key: cord-102808-c7ajfvt5
authors: Sundqvist, Martina; Holdfeldt, André; Wright, Shane C.; Møller, Thor C.; Siaw, Esther; Jennbacken, Karin; Franzyk, Henrik; Bouvier, Michel; Dahlgren, Claes; Forsman, Huamei
title: Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis
date: 2020-05-01
journal: bioRxiv
DOI: 10.1101/2020.04.30.070011
sha: 
doc_id: 102808
cord_uid: c7ajfvt5

Formyl 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 β-arrestin recruitment. β-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 β- arrestin/AP2 interaction and β-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/β-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 β-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in β-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 β-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of β-arrestin, while Barbadin selectively augments FPR2-mediated neutrophil ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/β-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from human neutrophils.

Neutrophils, the most abundant leukocytes in human peripheral blood, form the frontline of our innate host immune defense, and are rapidly recruited from the circulation to damaged or infected body tissues, where they contribute to bacterial clearance and tissue repair [1] [2] [3] [4] . The formyl peptide receptor 2 (FPR2), belonging to the family of G protein-coupled receptors (GPCRs), regulates directional neutrophil migration (chemotaxis), granule secretion (degranulation), formation of F-actin filaments (through polymerization of G-actin), and activation of the reactive oxygen species (ROS) producing NADPH-oxidase [3, 5, 6] . FPR2 recognizes not only N-formyl peptides of both bacterial and host mitochondrial origin, but also Neutrophil surface expression of FPR2 or CD11b was examined by flow cytometry. Neutrophils (5 × 10 6 /mL) were equilibrated for 20 min at 37°C, and then stimulated with either KRG (control), an FPR2 agonist or with barbadin with or without an FPR2 agonist in the presence of catalase (2000 Units/mL) to avoid potential agonist inactivation via oxidation. Cells 

The plasmid encoding human FPR2 with an N-terminal SNAP-tag was obtained from Genscript (Piscataway, NJ, USA); it was constructed by replacing GLP1R in the previously described pcDNA3.1(+)-Flag-SNAP-GLP1R plasmid [26] with human FPR2. HEK 293A cells (Thermo Neutrophil NADPH-oxidase activity NADPH-oxidase activity was determined by using isoluminol/luminol-enhanced chemiluminescence (CL) [5] in a six-channel Biolumat LB 9505 (Berthold Co., Wildbad, Germany). Polypropylene tubes containing a 900 µL reaction mixture of 10 5 neutrophils in KRG, isoluminol (2 × 10 -5 M) and HRP (4 Units/mL), were equilibrated at 37°C for 5 min before addition of 100 µL of stimulus. For latrunculin A or Barbadin dilution experiments, neutrophils (10 7 /mL) were pre-incubated at 37°C with latrunculin A (25 ng/mL) or Barbadin (10 µM) in a separate tube and then 10 µL cell suspension were transferred to polypropylene tubes containing 890 µL 37°C pre-heated reaction mixture (to obtain 10 5 cells in the assay system) of KRG, HRP and isoluminol in the absence or presence of latrunculin A (25 ng/mL) or Barbadin (10 µM) followed by agonist stimulation (100 µL). For phagocytosis-induced intracellular NADPH-oxidase activity, yeast particles (1 x 10 8 /mL) were opsonized in 25% normal human serum (30 min, 37°C) followed by washing and dilution in KRG to a concentration of 5 x 10 7 yeast particles/mL. Opsonized yeast solution (100 µL) was added to polypropylene tubes containing 900 µL reaction mixture containing 5 x 10 5 neutrophils in KRG, luminol (2 × 10 -5 M), superoxide dismutase (50 Units/mL) and catalase (2000 Units/mL) in the absence and presence of Barbadin (10 µM) or latrunculin A (25 ng/mL) that had been equilibrated at 37°C for 5 min; multiplicity of infection (MOI): 10:1. The light emission of yeast phagocytosis-induced ROS production is expressed as Mega counts per min (Mcpm).

Neutrophils (10 7 /mL) were equilibrated for 5 min in the absence and presence of Barbadin (10 µM) at 37°C followed by agonist stimulation. After 10 s of treatment, 100 μL cell suspension were transferred to ice-cold fixation/permeabilization solution (BD Cytofix/Cytoperm solution, 0.5 mL) and incubated on ice for 20 min. The cells were then washed twice with BD wash buffer before staining with AF647-conjugated phalloidin (30 min, 4°C). A minimum of 10,000 gated neutrophils (forward scatter; size versus side scatter; density) per sample were collected on an Accuri C6 flow cytometer (BD Biosciences, Sparks, MD, USA). The AF647-conjugated phalloidin intensity was determined by the geometric mean fluorescence intensity (MFI) as analyzed by FlowJo Software Version 10.3 (Tree star Inc., Ashland, OR, USA).

Neutrophils were loaded with Fura-2-AM (5 µM) (30 min, RT, in darkness) before washing and resuspension in KRG. Measurements of the transient rise in [Ca 2+ ]i were carried out at 37°C by using a PerkinElmer LC50 fluorescence spectrophotometer with excitation wavelengths of 340 nm and 380 nm, an emission wavelength of 509 nm, and slit widths of 5 nm and 10 nm.

The transient rise in intracellular calcium is presented as the fluorescence intensities for both the excitation wavelengths (340 and 380 nm) as measured in parallel. For reactivation experiments (Fig 6C) , isoluminol (2 × 10 -5 M) and HRP (4 Units/mL) were included in the assay system to avoid agonist inactivation by the radicals generated through the myeloperoxidase (MPO)-H2O2 enzyme system [28] .

Neutrophils (2 × 10 6 /mL) in KRG supplemented with BSA (0.3%) were loaded on top of a 3µm pore size filter and allowed to migrate towards stimuli loaded into the bottom wells of a chemotaxis plate (ChemoTx, Neuro probe, UK) for 90 min at 37°C, 5% CO2. Barbadin was present in both upper and lower chambers to avoid any gradient effect. The migrated cells were quantified by measuring myeloperoxidase activity of cell lysates [11] . All migration (myeloperoxidase activity) values were subtracted from the level of migration without any attractant in the lower compartment (negative control), and the resulting data are presented as the number of cells recovered in the lower compartment, relative to the total number of cells applied to the migration system (neutrophils were added directly to the bottom well of the chemotaxis plate).

Data analysis was performed using GraphPad Prism 8.1.0a (Graphpad Software, San Diego, CA, USA). Curve fitting was performed by non-linear regression using the sigmoidal doseresponse equation (variable-slope). Independent BRET experiments were normalized to the span of reference compound (WKYMVM) as determined by non-linear regression analysis and pooled together. Data represent mean values ± SEM. Statistical analysis was performed using a paired Student's t-test (Fig 2D; 3D, F and H; 4C and F; 5B and C; 6B and D and 7F) or a repeated measurement one-way ANOVA followed by Tukey's multiple comparison test ( Fig  4A, 7A and 7H ). Statistically significant differences are indicated by *p < 0.05, **p < 0.01, ***p < 0.001.

Barbadin (structure is shown in Fig 1) was recently described as a selective inhibitor of the machinery responsible for AP2/-arrestin-dependent endocytic internalization of some GPCRs including the vasopressin receptor 2 (V2R), the 2-adrenergic receptor (2AR) and the angiotensin II receptor type 1 (AT1R) [19] . To study the effect of Barbadin on agonist-induced FPR2 internalization, we used the peptide agonist WKYMVM and two lipopeptides, the peptidomimetic Cmp 14 and the pepducin F2Pal10 that are functionally biased (structures are shown in Fig 1) . All three agonists (WKYMVM, Cmp 14 and F2Pal10) are potent in inducing an FPR2-mediated transient rise in [Ca 2+ ]i, ERK1/2 phosphorylation, and ROS production in human neutrophils, but Cmp 14 and F2Pal10 are biased away from -arrestin recruitment and chemotaxis [9-11]. By using an enhanced bystander bioluminescence resonance energy transfer (ebBRET) assay system, we here confirmed that Cmp 14 and F2Pal10 are poor inducers of arrestin recruitment (Fig 2A) . Both WKYMVM and F2Pal10 triggered FPR2 internalization but Barbadin lacked effect on FPR2 internalization as determined by ebBRET ( Fig 2B) . The inability of Barbadin to inhibit agonist-triggered removal of FPR2 from the HEK 293 cell surface was also evident when time resolved-Förster Resonance Energy Transfer assay (TR-FRET) [26] was used to study FPR2 internalization (Fig 2C, D) . These data clearly show that internalization of FPR2 induced by agonists is unaffected by the presence of Barbadin.

In contrast to FPR2, and in agreement with previously published data [19] , Barbadin reduced V2R internalization as measured by ebBRET in HEK 293 cells that had been transiently transfected with donor-tagged V2R (V2R-RlucII) and an acceptor anchored to the membrane (rGFP-CAAX) (Fig 2E) . Although Barbadin did not appear to block agonist-induced FPR2 internalization, ebBRET experiments investigating FPR2 trafficking from the plasma membrane (rGFP-CAAX) to early endosomes (rGFP-FYVE) revealed that the internalization was largely (but not completely for WKYMVM) dependent on the presence of -arrestin1/2 as illustrated by the reduced responses in HEK 293 cells devoid of -arrestin1/2 (ARR, Fig 2F, G). In order to confirm that Barbadin did indeed block the interaction between -arrestin-2 and AP2, we performed BRET experiments to determine the WKYMVM and F2Pal10 induced interaction between the two proteins. Barbadin clearly blocked the WKYMVM-induced interaction between -arrestin-2 and AP2, whereas the low level of interaction induced by F2Pal10 alone makes it hard to determine the ability/inability of Barbadin to inhibit its effect ( Fig 2H) . Taken together, these findings demonstrate that Barbadin does not affect agonisttriggered internalization of surface-exposed FPR2s and that a mechanism independent of arrestin/AP2 interaction can sustain WKYMVM-induced receptor endocytosis.

We next determined whether Barbadin affects agonist-induced internalization of FPR2 when it is endogenously expressed in human neutrophils. Although AP2 was clearly present in neutrophils (Fig 3A) , the reduction in the number of surface-exposed FPR2s was identical in WKYMVM-activated control neutrophils (not treated with Barbadin) and in WKYMVMactivated cells treated with Barbadin ( Fig 3B) . Hence, these data strongly imply that Barbadin has no effect on agonist-induced FPR2 internalization in primary human neutrophils in which the receptor is endogenously expressed.

Neutrophils possess an electron transporting enzyme system, the neutrophil NADPH-oxidase, which upon activation generates ROS [5] . Despite the insensitivity of the agonist-triggered FPR2 internalization to Barbadin, pre-incubation of neutrophils with Barbadin substantially increased the neutrophil release of ROS upon WKYMVM stimulation (Fig 3C, D) . Expectedly, the FPR2-selective antagonist RhB-(Lys-βNPhe)6-NH2 (structure is shown in Fig 1, where it is denoted as FPR2 ant.) completely blocked the neutrophil response ( Fig 3C) . Also, the priming effect (i.e., potentiation of ROS production) of Barbadin was evident at a concentration of the peptide agonist WKYMVM (10 nM) too low to trigger ROS release in non-primed neutrophils ( Fig 3D) . The priming effect was concentration-dependent, reaching its full effect at 10 M of Barbadin with an EC50 value of 2 M (Fig 3E) . Similarly, Barbadin also significantly primed the ROS production induced by F2Pal10 and Cmp 14 ( Fig 3F) . The fact that F2Pal10 and Cmp 14 cannot induce -arrestin recruitment at these concentrations ([9-11], Fig 2A) , strongly suggests that the priming effect of Barbadin on FPR2-mediated ROS production is independent of the ability of the activating agonist to recruit -arrestin. In addition, the ROS release following activation with PMA, a compound that bypasses receptors and directly activate protein kinase C (PKC), was unaffected by Barbadin ( Fig 3G, H) , which suggests that Barbadin lacks a direct effect on the ROS-producing NADPH-oxidase machinery. It is also important to note that activation of the NADPH-oxidase could not be triggered by Barbadin alone (Fig 3G, H).

Collectively, these results suggest that Barbadin primes neutrophils in their response to FPR2 agonists, and the increased NADPH-oxidase activation is regulated independently of FPR2 internalization and FPR2-induced -arrestin recruitment.

Barbadin treatment significantly primed FPR2 agonist-induced ROS production in human neutrophils and further characterization revealed that a very short interaction time between neutrophils and Barbadin was needed for Barbadin to exert its priming effect. In fact, the increase in neutrophil ROS production was the same when Barbadin and the FPR2 agonist WKYMVM were added simultaneously, as when the cells were incubated with Barbadin prior to addition of the activating peptide WKYMVM (Fig 4A) . In addition, we found that the priming effect of Barbadin on neutrophils for increased WKYMVM response was also rapidly reduced when the Barbadin concentration was lowered significantly. Neutrophils first incubated for 5 min with an effective priming concentration of Barbadin (10 µM) and then transferred to two different ROS measurement reagents, one containing a new addition of Barbadin (10 µM) and the other not (the Barbadin concentration was thus lowered 100 x to 0.1 µM), followed by stimulation with WKYMVM. A significantly lower degree of priming effect was observed in cells that were exposed to a reduced concentration (from 10 µM to 0.1 µM) than cells incubated constantly to an effective priming concentration (10 µM) of Barbadin (Fig 4 B and C). This suggests that the neutrophil priming induced by Barbadin is a process that is reversible, unlike many other priming processes that involve an irreversible process of neutrophil granule secretion [17, 29, 30] . The effect of Barbadin on FPR2-mediated ROS production resembled the effect induced by the F-actin-disrupting agent latrunculin A. Barbadin and latrunculin A both prolonged and increased the magnitude of FPR2-mediated neutrophil ROS production as compared to the corresponding ROS production in non-treated control cells ( Fig 4D) . In addition, very similar to Barbadin, the priming effect of latrunculin A was rapidly reduced as shown when latrunculin A was removed prior to activation with the FPR2 agonist WKYMVM ( Fig 4E, F ), using the same dilution protocol, to show that the priming effect of Barbadin is reversible, as described above.

FPR2-induced ROS production is a process rapidly initiated after addition of an activating agonist, and within a time period of minutes, the ROS production is terminated and the cells are homologously desensitized. The desensitized cells are non-responsive to a second stimulation with FPR2 agonists but fully responsive to an FPR1 selective agonist or PMA [11, 31] . The desensitized state could be transferred to an active signaling state to produce ROS again (i.e., FPR2 resensitization or reactivation) when the cytoskeleton was disrupted by latrunculin A (Fig 5A) . The ROS production induced by latrunculin A in FPR2-desensitized cells was completely abolished by the FPR2-selective antagonist RhB-(Lys-βNPhe)6-NH2 ( Fig   5A) . To determine the ability of Barbadin to resensitize desensitized FPR2, we reversed the order by which the sensitizer (Barbadin) and FPR2 agonist (WKYMVM) were added to the neutrophils. Barbadin was added to WKYMVM-activated neutrophils at a time point when ROS production had returned to a background level; interestingly, these FPR2-desensitized cells could be resensitized to produce ROS also by Barbadin, similar to the effect of latrunculin A ( Fig 5A) . This response was also inhibited by an FPR2-selective antagonist (Fig 5A) , clearly demonstrating an FPR2-mediated neutrophil resensitization. Very similar results were obtained in FPR2-desensitized cells when F2Pal10 or Cmp 14 (at concentrations that could not recruit - Fig 2A) replaced WKYMVM as the agonist used to desensitize FPR2 (Fig 5A) .

Although the resensitization effects of latrunculin A and Barbadin appeared very similar, it should be noted that the lag phase before any ROS were generated was shorter when Barbadin was used for resensitization, and the time to reach maximal (peak) ROS production was also shorter as compared to the response following addition of latrunculin A ( Fig 5B) . However, the amounts of ROS produced during resensitization (as measured by the area under the curve) were comparable for Barbadin and latrunculin A (Fig 5C) .

In summary, we show that Barbadin, similarly to latrunculin A, not only potentiates the ROS production induced by the different FPR2 agonists, but also resensitizes FPR2 signaling when added to FPR2 desensitized neutrophils.

Barbadin treatment potentiated and resensitized FPR2-mediated signaling leading to ROS production. To further investigate the effect of Barbadin on FPR2 signaling and function in neutrophils, we measured the transient rise in cytosolic calcium [Ca 2+ ]i mediated through FPR2.

In contrast to the potentiating effect on ROS production, the rise in [Ca 2+ ]i induced by WKYMVM was not affected by Barbadin (Fig 6A, B) . Similarly, resensitization by Barbadin of agonist-desensitized FPR2 leading to an activation of the ROS producing NADPH-oxidase, was not associated with a corresponding rise in [Ca 2+ ]i (Fig 6C) .

At the functional level, we also observed a biased activity of Barbadin in favor of neutrophil ROS production over directional cell migration/chemotaxis. Neutrophil chemotaxis was measured by using a transwell migration system; neutrophils were placed on top of the filter and allowed to migrate towards different concentrations of FPR2 agonists that were placed in the bottom well of the chambers. Barbadin (10 M) was added to both compartments, so that it was present in both the upper chamber together with the cells and the lower chamber containing the agonist. In line with earlier data [9, 11], WKYMVM, but not F2Pal10, triggered a chemotactic migration of neutrophils. Further, neutrophil chemotaxis towards WKYMVM was unaffected by Barbadin, and the inability of F2Pal10 to trigger chemotaxis was retained in the presence of Barbadin (Fig 6D) . In summary, these data demonstrate that the effect of Barbadin in neutrophils is in favor of FPR2-mediated ROS over the rise in [Ca 2+ ]i and chemotaxis.

The functional similarities between Barbadin and latrunculin A, both being priming agents affecting the response induced by FPR2 agonists and agents that resensitize desensitized FPR2, promoted us to examine whether Barbadin could directly affect the integrity of the actin cytoskeleton and granule secretion. WKYMVM induced a rapid polymerization of G-actin monomers into polymerized filamentous actin (F-actin) in human neutrophils (Fig 7A) .

However, the presence of Barbadin did not affect the formation of F-actin induced by WKYMVM (Fig 7A) , indicating that Barbadin does not affect the integrity of the actin cytoskeleton.

The observation that Barbadin does not directly interfere with the integrity of the actin cytoskeleton gained further support from the results obtained with Barbadin on two neutrophil responses previously found to be regulated by the actin cytoskeleton, i.e., the ATP receptor P2Y2R-mediated ROS production and the phagocytosis process [32, 33] . It is known that ATP upon binding to its neutrophil receptor P2Y2R, triggers ROS production provided that the Factin structure is disrupted (Fig 7B) . In line with this, latrunculin A treated neutrophils produce ROS when activated with ATP, but no such effect was obtained with Barbadin ( Fig 7B) .

Activation of the ROS-generating NADPH-oxidase system during uptake (phagocytosis) of microbes is a process regulated by the cytoskeleton, and accordingly, latrunculin A inhibited the activation process ( Fig 7C) . However, Barbadin exerted no inhibitory effect, neither when added before addition of the phagocytosis prey nor when added during the ongoing activation process (Fig 7C, D) . Taken together, these data show that Barbadin has no direct effects on basic neutrophil functions regulated by the actin cytoskeleton, supporting the conclusion that Barbadin lacks a general effect on the assembly of the ROS-producing oxidase.

Our data reveal that Barbadin is able to prime neutrophils for enhanced FPR2-mediated ROS production. Neutrophil priming is a well-known process both in vitro and in vivo [17, 29, 30, 34, 35] , and an increased exposure of intracellular granule-localized receptors to the plasma membrane as a result of granule secretion has been suggested to be one of the main mechanisms that augments neutrophil NADPH-oxidase activity [17, 29, 30] . We next determined whether the receptor mobilizing effect with increased surface FPR2 expression could account for the Barbadin-induced priming effect. However, no increased surface exposure of FPR2 was induced by incubation of neutrophils with Barbadin for up to ten minutes (Fig 7E, F) . In addition to FPR2 expression, the ability of Barbadin to upregulate the surface expression of CD11b (complement receptor 3; CR3), a marker protein stored in easily mobilized neutrophil granule compartments that can be mobilized to the surface by many secretagogues or priming agents [36] was investigated. However, similar to the FPR2 expression, Barbadin also lacked effect to upregulate CD11b on the plasma membrane, whereas a profound increase of CD11b surface expression was induced by the classical secretagogue fMLF (Fig 7G, H) .

In summary, our data show that even though Barbadin affects FPR2 signaling in a way that resembles actin cytoskeleton-disrupting agents, Barbadin lacks direct effects on the reorganization of the actin cytoskeleton in neutrophils and on receptor mobilization from intracellular granule stores.

In the present study, we assessed the role of -arrestin in endocytosis of FPR2 and in receptor down-stream functional responses in human neutrophils using Barbadin, an AP2-binding inhibitor that blocks the interaction between -arrestin and AP2 and prevents agonist triggered endocytosis of many GPCRs [19] . Our data show that the AP2 protein targeted by Barbadin indeed is expressed in neutrophils, yet, Barbadin did not block FPR2 endocytosis. These results imply that FPR2 can be internalized through a -arrestin/AP2-independent process, an assumption in line with the observation that only residual endocytosis of FPR2 occurs in cells lacking -arrestin. Interestingly, Barbadin treatment potentiated FPR2-mediated ROS production and resensitization of FPR2-desensitized human neutrophils in a manner similar as an inhibitor of actin polymerization (i.e., latrunculin A). However, Barbadin did not interfere with other processes in neutrophils involving the actin cytoskeleton machinery. In addition, the potentiating effect of Barbadin on FPR2-mediated ROS production was found to involve biased functional/signaling as neither FPR2-promoted intracellular calcium mobilization nor chemotaxis was affected when AP2-binding was inhibited by Barbadin.

Previously, Barbadin has been shown to affect several GPCR-mediated functions, including hormone secretion mediated by gonadotropin-releasing hormone (GnRH) receptors [37] , and uptake/entry of influenza A viruses facilitated by short chain fatty acid receptor 2 (FFAR2) signaling [20] . As described, Barbadin prevents AP2/β-arrestin-mediated receptor endocytosis, which has been deemed to be the canonical molecular mechanism behind the functional effects of this AP2 inhibitor. However, the data presented in the current study suggest that alternative endocytosis-and β-arrestin-independent mechanisms can mediate the effects by this AP2binding inhibitor with regards to FPR2 expressing human neutrophils. Recent data infer that arrestin appears to be involved in non-canonical and endosomal signaling, besides playing roles in receptor desensitization and endocytosis [14, 38] . However, the exact functional role of β-arrestin in FPR2 signaling needs to be further investigated. It has been suggested that polymerized actin rather than -arrestin constitutes the basis for physical separation of G proteins from activated FPRs, resulting in termination of signaling and receptor desensitization [15, 16, 18] . The role of the actin cytoskeleton in the regulation of GPCR signaling in neutrophils was originally defined by measurements of ROS generated by the phagocyte NADPH-oxidase. Involvement of the actin cytoskeleton in the termination/desensitization of FPR signaling became evident from experiments in which actin cytoskeleton disrupting agents prolong FPR signaling and have the ability to resensitize the desensitized receptors [11, 17, 18] .

We now show that Barbadin lacks effect on agonist-induced endocytosis of FPR2 as examined in several assay systems. Intriguingly, these data strongly indicate that FPR2 can undergo endocytosis through a -arrestin/AP2-independent process. This is an internalization pattern shared with receptors for transferrin and endothelin-A, which are both endocytosed independently of β-arrestin and AP2, respectively [19] . Although Barbadin did not affect FPR2 internalization, it convincingly potentiated FPR2-mediated ROS production and promoted resensitization of desensitized FPR2s. A similar augmentation of the ROS response was also obtained at concentrations of FPR2 agonists (WKYMVM) that do not recruit -arrestin or by FPR2 agonists (F2Pal10 and Cmp 14) that are very poor in recruiting -arrestin, suggesting that this novel priming effect of Barbadin is achieved without -arrestin recruitment.

As mentioned above, the effect of Barbadin on FPR2-mediated ROS production resembled the effect induced by actin cytoskeleton-disrupting agents [11, 17, 18] . Despite this deviation from the prototypical mode of action for Barbadin, several lines of evidence suggest that Barbadin does not directly disrupt the actin cytoskeleton. These findings include that in contrast to latrunculin A, Barbadin had no effect on (i) the increase in F-actin polymerization induced by the FPR2 agonist WKYMVM, (ii) the actin cytoskeleton-dependent ROS production induced during phagocytic uptake of yeast particles was not affected by Barbadin, and (iii) the signals downstream of ATP-activated P2Y2Rs generated only when the actin cytoskeleton has been disrupted. Altogether, these observations indicate that Barbadin primes neutrophils and resensitizes/reactivates desensitized receptors through a mechanism resembling that of actin cytoskeleton-disruptive agents. However, as compared to actin cytoskeleton-disruptive agents, the effects exerted by Barbadin do not appear to involve a direct effect on the integrity of the actin cytoskeleton. At present, the precise mechanism underlying the influence of Barbadin on FPR2 activity is not known, but as Barbadin lacks effect on the FPR2-induced transient rise in [Ca 2+ ]i, a general modulation of downstream signaling of agonist-occupied FPR2 is unlikely.

Regarding assembly and activation of the electron-transporting NADPH-oxidase, a large number of stimuli (including many GPCR agonists) can induce ROS production in neutrophils, but not all signaling pathways that regulate these activation processes have been identified yet.

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. Furthermore, our data support the notion that the neutrophil NADPH-oxidase can be activated in the absence of arrestin recruitment [9-11]. It follows that Barbadin is a biased and functionally selective regulator of FPR2 signaling as it influences ROS production by activating NADPH-oxidase without affecting calcium mobilization and neutrophil granule secretion.

Although -arrestin modulated functions are inhibited by Barbadin, the AP2 inhibitor lacks direct effects on receptor-mediated -arrestin recruitment [19] . Our earlier reports have demonstrated that FPR2 agonists that are potent stimuli in triggering calcium signaling, ERK1/2 phosphorylation and ROS production, but differ in their ability to recruit -arrestin, also vary in their ability to induce neutrophils chemotaxis [9] [10] [11] . The present study shows that the functionally selective deviation linked to the ability to recruit -arrestin is retained in the presence of Barbadin, further supporting the proposed mode of action of Barbadin in that it lacks a direct effect on receptor mediated -arrestin recruitment [19] . The observation that Barbadin potentiates FPR2-mediated ROS production, no matter whether this was caused by the -arrestin recruiting WKYMVM peptide or FPR2 agonists that are very poor in recruiting -arrestin, suggests that the effects of Barbadin on FPR2-mediated ROS production is not dependent on -arrestin. Several in vitro as well as in vivo processes potentiate FPR-mediated ROS production, and increased surface receptor expression as a result of granule secretion has been suggested as an important mechanism underlying the potentiation [17, 29, 30] . However, this is not the mechanism involved in the priming effect of Barbadin, demonstrated by its inability to induce the mobilization of granules. This conclusion is supported by the fact that while granule mobilization is an irreversible process the effects of Barbadin are reversible. Thus, the mechanism by which Barbadin potentiates FPR2-mediated ROS remains to be elucidated. Given that Barbadin binds to AP2 to prevent -arrestin binding, the role of AP2 in the observed effects on ROS priming needs to be further investigated. With respect to the role of AP2, it is interesting to note that a comparison between EC50 values reveals that the potency of Barbadin mediated augmentation of ROS production in neutrophils is the same as that found for its inhibition of the -arrestin-AP2 interaction. This suggests that the Barbadin-mediated effect on ROS production could be a result of its action on AP2 (this study and [19] ). However, other target proteins including other binding partners for AP2, such as AP180, ARH and Scr [19] can, at this point, not be excluded until the modulating effect (if any) of Barbadin on these AP2 binding molecules has been be determined.

In summary, this study demonstrates some novel effects of the AP2 binding compound Barbadin. Although Barbadin did not affect agonist-induced endocytosis of FPR2, a process shown to be independent of whether the agonist recruits -arrestin or not, Barbadin both increased FPR2 agonist induced ROS production, and resensitized agonist-desensitized FPR2 to produce ROS. Notably, these effects of Barbadin on FPR2 also proved independent of whether the agonist recruited -arrestin or not. The effect of Barbadin on FPR2 induced neutrophil ROS production is very similar to the actin cytoskeleton-disrupting agent latrunculin A, albeit without altering other neutrophil functions regulated by a dynamic polymerization of the actin cytoskeleton. Elucidation of the precise mechanism(s) of Barbadin regarding its priming effect on the FPR2-mediated ROS production in neutrophils would lead to an increased understanding of the underlying molecular mechanisms regulating inflammatory reactions that are dependent on redox reactions. Barbadin and structurally related analogs of this AP2

inhibitor are expected to serve as useful molecular tools for further mechanistic studies of GPCR regulation in neutrophils. 

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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

Reactivation of formyl peptide receptors triggers the neutrophil NADPH-oxidase but not a transient rise in intracellular calcium

A new inhibitor of the betaarrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry

Combining Elements from Two Antagonists of Formyl Peptide Receptor 2 Generates More Potent Peptidomimetic Antagonists

Studies on acid stability and solid-phase block synthesis of peptide-peptoid hybrids: ligands for formyl peptide receptors

Monitoring G protein-coupled receptor and beta-arrestin trafficking in live cells using enhanced bystander BRET

Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g

Isolation of lymphocytes, granulocytes and macrophages

Real-time trafficking and signaling of the glucagon-like peptide-1 receptor

Translating in vitro ligand bias into in vivo efficacy

A methodological approach to studies of desensitization of the formyl peptide receptor: Role of the read out system, reactive oxygen species and the specific agonist used to trigger neutrophils

Lipopolysaccharide-induced granule mobilization and priming of the neutrophil response to Helicobacter pylori peptide Hp(2-20), which activates formyl peptide receptor-like 1

Galectin-3 activates the NADPH-oxidase in exudated but not peripheral blood neutrophils

The synthetic peptide Trp-Lys-Tyr-Met-Val-Met-NH2 specifically activates neutrophils through FPRL1/lipoxin A4 receptors and is an agonist for the orphan monocyte-expressed chemoattractant receptor FPRL2

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

An intact cytoskeleton is required for prolonged respiratory burst activity during neutrophil phagocytosis

Multiple Phenotypic Changes Define Neutrophil Priming

Priming and de-priming of neutrophil responses in vitro and in vivo

Granulopoiesis and granules of human neutrophils

beta-Arrestin-dependent signaling in GnRH control of hormone secretion from goldfish gonadotrophs and somatotrophs

GPCR-G Proteinbeta-Arrestin Super-Complex Mediates Sustained G Protein Signaling

Reactivation of desensitized formyl peptide receptors by platelet activating factor: a novel receptor cross talk mechanism regulating neutrophil superoxide anion production

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

After 15 min stimulation, Barbadin (10 M) or LA (25 ng/mL) was added (indicated by an arrow). (E-H) Analysis of FPR2 and CD11b surface expression was performed by flow cytometry. (E-F) Neutrophils were incubated in the absence (buffer) and presence of Barbadin (10 M) at 37°C for different time points as indicated on the x-axis, prior fixation and staining with an anti-FPR2 antibody. (G-H) Neutrophils were incubated in the absence (buffer, 20 min) and presence of Barbadin (10 M; 20 min) or fMLF (10 nM; 10 min) at 37°C prior staining with an anti-CD11b antibody