Photoaffinity labeling of the N-formyl peptide receptor of human polymorphonuclear leukocytes.
Quantitative analysis of ligand-occupied receptor interactions with elements of the cytoskeleton and with intracellular compartments requires a sensitive and simple method of identifying the receptor-ligand complex in living cells. Toward this goal, we have prepared a photoactivatable arylazide derivative of the chemotactic peptide N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys, which can be radiolabeled to high specific activity with 125I. This derivative was biologically active as judged by its ability to elicit superoxide anion production by human PMNL at nanomolar concentrations (ED50 approximately 0.7 nM). When incubated at 0 degree C with whole PMNL, radioactive ligand became specifically and saturably associated with a 60-70,000-dalton species (as assessed by SDS-PAGE) after exposure to UV light. Addition of 10-100-fold excess of unlabeled parent or unlabeled azidopeptide derivative completely blocked uptake into this species. Approximately 20-40% of the available surface receptor-binding sites were covalently labeled under these conditions. Subcellular fractionation of the labeled cells on sucrose gradients after homogenization showed that the labeled species was primarily associated with plasma membrane-rich fractions. The labeled receptor could be completely solubilized with Triton X-100 in a form which eluted as a single species with a Stoke's radius of less than 50 A on Sepharose 4B columns. In addition, the solubilized receptor-ligand complex bound specifically to wheat germ agglutinin, indicating that it is probably a glycoprotein. The ability to label the receptor in living PMNL with a high efficiency should facilitate the study of receptor dynamics and receptor physiochemical properties in this system.
D000345:Affinity Labels; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D037102:Lectins; D009504:Neutrophils / Q000032:analysis* / Q000378:metabolism / Q000648:ultrastructure; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000378:metabolism; D044042:Receptors, Formyl Peptide; D013481:Superoxides / Q000378:metabolism; D014909:Wheat Germ Agglutinins
D000345:Affinity Labels; D006023:Glycoproteins; D037102:Lectins; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D044042:Receptors, Formyl Peptide; D014909:Wheat Germ Agglutinins; D013481:Superoxides; C021642:F-chemotactic peptide