Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils.
The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli.
Sengeløv
H
H
Department of Hematology, State University Hospital, Copenhagen, Denmark.
Boulay
F
F
Kjeldsen
L
L
Borregaard
N
N
D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D018000:Receptors, Peptide / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D009240:N-Formylmethionine Leucyl-Phenylalanine