Ligand/receptor internalization: a spectroscopic analysis and a comparison of ligand binding, cellular response, and internalization by human neutrophils.
We have compared the kinetics of the responses of neutrophils to the kinetics of ligand-receptor interaction and internalization, using as a model ligand the fluoresceinated hexapeptide N-CHO-Nle-Leu-Phe-Nle-Tyr-Lys-Fluorescein (Nle, norleucine). Cellular responses, ie, membrane depolarization, enzyme (elastase) secretion, and superoxide anion (O-2) generation, are all initiated within 10 sec of the exposure of cells to stimulus. In the cases of membrane depolarization and secretion (in cytochalasin B-treated cells), full responses are elicited by binding which occurs within 15 sec of peptide addition. Ligand binding and internalization have been analyzed over the same time frame with new spectroscopic techniques. The association of ligand and receptor is monitored using an antibody to fluorescein. The antibody to fluorescein specifically quenches the ligand which is in solution, but receptor-bound ligand is inaccessible to the antibody. The internalization of the receptor-bound ligand is monitored by the accessibility of the fluoresceinated peptide to quenching by an external pH change (7.4 leads to 4.0). Ligand which is either outside or on the cell surface is instantaneously quenched while intracellular peptide (or intracellular fluorescein derived from fluorescein diacetate) is only slowly quenched. No internalization is observed until 1 min after binding begins and internalization proceeds at a rate of up to 5,000 receptors/min/cell following a near optimal stimulatory ligand concentration (approximately 1 nM) while the occupied receptors are being cleared from the surface. A comparison of the kinetics of internalization and the cellular responses suggests that internalization of the ligand is too slow to be involved in the triggering of the cellular responses.
D002462:Cell Membrane / Q000502:physiology; D005434:Flow Cytometry; D019793:Fluorescein; D005452:Fluoresceins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009504:Neutrophils / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000378:metabolism
D005452:Fluoresceins; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D044042:Receptors, Formyl Peptide; D013481:Superoxides; C021642:F-chemotactic peptide; D010196:Pancreatic Elastase; D019793:Fluorescein