LOW-AFFINITY RECEPTOR-MEDIATED INDUCTION OF SUPEROXIDE BY N-FORMYL-METHIONYL-LEUCYL-PHENYLALANINE AND WKYMVm IN IMR90 HUMAN FIBROBLASTS

Abstract

We investigated in IMR90 cells the effects of N -formyl-Met-Leu-Phe ( N -fMLP) and WKYMVm (W peptide) on activation of the NADPH oxidase-like enzyme. In serum-deprived human fibroblasts, exposure to 100 μM N -fMLP or 10 μM peptide W for 1 min induced both p47 phox translocation and NADPH-dependent superoxide generation. These effects were in large part mediated by prevention of the rapid activation of extracellular signal-regulated kinases (ERKs) by preincubation with the MEK1 inhibitor PD098059. Furthermore, responses to N -fMLP or W peptide were inhibited by pertussis toxin, suggesting the involvement of a seven-transmembrane G protein-coupled receptor(s) for peptides. RT-PCR experiments demonstrated the expression in these cells of the low-affinity receptor FPRL1, but not the high-affinity receptor FPR. Incubation with radiolabeled WKYMVm, which had a higher efficiency on FPRL1, revealed that human fibroblasts express binding sites for 125 I-WKYMVm that are specifically displaced by increasing concentrations of unlabeled ligand. Analysis of the binding data predicted a K d of 155.99 nM and a receptor density of about 16,200 molecules/cell. HEK293 cells, which express a NADPH oxidase-like enzyme but not formyl peptide receptors, transiently transfected with FPRL1 cDNA produced superoxide on stimulation with N -fMLP or W peptide, demonstrating that this receptor is biologically functional.