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OBJECTIVE To identify the mechanisms by which the formyl peptide receptor 2(FPR2)mediates both inflammatory and anti-inflammatory signaling in an agonist-dependent manner.METHODS Cells expressing FPR2 were incubated with weak agonists,Aβ42 and Ac2-26,before stimulation with a strong agonist,WKYMVm.Calcium mobilization,c AMP inhibition and MAP kinase activation were measured.Intramolecular FRET were determined using FPR2 constructs with an ECFP attached to the C-terminus and a Fl As H binding motif embedded in the first or third intracellular loop(IL1 or IL3,respectively).RESULTS Aβ42 did not induce significant Ca~(2+) mobilization,but positively modulated WKYMVm-induced Ca~(2+) mobilization and c AMP reduction in a dose-variable manner within a narrow range of ligand concentrations.Treating FPR2-expressing cells with Ac2-26,a peptide with anti-inflammatory activity,negatively modulated WKYMVm-induced Ca~(2+) mobilization and c AMP reduction.Intramolecular FRET assay showed that stimulation of the receptor constructs with Aβ42 brought the C-terminal domain closer to IL1 but away from IL3.An opposite conformational change was induced by Ac2-26.The FPR2 conformation induced by Aβ42 corresponded to enhanced ERK phosphorylation and attenuated p38 MAPK phosphorylation,whereas Ac2-26 induced FPR2 conformational change corresponding to elevated p38 MAPK phosphorylation and reduced ERK phosphorylation.CONCLUSION Aβ42 and Ac2-26 induce different conformational changes in FPR2.These findings provide a structural basis for FPR2 mediation of inflammatory vs anti-inflammatory functions and identify a type of receptor modulation that differs from the classic positive and negative allosteric modulation.
OBJECTIVE To identify the mechanisms by which the formyl peptide receptor 2 (FPR2) mediates both inflammatory and anti-inflammatory signaling in an agonist-dependent manner. METHODS Cells expressing FPR2 were incubated with weak agonists, Aβ42 and Ac2-26, before stimulation with a strong agonist, WKYMVm.Calcium mobilization, c AMP inhibition and MAP kinase activation were measured. Intramolecular FRET were determined using FPR2 constructs with an ECFP attached to the C-terminus and a Fl As H binding motif embedded in the first or third intracellular loop ( IL1 or IL3, respectively) .RESULTS Aβ42 did not induce significant Ca 2+ mobilization, but positively modulated WKYMVm-induced Ca 2+ mobilization and c AMP reduction in a dose-variable manner within a narrow range of ligand concentrations.Treating FPR2-expressing cells with Ac2-26, a peptide with anti-inflammatory activity, negatively modulated WKYMVm-induced Ca 2+ mobilization and c AMP reduction. Intramolecular FRET assay showed that stimu lation of the receptor constructs with Aβ42 brought the C-terminal domain closer to IL1 but away from IL3.An opposite conformational change was induced by Ac2-26. The FPR2 conformation induced by Aβ42 was associated with enhanced ERK phosphorylation and attenuated p38 MAPK phosphorylation, while Ac2-26 induced FPR2 conformational change corresponding to elevated p38 MAPK phosphorylation and reduced ERK phosphorylation. CONCLUSION Aβ42 and Ac2-26 induce different conformational changes in FPR2. The findings provide a structural basis for FPR2 mediation of inflammatory vs anti-inflammatory functions and identify a type of receptor modulation that differs from the classic positive and negative allosteric modulation.