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.

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.

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.

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.

CFP-10 from Mycobacterium tuberculosis selectively activates human neutrophils through a pertussis toxin-sensitive chemotactic receptor.

Upon infection with Mycobacterium tuberculosis, neutrophils are massively recruited to the lungs, but the role of these cells in combating the infection is poorly understood. Through a type VII secretion system, M. tuberculosis releases a heterodimeric protein complex, containing a 6-kDa early secreted antigenic target (ESAT-6) and a 10-kDa culture filtrate protein (CFP-10), that is essential for virulence. Whereas the ESAT-6 component possesses multiple virulence-related activities, no direct biological activity of CFP-10 has been shown, and CFP-10 has been described as a chaperone protein for ESAT-6. We here show that the ESAT-6:CFP-10 complex induces a transient release of Ca(2+) from intracellular stores in human neutrophils. Surprisingly, CFP-10 rather than ESAT-6 was responsible for triggering the Ca(2+) response, in a pertussis toxin-sensitive manner, suggesting the involvement of a G-protein-coupled receptor. In line with this, the response was accompanied by neutrophil chemotaxis and activation of the superoxide-producing NADPH-oxidase. Neutrophils were unique among leukocytes in responding to CFP-10, as monocytes and lymphocytes failed to produce a Ca(2+) signal upon stimulation with the M. tuberculosis protein. Hence, CFP-10 may contribute specifically to neutrophil recruitment and activation during M. tuberculosis infection, representing a novel biological role for CFP-10 in the ESAT-6:CFP-10 complex, beyond the previously described chaperone function.

Antibacterial activity of pepducins, allosterical modulators of formyl peptide receptor signaling.

Pepducins containing a fatty acid linked to an amino acid sequence derived from cytosolic parts of a G-protein-coupled receptor (GPCR) constitute a new group of lipopeptide tools in GPCR studies. Pepducins corresponding to the third intracellular loop of formyl peptide receptor 2 (FPR2) activate human neutrophils, and we show here that, in addition, these allosteric modulators of receptor activity also kill bacteria. The functional dualism of FPR2 pepducins could potentially be explored as a novel class of antibacterial drugs with immunomodulatory properties.

Formyl Peptide Receptors in Mice and Men: Similarities and Differences in Recognition of Conventional Ligands and Modulating Lipopeptides.

The pattern recognition formyl peptide receptors (FPRs) belong to the class of G-protein-coupled receptors (GPCRs), the largest group of cell surface receptors involved in a range of physiological processes and pathologies. The FPRs have regulatory function in the initiation as well as resolution of inflammatory reactions, making them highly interesting as targets for drug development. Recent research in the GPCR/FPR fields has uncovered novel receptor biology concepts, including biased signalling/functional selectivity, allosteric modulation, receptor reactivation and receptor cross-talk. When it comes to allosteric modulators, 'tailor-made' lipopeptides (pepducins and lipopeptoids) represent a novel concept of GPCR/FPR regulation. This MiniReview is focused on the basis for recognition of conventional ligands and immunomodulating lipopeptides, novel allosteric modulators for the FPRs, receptors that are highly expressed by both human and mouse neutrophils. The FPRs play key roles in host defence against microbial infections, tissue homeostasis and the initiation as well as resolution of inflammation but there are both similarities and differences in ligand recognition between mice and men. Thus, identification and functional characterization of activating and inhibiting ligands should provide insights into future design of FPR-based animal models of human diseases and development of therapeutics for treating inflammatory diseases.

NCAM2 Fibronectin type-III domains form a rigid structure that binds and activates the Fibroblast Growth Factor Receptor.

NCAM1 and NCAM2 have ectodomains consisting of 5 Ig domains followed by 2 membrane-proximal FnIII domains. In this study we investigate and compare the structures and functions of these FnIII domains. The NCAM1 and -2 FnIII2 domains both contain a Walker A motif. In NCAM1 binding of ATP to this motif interferes with NCAM1 binding to FGFR. We obtained a structural model of the NCAM2 FnIII2 domain by NMR spectroscopy, and by titration with an ATP analogue we show that the NCAM2 Walker A motif does not bind ATP. Small angle X-ray scattering (SAXS) data revealed that the NCAM2 FnIII1-2 double domain exhibits a very low degree of flexibility. Moreover, recombinant NCAM2 FnIII domains bind FGFR in vitro, and the FnIII1-2 double domain induces neurite outgrowth in a concentration-dependent manner through activation of FGFR. Several synthetic NCAM1-derived peptides induce neurite outgrowth via FGFR. Only 2 of 5 peptides derived from similar regions in NCAM2 induce neurite outgrowth, but the most potent of these peptides stimulates neurite outgrowth through FGFR-dependent activation of the Ras-MAPK pathway. These results reveal that the NCAM2 FnIII domains form a rigid structure that binds and activates FGFR in a manner related to, but different from NCAM1.

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.

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.

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.

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 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.

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.

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.

Neural cell adhesion molecules belonging to the family of leucine-rich repeat proteins.

Leucine-rich repeats (LRRs) are motifs that form protein-ligand interaction domains. There are approximately 140 human genes encoding proteins with extracellular LRRs. These encode cell adhesion molecules (CAMs), proteoglycans, G-protein-coupled receptors, and other types of receptors. Here we give a brief description of 36 proteins with extracellular LRRs that all can be characterized as CAMs or putative CAMs expressed in the nervous system. The proteins are involved in multiple biological processes in the nervous system including the proliferation and survival of cells, neuritogenesis, axon guidance, fasciculation, myelination, and the formation and maintenance of synapses. Moreover, the proteins are functionally implicated in multiple diseases including cancer, hearing impairment, glaucoma, Alzheimer's disease, multiple sclerosis, Parkinson's disease, autism spectrum disorders, schizophrenia, and obsessive-compulsive disorders. Thus, LRR-containing CAMs constitute a large group of proteins of pivotal importance for the development, maintenance, and regeneration of the nervous system.

A pepducin derived from the third intracellular loop of FPR2 is a partial agonist for direct activation of this receptor in neutrophils but a full agonist for cross-talk triggered reactivation of FPR2.

We recently described a novel receptor cross-talk mechanism in neutrophils, unique in that the signals generated by the PAF receptor (PAFR) and the ATP receptor (P2Y2R) transfer formyl peptide receptor 1 (FPR1) from a desensitized (non-signaling) state back to an actively signaling state (Forsman H et al., PLoS One, 8:e60169, 2013; Önnheim K, et al., Exp Cell Res, 323∶209, 2014). In addition to the G-protein coupled FPR1, neutrophils also express the closely related receptor FPR2. In this study we used an FPR2 specific pepducin, proposed to work as an allosteric modulator at the cytosolic signaling interface, to determine whether the cross-talk pathway is utilized also by FPR2. The pepducin used contains a fatty acid linked to a peptide sequence derived from the third intracellular loop of FPR2, and it activates as well as desensensitizes this receptor. We now show that neutrophils desensitized with the FPR2-specific pepducin display increased cellular responses to stimulation with PAF or ATP. The secondary PAF/ATP induced response was sensitive to FPR2-specific inhibitors, disclosing a receptor cross-talk mechanism underlying FPR2 reactivation. The pepducin induced an activity in naïve cells similar to that of a conventional FPR2 agonist, but with lower potency (partial efficacy), meaning that the pepducin is a partial agonist. The PAF- or ATP-induced reactivation was, however, much more pronounced when neutrophils had been desensitized to the pepducin as compared to cells desensitized to conventional agonists. The pepducin should thus in this respect be classified as a full agonist. In summary, we demonstrate that desensitized FPR2 can be transferred back to an actively signaling state by receptor cross-talk signals generated through PAFR and P2Y2R, and the difference in agonist potency with respect to pepducin-induced direct receptor activation and cross-talk reactivation of FPR2 puts the concept of functional selectivity in focus.

NCAM2/OCAM/RNCAM: cell adhesion molecule with a role in neuronal compartmentalization.

Neural cell adhesion molecules 2 (NCAM2/OCAM/RNCAM), is a paralog of NCAM1. The protein exists in a transmembrane and a lipid-anchored isoform, and has an ectodomain consisting of five immunoglobulin modules and two fibronectin type 3 homology modules. Structural models of the NCAM2 ectodomain reveal that it facilitates cell adhesion through reciprocal interactions between the membrane-distal immunoglobulin modules. There are no known heterophilic NCAM2 binding partners, and NCAM2 is not glycosylated with polysialic acid, a posttranslational modification known to be a major modulator of NCAM1-mediated processes. This suggests that NCAM2 has a function or mode of action distinctly different from that of NCAM1. NCAM2 is primarily expressed in the brain, where it is believed to stimulate neurite outgrowth and to facilitate dendritic and axonal compartmentalization.