Regulation of N ‐formyl‐methionyl‐leucyl‐phenylalanine receptor recycling by surface membrane neutral endopeptidase–‐mediated degradation of ligand

Neutrophil responses to α‐ N ‐formyl‐L‐Met‐ L‐Leu‐L‐Phe (fMLF) are modulated by inhibitors of surface membrane neutral endopeptidase (NEP), such as phosphoramidon (PPAD). Because receptor recycling is presumably required for a sustained cellular response, the effect of PPAD on receptor reexpression was examined. After down‐regulation of surface fMLF receptors by fMLF, PPAD blocked the normal reexpression of surface receptors in a manner that was related to the time of prior exposure to fMLF. Internalized fML[ 3 H]F was hydrolyzed by NEP at a rate comparable to the rate of receptor reexpression at the cell surface, suggesting that ligand hydrolysis is rate limiting. To test this hypothesis, cells were incubated with fluorescein‐labeled formyl‐Met‐Leu‐Phe‐Nle‐Tyr‐Lys at 15°C. After binding was complete, but before internalization of receptor‐ligand complexes, high‐affinity antifluorescein antibody F(ab') 2 fragments were added and the cells incubated at 37°C for 60 min in the presence of PPAD. Under these conditions, the inhibitory effects of PPAD were largely reversed and nonimmune F(ab') 2 fragments were without effect.