Biased allosteric modulation of formyl peptide receptor 2 leads to distinct receptor conformational states for pro- and anti-inflammatory signaling

Background and purpose: Formyl peptide receptor 2 (FPR2) is a Class A G protein-coupled receptor (GPCR) that interacts with multiple ligands and transduces both proinflammatory and anti-inflammatory signals. These ligands include weak agonists and modulators that are produced during inflammation. The present study investigates how prolonged exposure to FPR2 modulators influence receptor signaling. Experimental approach: Fluorescent biosensors of FPR2 were constructed based on single-molecule fluorescent resonance energy transfer (FRET) and used for measurement of ligand-induced receptor conformational changes. These changes were combined with FPR2-mediated signaling events and used as parameters for the conformational states of FPR2. Ternary complex models were developed to interpret ligand concentration-dependent changes in FPR2 conformational states. Key results: Incubation with Ac2-26, an anti-inflammatory ligand of FPR2, decreased FRET intensity at picomolar concentrations. In comparison, WKYMVm (W-pep) and A beta(42), both proinflammatory agonists of FPR2, increased FRET intensity. Preincubation with Ac-2-(26) at 10 pM diminished W-pep-induced Ca2+ flux but potentiated W-pepstimulated beta-arrestin2 membrane translocation and p38 MAPK phosphorylation. The opposite effects were observed with 10 pM of A beta(42). Neither Ac2-26 nor A beta(42 )competed for W-pep binding at the picomolar concentrations. Conclusions and implications: The results support the presence of two allosteric binding sites on FPR2, each for AC(2-26) and A beta(42), with high and low affinities. Sequential binding of the two allosteric ligands at increasing concentrations induce different conformational changes in FPR2, providing a novel mechanism by which biased allosteric modulators alter receptor conformations and generate pro- and anti-inflammatory signals.