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

Dahlgren, Cláes; Holdfeldt, André; Lind, Simon; Mårtensson, Jonas; Gabl, Michael; Björkman, Lena; Sundqvist, Martina; Forsman, Huamei

doi:10.1021/acsptsci.0c00004

Cited by 38 publications

(72 citation statements)

References 117 publications

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“…Our finding that the ability of FPR1 agonists to trigger neutrophil migration is linked to the ability to recruit -arrestin is in agreement with the emerging concept of biased FPR agonism and functional selectivity shown to be valid also for FPR2. [29][30][31]45,68,69 Our data are also in line with the documented role of -arrestin in regulating cell migration in studies performed with other cell types, including neutrophillike HL60 cells. [70][71][72][73] At the structural level, these data suggest that is not reduced by a G q inhibitor, and this is in line with earlier studies that have identified the part of a G i containing G protein downstream of FPR1, to be the link between the receptor an activation of the PLC-PIP 2 -IP 3 -Ca 2+ pathway.…”

Section: Discussionsupporting

confidence: 87%

“…Recent research suggests that the receptor crosstalk hierarchy is complex and not only desensitized receptors but also allosteric modulated GPCRs can communicate with other receptors. 45,49,50 A prominent example of such a crosstalk is that FPRs signaling can be positively regulated by free fatty acid receptor 2 (FFAR2) as illustrated by the fact that neutrophils with their FFARs allosterically modulated are primed when activated by low (normally nonactivating) concentrations of FPR agonists. 51,52 The fact that RE-04-001 response induced in neutrophils in the presence of an allosteric FFAR2 modulator, is inhibited not only to an FPR1 antagonist but also by an antagonist specific for FFAR2 (Fig.…”

Section: Re-04-001 Promotes Fpr1 To Crosstalk With Other Neutrophil Rmentioning

confidence: 99%

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Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2 complex

Lind

Dahlgren

Holmdahl

et al. 2020

Journal of Leukocyte Biology

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The formyl peptide receptors FPR1 and FPR2 are abundantly expressed by neutrophils, in which they regulate proinflammatory tissue recruitment of inflammatory cells, the production of reactive oxygen species (ROS), and resolution of inflammatory reactions. The unique dual functionality of the FPRs makes them attractive targets to develop FPR-based therapeutics as novel anti-inflammatory treatments. The small compound RE-04-001 has earlier been identified as an inducer of ROS in differentiated HL60 cells but the precise target and the mechanism of action of the compound was has until now not been elucidated. In this study, we reveal that RE-04-001 specifically targets and activates FPR1, and the concentrations needed to activate the neutrophil NADPH-oxidase was very low (EC 50 ∼1 nM). RE-04-001 was also found to be a neutrophil chemoattractant, but when compared to the prototype FPR1 agonist N-formyl-Met-Leu-Phe (fMLF), the concentrations required were comparably high, suggesting that signaling downstream of the RE-04-001-activated-FPR1 is functionally selective. In addition, the RE-04-001-induced response was strongly biased toward the PLC-PIP 2-Ca 2+ pathway and ERK1/2 activation but away from-arrestin recruitment. Compared to the peptide agonist fMLF, RE-04-001 is more resistant to inactivation by the MPO-H 2 O 2-halide system. In summary, this study describes RE-04-001 as a novel small molecule agonist specific for FPR1, which displays a biased signaling profile that leads to a functional selective activating of human neutrophils. RE-04-001 is, therefore, a useful tool, not only for further mechanistic studies of the regulatory role of FPR1 in inflammation in vitro and in vivo, but also for developing FPR1-specific drug therapeutics.

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Section: Discussionsupporting

confidence: 87%

Section: Re-04-001 Promotes Fpr1 To Crosstalk With Other Neutrophil Rmentioning

confidence: 99%

Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2 complex

Lind

Dahlgren

Holmdahl

et al. 2020

Journal of Leukocyte Biology

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“…However, it has been established that there is no direct link between GPCR-mediated activation of the oxidase and the transient rise in [Ca 2+ ]i [5,18,32,39,40], which gains further support from the data presented in this study. Hence, even although it is difficult to directly correlate NADPH-oxidase activity and calcium signaling during receptor reactivation induced by Barbadin or latrunculin A, our data corroborate previous studies demonstrating that a rise in [Ca 2+ ]i is not a requirement for activation of the NADPH-oxidase.…”

Section: Discussionsupporting

confidence: 68%

Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis

Sundqvist

Holdfeldt

Wright

et al. 2020

Preprint

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Running title: Barbadin modulates specific neutrophil functions Abbreviations: Aoc = 2-aminooctanoic acid; AF = Alexa Fluor; AP2 = clathrin adaptor protein 2; AUC = area under the curve; BRET = bioluminescence resonance energy transfer; BSA = bovine serum albumin; CL = chemiluminescence; DMEM = Dulbecco's modified Eagle medium; DMSO = dimethyl sulfoxide; DPBS = Dulbecco's phosphate-buffered saline; FPR = formyl peptide receptor; GPCR = G protein-coupled receptor; HBSS = Hank's balanced salt solution; HRP = horseradish peroxidase; Lau = Lauroyl; KRG = Krebs-Ringer phosphate buffer: MOI = multiplicity of infection; MPO = myeloperoxidase; βNPhe = N-phenylmethylβ-alanine; βNrpe = N-(R)-1-phenylethyl-β-alanine; PFA = paraformaldehyde; PKC = protein kinase C; PLC = phospholipase C; PMA = phorbol 12-myristate 13-acetate; RhB = rhodamine B; RT = room temperature; ROS = reactive oxygen species; SOD = superoxide dismutase; TR-FRET = time-resolved förster resonance energy transfer AbstractFormyl peptide receptor 2 (FPR2), a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to -arrestin recruitment. -Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of β-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study.Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent arrestin/AP2 interaction and -arrestin-mediated GPCR endocytosis. In agreement with this, AP2/-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin.Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of -arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in -arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis reveled that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of -arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of -arrestin, while Barbadin selectively augments FPR2-mediated neutrophil ROS production independently of receptor endocytosis.Given that Barbadin binds to AP2 and prevents the AP2/-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from human neutrophils. a large number of ligands belonging to different chemical classes [5][6][7]. Depending on the agonists examined, the signals generated by FPR2 mediate primarily pro-inflammatoryactivities, yet anti-in...

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“…Such an allosteric GPCR modulation is, according to the dogma for allosteric GPCR-modulation, solely affecting the signaling properties of agonists that specifically interact with the receptor that is allosterically modulated (6,7). This GPCR-modulation dogma has, however, been challenged and shown to be more complex than initially anticipated, when it was shown that allosteric modulators specific for Free Fatty Acid Receptor 2 (FFAR2) affect signaling mediated not only by orthosteric FFAR2 agonists but also by specific agonists for the ATP-receptor P2Y2R and for formyl peptide receptors (FPRs; (8)(9)(10).…”

Section: Introductionmentioning

confidence: 99%

Multiple ligand recognition sites in free fatty acid receptor 2 (FFAR2) direct distinct neutrophil activation patterns

Lind

Holdfeldt

Mårtensson

et al. 2020

Preprint

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Non-activating positive allosteric modulators specific for free fatty acid receptor 2 (FFAR2) increased the activity induced by orthosteric agonists to trigger a rise in intracellular Ca2+ and activate the superoxide producing neutrophil NADPH-oxidase. In addition, two allosteric modulators (Cmp58 and AZ1729) recognized by different receptor domains on FFAR2, cooperatively triggered activation without any rise in intracellular calcium ions. To gain insights into FFAR2 modulation and signaling, we set out to identify structurally diverse allosteric FFAR2 modulators. Initially, we identified two molecules that directly activate neutrophils and these were classified as an allosteric FFAR2 agonists and an orthosteric agonist, respectively. Based on the sensitizing effect on the neutrophil response to propionate, ten non-direct-activating molecules were classified as allosteric FFAR2 modulators. One of these synergistically activated neutrophils when combined with AZ1729, but not when combined with Cmp58. The remaining nine compounds synergistically induced the same type of biased neutrophil signaling but only when combined with Cmp58. The activation signals down-stream of FFAR2 when stimulated by two allosteric modulators with different binding sites were in most cases biased in that two complementary modulators together triggered an activation of the NADPH-oxidase, but no increase in intracellular calcium ions. The neutrophil activation pattern achieved when two functionally AZ1729- or Cmp58-like allosteric FFAR2 modulators were combined, supporting a model for activation in which FFAR2 has two different sites that selectively bind allosteric modulators. The novel neutrophil activation patterns and receptor down-stream signaling mediated by two cross-sensitizing allosteric modulators represent a new regulatory mechanism that controls FFAR2 receptor function.

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

Cited by 38 publications

References 117 publications

Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2 complex

Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2 complex

Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis

Multiple ligand recognition sites in free fatty acid receptor 2 (FFAR2) direct distinct neutrophil activation patterns

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