Formyl peptide chemotactic receptor. Evidence for an active proteolytic fragment.

Abstract

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.