptor: characterization and chromosome mapping of a peptide chemoattractant receptor family.
J. Biol. Chem.
267
:
7637
13
Ye, R. D., S. L. Cavanagh, O. Quehenberger, E. R. Prossnitz, C. G. Cochrane.
1992
. Isolation of a cDNA that encodes a novel granulocyte N-formyl peptide receptor.
Biochem. Biophys. Res. Commun.
184
:
582
14
Bao, L., N. P. Gerard, R. L. Eddy, T. B. Shows, C. Gerard.
1992
. Mapping genes for the human C5a receptor (C5AR), human FMLP receptor (FPR), and two FMLP receptor homologue orphan receptors (FPRH1, FPRH2) to chromosome 19.
Genomics
13
:
437
15
Ye, R. D., O. Quehenberger, K. M. Thomas, J. Navarro, S. L. Cavanagh, E. R. Prossnitz, C. G. Cochrane.
1993
. The rabbit neutrophil N-formyl peptide receptor: cDNA cloning, expression, and structure/function implications.
J. Immunol.
150
:
1383
16
Gao, L. J., P. M. Murphy.
1993
. Species and subtype variants of the N-formyl peptide chemotactic receptor reveal multiple important functional domains.
J. Biol. Chem.
268
:
25395
17
Hartt, J. K., G. Barish, P. M. Murphy, J.-L. Gao.
1999
. N-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two N-formylpeptide receptor (FPR) subtypes: molecular characterization of FPR2, a second mouse neutrophil FPR.
J. Exp. Med.
190
:
741
18
Gao, J.-L., H. Chen, J. D. Filie, C. A. Kozak, P. M. Murphy.
1998
. Differential expansion of the N-formylpeptide receptor gene cluster in human and mouse.
Genomics
51
:
270
19
Gao, J.-L., E. J. Lee, P. M. Murphy.
1999
. Impaired anti-bacterial host defense in mice lacking the N-formylpeptide receptor.
J. Exp. Med.
189
:
657
20
Fiore, S., J. F. Maddox, H. D. Perez, C. N. Serhan.
1994
. Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor.
J. Exp. Med.
180
:
253
21
Su, S. B., W. Gong, J. L. Gao, W. Shen, P. M. Murphy, J. J. Oppenheim, J. M. Wang.
1999
. A seven-transmembrane, G protein-coupled receptor, FPRL1, mediates the chemotactic activity of serum amyloid A for human phagocytic cells.
J. Exp. Med.
189
:
395
22
Su, S. B., W. H. Gong, J. L. Gao, W. P. Shen, M. C. Grimm, X. Deng, P. M. Murphy, J. J. Oppenheim, J. M. Wang.
1999
. T20/DP178, an ectodomain peptide of human immunodeficiency virus type 1 gp41, is an activator of human phagocyte N-formyl peptide receptor.
Blood
93
:
3885
23
Su, S. B., J. Gao, W. Gong, N. M. Dunlop, P. M. Murphy, J. J. Oppenheim, J. M. Wang.
1999
. T21/DP107, a synthetic leucine zipper-like domain of the HIV-1 envelope gp41, attracts and activates human phagocytes by using G protein- coupled formyl peptide receptors.
J. Immunol.
162
:
5924
24
Gao, J.-L., E. L. Becker, R. J. Freer, N. Muthukumaraswamy, P. M. Murphy.
1994
. A high potency nonformylated peptide agonist for the phagocyte N-formylpeptide chemotactic receptor.
J. Exp. Med.
180
:
2191
25
Derian, C. K., H. Solomon, J. D. Higgins, M. J. Beblavy, R. J. Santuli, G. J. Bridger, M. C. Pike, D. J. Kroon, A. J. Fischman.
1996
. Selective inhibition of N-formylpeptide-induced neutrophil activation by carbamate-modified peptide analogues.
Biochemistry
35
:
1265
26
Higgins, J. D. I., G. J. Bridger, C. K. Derian, M. J. Beblavy, P. E. Hernandez, F. E. Gaul, M. J. Abrams, M. C. Pike, H. F. Solomon.
1996
. N-terminus urea-substituted chemotactic peptides: new potent agonists and antagonists toward the neutrophil fMLF receptor.
J. Med. Chem.
39
:
1013
27
Seo, J. K., S. Y. Choi, Y. Kim, S. H. Baek, K. T. Kim, C. B. Chae, J. D. Lambeth, P. G. Suh, S. H. Ryu.
1997
. A peptide with unique receptor specificity: stimulation of phosphoinositide hydrolysis and induction of superoxide generation in human neutrophils.
J. Immunol.
158
:
1895
28
Bae, Y. S., S. A. Ju, J. Y. Kim, J. K. Seo, S. H. Baek, J. Y. Kwak, B. S. Kim, P. G. Suh, S. H. Ryu.
1999
. Trp-Lys-Tyr-Met-Val-d-Met stimulates superoxide generation and killing of Staphylococcus aureus via phospholipase D activation in human monocytes.
J. Leukocyte Biol.
65
:
241
29
Baek, S. H., J. K. Seo, C. B. Chae, P. G. Suh, S. H. Ryu.
1996
. Identification of the peptides that stimulate the phosphoinositide hydrolysis in lymphocyte cell lines from peptide libraries.
J. Biol. Chem.
271
:
8170
30
Le, Y., W. Gong, B. Li, N. M. Dunlop, W. Shen, S. B. Su, R. D. Ye, P. M. Murphy, J. M. Wang.
1999
. Utilization of two seven-transmembrane, G protein-coupled receptors, formyl peptide receptor-like 1 and formyl peptide receptor, by the synthetic hexapeptide WKYMVm for human phagocyte activation.
J. Immunol.
163
:
6777
31
Fuhlbrigge, R. C., S. M. Fine, E. R. Unanue, D. D. Chaplin.
1988
. Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1a cDNA.
Proc. Natl. Acad. Sci. USA
85
:
5649
32
Quehenberger, O., E. R. Prossnitz, S. L. Cavanagh, C. G. Cochrane, R. D. Ye.
1993
. Multiple domains of the N-formyl peptide receptor are required for high-affinity ligand binding: construction and analysis of chimeric N-formyl peptide receptors.
J. Biol. Chem.
268
:
18167
33
Ali, H., R. M. Richardson, E. D. Tomhave, J. R. Didsbury, R. Snyderman.
1993
. Differences in phosphorylation of formylpeptide and C5a chemoattractant receptors correlate with differences in desensitization.
J. Biol. Chem.
268
:
24247
34
Hsu, M. H., J. A. Ember, M. Wang, E. R. Prossnitz, T. E. Hugli, R. D. Ye.
1997
. Cloning and functional characterization of the mouse C3a anaphylatoxin receptor gene.
Immunogenetics
47
:
64
35
Wickman, K., D. E. Clapham.
1995
. Ion channel regulation by G proteins.
Physiol. Rev.
75
:
865
36
Grinstein, S., W. Furuya.
1992
. Chemoattractant-induced tyrosine phosphorylation and activation of microtubule-associated protein kinase in human neutrophils.
J. Biol. Chem.
267
:
1812
37
Torres, M., F. L. Hall, K. O’Neill.
1993
. Stimulation of human neutrophils with formyl-methionyl-leucyl-phenylalanine induces tyrosine phosphorylation and activation of two distinct mitogen-activated protein-kinases.
J. Immunol.
150
:
1563
38
Takano, T., S. Fiore, J. F. Maddox, H. R. Brady, N. A. Petasis, C. N. Serhan.
1997
. Aspirin-triggered 15-epi-lipoxin A4 (LXA4) and LXA4 stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors.
J. Exp. Med.
185
:
1693