Abstract
We recently described a novel receptor cross-talk mechanism in neutrophils, unique in that the signals generated by the PAF receptor (PAFR) and the ATP receptor (P2Y2R) transfer formyl peptide receptor 1 (FPR1) from a desensitized (non-signaling) state back to an actively signaling state (Forsman H et al., PLoS One, 8:e60169, 2013; Önnheim K, et al., Exp Cell Res, 323∶209, 2014). In addition to the G-protein coupled FPR1, neutrophils also express the closely related receptor FPR2. In this study we used an FPR2 specific pepducin, proposed to work as an allosteric modulator at the cytosolic signaling interface, to determine whether the cross-talk pathway is utilized also by FPR2. The pepducin used contains a fatty acid linked to a peptide sequence derived from the third intracellular loop of FPR2, and it activates as well as desensensitizes this receptor. We now show that neutrophils desensitized with the FPR2-specific pepducin display increased cellular responses to stimulation with PAF or ATP. The secondary PAF/ATP induced response was sensitive to FPR2-specific inhibitors, disclosing a receptor cross-talk mechanism underlying FPR2 reactivation. The pepducin induced an activity in naïve cells similar to that of a conventional FPR2 agonist, but with lower potency (partial efficacy), meaning that the pepducin is a partial agonist. The PAF- or ATP-induced reactivation was, however, much more pronounced when neutrophils had been desensitized to the pepducin as compared to cells desensitized to conventional agonists. The pepducin should thus in this respect be classified as a full agonist. In summary, we demonstrate that desensitized FPR2 can be transferred back to an actively signaling state by receptor cross-talk signals generated through PAFR and P2Y2R, and the difference in agonist potency with respect to pepducin-induced direct receptor activation and cross-talk reactivation of FPR2 puts the concept of functional selectivity in focus.
Highlights
Human neutrophils, the most prominent effector cells in innate immune reactions and inflammation, express a number of different chemoattractant receptors including the receptors for the complement component C5a (C5aR), the leukotriene LTB4 (BLT1), the chemokine IL-8 (CXCR1 and CXCR2), the platelet activating factor (PAFR), the nucleotide ATP (P2Y2R), and two members of the formyl peptide receptor family (FPR1 and FPR2) [1,2,3]
The response was completely abolished by the FPR2 selective inhibitor PBP10 or the WRW4 peptide antagonist but it was not affected by cyclosporin H, a reversed agonist that inhibits the closely related formyl peptide receptor 1 (FPR1). We conclude from these data that the neutrophil response induced by F2Pal10 is mediated through FPR2, and the third intrallular loop of FPR2 differs in only two amino acids from the corresponding part of FPR1, the pepducin did not cross-react with FPR1
The receptor preference for FPR2 was further supported by desensitization data showing that neutrophils preactivated with F2Pal10 were non-responsive to a second stimulation with the established FPR2 specific peptide agonist WKYMVM and vice versa (Fig 1B)
Summary
The most prominent effector cells in innate immune reactions and inflammation, express a number of different chemoattractant receptors including the receptors for the complement component C5a (C5aR), the leukotriene LTB4 (BLT1), the chemokine IL-8 (CXCR1 and CXCR2), the platelet activating factor (PAFR), the nucleotide ATP (P2Y2R), and two members of the formyl peptide receptor family (FPR1 and FPR2) [1,2,3] All these receptors belong to the family of seven transmembrane G protein-coupled receptors (GPCRs), a large and diverse group of cell surface receptors important for many cellular activities in health and disease [4,5,6,7]. Signaling is terminated (or directed towards endocytic uptake of the receptor-ligand complex) and the occupied receptor becomes refractory to further stimulation with the same agonist and to other agonists that bind the same receptor, an effect commonly termed homologous receptor desensitization [10,11]
Published Version (Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have