Covalent cross-linking of radiolabeled N-formylated hexapeptide to its specific receptor on rat and human neutrophils: evidence for a ligand induced complex formation.
The structure of the rat and human neutrophil receptor for N-formylated chemotactic peptides was characterized using 125I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys hexapeptide as a ligand and an affinity cross-linking technique. 125I-hexapeptide bound to purified rat peritoneal neutrophils was time, temperature and pH-dependent. The average receptor number per cell was about 67,000 and dissociation constant (Kd) 0.41 nM. Formyl-MLLP, fMLP, fNLP, were 750%, 15%, 8.6% respectively and Boc-MLP, Boc-NLP, and MLP 0.6% as potent as the hexapeptide in inhibiting the binding of 125I-hexapeptide to rat neutrophils. The same correlation was found between these peptides in their potency to induce chemotaxis. This indicates that the rat neutrophil chemotactic receptor is like human receptor also a highly stereoselective and requires a N-formylated ligand for high affinity binding. Affinity cross-linking of 125I-hexapeptide to rat and human neutrophil chemotactic receptor with glutaraldehyde revealed on SDS-PAGE a 85-kDa and 62-kDa major complex and a 170-kDa and 120-kDa minor complex, respectively. The 120-kDa complex was absent in human neutrophils if the cells were treated with glutaraldehyde prior to cross-linking of 125I-hexapeptide to its receptor. Likewise, the larger complex was absent if neutrophils were exposed to heterologous ligand (C5a) prior to glutaraldehyde treatment and cross-linking of 125I-hexapeptide to its specific receptor. These results demonstrate that the rat neutrophils possess a functional high-affinity receptor for N-formylated chemotactic peptides and that the size of the monomeric receptor is 85-kDa and about 23-kDa larger than that of the human receptor. Upon homologous ligand binding the receptor seems to form a larger complex.
Remes
J
J
Biocenter, University of Oulu, Finland.
Keinänen
K
K
Petäjä-Repo
U
U
Rajaniemi
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002630:Chemotactic Factors / Q000737:chemistry*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral; D006801:Humans; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry*; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011971:Receptors, Immunologic / Q000737:chemistry*
D002630:Chemotactic Factors; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; C078594:N-formylnorleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine receptor; D009842:Oligopeptides; D011971:Receptors, Immunologic; C021642:F-chemotactic peptide; D005976:Glutaral