title: The fate of the N-formyl-chemotactic peptide receptor in stimulated human granulocytes: subcellular fractionation studies.
abstract: Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4 degrees C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[125I] Tyr-N epsilon (6-(4'-azido-2'-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37 degrees C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor-associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4 degrees C or incubated at 37 degrees C for 2 min or less. Fractionation of cells incubated at 37 degrees C for longer times revealed that the radioactivity sedimented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl-chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N-formyl-Met-Leu-Phe at 37 degrees C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N-formyl-Met-Leu-[3H]Phe decreased 76% +/- 9% in plasma membrane fractions. N-formyl-Met-Leu-[3H]Phe-binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N-formyl-Met-Leu-Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N-formyl-chemotactic peptides to a Golgi-enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction cosedimenting with dense granules.
mesh_terms: D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D004705:Endocytosis; D006056:Golgi Apparatus / Q000378:metabolism; D006098:Granulocytes / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008715:Methionine / Q000031:analogs & derivatives*; D009239:N-Formylmethionine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D044042:Receptors, Formyl Peptide
chemical_list: D009842:Oligopeptides; D011956:Receptors, Cell Surface; D044042:Receptors, Formyl Peptide; D009239:N-Formylmethionine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D008715:Methionine