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International audience; Pathogenic Staphylococcus aureus strains produce N-formylmethionyl containing peptides, of which the tetrapeptide fMIFL is a potent activator of the neutrophil formyl peptide receptor 1 (FPR1) and the PSMα2 peptide is a potent activator of the closely related FPR2. Variants derived from these two peptide activators were used to disclose the structural determinants for receptor interaction. Removal of five amino acids from the C-terminus of PSMα2 gave rise to a peptide that had lost the receptor-independent neutrophil permeabilizing effect, whereas neutrophil activation capacity as well as its preference for FPR2 was retained. Shorter peptides, PSMα21-10 and PSMα21-5, activate neutrophils, but the receptor preference for these peptides was switched to FPR1. The fMIFL-PSM5-16 peptide, in which the N-terminus of PSMα21-16 was replaced by the sequence fMIFL, was a dual agonist for FPR1/FPR2, whereas fMIFL-PSM5-10 preferred FPR1 to FPR2. Further, an Ile residue was identified as a key determinant for interaction with FPR2. A chimeric receptor in which the cytoplasmic tail of FPR1 was replaced by the corresponding part of FPR2 lost the ability to recognize FPR1 agonists, but gained function in relation to FPR2 agonists. Taken together, our data demonstrate that the C-terminus of the PSMα2 peptide plays a critical role for its cytotoxicity, but is not essential for the receptor-mediated pro-inflammatory activity. More importantly, we show that the amino acids present in the C-terminus, which are not supposed to occupy the agonist-binding pocket in the FPRs, are of importance for the choice of receptor.

Serum amyloid A (SAA) is one of the acute-phase reactants, a group of plasma proteins that increases immensely in concentration during microbial infections and inflammatory conditions, and a close relationship between SAA levels and disease activity in rheumatoid arthritis (RA) has been observed. RA is an inflammatory disease, where neutrophils play important roles, and SAA is thought to participate in the inflammatory reaction by being a neutrophil chemoattractant and inducer of proinflammatory cytokines. The biological effects of SAA are reportedly mediated mainly through formyl peptide receptor like-1 (FPRL1), a G protein-coupled receptor (GPCR) belonging to the formyl peptide receptor family. Here, we confirmed the affinity of SAA for FPRL1 by showing that stably transfected HL-60 cells expressing FPRL1 were activated by SAA and that the response was inhibited by the use of the FPRL1-specific antagonist WRWWWW (WRW4). We also show that SAA activates the neutrophil NADPH-oxidase and that a reserve pool of receptors is present in storage organelles mobilized by priming agents such as TNF-α and LPS from Gram-negative bacteria. The induced activity was inhibited by pertussis toxin, indicating the involvement of a GPCR. However, based on FPRL1-specific desensitization and use of FPRL1 antagonist WRW4, we found the SAA-mediated effects in neutrophils to be independent of FPRL1. Based on these findings, we conclude that SAA signaling in neutrophils is mediated through a GPCR, distinct from FPRL1. Future identification and characterization of the SAA receptor could lead to development of novel, therapeutic targets for treatment of RA.

International audience; The objective was to evaluate which receptors house dust mite (HDM) and birch pollen extracts engage to activate human eosinophils. Chemotaxis and degranulation were studied in eosinophils pretreated with pertussis toxin and other antagonists of G protein-coupled receptors, e.g. the formyl peptide receptor (FPR), CC chemokine receptor 3 (CCR3) and leukotriene receptor B4 (LTB(4)R). Inhibition of the FPR as well as desensitization of the receptor rendered eosinophils anergic to activation by the allergens. Blockade of CCR3 or LTB(4)R did not affect eosinophilic reactivity. It was determined by PCR that human eosinophils express the FPR family members FPR and FPR-like 1 (FPRL1). HDM, unlike birch pollen, evoked calcium fluxes in HL-60 cells transfected with FPR or FPRL1. Although both allergens gave rise to calcium transients in neutrophils, which also express FPR and FPRL1, only the HDM response was decreased by the FPR antagonist. Moreover, neutrophils migrated toward HDM but not to birch pollen. Eosinophils pretreated with inhibitors of MAPK p38, ERK1/2 or protein kinase C exhibited diminished responsiveness to the aeroallergens. This study indicates that FPR and FPRL1 mediate the activation of eosinophils by HDM, whereas birch pollen employs other pathways shared with FPR to activate human eosinophils.

International audience; Neutrophils express the G protein-coupled N-formyl peptide receptor (FPR) and its homologue FPRL1. The hexapeptide Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm) activates HL-60 cells transfected either with FPRL1 or with FPR. The signaling through the stably expressed receptors was inhibited by specific receptor antagonists, cyclosporine H and WRWWWW (WRW4) for FPR and FPRL1, respectively. The neutrophil release of superoxide was used to determine receptor preference, when these cells were triggered with WKYMVm. The response was not affected by the FPR specific antagonist suggesting that no signals are transduced through this receptor. The response was only partly inhibited by WRW4, but this antagonist induced a receptor switch, perceptible as a change in sensitivity to the FPR antagonist. The activity remaining in the presence of WRW4 was inhibited by cyclosporine H. A cell permeable peptide (PBP10) corresponding to the phosphatidyl-inositol-bisphosphate binding region of gelsolin, inhibited the FPRL1-, but not the FPR-induced cellular response and induced the same type of receptor switch. We show that an agonist that has the potential to bind and activate neutrophils through FPRL1 as well as through FPR, uses the latter receptor and its signaling route, only when the activating signal generated through FPRL1 is blocked. The receptor switch is achieved when signaling through FPRL1 is inhibited both by a receptor antagonist, and by an inhibitor operating from the inside of the plasma membrane. The phenomenon described is of general importance for proper interpretation of results generated through the use of different "silencing technologies" in receptor operated signaling transduction research.