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

Neutrophil responses to alpha-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[3H]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 degrees 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 degrees 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.