Low-affinity receptor-mediated induction of superoxide by N-formyl-methionyl-leucyl-phenylalanine and WKYMVm in IMR90 human fibroblasts.
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 microM N-fMLP or 10 microM peptide W for 1 min induced both p47phox 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 125I-WKYMVm that are specifically displaced by increasing concentrations of unlabeled ligand. Analysis of the binding data predicted a Kd 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.
Ammendola
Rosario
R
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via Pansini 5, 80131, Naples, Italy. ammendola@dbbm.unina.it
Russo
Lucia
L
De Felice
Carmela
C
Esposito
Franca
F
Russo
Tommaso
T
Cimino
Filiberto
F
D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D019255:NADPH Oxidases / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology*; D037342:Pertussis Toxin / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D044042:Receptors, Formyl Peptide / Q000096:biosynthesis / Q000378:metabolism*; D044067:Receptors, Lipoxin / Q000096:biosynthesis / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
C105101:FPR2 protein, human; D005419:Flavonoids; D006133:Growth Substances; D009842:Oligopeptides; D010750:Phosphoproteins; D044042:Receptors, Formyl Peptide; D044067:Receptors, Lipoxin; C113617:Trp-Lys-Tyr-Met-Val-Met; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D037342:Pertussis Toxin; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one