Intracellular signaling cascades activated by Formyl Peptide Receptors

The formyl peptide receptors FPR1, FPR2 and FPR3 are seven transmembrane Gi-protein coupled receptors. They were first identified as mediators of chemotaxis and activation of leukocytes in response to bacterial formylated peptides. To date, expression of FPRs was described also in non-myeloid cells, together with the ability of these receptors to recognize an heterogenous range of ligands of different origin. In the last few years, FPRs activation or their overexpression has been correlated to cell tumorigenicity, inflammation, cell proliferation, invasion and tumour progression. Furthermore, increasing evidence have highlighted the ability of FPRs to transactivate receptor tyrosine kinase (RTKs) through NADPH oxidase-dependent production of reactive oxygen species (ROS). Herein, we investigated (i) the ability of FPR1 to transactivate the nerve growth factor receptor TrkA, in SH-SY5Y human neuroblastoma cell line; (ii) the involvement of NADPH oxidase-derived ROS in mediating TrkA transactivation; (iii) the downstream signaling cascades triggered by FPR1 stimulation and, in turn, by TrkA transactivation; (iv) the biological effects of FPR1 activation. Western blotting experiments demonstrated that FPR1 stimulation mediates NADPH oxidase-dependent phosphorylation of cytosolic residues Y490, Y751, and Y785 of TrkA, that represent docking sites for Erk, Akt and PKC pathway activation. Cell count assay, neurite outgrowth assay and wound-healing assay indicated that FPR1-mediated TrkA phosphorylation enhances cell proliferation, growth and migration. Furthermore, to characterize phosphorylations of intracellular signaling molecules triggered by FPR activation, we performed a TiO2-based affinity chromatography to obtain an enrichment of phosphoproteins derived from FPR2 stimulation. Mass spectrometry analysis identified 290 differentially phosphorylated proteins and 53 unique phosphopeptides. Phosphorylations of five selected phosphoproteins (HSP-27, MCM2, OSR1, Rb and MARCKS) were further validated by western blotting experiments, confirming their dependence on FPR2 activation. Furthermore, we show that FPR2 stimulation with two anti-inflammatory agonists (Annexin A1 or Lipoxin A4) induces the phosphorylation of selected differentially phosphorylated proteins, suggesting their role in the resolution of inflammation. Taken together, these data represent a promising resource for further studies on new signaling networks established by FPRs that could lead to the identification of novel molecular drug targets for human diseases