Formyl peptide chemotactic receptor. Evidence for an active proteolytic fragment.
We have developed a radioiodinated photoaffinity label, N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys-N-6-(4'-azido-2'-nitrophenylamino) hexanoate (where Nle represents norleucine) (125I-PAL), which forms a covalent complex with the formyl peptide chemotactic receptor of living human neutrophils. Labeling was 12 to 16% efficient and did not alter cell viability. The receptor on live neutrophils and neutrophil membranes has an apparent molecular weight of 50,000 to 70,000 by sodium dodecyl sulfate-polyacrylamide electrophoresis. The receptor on intact cells possesses one predominant papain cleavage site, yielding a 35,000-Da fragment. This receptor fragment retains an affinity for N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys indistinguishable from the receptor on control cells (KD = 1.9 and 1.8 nM, respectively). The 35,000-Da papain fragment was biologically active as evidenced by an unchanged dose-response curve for peptide-stimulated beta-glucuronidase release and fluorescent peptide uptake. Papain treatment of 125I-PAL-labeled neutrophil membranes or of digitonin-soluble 125I-PAL-labeled receptors produced a predominant 28,000-Da fragment without evidence of the 35,000-Da fragment seen with whole cells. Pronase, which did not cleave the receptor on intact cells, produced multiple receptor fragments when used to treat 125I-PAL-labeled membranes.
D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D005966:Glucuronidase / Q000097:blood; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010206:Papain / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D044042:Receptors, Formyl Peptide
D000345:Affinity Labels; D001386:Azides; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D044042:Receptors, Formyl Peptide; C036764:NPH-peptide; D005966:Glucuronidase; D010206:Papain