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.
Remes
J J
JJ
Biocenter, University of Oulu, Finland.
Petäjä-Repo
U E
UE
Tuukkanen
K J
KJ
Rajaniemi
H J
HJ
D002118:Calcium / Q000378:metabolism; D002241:Carbohydrates / Q000502:physiology*; D016326:Extracellular Matrix Proteins / Q000502:physiology; D006801:Humans; D009504:Neutrophils / Q000502:physiology*; D009842:Oligopeptides / Q000494:pharmacology; D010206:Papain / Q000494:pharmacology; D009195:Peroxidase / Q000378:metabolism; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000737:chemistry / Q000502:physiology*; D018000:Receptors, Peptide / Q000737:chemistry / Q000502:physiology*; D015398:Signal Transduction
D002241:Carbohydrates; D016326:Extracellular Matrix Proteins; D009842:Oligopeptides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; C021642:F-chemotactic peptide; D009195:Peroxidase; D010206:Papain; D002118:Calcium