Ligand/receptor internalization: a spectroscopic analysis and a comparison of ligand binding, cellular response, and internalization by human neutrophils

Abstract

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.

Rapid modulation of N-formyl chemotactic peptide receptors on the surface of human granulocytes: formation of high-affinity ligand-receptor complexes in transient association with cytoskeleton.

“…smaller fraction remaining at the cell surface. This observation is in agreement with the extent of internalization in this system (>60% ; 16,28,60) . The distribution of grains in the 1384 THE JOURNAL OF CELL BIOLOGY " VOLUME 98, 1984 cytoskeleton obtained from such cells reflects a higher degree of surface localization than in the intact cells (1 .6-fold vs. 0.7fold; respectively).…”

“…Of importance is the observation that uptake is detected as early as 5 s and continues for -1 min at the same rate as in the whole cell . During this time internalization of the ligand has not occurred (16,27,28,60) .…”

Secretagogue-induced phosphoinositide metabolism in human leucocytes

“…Interestingly, the receptor binding exhibits apparently slower kinetics, with binding still increasing at a time when the biological response and the InsP3 formation are complete. Similar results have been reported by Sklar et al (1982) and Radin et al (1982). It should be noted, however, that the binding kinetics are complicated by the time-dependent decrease in displaceable specific binding that occurs at 37°C (Fig.…”

DNP‐phycobiliproteins, fluorescent antigens to study dynamic properties of antigen‐IgE‐receptor complexes on RBL‐2H3 rat mast cells

“…Sklar and his colleagues have demonstrated the utility of the flow cytometer for kinetic analysis of ligandreceptor interaction in neutrophils (27,28). Our data establish the value of flow cytometry for analyses of dynamic membrane events associated with antigenstimulated mediator release in the RBL-2H3 mast cell model.…”