Neutrophil Signaling That Challenges Dogmata of G Protein-Coupled Receptor Regulated Functions.

Activation as well as recruitment of neutrophils, the most abundant leukocyte in human blood, to sites of infection/inflammation largely rely on surface-exposed chemoattractant receptors. These receptors belong to the family of 7-transmembrane domain receptors also known as G protein-coupled receptors (GPCRs) due to the fact that part of the downstream signaling relies on an activation of heterotrimeric G proteins. The neutrophil GPCRs share significant sequence homologies but bind many structurally diverse activating (agonistic) and inhibiting (antagonistic) ligands, ranging from fatty acids to purines, peptides, and lipopeptides. Recent structural and functional studies of neutrophil receptors have generated important information on GPCR biology in general; this knowledge aids in the overall understanding of general pharmacological principles, governing regulation of neutrophil function and inflammatory processes, including novel leukocyte receptor activities related to ligand recognition, biased/functional selective signaling, allosteric modulation, desensitization mechanisms and reactivation, and communication (cross-talk) between GPCRs. This review summarizes the recent discoveries and pharmacological hallmarks with focus on neutrophil GPCRs. In addition, unmet challenges are dealt with, including recognition by the receptors of diverse ligands and how biased signaling mediates different biological effects.

Porphyromonas gingivalis Produce Neutrophil Specific Chemoattractants Including Short Chain Fatty Acids.

Neutrophil migration from blood to tissue-residing microbes is governed by a series of chemoattractant gradients of both endogenous and microbial origin. Periodontal disease is characterized by neutrophil accumulation in the gingival pocket, recruited by the subgingival biofilm consisting mainly of gram-negative, anaerobic and proteolytic species such as Porphyromonas gingivalis. The fact that neutrophils are the dominating cell type in the gingival pocket suggests that neutrophil-specific chemoattractants are released by subgingival bacteria, but characterization of chemoattractants released by subgingival biofilm species remains incomplete. In the present study we characterized small (< 3 kDa) soluble chemoattractants released by growing P. gingivalis, and show that these are selective for neutrophils. Most neutrophil chemoattractant receptors are expressed also by mononuclear phagocytes, the free fatty acid receptor 2 (FFAR2) being an exception. In agreement with the selective neutrophil recruitment, the chemotactic activity found in P. gingivalis supernatants was mediated in part by a mixture of short chain fatty acids (SCFAs) that are recognized by FFAR2, and other leukocytes (including monocytes) did not respond to SCFA stimulation. Although SCFAs, produced by bacterial fermentation of dietary fiber in the gut, has previously been shown to utilize FFAR2, our data demonstrate that the pronounced proteolytic metabolism employed by P. gingivalis (and likely also other subgingival biofilm bacteria associated with periodontal diseases) may result in the generation of SCFAs that attract neutrophils to the gingival pocket. This finding highlights the interaction between SCFAs and FFAR2 in the context of P. gingivalis colonization during periodontal disease, but may also have implications for other inflammatory pathologies involving proteolytic bacteria.

Staphylococcus aureus-Derived PSMα Peptides Activate Neutrophil FPR2 but Lack the Ability to Mediate ß-Arrestin Recruitment and Chemotaxis.

Formyl peptide receptor 2 (FPR2) is a G protein-coupled pattern recognition receptor sensing both mitochondrial- and bacterial-derived formylated peptides, including the PSMα toxins secreted by community-associated methicillin-resistant Staphylococcus aureus strains. Similar to many other FPR2 agonistic peptides, nanomolar concentrations of both PSMα2 and PSMα3 activate neutrophils to increase the cytosolic concentration of Ca2+ and release NADPH oxidase-derived reactive oxygen species. In addition, the PSMα peptides induce FPR2 homologous desensitization, actin polymerization, and neutrophil reactivation through a receptor cross-talk mechanism. However, in contrast to conventional FPR2 agonistic peptides, including the host-derived formyl peptide MCT-ND4, we found that the PSMα peptides lacked the ability to recruit ß-arrestin and induce neutrophil chemotaxis, supporting the previous notion that ß-arrestin translocation is of importance for cell migration. Despite the lack of ß-arrestin recruitment, the PSMα peptides induced an FPR2-dependent ERK1/2 phosphorylation and internalization. Furthermore, structure-activity relationship analysis with PSMα2 derivatives revealed critical roles of the first 3 aa linked to N-fMet as well as the C terminus of PSMα2 in promoting FPR2 to recruit ß-arrestin. In summary, our data demonstrate a novel neutrophil activation pattern upon FPR2 sensing of PSMα peptides, signified by the ability to induce increased intracellular Ca2+, ERK1/2 phosphorylation, internalization, and NADPH oxidase activity, yet lack of ß-arrestin recruitment and neutrophil chemoattraction. These novel features adopted by the PSMα peptides could be of importance for S. aureus virulence and might facilitate identification of new therapeutic strategies for treating S. aureus infections.

Identification of Residues Critical for FPR2 Activation by the Cryptic Peptide Mitocryptide-2 Originating from the Mitochondrial DNA-Encoded Cytochrome b.

Similar to bacteria, synthesis of mitochondrial DNA-encoded proteins requires an N-formylated methionine to initiate translation. Thus, the N-formylated methionine peptides originating from mitochondria should be recognized as danger signals. To date, only one such peptide, denoted as mitocryptide-2 (MCT-2), originating from the N-terminal of the mitochondrial cytochrome b, has been isolated from mammalian tissues. Human neutrophils express FPR1 and FPR2 that detect formyl peptides, and the precise structural determinants for receptor recognition remain to be elucidated. MCT-2 is known to activate neutrophils through FPR2 but not FPR1. The aim of this study was to elucidate the structural determinants of importance for receptor preference and human neutrophil activation in MCT-2 by generating a series of MCT-2 variants. We show that there is an absolute requirement for the N-formyl group and the side chain of Met1 at position 1 of MCT-2 but also the C terminus is of importance for MCT-2 activity. We also uncovered individual side chains that positively contribute to MCT-2 activity as well as those suppressed in the response. The MCT-2 peptide and its two polymorphic variants ([Thr7]MCT-2 and [Ser8]MCT-2) all activated neutrophils, but MCT-2 containing Ile7 and Asn8 was the most potent. We also show that some peptide variants displayed a biased FPR2-signaling property related to NADPH oxidase activation and ß-arrestin recruitment, respectively. In conclusion, we disclose several critical elements in MCT-2 that are required for neutrophil activation and disclose structural insights into how FPR2 recognition of this mitochondrial DNA-derived peptide may increase our understanding of the role of FPR2 in aseptic inflammation.

Neutrophil priming that turns natural FFA2R agonists into potent activators of the superoxide generating NADPH-oxidase.

Acetate, an agonist for the free fatty acid receptor 2 (FFA2R/GPR43), triggers an increase in the cytosolic concentration of free Ca2+ in neutrophils without any assembly of the superoxide generating NADPH-oxidase. We show that the phenylacetamide compound 58 (Cmp 58; (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide), lacking a direct activating effect on neutrophils, acts as a positive FFA2R modulator that turns acetate into a potent activating agonist that triggers an assembly of the NADPH-oxidase. The NADPH-oxidase activity could be further increased in neutrophils treated with the pro-inflammatory cytokine TNF-α. Many neutrophil chemoattractant receptors are stored in secretory organelles but no FFA2R mobilization was induced in neutrophils treated with TNF-α. The receptor selectivity was demonstrated through the inhibition of the neutrophil response induced by the combined action of acetate and Cmp 58 by the FFA2R antagonist CATPB. Receptor modulators that positively co-operate with natural FFA2R agonists and prime neutrophils in their response to such agonists, may serve as good tools for further unraveling the physiological functions of FFA2R and its involvement in various diseases. In this study, we show that neutrophils primed with a presumed allosteric FFA2R modulator produce increased amounts of reactive oxygen species when activated by receptor specific agonists.

Mitocryptides from Human Mitochondrial DNA-Encoded Proteins Activate Neutrophil Formyl Peptide Receptors: Receptor Preference and Signaling Properties.

Phagocytic neutrophils express formyl peptide receptors (FPRs; FPR1 and FPR2) that distinctly recognize peptides starting with an N-formylated methionine (fMet). This is a hallmark of bacterial metabolism; similar to prokaryotes, the starting amino acid in synthesis of mitochondrial DNA-encoded proteins is an fMet. Mitochondrial cryptic peptides (mitocryptides; MCTs) with an N-terminal fMet could be identified by our innate immune system; however, in contrast to our knowledge about bacterial metabolites, very little is known about the recognition profiles of MCTs. In this study, we determined the neutrophil-recognition profiles and functional output of putative MCTs originating from the N termini of the 13 human mitochondrial DNA-encoded proteins. Six of the thirteen MCTs potently activated neutrophils with distinct FPR-recognition profiles: MCTs from ND3 and ND6 have a receptor preference for FPR1; MCTs from the proteins ND4, ND5, and cytochrome b prefer FPR2; and MCT-COX1 is a dual FPR1/FPR2 agonist. MCTs derived from ND2 and ND4L are very weak neutrophil activators, whereas MCTs from ND1, ATP6, ATP8, COX2, and COX3, do not exert agonistic or antagonistic FPR effects. In addition, the activating MCTs heterologously desensitized IL-8R but primed the response to the platelet-activating factor receptor agonist. More importantly, our data suggest that MCTs have biased signaling properties in favor of activation of the superoxide-generating NADPH oxidase or recruitment of ß-arrestin. In summary, we identify several novel FPR-activating peptides with sequences present in the N termini of mitochondrial DNA-encoded proteins, and our data elucidate the molecular basis of neutrophil activation by MCTs.

Similarities and differences between the responses induced in human phagocytes through activation of the medium chain fatty acid receptor GPR84 and the short chain fatty acid receptor FFA2R.

GPR84 is a recently de-orphanized member of the G-protein coupled receptor (GPCR) family recognizing medium chain fatty acids, and has been suggested to play important roles in inflammation. Due to the lack of potent and selective GPR84 ligands, the basic knowledge related to GPR84 functions is very limited. In this study, we have characterized the GPR84 activation profile and regulation mechanism in human phagocytes, using two recently developed small molecules that specifically target GPR84 agonistically (ZQ16) and antagonistically (GLPG1205), respectively. Compared to our earlier characterization of the short chain fatty acid receptor FFA2R which is functionally expressed in neutrophils but not in monocytes, GPR84 is expressed in both cell types and in monocyte-derived macrophages. In neutrophils, the GPR84 agonist had an activation profile very similar to that of FFA2R. The GPR84-mediated superoxide release was low in naïve cells, but the response could be significantly primed by TNFα and by the actin cytoskeleton disrupting agent Latrunculin A. Similar to that of FFA2R, a desensitization mechanism bypassing the actin cytoskeleton was utilized by GPR84. All ZQ16-mediated cellular responses were sensitive to GLPG1205, confirming the GPR84-dependency. Finally, our data of in vivo transmigrated tissue neutrophils indicate that both GPR84 and FFA2R are involved in neutrophil recruitment processes in vivo. In summary, we show functional similarities but also some important differences between GPR84 and FFA2R in human phagocytes, thus providing some mechanistic insights into GPR84 regulation in blood neutrophils and cells recruited to an aseptic inflammatory site in vivo.

FPR2 signaling without ß-arrestin recruitment alters the functional repertoire of neutrophils.

G-protein coupled receptor (GPCR) biased agonism or functional selectivity has become an essential concept in GPCR research over the last years. Receptor-specific biased agonists selectively trigger one signaling pathway over another and induce a restricted/directed functional response. In this study, we aimed to characterize the concept of biased agonism for FPR2, a member of the formyl peptide receptor (FPR) subfamily of GPCRs. We show that the earlier described FPR2-activating pepducin F2Pal10 is a biased FPR2 agonist. The effects of F2Pal10 on neutrophil function differed in several aspects compared to those mediated by WKYMVM, a conventional FPR2-specific peptide agonist. Upon interaction with FPR2 expressed by neutrophils both F2Pal10 and WKYMVM activated the PLC-PIP2-Ca2+ signaling pathway and the superoxide-generating NADPH-oxidase, but only WKYMVM activated the receptor to recruit ß-arrestin. The functional consequences linked to a lack of ß-arrestin recruitment were further explored, and we demonstrate that FPR2 desensitization occurred independent of ß-arrestin. Despite this, reactivation of desensitized receptors achieved through a disruption of the cytoskeleton and through a novel FPR2 cross-talk mechanism with P2Y2R (the ATP receptor) and PAFR (the receptor for PAF) differed between F2Pal10-desensitized and WKYMVM-desensitized neutrophils. Further, the inability to recruit ß-arrestin was found to be associated with a reduced rate of receptor internalization and impaired chemotaxis in neutrophils. In summary, we provide experimental evidence of biased agonism for FPR2 and our data disclose critical roles of ß-arrestin in neutrophil chemotaxis and reactivation of desensitized receptors.

Formyl peptide derived lipopeptides disclose differences between the receptors in mouse and men and call the pepducin concept in question.

A pepducin is a lipopeptide containing a peptide sequence that is identical to one of the intracellular domains of the G-protein coupled receptor (GPCR) assumed to be the target. Neutrophils express two closely related formyl peptide receptors belonging to the family of GPCRs; FPR1 and FPR2 in human and their respective orthologue Fpr1 and Fpr2 in mouse. By applying the pepducin concept, we have earlier identified FPR2 activating pepducins generated from the third intracellular loop of FPR2. The third intracellular loop of FPR2 differs in two amino acids from that of FPR1, seven from Fpr2 and three from Fpr1. Despite this, we found that pepducins generated from FPR1, FPR2, Fpr1 and Fpr2 all targeted FPR2 in human neutrophils and Fpr2 in mouse, but with different modulating outcomes. Whereas the FPR1/Fpr1 derived pepducins inhibited the FPR2 function in human neutrophils, they activated Fpr2 in mouse. The FPR2 derived pepducin activated FPR2/Fpr2, whereas the pepducin generated from Fpr2 inhibited both FPR2 and Fpr2. In summary, our data demonstrate that pepducins generated from the third intracellular loop of human FPR1/2 and mouse Fpr1/2, all targeted FPR2 in human and Fpr2 in mouse. With respect to the modulating outcomes, pepducin inhibitors identified for FPR2 are in fact activators for Fpr2 in mouse neutrophils. Our data thus questions the validity of pepducin concept regarding their receptor selectivity but supports the notion that FPR2/Fpr2 may recognize a lipopeptide molecular pattern, and highlight the differences in ligand recognition profile between FPR2 and its mouse orthologue Fpr2.

Reactivation of Gαi-coupled formyl peptide receptors is inhibited by Gαq-selective inhibitors when induced by signals generated by the platelet-activating factor receptor.

Formyl peptide receptor (FPR)-desensitized neutrophils display increased production/release of superoxide (O2-) when activated by platelet-activating factor (PAF), a priming of the response achieved through a unique receptor crosstalk mechanism. The aim of this study was to determine the effect of an inhibitor selective for small, heterotrimeric G proteins belonging to the Gαq subclass on that receptor crosstalk. We show that signals generated by FPRs and the PAF receptor (PAFR) induce activation of the neutrophil O2-, producing NADPH-oxidase, and that response was sensitive to Gαq inhibition in cells activated by PAF, but no inhibition was obtained in cells activated by FPR agonists. Signaling in naive neutrophils is terminated fairly rapidly, and the receptors become homologously desensitized. The downstream sensitivity to Gαq inhibition in desensitized cells displaying increased production/release of O2- through the PAFR receptor crosstalk mechanism also comprised the reactivation of the FPRs, and the activation signals were redirected from the PAFR to the desensitized/reactivated FPRs. The Gαq-dependent activation signals generated by the PAFRs activate the Gαi-coupled FPRs, a receptor crosstalk that represents a novel pathway by which G protein-coupled receptors can be regulated and signaling can be turned on and off.

Formylated MHC Class Ib Binding Peptides Activate Both Human and Mouse Neutrophils Primarily through Formyl Peptide Receptor 1.

Two different immune recognition systems have evolved in parallel to recognize peptides starting with an N-formylated methionine, and recognition similarities/differences between these two systems have been investigated. A number of peptides earlier characterized in relation to the H2-M3 complex that presents N-formylated peptides to cytotoxic T cells, have been characterized in relation to the formyl peptide receptors expressed by phagocytic neutrophils in both men (FPRs) and mice (Fprs). FPR1/Fpr1 was identified as the preferred receptor for all fMet-containing peptides examined, but there was no direct correlation between H2-M3 binding and the neutrophil activation potencies. Similarly, there was no direct correlation between the activities induced by the different peptides in human and mouse neutrophils, respectively. The formyl group was important in both H2-M3 binding and FPR activation, but FPR2 was the preferred receptor for the non-formylated peptide. The structural requirements differed between the H2-M3 and FPR/Fpr recognition systems and these data suggest that the two recognition systems have different evolutionary traits.

The peptidomimetic Lau-(Lys-ßNSpe)6-NH2 antagonizes formyl peptide receptor 2 expressed in mouse neutrophils.

The formyl peptide receptor (FPR) gene family has a complex evolutionary history and comprises eight murine members but only three human representatives. To enable translation of results obtained in mouse models of human diseases, more comprehensive knowledge of the pharmacological similarities/differences between the human and murine FPR family members is required. Compared to FPR1 and FPR2 expressed by human neutrophils, very little is known about agonist/antagonist recognition patterns for their murine orthologues, but now we have identified two potent and selective formylated peptide agonists (fMIFL and PSMα2) for Fpr1 and Fpr2, respectively. These peptides were used to determine the inhibition profile of a set of antagonists with known specificities for the two FPRs in relation to the corresponding murine receptors. Some of the most potent and selective antagonists for the human receptors proved to be devoid of effect on their murine orthologues as determined by their inability to inhibit superoxide release from murine neutrophils upon stimulation with receptor-specific agonists. The Boc-FLFLF peptide was found to be a selective antagonist for Fpr1, whereas the lipidated peptidomimetic Lau-(Lys-ßNSpe)6-NH2 and the hexapeptide WRW4 were identified as Fpr2-selective antagonists.

The Neutrophil Response Induced by an Agonist for Free Fatty Acid Receptor 2 (GPR43) Is Primed by Tumor Necrosis Factor Alpha and by Receptor Uncoupling from the Cytoskeleton but Attenuated by Tissue Recruitment.

Ligands with improved potency and selectivity for free fatty acid receptor 2 (FFA2R) have become available, and we here characterize the neutrophil responses induced by one such agonist (Cmp1) and one antagonist (CATPB). Cmp1 triggered an increase in the cytosolic concentration of Ca(2+), and the neutrophils were then desensitized to Cmp1 and to acetate, a naturally occurring FFA2R agonist. The antagonist CATPB selectively inhibited responses induced by Cmp1 or acetate. The activated FFA2R induced superoxide anion secretion at a low level in naive blood neutrophils. This response was largely increased by tumor necrosis factor alpha (TNF-α) in a process associated with a recruitment of easily mobilizable granules, but neutrophils recruited to an aseptic inflammation in vivo were nonresponding. Superoxide production induced by Cmp1 was increased in latrunculin A-treated neutrophils, but no reactivation of desensitized FFA2R was induced by this drug, suggesting that the cytoskeleton is not directly involved in terminating the response. The functional and regulatory differences between the receptors that recognize short-chain fatty acids and formylated peptides, respectively, imply different roles of these receptors in the orchestration of inflammation and confirm the usefulness of a selective FFA2R agonist and antagonist as tools for the exploration of the precise role of the FFA2R.

Data on human neutrophil activation induced by pepducins with amino acid sequences derived from ß2AR and CXCR4.

The data described here is related to the research article titled (Gabl et al., 2016) [1]. Pepducins with peptide sequence derived from one of the intracellular domains of a given G-protein coupled receptor (GPCR) can either activate or inhibit cell functions. Here we include data on human neutrophil function induced by pepducins derived from ß2AR (ICL3-8) and CXCR4 (ATI-2341), respectively. ICL3-8 exerts neither direct activating effect on the NADPH-oxidase as measured by superoxide release nor inhibitory effect on FPR signaling. ATI-2341 dose-dependently triggers neutrophil activation and these cells were subsequently desensitized in their response to FPR2 specific agonists F2Pal10 and WKYMVM. Moreover, the ATI-2341 response is inhibited by PBP10 and the peptidomimetic Pam-(Lys-betaNSpe)6-NH2 (both are FPR2 specific inhibitors), but not to the FPR1 specific inhibitor cyclosporine H.

The Lipidated Peptidomimetic Lau-((S)-Aoc)-(Lys-ßNphe)6-NH2 Is a Novel Formyl Peptide Receptor 2 Agonist That Activates Both Human and Mouse Neutrophil NADPH Oxidase.

Neutrophils expressing formyl peptide receptor 2 (FPR2) play key roles in host defense, immune regulation, and resolution of inflammation. Consequently, the search for FPR2-specific modulators has attracted much attention due to its therapeutic potential. Earlier described agonists for this receptor display potent activity for the human receptor (FPR2) but low activity for the mouse receptor orthologue (Fpr2), rendering them inapplicable in murine models of human disease. Here we describe a novel FPR2 agonist, the proteolytically stable α-peptide/ß-peptoid hybrid Lau-((S)-Aoc)-(Lys-ßNphe)6-NH2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular Ca(2+) concentration and assembly of the superoxide-generating NADPH oxidase. This FPR2/Fpr2 agonist contains a headgroup consisting of a 2-aminooctanoic acid (Aoc) residue acylated with lauric acid (C12 fatty acid), which is linked to a peptide/peptoid repeat ((Lys-ßNphe)6-NH2). Both the fatty acid moiety and the (S)-Aoc residue were required for FPR2/Fpr2 activation. This type of proteolytically stable FPR2-specific peptidomimetics may serve as valuable tools for future analysis of FPR2 signaling as well as for development of prophylactic immunomodulatory therapy. This novel class of cross-species FPR2/Fpr2 agonists should enable translation of results obtained with mouse neutrophils (and disease models) into enhanced understanding of human inflammatory and immune diseases.

Basic characteristics of the neutrophil receptors that recognize formylated peptides, a danger-associated molecular pattern generated by bacteria and mitochondria.

Proper recruitment and activation of neutrophils to/at sites of infection/inflammation relies largely on the surface expression of chemoattractant receptors of which a formyl peptide receptor (FPR1) was the first to be cloned and characterized in more detail. This receptor displays high affinity for bacterial- or mitochondrial-derived peptides that contain a formylated methionine in the N-terminus. The neutrophil chemoattractant receptors belong to the group of 7-transmembrane domain receptors that signal through activation of heterotrimeric G proteins. These receptors have been shown to be important in host defense against microbial intruders and in regulating inflammatory reactions. The two FPRs (FPR1, FPR2) expressed in neutrophils share significant sequence homology and bind many structurally diverse activating (agonistic) and inhibiting (antagonistic) ligands, ranging from peptides to lipopeptides containing peptide sequences derived from intracellular regions of the FPRs. Recent structural and functional studies of the two neutrophil FPRs have generated important information for our understanding of general pharmacological principles, governing regulation of neutrophil function and inflammation and increased knowledge of more general G-protein coupled receptor features, such as ligand recognition, biased signaling, allosteric modulation, and a unique receptor cross-talk phenomenon. This article aims to summarize recent discoveries and pharmacological characterization of neutrophil FPRs and to discuss unmet challenges, including recognition by the receptors of diverse ligands and how biased signals mediate different biological effects.

A pepducin designed to modulate P2Y2R function interacts with FPR2 in human neutrophils and transfers ATP to an NADPH-oxidase-activating ligand through a receptor cross-talk mechanism.

Several G-protein-coupled receptors (GPCRs) can be activated or inhibited in a specific manner by membrane-permeable pepducins, which are short palmitoylated peptides with amino acid sequences identical to an intracellular domain of the receptor to be targeted. Unlike the endogenous P2Y2R agonist ATP, the P2Y2PalIC2 pepducin, which has an amino acid sequence corresponding to the second intracellular loop of the human ATP receptor (P2Y2R), activated the superoxide anion-generating NADPH-oxidase in neutrophils. In addition to having a direct effect on neutrophils, the P2Y2R pepducin converted naïve neutrophils to a primed state, which secondarily responded to ATP by producing superoxide. A pepducin with a peptide identical to the third intracellular loop of P2Y2R (P2Y2PalIC3) exhibited the same basic functions as P2Y2PalIC2, whereas one with a peptide that was identical to the first intracellular loop (P2Y2PalIC1) lacked these functions. The responses induced in neutrophils by the P2Y2R pepducins were not inhibited by the P2Y2R antagonist AR-C118925, and the receptor desensitization profile suggested the involvement of FPR2 rather than P2Y2R. Accordingly, antagonists/inhibitors of FPR2 attenuated the activities of the P2Y2R pepducins, which also selectively activated FPR2-overexpressing cells. In summary, we show that pepducins supposed to target P2Y2R activate human neutrophils through FPR2. We also show that the P2Y2PalIC2 pepducin can convert ATP from a non-activating agent to a potent neutrophil NADPH-oxidase activator. The molecular basis of this phenomenon involves cross-talk between the receptor/ligand pairs of P2Y2R/ATP and FPR2/P2Y2-pepducin.

P2Y2 receptor signaling in neutrophils is regulated from inside by a novel cytoskeleton-dependent mechanism.

Functional selectivity, a process by which G-protein coupled receptors (GPCRs) can activate one signaling route while avoiding another, is regulated by ligand-mediated stabilization of specific receptor states that modulate different downstream signaling events. We propose a novel mechanism for functional selectivity, induced by the endogenous P2Y2R agonist ATP and regulated at the signaling interface by the cytoskeleton. Upon ATP stimulation of human neutrophils, a transient rise in the cytosolic concentration of free Ca(2+) was not followed by activation of the superoxide anion-generating NADPH-oxidase. This was in contrast to signals generated through the formyl peptide receptor 1 (FPR1), as its activation was accompanied by both a mobilization of Ca(2+) and activation of the NADPH-oxidase. The phospholipase C/Ca(2+) signaling route is not modulated by the cytoskeleton-disrupting drug latrunculin A, but this drug was able to launch a new signaling route downstream of P2Y2R that led to NADPH-oxidase activation. The signaling downstream of P2Y2R was rapidly terminated and the receptors were desensitized; however, in contrast to desensitized FPR1, no P2Y2 receptor reactivation could be induced by latrunculin A. Thus, P2Y2R desensitization does not appear to involve the cytoskeleton, contrary to FPR1 desensitization. In summary, we hereby describe how ATP regulates functional selectivity via the cytoskeleton, leading to intracellular Ca(2+) increase, alone or with simultaneous NADPH-oxidase activation in neutrophils.

A neutrophil inhibitory pepducin derived from FPR1 expected to target FPR1 signaling hijacks the closely related FPR2 instead.

Pepducins constitute a unique class of G-protein coupled receptor (GPCR) modulating lipopeptides. Pepducins with inhibitory effects on neutrophils could potentially be developed into anti-inflammatory pharmaceuticals. A pepducin with a peptide sequence identical to the third intracellular loop of FPR1 was found to inhibit neutrophil functions including granule mobilization and superoxide production. This FPR1-derived pepducin selectively inhibited signaling and cellular responses through FPR2, but not FPR1 as expected. Binding to the neutrophil surface of a conventional FPR2 agonist is also inhibited. The fatty acid is essential for inhibition and pepducins with shorter peptides lose in potency. In summary, a pepducin designed to target FPR1 was found to hijack FPR2 and potently inhibit neutrophil functions.

Structural changes of the ligand and of the receptor alters the receptor preference for neutrophil activating peptides starting with a formylmethionyl group.

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.