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.

A nonactivating allosteric modulator of free fatty acid receptor 2 (FFA2R, also called GPCR 43) turns both propionate (an orthosteric FFA2R agonist) and ATP (an agonist for the purinergic P2Y2 receptor), into potent activating ligands that trigger an assembly of the superoxide‐generating neutrophil NADPH oxidase. The ATP‐induced activation requires the participation of FFA2R, and the signaling is biased toward oxidase activation, leaving the ATP‐induced rise in intracellular Ca2+ unaffected. No NADPH oxidase activity was induced by ATP when propionate replaced the allosteric modulator. Signaling downstream of propionate‐activated FFA2Rs was insensitive to Gαq inhibition, but the crosstalk activation involving both FFA2R and P2Y2R relied on Gαq signaling. The receptor crosstalk, by which allosterically modulated FFA2Rs communicate with P2Y2Rs and generate NADPH oxidase activating signals downstream of Gαq, represent a novel mechanism by which GPCR activities can be regulated from inside the plasma membrane. Further, the finding that an allosteric FFA2R modulator sensitizes not only the response induced by orthosteric FFA2R agonists, but also the response induced by ATP (P2Y2R‐specific agonist) and formyl peptide receptor‐specific agonists, violates the receptor restriction characteristics normally defining the selectivity of allosteric GPCR modulators.—Lind, S., Holdfeldt, A., Mårtensson, J., Sundqvist, M., Bjórkman, L., Forsman, H., Dahlgren, C. Functional selective ATP receptor signaling controlled by the free fatty acid receptor 2 through a novel allosteric modulation mechanism. FASEB J. 33, 6887–6903 (2019). http://www.fasebj.org

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Summary Neutrophils, the most abundant white blood cell in human blood, express receptors that recognize damage/microbial associated pattern molecules of importance for cell recruitment to sites of inflammation. Many of these receptors belong to the family of G protein coupled receptors (GPCRs). These receptor‐proteins span the plasma membrane in expressing cells seven times and the down‐stream signaling rely in most cases on an activation of heterotrimeric G proteins. The GPCRs expressed in neutrophils recognize a number of structurally diverse ligands (activating agonists, allosteric modulators, and inhibiting antagonists) and share significant sequence homologies. Studies of receptor structure and function have during the last 40 years 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, and reactivation mechanisms as well as communication (receptor transactivation/cross‐talk) between GPCRs. This review summarizes the recent discoveries and pharmacological hallmarks with focus on some of the neutrophil expressed pattern recognition GPCRs. In addition, unmet challenges, including recognition by the receptors of diverse ligands and how biased signaling mediate different biological effects are described/discussed.

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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 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 β-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.

Despite the steadily increased numbers of formyl peptide receptor (FPR) ligands identified over the years, few have been characterized in studies using animal disease models and even less have entered clinical trials in human subjects. A small-molecule compound, Act-389949, was however recently tested in a phase I clinical trial and found to be safe and well tolerated in healthy human subjects. The desired anti-inflammatory property of Act-389949 was proposed to be mediated through FPR2, one of the FPRs expressed in neutrophils, but no basic characterization was included in the study. To gain more insights into FPR2 recognition of this first-in-class compound for future utility of the agonist, we have in this study determined the receptor preference and down-stream signaling characteristics induced by Act-389949 in human blood neutrophils isolated from healthy donors. Our data demonstrate that Act-389949 is an agonist for FPR2 that triggers functional/signaling repertoires comparable to what has been earlier described for other FPR2 agonists, including neutrophil chemotaxis, granule mobilization and activation of the NADPH-oxidase. In fact, Act-389949 was found to be as potent as the prototype FPR2 peptide agonist WKYMVM and had the advantage of being resistant to oxidation by the MPO-H2O2-halide derived oxidants, as compared to the sensitive WKYMVM. The down-stream signals generated by Act-389949 include an FPR2-dependent and Gαq-independent transient rise in intracellular Ca 2+ and recruitment of β-arrestin. In summary, our data show that Act-389949 serves as an excellent tool-compound for further dissection of FPR2-regulated activities in vitro and in vivo. Potent and stable FPR ligands such as Act-389949 may therefore be used to develop the next generation of FPR signaling regulating anti-inflammatory therapeutics.

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