Small, Mobile FcɛRI Receptor Aggregates Are Signaling Competent

Abstract

SUMMARY Crosslinking of IgE-bound FcεRI triggers mast cell degranulation. Previous FRAP and phosphorescent anisotropy studies suggested that FcεRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods are used to redefine relationships between receptor mobility and signaling. QD-IgE-FcεRI aggregates of at least three receptors remain highly mobile over extended times at low concentrations of antigen that induce Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remains mobile at low doses that support secretion. FcεRI immobilization is marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocks secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates, is a trigger for receptor internalization, and is not required for tyrosine kinase activation leading to secretion.

The architectural relationship of components controlling mast cell endocytosis

“…On the basis of the mutagenesis studies of Metzger and colleagues, FceRI cytoplasmic tails lack specific endocytic sorting signals (Mao et al, 1993;Mao et al, 1991). Instead, the extent of aggregation is the key criteria regulating the internalization of FceRI (Andrews et al, 2009). This is a feature shared with glycolipids, such as gangliosides and glycosphingolipids, whose endocytosis by several routes can be activated by crosslinking Torgersen et al, 2001).…”

“…Prior evidence suggests that the endocytic efficiency and/or route for specific receptors depends on complex factors such as aggregation state (Andrews et al, 2009;Liu et al, 2010;Mellman and Plutner, 1984) or ligand dose (Sigismund et al, 2005). The best-known example of a receptor that utilizes both CME and CIE pathways is the epidermal growth factor receptor (EGFR) (Sigismund et al, 2005;Sorkin and von Zastrow, 2009).…”

Mathematical Modeling of Syk Activation in Allergen-Stimulated Mast Cells and Basophils

“…This is a reasonable assumption as long as receptors remain mobile on the cell surface. Receptors in small aggregates on the RBL cell surface remain mobile (Andrews et al, 2009), but not the receptors in large aggregates (Andrews et al, 2008;Menon et al, 1986). We expect serial engagement to be significantly reduced and exert little effect on cell signaling when the surface receptors are immobile.…”

“…This observation suggests the possibility that a site on a bound ligand can dissociate from, and rebind to the same immobilized receptor many times, effectively increasing its off rate constant, before all its sites on the ligand become free and the ligand dissociates from the cell. Recent multi-color tracking experiments indicate that small aggregates of IgE-FcǫRI complexes, comprising two to four receptors, remain mobile on the RBL surface even at ligand doses that result in degranulation (Andrews et al, 2009).…”

Quantum Dots in Cell Biology

“…To be successful, the conjugation reaction conditions should be carefully monitored, and a thorough evaluation of the QD valency should be performed (Andrews et al 2008;Andrews et al 2009;Clarke et al 2008;Howarth et al 2008). This approach creates a large amount of unconjugated QDs, which could lead to significant nonspecific background in cell surface binding experiments.…”