Receptor FPRL1 Research Articles

Protein kinase C-α and -δ are required for NADPH oxidase activation in WKYMVm-stimulated IMR90 human fibroblasts

The regulation of the activation of non phagocytic NADPH oxidase is poorly understood. Previously we demonstrated that in fibroblasts the exposure to WKYMVm induced p47 phox phosphorylation and translocation and that these effects were mediated by ERKs activation. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in polymorphonucleate cells stimulated via FPRL1 receptor, but its involvement in fibroblasts was not demonstrated. Therefore, we investigated in IMR90 cells exposed to WKYMVm the role of PKC isoenzymes in the activation of NADPH oxidase-like enzyme. Preincubation with general pharmacological inhibitors of PKC, before stimulation with WKYMVm, prevented the ERKs activation, p47 phox phosphorylation and translocation. The analysis of cellular partitioning of PKC isoenzymes demonstrated that PKCα and PKCδ translocated from the cytosolic to the membrane fraction upon stimulation with WKYMVm. Preincubation with Gö6976 or with rottlerin prevented the phosphorylation and translocation of NADPH oxidase regulatory subunit.

An Uncleavable uPAR Mutant Allows Dissection of Signaling Pathways in uPA-dependent Cell Migration

Urokinase-type plasminogen activator (uPA) binding to uPAR induces migration, adhesion, and proliferation through multiple interactions with G proteins-coupled receptor FPRL1, integrins, or the epidermal growth factor (EGF) receptor (EGFR). At least two forms of uPAR are present on the cell surface: full-length and cleaved uPAR, each specifically interacting with one or more transmembrane proteins. The connection between these interactions and the effects on the signaling pathways activation is not clear. We have exploited an uPAR mutant (hcr, human cleavage resistant) to dissect the pathways involved in uPA-induced cell migration. This mutant is not cleaved by proteases, is glycosylphosphatidylinositol anchored, and binds uPA with a normal K(d). Both wild-type (wt) and hcr-uPAR are able to mediate uPA-induced migration, are constitutively associated with the EGFR, and associate with alpha3beta1 integrin upon uPA binding. However, they engage different pathways in response to uPA. wt-uPAR requires both integrins and FPRL1 to mediate uPA-induced migration, and association of wt-uPAR to alpha3beta1 results in uPAR cleavage and extracellular signal-regulated kinase (ERK) activation. On the contrary, hcr-uPAR does not activate ERK and does not engage FPRL1 or any other G protein-coupled receptor, but it activates an alternative pathway initiated by the formation of a triple complex (uPAR-alpha3beta1-EGFR) and resulting in the autotyrosine phosphorylation of EGFR.

Orally administered FPRL1 receptor agonist peptide MMK-1 inhibits etoposide-induced alopecia by a mechanism different from intraperitoneally administered MMK-1

Oral administration for 6 days of 100 mg/kg MMK-1, an agonist peptide selective for the FPRL1 receptor, suppressed alopecia induced by the anticancer drug etoposide in neonatal rats. The anti-alopecia effect of orally administered MMK-1 was not inhibited by pyrilamine or cimetidine, antagonists for histamine H 1 and H 2 receptors, respectively, which blocked the anti-alopecia effect of intraperitoneally administered MMK-1 at a dose of 10 mg/kg for 4 days. However, the anti-alopecia effect of orally administered MMK-1 was inhibited by indomethacin, an inhibitor of cyclooxygenase (COX), or AH-23848B, an antagonist of the EP 4 receptor for prostaglandin (PG) E 2, suggesting involvement of PGE 2 release and the EP 4 receptor in the oral MMK-1 anti-alopecia mechanism. The anti-alopecia effect of orally administered MMK-1 was also blocked by an inhibitor of nuclear factor-κB (NF-κB), pyrrolidine dithiocarbamate, suggesting that the oral anti-alopecia effect of MMK-1 may be mediated by activation of NF-κB. These results suggest that MMK-1 bound to FPRL1 receptor might suppress etoposide-induced apoptosis of hair follicle cells and alopecia by way of PGE 2 release and NF-κB activation.

The Immunostimulatory Peptide WKYMVm‐NH2 Activates Bone Marrow Mouse Neutrophils via Multiple Signal Transduction Pathways

G-protein-coupled receptors play a major role in the activation of the innate immune system, such as polymorphonuclear neutrophils. Members of the formyl peptide receptor family recognize chemotactic peptides as well the amyloïd-beta peptide and fragments of the human immunodeficiency virus envelope and may thus be implicated in major pathologies. The peptide WKYMVm-NH2 probably activates the receptor FPRL1 and its mouse orthologues Fpr-rs1 and Fpr-rs2. We examined the stimulation of C57BL6 mouse neutrophils by WKYMVm-NH2 and the effects of several inhibitors for intracellular signalling pathways (wortmannin, LY 294002, staurosporin, H-89, U 73122, thapsigargin and SKF 96365). We show here that WKYMVm-NH2 is a powerful stimulator of primary and secondary granule exocytosis as well as superoxide production. The signalling pathway involves phosphoinositide 3-kinase, protein kinase C, phospholipase C and store-operated calcium influx. Studies with peptide antagonists suggest that WKYMVm-NH2 preferentially activates exocytosis via FPRL1 and not FPR, the major receptor for N-formylated peptides such as fMLF. However, the signalling pathways activated by WKYMVm-NH2 in mouse neutrophils are similar to those activated by fMLF in human neutrophils. Thus, the effect and the signalling pathways of the two agonists and their receptors are at least partially overlapping.

The Chemoattractant Receptors FPR and C5aR: Same Functions – Different Fates

The Chemoattractant Receptors FPR and C5aR: Same Functions – Different Fates

Agonist-induced trafficking of the low-affinity formyl peptide receptor FPRL1.

The formyl peptide-like receptor FPRL1 is a member of the chemoattractant subfamily of G protein- coupled receptors involved in regulating leukocyte migration in inflammation. To elucidate mechanisms underlying the internalization of ligand-bound FPRL1 and possible receptor recycling, we characterized the endocytic itinerary of FPRL1. We show that agonist-triggered internalization from the plasma membrane into intracellular compartments is prevented by perturbation of clathrin-mediated endocytosis, such as expression of the dominant-negative clathrin Hub mutant, siRNA-mediated depletion of cellular clathrin and expression of a dominant-negative mutant of the large GTPase dynamin. Internalized FPRL1 co-localized with endocytosed transferrin and the small GTPases Rab4 and Rab11 in vesicular structures most resembling recycling endosomes. Recycling of FPRL1 was significantly reduced by pretreatment with PI3-kinase inhibitors. Thus, ligand-bound FPRL1 undergoes primarily clathrin-mediated and dynamin-dependent endocytosis and the receptor recycles via a rapid PI3-kinase-sensitive route as well as pathways involving perinuclear recycling endosomes.

Inhibition of pulmonary inflammatory diseases, asthma and pulmonary fibrosis, by lipoxin A4 and a novel series of selective small molecule FPRL1 agonists

Rationale Administration of a lipoxin A4 stable analog blocks airway hyperresponsiveness (AHR) and pulmonary inflammation. The lipoxin A4 receptor, LXA4R/FPRL1, is a G protein-couple receptor expressed on leukocytes. In this study, we examined the effect of lipoxin A4 and a novel series of small molecule FPRL1 agonists on inflammatory responses. Small molecule compounds with an anti-inflammatory profile in vitro were then assessed in animal models of asthma and pulmonary fibrosis in vivo. Methods Eosinophils and neutrophils were incubated with lipoxin A4 or FPRL1 agonists prior to induction of LL37-induced eosinophil degranulation and IL-8 synthesis from neutrophils in response to TNFα. LL37, an antimicrobial peptide, can cause eosinophil degranulation and results in high levels of eosinophil cationic protein (ECP) in cell culture supernatants. In addition, active compounds were further assessed for their ability to inhibit ovalbumin-induced AHR in BALB/c mice and bleomycin-induced pulmonary fibrosis in C57BL/6 mice. Results LL37-induced ECP was suppressed by lipoxin A4 and some receptor specific agonists. Furthermore, AR232925 suppresses ovalbumin-induced AHR in vivo. In neutrophils, TNFα-induced IL-8 was suppressed by lipoxin A4 and several novel FPRL1 agonists. One of these FPRL1 agonists, AR234245, decreased mortality in the mouse model of bleomycin-induced pulmonary fibrosis. Conclusions Here we show that a novel series of selective FPRL1 receptor agonists inhibit eosinophil degranulation and IL-8 responsiveness in neutrophils in vitro. In addition, these agonists inhibit AHR and pulmonary fibrosis in vivo. These data suggest the use of a novel series of FPRL1 agonists for the treatment of human asthma and COPD.

Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils

BackgroundCytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions.ResultsTumor necrosis factor alpha (TNF-alpha) primes neutrophils for subsequent activation by cytochalasin B. Pretreatment with TNF-alpha induced mobilization of receptor-storing neutrophil organelles, suggesting that receptor up-regulation significantly contributes to the response, but the receptor mobilization was not sufficient for induction of the cytochalasin B sensitive state. The TNF-alpha primed state resembled that of the desensitized non-signaling state of agonist-occupied neutrophil formyl peptide receptors. The fact that the TNF-alpha primed, cytochalasin B-triggered activation process was pertussis toxin sensitive suggests that the activation process involves a GPCR. Based on desensitization experiments the unidentified receptor was found to be distinct from the C5a receptor as well as the formyl peptide receptor family members FPR and FPRL1. Based on the fact the occupied and desensitized receptors for interleukin-8 and platelet activating factor could not be reactivated by cytochalasin B, also these could be excluded as receptor candidates involved in the TNF-alpha primed state.ConclusionsThe TNF-alpha-induced priming signals could possibly trigger a release of an endogenous GPCR-agonist, amplifying the response to the receptor-uncoupling effect of cytochalasin B. However, no such substance could be found, suggesting that TNF-alpha can transfer G-protein coupled receptors to a signaling state independently of agonist binding.

The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide

BackgroundThe formylpeptide receptor family members FPR and FPRL1, expressed in myeloid phagocytes, belong to the G-protein coupled seven transmembrane receptor family (GPCRs). They share a high degree of sequence similarity, particularly in the cytoplasmic domains involved in intracellular signaling. The established model of cell activation through GPCRs states that the receptors isomerize from an inactive to an active state upon ligand binding, and this receptor transformation subsequently activates the signal transducing G-protein. Accordingly, the activation of human neutrophil FPR and FPRL1 induces identical, pertussis toxin-sensitive functional responses and a transient increase in intracellular calcium is followed by a secretory response leading to mobilization of receptors from intracellular stores, as well as a release of reactive oxygen metabolites.ResultsWe report that a cell permeable ten amino acid peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate (PIP2) binding region of gelsolin (an uncapper of actin filaments) blocks granule mobilization as well as secretion of oxygen radicals. The inhibitory effect of PBP10 is, however, receptor specific and affects the FPRL1-, but not the FPR-, induced cellular response. The transient rise in intracellular calcium induced by the active receptors is not affected by PBP10, suggesting that the blockage occurs in a parallel, novel signaling pathway used by FPRL1 to induce oxygen radical production and secretion. Also the FPR can activate neutrophils through a PBP10-sensitive signaling pathway, but this signal is normally blocked by the cytoskeleton.ConclusionsThis study demonstrates that the two very closely related chemoattractant receptors, FPR and FPRL1, use distinct signaling pathways in activation of human neutrophils. The PIP2-binding peptide PBP10 selectively inhibits FPRL1-mediated superoxide production and granule mobilization. Furthermore, the activity of this novel PBP10 sensitive pathway in neutrophils is modulated by the actin cytoskeleton network.

Low-affinity receptor-mediated induction of superoxide by N-formyl-methionyl-leucyl-phenylalanine and WKYMVm in IMR90 human fibroblasts

We investigated in IMR90 cells the effects of N-formyl-Met-Leu-Phe ( N-fMLP) and WKYMVm (W peptide) on activation of the NADPH oxidase-like enzyme. In serum-deprived human fibroblasts, exposure to 100 μM N-fMLP or 10 μM peptide W for 1 min induced both p47 phox translocation and NADPH-dependent superoxide generation. These effects were in large part mediated by prevention of the rapid activation of extracellular signal-regulated kinases (ERKs) by preincubation with the MEK1 inhibitor PD098059. Furthermore, responses to N-fMLP or W peptide were inhibited by pertussis toxin, suggesting the involvement of a seven-transmembrane G protein-coupled receptor(s) for peptides. RT-PCR experiments demonstrated the expression in these cells of the low-affinity receptor FPRL1, but not the high-affinity receptor FPR. Incubation with radiolabeled WKYMVm, which had a higher efficiency on FPRL1, revealed that human fibroblasts express binding sites for 125I-WKYMVm that are specifically displaced by increasing concentrations of unlabeled ligand. Analysis of the binding data predicted a K d of 155.99 nM and a receptor density of about 16,200 molecules/cell. HEK293 cells, which express a NADPH oxidase-like enzyme but not formyl peptide receptors, transiently transfected with FPRL1 cDNA produced superoxide on stimulation with N-fMLP or W peptide, demonstrating that this receptor is biologically functional.

LOW-AFFINITY RECEPTOR-MEDIATED INDUCTION OF SUPEROXIDE BY N-FORMYL-METHIONYL-LEUCYL-PHENYLALANINE AND WKYMVm IN IMR90 HUMAN FIBROBLASTS

We investigated in IMR90 cells the effects of N -formyl-Met-Leu-Phe ( N -fMLP) and WKYMVm (W peptide) on activation of the NADPH oxidase-like enzyme. In serum-deprived human fibroblasts, exposure to 100 μM N -fMLP or 10 μM peptide W for 1 min induced both p47 phox translocation and NADPH-dependent superoxide generation. These effects were in large part mediated by prevention of the rapid activation of extracellular signal-regulated kinases (ERKs) by preincubation with the MEK1 inhibitor PD098059. Furthermore, responses to N -fMLP or W peptide were inhibited by pertussis toxin, suggesting the involvement of a seven-transmembrane G protein-coupled receptor(s) for peptides. RT-PCR experiments demonstrated the expression in these cells of the low-affinity receptor FPRL1, but not the high-affinity receptor FPR. Incubation with radiolabeled WKYMVm, which had a higher efficiency on FPRL1, revealed that human fibroblasts express binding sites for 125 I-WKYMVm that are specifically displaced by increasing concentrations of unlabeled ligand. Analysis of the binding data predicted a K d of 155.99 nM and a receptor density of about 16,200 molecules/cell. HEK293 cells, which express a NADPH oxidase-like enzyme but not formyl peptide receptors, transiently transfected with FPRL1 cDNA produced superoxide on stimulation with N -fMLP or W peptide, demonstrating that this receptor is biologically functional.

β Amyloid peptide (Aβ42) is internalized via the G‐protein‐coupled receptor FPRL1 and forms fibrillar aggregates in macrophages1

The 42 amino acid form of beta amyloid (Abeta42) plays a pivotal role in neurotoxicity and the activation of mononuclear phagocytes in Alzheimer's disease (AD). Our recent study revealed that FPRL1, a G-protein-coupled receptor, mediates the chemotactic and activating effect of Abeta42 on mononuclear phagocytes (monocytes and microglia), suggesting that FPRL1 may be involved in the proinflammatory responses in AD. We investigated the role of FPRL1 in cellular uptake and the subsequent fibrillar formation of Abeta42 by using fluorescence confocal microscopy. We found that upon incubation with macrophages or HEK293 cells genetically engineered to express FPRL1, Abeta42 associated with FPRL1 and the Abeta42/FPRL1 complexes were rapidly internalized into the cytoplasmic compartment. The maximal internalization of Abeta42/FPRL1 complexes occurred by 30 min after incubation. Removal of free Abeta42 from culture supernatants at 30 min resulted in a progressive recycling of FPRL1 to the cell surface and degradation of the internalized Abeta42. However, persistent exposure of the cells to Abeta42 over 24 h resulted in retention of Abeta42/FPRL1 complexes in the cytoplasmic compartment and the formation of Congo red positive fibrils in macrophages but not in HEK 293 cell transfected with FPRL1. These results suggest that besides mediating the proinflammatory activity of Abeta42, FPRL1 is also involved in the internalization of Abeta42, which culminates in the formation of fibrils only in macrophages.

N36, a synthetic N-terminal heptad repeat domain of the HIV-1 envelope protein gp41, is an activator of human phagocytes.

Human immunodeficiency virus type 1 (HIV-1) envelope protein gp41 mediates viral fusion with human host cells. In this study we show that N36, a synthetic peptide derived from the N-terminus of gp41, induced directional migration and calcium mobilization in human monocytes and neutrophils. The activity of N36 on phagocytes was pertussis toxin sensitive, suggesting involvement of a Gi-coupled seven-transmembrane receptor(s). Since high concentrations of the bacterial chemotactic peptide fMet-Leu-Phe (fMLF) partially desensitized the calcium mobilizing activity of N36 in phagocytes, we postulated that N36 might use a low-affinity fMLF receptor. By using cells stably expressing fMLF receptor FPR or FPRL1, we demonstrate that N36 uses FPRL1 as a functional receptor. Our results suggest that HIV-1 gp41 may contain a fragment(s) that activates the innate host immune cells through FPRL1. Since the activation of FPRL1 in monocytes has been shown to heterologously desensitize chemokine receptors, the reduced phagocyte response to chemoattractants seen in AIDS patients may be attributed, at least in part, to heterologous desensitization.

Activation of the Chemotactic Peptide Receptor FPRL1 in Monocytes Phosphorylates the Chemokine Receptor CCR5 and Attenuates Cell Responses to Selected Chemokines

FPRL1 is a seven-transmembrane (STM), G-protein coupled receptor which was originally identified as a low affinity receptor for the bacterial chemotactic formyl peptide and a high affinity receptor for the lipid metabolite lipoxin A4. We recently discovered that a number of peptides, including several synthetic domains of the HIV-1 envelope proteins and the serum amyloid A, use FPRL1 to induce migration and calcium mobilization in human monocytes and neutrophils. In this study, we report that a synthetic peptide domain of the V3 region of the HIV-1 envelope gp120, activates the FPRL1 receptor in monocytes and neutrophils. Furthermore, monocytes prestimulated with V3 peptide showed reduced response to several chemokines that use multiple cell receptors. This is associated with a rapid phosphorylation of the chemokine receptor CCR5 on the serine residues. Our study suggests that FPRL1, as a classical chemoattractant receptor, may play an important role in modulating monocyte activation in the presence of multiple stimuli.

Differential Expansion of the N-Formylpeptide Receptor Gene Cluster in Human and Mouse

The human formylpeptide receptor (FPR) gene cluster has three members:FPR1andFPRL1, which are expressed in neutrophils and monocytes and encode seven-transmembrane-domain chemotactic receptors specific for N-formylpeptides, andFPRL2,whose function is unknown. The FPRL1 receptor is also a lipoxin A4 receptor. Using probes for the three human genes we have cloned six distinct mouse genes, designatedFpr1andFpr-rs1throughFpr-rs5,which form a cluster on chromosome 17 in a region of conserved synteny with human chromosome 19.Fpr1encodes a functional receptor and is clearly the orthologue ofFPR1.BothFpr-rs1andFpr-rs2have higher sequence homology toFPRL1than toFPRL2; Fpr-rs1is 97% identical in amino acid sequence to a previously reported cDNA that encodes a lipoxin A4 receptor, whereas the putative ligand forFpr-rs2is unknown.Fpr-rs3, Fpr-rs4,andFpr-rs5appear to lack human counterparts and are most similar in sequence toFPRL1.RNA forFpr1, Fpr-rs1,andFpr-rs2is present in leukocytes, spleen, and lung, whereas RNA forFpr-rs3was detected only in skeletal muscle. We did not detectFpr-rs4orFpr-rs5RNA in any tissue tested. Moreover,Fpr-rs5has a stop codon in the protein-coding region corresponding to transmembrane domain VI and may not encode a functional receptor. These results suggest that the FPR gene cluster has undergone differential expansion in mammals withFPRL2, Fpr-rs2, Fpr-rs3, Fpr-rs4,andFpr-rs5arising after divergence of human and mouse.

A structural homologue of the N-formyl peptide receptor. Characterization and chromosome mapping of a peptide chemoattractant receptor family.

Phagocytic cells of many higher species express calcium mobilizing G protein-coupled receptors for bacterial N-formyl peptides which mediate chemotaxis, degranulation, and the respiratory burst. cDNA encoding an N-formyl peptide receptor (FPR) has been reported. We now report the isolation of a closely related cDNA, 2.6 kilobase pairs in length, which we have designated as the FPRL1 receptor cDNA (FPRL1 = formyl peptide receptor like-1). FPR and the FPRL1 receptor derive from small, single-copy genes, both of which are located on human chromosome 19. The gene loci are designated FPR1 and FPRL1, respectively. Both FPR and FPRL1 cDNA cross-hybridize under high stringency conditions with a third gene, designated as FPRL2, which does not appear to be expressed in neutrophils. In contrast, transcripts for both the FPRL1 receptor and FPR are detected only in differentiated myeloid cells; the distribution of N-formyl peptide binding sites is also restricted to mature myeloid cells. FPRL1 cDNA encodes a 351-amino acid polypeptide whose sequence is 69% identical to that of FPR. G protein-coupled receptors that exhibit this degree of structural similarity typically possess a conserved ligand specificity. However, the FPRL1 receptor does not bind prototype N-formyl peptide ligands when expressed in heterologous cell types. These results suggest that FPR1 may be the only gene that is expressed by neutrophils that encodes a receptor capable of binding prototype N-formyl peptides. Moreover, discovery of the FPRL1 receptor indicates the existence of another as yet unidentified peptide that may recruit neutrophils to sites of inflammation.