Significance of the extracellular domain and the carbohydrates of the human neutrophil N-formyl peptide chemotactic receptor for the signal transduction by the receptor.

The N-formyl peptide chemotactic receptor of human neutrophils possesses a 2-kDa papain-removable extracellular domain that contains two N-linked oligosaccharide side chains and is not required for the high-affinity ligand binding. In the present study, the significance of the extracellular domain and the carbohydrates for signal transduction was elucidated by measuring the N-formyl hexapeptide-induced intracellular free calcium ([Ca2+]i) and the change in myeloperoxidase secretion in the control and papain-treated human neutrophils. [Ca2+]i was monitored both in cell suspension and individual cells with intracellularly trapped Fura 2 acetoxymethyl ester, using spectrofluorometric analysis and fluorescence ratio image analysis, respectively. The exposure of the cells in suspension to N-formyl hexapeptide resulted in an immediate, dose-dependent burst of elevated [Ca2+]i, which was virtually identical in both control and papain-treated cells with respect to the extent and kinetics. The maximum burst was 1.6-fold and was obtained at 10(-6) M hexapeptide. The individual control and papain-treated cells responded to 10(-6) M hexapeptide in a similar manner with several successive transients of [Ca2+]i, the maximum level being 3.0-3.5 microM. In both groups the [Ca2+]i transient began initially in the cell periphery, expanding rapidly throughout the cells. Concomitantly, the cells became polarized, and their chemokinesis increased. The secretion of myeloperoxidase was monitored as a physiological end response to N-formyl chemotactic peptides. The exposure of the control and papain-treated cells in suspension to hexapeptide resulted in a dose-dependent secretion of myeloperoxidase. The maximum secretion after exposure to 10(-8)-10(-6) M hexapeptide was equal in control and papain-treated neutrophils. These results indicate that the functional properties of the membrane-inserted N-formyl peptide chemotactic receptor are inherent to the receptor's transmembrane and intracellular domains, as far as binding of the ligand and subsequent receptor activation are concerned.

Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores.

Rat and human neutrophil N-formyl-peptide chemotactic receptors were subjected to glycosidase and proteinase treatments to determine the extent and species differences of glycosylation and the carbohydrate requirement in the high-affinity ligand binding. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was attached to rat and human neutrophils either before or after glycosidase and proteinase treatments, and the labelled receptors were solubilized after glutaraldehyde cross-linking and analysed by SDS/PAGE and autoradiography. Both the rat and human N-formyl-peptide chemotactic receptors contain only N-linked oligosaccharides, as demonstrated by their sensitivity to peptide N-glycosidase F (PNGase F) and resistance to O-glycanase treatment. The N-linked oligosaccharides seem to be of the complex type rather than the high-mannose or hybrid type and lack terminal sialic acid, as demonstrated by their resistance to endoglycosidases D and H and neuraminidase treatments. This sensitivity pattern was similar in both species, and the shift in the molecular size of the receptors to 35-38 kDa after PNGase F treatment occurred through one intermediate product, suggesting that both receptors contain a similar 35-38 kDa polypeptide core with two N-linked complex-type oligosaccharides, the heterogeneity of which is responsible for the species difference in receptor size. Papain treatment alone or followed by PNGase F produced in both species a 33-36 kDa membrane-bound fragment that was still able to bind the ligand, suggesting that the oligosaccharides are located on the approx. 2 kDa papain-cleavable polypeptide fragment of the receptors. The cleavage sites for both papain and PNGase F were hidden in occupied receptors, suggesting a conformational or topographical change in these upon ligand binding. Scatchard analyses and cross-linking experiments demonstrated that carbohydrates are not required for high-affinity ligand binding and that the 33-36 kDa membrane-bound papain fragment of both receptors contains the ligand-binding site.