Identification of C-terminal Phosphorylation Sites of N-Formyl Peptide Receptor-1 (FPR1) in Human Blood Neutrophils

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 M_r) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu³¹²–Arg³²² and Arg³²³–Lys³⁵⁰) and extracellular FPR1 peptide (Ile¹⁹¹–Arg²⁰¹) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala³²³–Lys³⁵⁰ only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr³²⁵, Ser³²⁸, Thr³²⁹, Thr³³¹, Ser³³², Thr³³⁴, and Thr³³⁹. No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC₅₀ = 33 ± 8 to 74 ± 21 nm. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.     

Synthesis and evaluation of a [18F]formyl–Met–Leu–Phe derivative: A positron emission tomography imaging probe for bacterial infections

The tripeptide formyl–Met–Leu–Phe (fMLF) is a prototype of N-formylated chemotactic peptides for neutrophils owing to its ability to bind and activate the G protein-coupled formyl peptide receptor (FPR). Here, we developed an ¹⁸F-labeled fMLF derivative targeting FPR as a positron emission tomography (PET) imaging probe for bacterial infections. The study demonstrates that the fMLF derivative fMLFXYk(FB)k (X?=?Nle) has a high affinity for FPR (Ki?=?0.62?±?0.13?nM). The radiochemical yield and purity of [¹⁸F]fMLFXYk(FB)k were 16% and >96%, respectively. The in vivo biodistribution study showed that [¹⁸F]fMLFXYk(FB)k uptake was higher in the bacterial infected region than in the non-infected region. We observed considerably higher infection-to-muscle ratio of 4.6 at 60?min after [¹⁸F]fMLFXYk(FB)k injection. Furthermore, small-animal PET imaging studies suggested that [¹⁸F]fMLFXYk(FB)k uptake in the bacterial infected region was clearly visualized 60?min after injection.     

Characterization of the binding site on the formyl peptide receptor using three receptor mutants and analogs of Met-Leu-Phe and Met-Met-Trp-Leu-Leu

The formyl peptide receptor (FPR) is a chemotactic G protein-coupled receptor found on the surface of phagocytes. We have previously shown that the formyl peptide binding site maps to the membrane-spanning region (Miettinen, H. M., Mills, J. S., Gripentrog, J. M., Dratz, E. A., Granger, B. L., and Jesaitis, A. J. (1997) J. Immunol. 159, 4045-4054). Recent reports have indicated that non-formylated peptides, such as MMWLL can also activate this receptor (Chen, J., Bernstein, H. S., Chen, M., Wang, L., Ishi, M., Turck, C. W., and Coughlin, S. R. (1995) J. Biol. Chem. 270, 23398-23401.) Here we show that the selectivity for the binding of different NH(2)-terminal analogs of MMWLL or MLF can be markedly altered by mutating Asp-106 to asparagine or Arg-201 to alanine. Both D106N and R201A produced a similar change in ligand specificity, including an enhanced ability to bind the HIV-1 peptide DP178. In contrast, the mutation R205A exhibited altered specificity at the COOH terminus of fMLF, with R205A binding fMLF-O-butyl > fMLF-O-methyl > fMLF, whereas wt FPR bound fMLF > fMLF-O-methyl approximately fMLF-O-butyl. These data, taken together with our previous finding that the leucine side chain of fMLF is probably bound to FPR near FPR (93)VRK(95) (Mills, J. S., Miettinen, H. M., Barnidge, D., Vlases, M. J., Wimer-Mackin, S., Dratz, E. A., and Jesaitis, A. J. (1998) J. Biol. Chem. 273, 10428-10435.), indicate that the most likely positioning of fMLF in the binding pocket of FPR is approximately parallel to the fifth transmembrane helix with the formamide group of fMLF hydrogen-bonded to both Asp-106 and Arg-201, the leucine side chain pointing toward the second transmembrane region, and the COOH-terminal carboxyl group of fMLF ion-paired with Arg-205.     

PKC-δ controls the fMLF-induced overproduction of superoxide by neutrophils

Antimicrobial defense by neutrophils implicates the production of reactive oxygen species. Neutrophil responses can be modulated by agonists such as bacterial peptides and proinflammatory factors that modulate neutrophil activity and survival. We evaluated the production of superoxide anions (O₂^?) in response to fMLF by normal human neutrophils after 3 days of preincubation with GM-CSF + IL-4 + TNF-α (survival medium). After 3 days of incubation in survival medium, long-lived neutrophils produced up to six times more O₂^? relative to control neutrophils in response to fMLF and WKYMVM. This augmented response to fMLF was preferentially linked to formyl peptide receptor (FPR), whereas the response to WKYMVM was dependent on formyl peptide receptor-like 1 (FPRL-1). Real-time RT-PCR revealed a diminution of FPR and FPRL-1 expression in neutrophils incubated in survival medium. fMLF-induced overproduction of O₂^? by long-lived neutrophils was independent of intracellular calcium mobilization. The protein tyrosine phosphorylation profile of long-lived neutrophils was modified with respect to control cells. Pharmacological inhibitors of intracellular signals indicated that mechanisms of the excessive fMLF-induced production of O₂^? by long-lived neutrophils implicated the protein kinase C (PKC) pathway, preferentially the PKC-δ isoform, whose protein was augmented in these cells. Thus, long-term cytokine exposure modifies molecular pathways and functional characteristics of the neutrophil.     

Small G-proteins Ras,Rac and Rho in the regulation of the neutrophil respiratory burst induced by formyl peptide

Formylated peptides specifically activate many of the neutrophil functions; their action is mediated via formyl peptide receptors (FPRs). FPRs belong to the family of receptors having seven transmembrane-spanning domains and coupled with G-proteins (GPCR). About a dozen of highly homologous genes of FPRs were found to be localized in mouse chromosome 17. By binding with labeled N-formyl-Met-Leu-Phe (fMLF), FPRs are classified as receptors with high (FPR1) and low (FPR2 and FPR3/FPRL1) affinity to formyl peptide. Binding of formyl peptide with FPRs triggers the complex signaling events, the most studied are: activation of phospholipase C (PLC) with subsequent calcium signaling; launching of mitogen activated protein kinases (MAPKs) cascade pathway, and activation of phosphoinositol-3-kinase (PI3K) cascades. As we have shown previously, the priming of the respiratory burst of mice neutrophils occurs under the cell activation by fMLF in high doses only, i.e., it is necessary to activate low affinity FPRs. Besides, the usage of the specific MEK and p38MAPK inhibitors induced significant suppression of the response to 1 μM fMLM, while the response to 50 μM fMLF increased in the presence of the inhibitors. We suggest that there is a signal divergence upon activation of high and low affinity fMLF receptors, and small G protein dependent signaling pathways could be alternative to activate NADPH oxidase. Here we demonstrate that Ras-proteins participate in the respiratory burst activation, especially in activation via the high affinity fMLF receptors. Activation of the Rho- and Rac-proteins induced the down-regulation of the respiratory burst under the stimulation of high affinity FPRs. The inhibition of the Rho-proteins almost completely suppressed the respiratory burst activated via the high and low affinity receptors, probably due to inability to assemble of the cytoskeleton proteins and NADPH oxidase components.     

Regulation of the Formyl Peptide Receptor 1 (FPR1) Gene in Primary Human Macrophages

The formyl peptide receptor 1 (FPR1) is mainly expressed by mammalian phagocytic leukocytes and plays a role in chemotaxis, killing of microorganisms through phagocytosis, and the generation of reactive oxygen species. A large number of ligands have been identified triggering FPR1 including formylated and non-formylated peptides of microbial and endogenous origin. While the expression of FPR1 in neutrophils has been investigated intensively, knowledge on the regulation of FPR1 expression in polarized macrophages is lacking. In this study we show that primary human neutrophils, monocytes and resting macrophages do express the receptor on their cell surface. Polarization of macrophages with IFNγ, LPS and with the TLR8 ligand 3M-002 further increases FPR1 mRNA levels but does not consistently increase protein expression or chemotaxis towards the FPR1 ligand fMLF. In contrast, polarization of primary human macrophages with IL-4 and IL-13 leading to the alternative activated macrophages, reduces FPR1 cell surface expression and abolishes chemotaxis towards fMLF. These results show that M2 macrophages will not react to triggering of FPR1, limiting the role for FPR1 to chemotaxis and superoxide production of resting and pro-inflammatory M1 macrophages.     

Identification of formyl peptides from Listeria monocytogenes and Staphylococcus aureus as potent chemoattractants for mouse neutrophils

The prototypic formyl peptide N-formyl-Met-Leu-Phe (fMLF) is a major chemoattractant found in Escherichia coli culture supernatants and a potent agonist at human formyl peptide receptor (FPR) 1. Consistent with this, fMLF induces bactericidal functions in human neutrophils at nanomolar concentrations. However, it is a much less potent agonist for mouse FPR (mFPR) 1 and mouse neutrophils, requiring micromolar concentrations for cell activation. To determine whether other bacteria produce more potent agonists for mFPR1, we examined formyl peptides from Listeria monocytogenes and Staphylococcus aureus for their abilities to activate mouse neutrophils. A pentapeptide (N-formyl-Met-Ile-Val-Ile-Leu (fMIVIL)) from L. monocytogenes and a tetrapeptide (N-formyl-Met-Ile-Phe-Leu (fMIFL)) from S. aureus were found to induce mouse neutrophil chemotaxis at 1-10 nM and superoxide production at 10-100 nM, similar to the potency of fMLF on human neutrophils. Using transfected cell lines expressing mFPR1 and mFPR2, which are major forms of FPRs in mouse neutrophils, we found that mFPR1 is responsible for the high potency of fMIVIL and fMIFL. In comparison, activation of mFPR2 requires micromolar concentrations of the two peptides. Genetic deletion of mfpr1 resulted in abrogation of neutrophil superoxide production and degranulation in response to fMIVIL and fMIFL, further demonstrating that mFPR1 is the primary receptor for detection of these formyl peptides. In conclusion, the formyl peptides from L. monocytogenes and S. aureus are approximately 100-fold more potent than fMLF in activating mouse neutrophils. The ability of mFPR1 to detect bacterially derived formyl peptides indicates that this important host defense mechanism is conserved in mice.     

The synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met is a potent chemotactic agonist for mouse formyl peptide receptor

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 (K(d) approximately 1 nM). The mouse FPR (mFPR) is a low-affinity receptor for fMLF (K(d) approximately 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 EC(50) 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 Galpha 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 [(3)H]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.     

Cathepsin G-regulated Release of Formyl Peptide Receptor Agonists Modulate Neutrophil Effector Functions

Neutrophil serine proteases play an important role in inflammation by modulating neutrophil effector functions. We have previously shown that neutrophils deficient in the serine proteases cathepsin G and neutrophil elastase (CG/NE neutrophils) exhibit severe defects in chemokine CXCL2 release and reactive oxygen species (ROS) production when activated on immobilized immune complex. Exogenously added active CG rescues these defects, but the mechanism remains undefined. Using a protease-based proteomic approach, we found that, in vitro, the addition of exogenous CG to immune complex-stimulated CG/NE neutrophils led to a decrease in the level of cell-associated annexin A1 (AnxA1) and cathelin-related antimicrobial peptide (CRAMP), both known inflammatory mediators. We further confirmed that, in vivo, CG was required for the extracellular release of AnxA1 and CRAMP in a subcutaneous air pouch model. In vitro, CG efficiently cleaved AnxA1, releasing the active N-terminal peptide Ac2-26, and processed CRAMP in limited fashion. Ac2-26 and CRAMP peptides enhanced the release of CXCL2 by CG/NE neutrophils in a dose-dependent manner via formyl peptide receptor (FPR) stimulation. Blockade of FPRs by an antagonist, Boc2 (t-Boc-Phe-d-Leu-Phe-d-Leu-Phe), abrogates CXCL2 release, whereas addition of FPR agonists, fMLF and F2L, relieves Boc2 inhibition. Furthermore, the addition of active CG, but not inactive CG, also relieves Boc2 inhibition. These findings suggest that CG modulates neutrophil effector functions partly by controlling the release (and proteolysis) of FPR agonists. Unexpectedly, we found that mature CRAMP, but not Ac2-26, induced ROS production through an FPR-independent pathway.     

Cyclization of the Urokinase Receptor-Derived Ser-Arg-Ser-Arg-Tyr Peptide Generates a Potent Inhibitor of Trans-Endothelial Migration of Monocytes

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR_88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR_88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC₅₀ value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.     

The N-formylpeptide receptor (FPR) and a second G(i)-coupled receptor mediate fMet-Leu-Phe-stimulated activation of NADPH oxidase in murine neutrophils

N-Formylypeptides such as fMet-Leu-Phe (fMLF) potently induce superoxide production through NADPH oxidase activation. The receptors that mediate this response have not been defined. Here, we provide definitive proof using a mouse model that formyl peptide receptor (FPR) is a receptor, but not the only receptor, that mediates fMLF-induced oxidase activation. In wild-type (FPR(+/+)) mouse neutrophils, superoxide production is dependent on the concentration of fMLF with an EC(50) of approximately 5 microM and a peak at approximately 50 microM. In contrast, FPR-deficient (FPR(-/-)) mouse neutrophils produced markedly less superoxide with an EC(50) of approximately 50 microM and a peak at approximately 200 microM. Yet, FPR(+/+) and FPR(-/-) neutrophils showed similar oxidase activation kinetics and G(i) protein-dependent pharmacological sensitivities. These results suggested that a second receptor, likely FPR2, mediates superoxide production at high concentrations of fMLF. This less sensitive second pathway may permit continued oxidant generation in response to formyl peptides when FPR is desensitized in high concentrations of the chemotactic gradient.     

Discovery of small molecule human FPR1 receptor antagonists

The identification of two novel series of formyl peptide receptor 1 (FPR1) antagonists are reported, represented by methionine benzimidazole 6 and diamide 7. Both series specifically inhibited the binding of labelled fMLF to hrFPR1 and selectively antagonized FPR1 function in human neutrophils, making them useful in vitro validation tools for the target.     

A formyl peptide substituted with a conformationally constrained phenylalanine residue evokes a selective immune response in human neutrophils

Formyl-Met-Leu-Phe-OH (fMLP) binds to formyl peptide receptors, FPR1 and FPR2, and evokes migration and superoxide anion production in human neutrophils. To obtain a more effective and selective ligand, fMLP analogs in which the Phe residue was substituted with four isomers of cyclopropanephenylalanine were synthesized. While Z-isomer peptides induced both migration and superoxide anion production, E-isomer peptides elicited only chemotaxis. Homologous receptor desensitization experiments revealed that E-isomer peptides bound to FPR2. Although a selective agonist of chemotaxis also binds to FPR2 without increasing intracellular calcium concentration, E-isomer peptide elevated the concentration to the same level as fMLP. Understanding of mechanisms responsible for the selectivity of the reported selective agonists and ?Phe-substituted analogs should prove useful for revealing the relationship between receptor–ligand interactions and biological responses of human neutrophils.     

Stimulation of human formyl peptide receptors by calpain inhibitors: homology modeling of receptors and ligand docking simulation

Calpain inhibitors, including peptide aldehydes (N-acetyl-Leu-Leu-Nle-CHO and N-acetyl-Leu-Leu-Met-CHO) and α-mercapto-acrylic acid derivatives (PD150606 and PD151746), have been shown to stimulate phagocyte functions via activation of human formyl peptide receptor (hFPR) and/or hFPR-like 1 (hFPRL1). Using the homology modeling of the receptors and the ligand docking simulation, here we show that these calpain inhibitors could bind to the putative N-formyl-Met-Leu-Phe (fMLF) binding site on hFPR and/or hFPRL1. The studies with HEK-293 cells stably expressing hFPR or hFPRL1 showed that the concentrations of calpain inhibitors required to induce an increase in cytoplasmic free Ca²⁺ ([Ca²⁺]_i) was much higher (>100 folds) than those of fMLF and Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm). HEK-293 cells expressing hFPR or hFPRL1 with the mutated fMLF binding site never exhibited the [Ca²⁺]_i response to calpain inhibitors. When the optimal concentrations of each stimulus were used, pretreatment of cells with fMLF or WKYMVm abolished an increase in [Ca²⁺]_i induced by calpain inhibitors as well as the same stimulus, whereas pretreatment of cells with calpain inhibitors significantly suppressed, but never abolished, the [Ca²⁺]_i response induced by fMLF or WKYMVm, suggesting that the binding affinity of the inhibitors to the putative fMLF binding site may be lower than that of fMLF or WKYMVm.     

The FPR2-induced rise in cytosolic calcium in human neutrophils relies on an emptying of intracellular calcium stores and is inhibited by a gelsolin-derived PIP<Subscript>2</Subscript>-binding peptide

Background  

The molecular basis for neutrophil recognition of chemotactic peptides is their binding to specific G-protein-coupled cell surface receptors (GPCRs). Human neutrophils express two pattern recognition GPCRs, FPR1 and FPR2, which belong to the family of formyl peptide receptors. The high degree of homology between these two receptors suggests that they share many functional and signal transduction properties, although they exhibit some differences with respect to signaling. The aims of this study were to determine whether FPR2 triggers a unique signal that allows direct influx of extracellular calcium without the emptying of intracellular calcium stores, and whether the gelsolin-derived PIP₂-binding peptide, PBP10, selectively inhibits FPR2-mediated transient rise in intracellular Ca²⁺.     

Expression and functional role of formyl peptide receptor in human bone marrow-derived mesenchymal stem cells

We investigated the expression of formyl peptide receptor (FPR) and its functional role in human bone marrow-derived mesenchymal stem cells (MSCs). We analyzed the expression of FPR by using ligand-binding assay with radio-labeled N-formyl-met-leu-phe (fMLF), and found that MSCs express FPR. FMLF stimulated intracellular calcium increase, mitogen-activated protein kinases activation, and Akt activation, which were mediated by G(i) proteins. MSCs were chemotactically migrated to fMLF. FMLF-induced MSC chemotaxis was also completely inhibited by pertussis toxin, LY294002, and PD98059, indicating the role of G(i) proteins, phosphoinositide 3-kinase, and extracellular signal regulated protein kinase. N-terminal fragment of annexin-1, Anx-1(2-26), an endogenous agonist for FPR, also induced chemotactic migration of MSCs. Thus MSCs express functional FPR, suggesting a new (patho)physiological role of FPR and its ligands in regulating MSC trafficking during induction of injured tissue repair.     

Human neutrophils are activated by a peptide fragment of Clostridium difficile toxin B presumably via formyl peptide receptor

Clostridium difficile may induce antibiotic‐associated diarrhoea and, in severe cases, pseudomembranous colitis characterized by tremendous neutrophil infiltration. All symptoms are caused by two exotoxins: TcdA and TcdB. We describe here the activation of isolated human blood neutrophils by TcdB and, moreover, by toxin fragments generated by limited proteolytical digestion. Kinetics and profiles of TcdB‐induced rise in intracellular‐free Ca²⁺ and reactive oxygen species production were similar to that induced by fMLF, which activates the formyl peptide receptor (FPR) recognizing formylated bacterial peptide sequences. Transfection assays with the FPR‐1 isoform hFPR26 in HEK293 cells, heterologous desensitization experiments and FPR inhibition via cyclosporine H strongly suggest activation of cells via FPR‐1. Domain analyses revealed that the N‐terminal glucosyltransferase domain of TcdB is a potent activator of FPR pointing towards an additional mechanism that might contribute to pathogenesis. This pro‐inflammatory ligand effect can be triggered even by cleaved and, thus, non‐cytotoxic toxin. In summary, we report (i) a ligand effect on neutrophils as completely new molecular mode of action, (ii) pathogenic potential of truncated or proteolytically cleaved ‘non‐cytotoxic’ fragments and (iii) an interaction of the N‐terminal glucosyltransferase domain instead of the C‐terminal receptor binding domain of TcdB with target cells.     

Antimicrobial peptide scolopendrasin VII,derived from the centipede Scolopendra subspinipes mutilans,stimulates macrophage chemotaxis via formyl peptide receptor 1

In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. [BMB Reports 2015; 48(8): 479-484]     

Formyl Peptide Receptor as a Novel Therapeutic Target for Anxiety-Related Disorders

Formyl peptide receptors (FPR) belong to a family of sensors of the immune system that detect microbe-associated molecules and inform various cellular and sensorial mechanisms to the presence of pathogens in the host. Here we demonstrate that Fpr2/3-deficient mice show a distinct profile of behaviour characterised by reduced anxiety in the marble burying and light-dark box paradigms, increased exploratory behaviour in an open-field, together with superior performance on a novel object recognition test. Pharmacological blockade with a formyl peptide receptor antagonist, Boc2, in wild type mice reproduced most of the behavioural changes observed in the Fpr2/3 ^-/- mice, including a significant improvement in novel object discrimination and reduced anxiety in a light/dark shuttle test. These effects were associated with reduced FPR signalling in the gut as shown by the significant reduction in the levels of p-p38. Collectively, these findings suggest that homeostatic FPR signalling exerts a modulatory effect on anxiety-like behaviours. These findings thus suggest that therapies targeting FPRs may be a novel approach to ameliorate behavioural abnormalities present in neuropsychiatric disorders at the cognitive-emotional interface.     

The Role of Water in Activation Mechanism of Human N-Formyl Peptide Receptor 1 (FPR1) Based on Molecular Dynamics Simulations

The Formyl Peptide Receptor 1 (FPR1) is an important chemotaxis receptor involved in various aspects of host defense and inflammatory processes. We constructed a model of FPR1 using as a novel template the chemokine receptor CXCR4 from the same branch of the phylogenetic tree of G-protein-coupled receptors. The previously employed template of rhodopsin contained a bulge at the extracellular part of TM2 which directly influenced binding of ligands. We also conducted molecular dynamics (MD) simulations of FPR1 in the apo form as well as in a form complexed with the agonist fMLF and the antagonist tBocMLF in the model membrane. During all MD simulation of the fMLF-FPR1 complex a water molecule transiently bridged the hydrogen bond between W254^6.48 and N108^3.35 in the middle of the receptor. We also observed a change in the cytoplasmic part of FPR1 of a rotamer of the Y301^7.53 residue (tyrosine rotamer switch). This effect facilitated movement of more water molecules toward the receptor center. Such rotamer of Y301^7.53 was not observed in any crystal structures of GPCRs which can suggest that this state is temporarily formed to pass the water molecules during the activation process. The presence of a distance between agonist and residues R201^5.38 and R205^5.42 on helix TM5 may suggest that the activation of FPR1 is similar to the activation of β-adrenergic receptors since their agonists are separated from serine residues on helix TM5. The removal of water molecules bridging these interactions in FPR1 can result in shrinking of the binding site during activation similarly to the shrinking observed in β-ARs. The number of GPCR crystal structures with agonists is still scarce so the designing of new ligands with agonistic properties is hampered, therefore homology modeling and docking can provide suitable models. Additionally, the MD simulations can be beneficial to outline the mechanisms of receptor activation and the agonist/antagonist sensing.