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

<div><p>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.</p></div

List of the four formyl peptide receptors and their corresponding pepducins investigated in this study.

<p>List of the four formyl peptide receptors and their corresponding pepducins investigated in this study.</p

The mF1Pal₁₆ pepducin generated from Fpr1 inhibits the FPR2 activity in human neutrophils.

<p><b>A)</b> Human neutrophils (10⁵ cells) were pre-incubated without (solid line) or with mF1Pal₁₆ (1 μM, dashed line) before stimulation with FPR2 agonist WKYMVM (50 nM, indicated by the arrow) and the release of superoxide anions was continuously measured. The figure shows one representative experiment out of four independent experiments with individual buffy coats. <b>B)</b> Human neutrophils (10⁵ cells) were pre-incubated without (solid line) or with mF1Pal₁₆ (1 μM, dashed line) before stimulation with the FPR1 agonist fMLF (50 nM, indicated by the arrow) and the release of superoxide anions was continuously measured. The figure shows one representative experiment out of four independent experiments with individual buffy coats.</p

The mF2Pal₁₆ pepducin generated from Fpr2 inhibits the activity of Fpr2 in mouse neutrophils.

<p><b>A)</b> WT mouse neutrophils (5 x 10⁴ cells) were pre-incubated without (solid line) or with mF2Pal₁₆ (500 nM) for 5 min before stimulation with the peptide PSMα2 (10 nM, indicated by the arrow) and the release of superoxide anions was continuously measured. <b>Inset:</b> The fMIFL response (10 nM) in the presence (dashed line) or absence (solid line) of mF2Pal₁₆ (1 μM). <b>B)</b> The percent of inhibition on the response induced by PSMα2 (10 nM), F2M2 (150 nM), F2Pal₁₆ (500 nM) and fMIFL (10 nM) was calculated from the each individual agonist peak response in the absence of mF2Pal₁₆ (500 nM, mean ± SD) from three independent experiments with individual mouse.</p

The F2Pal₁₆ pepducin generated from FPR2 activates both human and mouse neutrophils to produce superoxide.

<p><b>A)</b> Human neutrophils (10⁵ cells) were activated by the pepducin F2Pal₁₆ (1 μM, indicated by the arrow) in the presence of the FPR2 inhibitor PBP₁₀ (1μM, dashed line) or the FPR1 antagonist CysH (1 μM, dotted line) or left untreated (solid line). The antagonists were pre-incubated with human neutrophils for five minutes in the presence of HRP and isoluminol before F2Pal₁₆ addition and the release of superoxide anions was continuously measured. The figure shows one representative experiment out of five independent experiments performed with individual buffy coats. <b>B)</b> Mouse neutrophils (5 x 10⁴ cells) were stimulated with F2Pal₁₆ (1 μM, solid line) and the release of superoxide anions was continuously measured. The response induced by FPR1 agonist fMIFL (10 nM, dashed line) is shown for comparison. <b>Inset:</b> Dose-dependent activation of WT mouse neutrophils by F2Pal₁₆. The peak values obtained from different concentrations of F2Pal₁₆ were normalized to the max response and Curve fitting was performed by non-linear regression using the sigmoidal dose-response equation (variable-slope, HillSlope 2). EC₅₀ value and 95% confidence interval (CI) are calculated from three independent experiments with individual mouse (mean ± SD).</p

The mF2Pal₁₆ pepducin inhibits the activity of FPR2 in human neutrophils.

<p>Human neutrophils (10⁵ cells) were pre-incubated without (solid line) or with mF2Pal₁₆ (1 μM) for 5 min before stimulation with <b>A</b>) the FPR2 specific agonist WKYMVM (50 nM, indicated by the arrow). <b>Inset:</b> Dose-dependent activation of mouse neutrophils by mF2Pal₁₆. Data are normalized to the maximal response and curve fillting by non-linear regression using the sigmoidal dose-response equation (variable-slope, HillSlope of -1.3) was used. Curve fitting was performed by <b>B</b>) effect of mF2Pal₁₆ (1 μM) on the FPR1 specific agonist fMLF (50 nM, indicated by the arrow). The release of superoxide anions was measured continuously. Representative responses out of five experiments from individual buffy coats are shown.</p

The mF1Pal₁₆ pepducin activates primarily Fpr2 in mouse neutrophils.

<p><b>A)</b> Mouse neutrophils (5 x 10⁴ cells) were activated by addition of the mF1Pal₁₆ pepducin (1 μM). <b>Inset:</b> Dose-dependent activation of mouse neutrophils by mF1Pal₁₆. Data is normalized to the maximal response and curve fitting was performed by non-linear regression using the sigmoidal dose-response equation (variable slope, HillSlope 0.9). EC₅₀ value and 95% confidence interval (mean ± SD) were from three independent experiments with individual mouse. <b>B)</b> The mF1Pal₁₆ induced cell activation in mouse neutrophils (5 x 10⁴ cells) isolated from WT (black bar) and Fpr2^-/- (grey bar) mice. Cells were incubated for 5 min at 37°C before the addition of mF1Pal₁₆ (1 μM) and the NADPH-oxidase activity was measured over time. The peak values obtained were compared and the results are expressed as percent of the peak activity induced by fMIFL (10 nM) in cells derived from each individual mouse (mean ± SD) from three independent experiments with individual mouse. * <i>p</i> ≤ 0.05. <b>C)</b> Cross-desensitization between PSMα2 (50 nM) and mF1Pal₁₆ (1 μM) in WT mouse neutrophils. Cells first received (indicated by the first arrow) PSMα2 (solid line) or mF1Pal₁₆ (dashed line) and when the response had declined, a second stimulation (indicated by the second arrow) was induced by mF1Pal₁₆ (solid line) or PSMα2 (dashed line). <b>Inset:</b> Cross-desensitization between mF1Pal₁₆ and fMIFL in mouse neutrophils. Cells first received (indicated by the first arrow) fMIFL (10 nM, solid line) or mF1Pal₁₆ (1 μM, dashed line) and when the response had declined, a second stimulation (indicated by the second arrow) was induced by mF1Pal₁₆ (1 μM, solid line) or fMIFL (10 nM, dashed line). The release of superoxide anions was continuously measured. Representative curves out of three independent experiments from individual mouse are shown. <b>D)</b> Effect of Fpr1 inhibitor Boc2 (2 μM, black bar) and Fpr2 antagonist (1 μM, grey bar) on the mF1Pal₁₆ (500 nM) response. The data are from five independent experiments with individual mouse (mean ± SD) and One-way ANOVA with Dunnett’s multiple comparisons test in comparison to the mF1Pal₁₆ control response was used for statistics.</p

The F1Pal₁₆ pepducin generated from FPR1 inhibits the activity of FPR2 in human neutrophils but activates mouse neutrophils.

<p><b>A)</b> Human neutrophils (10⁵ cells) were pre-incubated with F1Pal₁₆ (1 μM, dashed line) or without (solid line) for 5 min before stimulation with the FPR2 agonist WKYMVM (50 nM, indicated by the arrow). The release of superoxide anions was continuously measured. The figure shows one representative experiment out of five independent experiments from individual buffy coats. <b>Inset</b>: Cells were pre-incubated with F1Pal₁₆ (1 μM, dashed line) for 5 min before stimulation with the FPR1 agonist, fMLF (50 nM, indicated by the arrow, dashed line) or left untreated (solid line). <b>B)</b> Mouse neutrophils (5 x 10⁴ cells) were activated by addition of the pepducin F1Pal₁₆ (1 μM, indicated by the arrow). The figure shows one representative experiment out of five. <b>Inset:</b> Dose-dependent activation of WT mouse neutrophils by F1Pal₁₆. Data are normalized to the maximal response and curve fitting was performed by non-linear regression using the sigmoidal dose-response equation (variable-slope, HillSlope 2.5). EC₅₀ value and 95% confidence interval (n = 3, mean ± SD). <b>C)</b> Mouse neutrophils (5x10⁴ cells) were activated with F1Pal₁₆ (1 μM) and the C-terminal truncated peptides F1Pal₁₄ and F1Pal₁₂, and the release of superoxide anions was continuously recorded. The relative activity of pepducins is expressed in percent of the peak value obtained by the fMIFL (10 nM) response (mean ± SD) from three independent experiments with individual mouse.</p

The superoxide release induced by the shorter F2Pal₁₆ derivatives in mouse neutrophils.

<p>WT mouse neutrophils (5x10⁴ cells) were activated with 1 μM of F2Pal₁₆ and its C-terminal truncated derivatives F2Pal₁₂ and F2Pal₁₀ after which the release of superoxide anions was continuously recorded. The relative activity of pepducins is expressed in percent of the peak value obtained by the fMIFL response (10 nM) (mean ± SD) from three independent experiments with individual mouse.</p