Identification of a human neutrophil protein of Mr 24 000 that binds N-formyl peptides: co-sedimentation with specific granules.
In assaying subcellular fractions of human neutrophils for N-formyl peptide binding sites using the photoaffinity ligand FMLPL-SASD-125I (125I-labelled N-formylmethionylleucylphenylalanyl-N epsilon- (2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionyl)-lysine) several molecular species were observed. We confirmed localization of the N-formyl peptide receptor of Mr 50 000-70 000 in the plasma membrane and specific granule fractions. A species of Mr 33 000-35 000 was detected in the light Golgi/endosomal fraction, whose size is consistent with the deglycosylated form of the receptor. In addition, a major binding species of Mr 24 000 was identified that co-localized on sucrose gradients with specific granule markers. This Mr 24 000 species, which was investigated further, was found to be released upon cell stimulation with phorbol myristate acetate or FMLP in the presence of dihydrocytochalasin B. It had an affinity for FMLPL-SASD of 145 nM (cf. 0.3 nM for the cell surface receptor). The specificity for the formyl group was lost as the nonformylated Met-Leu-Phe was as effective FMLPL in competing with FMLPL-SASD-125I for binding to th Mr 24 000 species. A structurally unrelated peptide, however, did not compete for the binding. The labelling of the Mr 24 000 species was dependent on the presence of Ca2+, as was its apparent Mr, which shifted from 24 000 to 50 000-70 000 in the presence of Ca2+. By incubating photoaffinity-labelled plasma membrane fractions with specific granule fractions, we could generate a receptor fragment of Mr 24 000, although the relationship to this fragment of the specific granule species is unknown at present. The N-terminal sequence of the Mr 24 000 species was determined and it appears to be a novel protein. Further work will allow its relationship to the receptor, if any, to be elucidated and allow assignment of a function to this potentially important molecule.
Allen
R A
RA
Research Institute of Scripps Clinic, Department of Immunology, La Jolla, CA.
Erickson
R W
RW
Jesaitis
A J
AJ
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001386:Azides; D001667:Binding, Competitive; D002413:Cations, Divalent; D002462:Cell Membrane / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives / Q000494:pharmacology; D009504:Neutrophils / Q000737:chemistry*; D009842:Oligopeptides / Q000378:metabolism*; D010447:Peptide Hydrolases; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000032:analysis*; D013347:Subcellular Fractions / Q000737:chemistry
D000345:Affinity Labels; D001386:Azides; D002413:Cations, Divalent; D009842:Oligopeptides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; C049721:N-formyl-methionylleucyl-phenylalanyl-N(epsilon)-(2-(4-azidosalicylamido)ethyl-1,3'-dithiopropionyl)lysine; D009240:N-Formylmethionine Leucyl-Phenylalanine; C021642:F-chemotactic peptide; C049813:N-formylmethionyl-leucyl-phenylalanyl-lysine; D010447:Peptide Hydrolases