Plasticity of B Cell Receptor Internalization upon Conditional Depletion of Clathrin

Abstract

B cell antigen receptor (BCR) association with lipid rafts, the actin cytoskeleton, and clathrin-coated pits influences B cell signaling and antigen presentation. Although all three cellular structures have been separately implicated in BCR internalization, the relationship between them has not been clearly defined. In this study, internalization pathways were characterized by specifically blocking each potential mechanism of internalization. BCR uptake was reduced by ϳ70% in B cells conditionally deficient in clathrin heavy chain expression. Actin or raft antagonists were both able to block the residual, clathrin-independent BCR internalization. These agents also affected clathrin-dependent internalization, indicating that clathrin-coated pits, in concert with mechanisms dependent on rafts and actin, mediate the majority of BCR internalization. Clustering G M1 gangliosides enhanced clathrin-independent BCR internalization, and this required actin. Thus, although rafts or actin independently did not mediate BCR internalization, they apparently cooperate to promote some internalization even in the absence of clathrin. Simultaneous inhibition of all BCR uptake pathways resulted in sustained tyrosine phosphorylation and activation of the extracellular signal-regulated kinase (ERK), strongly suggesting that downstream BCR signaling can occur without receptor translocation to endosomes and that internalization leads to signal attenuation. INTRODUCTIONThe B cell immune response is initiated by antigen binding to the B cell antigen receptor (BCR). This results in signaling, entry of B cells into the cell cycle, and antigen internalization leading to its presentation to helper T-cells (Lanzavecchia, 1985;Reth and Wienands, 1997). Although BCR internalization is a well-defined event during antigen processing, several different membrane traffic pathways have been implicated in this process. Here, we address the relative contributions of three cellular mechanisms to BCR internalization and the relationship of BCR internalization to downstream receptor signaling.Following engagement of the antigen receptor, the BCR can be observed in clathrin-coated pits and vesicles, suggesting a clathrin-mediated route of internalization (Salisbury et al., 1980;Brown and Song, 2001). The clathrin molecule has a triskelion or three-legged shape and is composed of three identical heavy chains, each with a tightly associated light chain. Clathrin triskelia, together with additional coat components, assemble into a polygonal lattice at the plasma membrane . The assembly of further triskelia accompanies invagination of the coated pit and transforms it into a clathrin-coated vesicle (CCV), which carries concentrated transmembrane receptors into the cell. In recent years, it has become increasingly apparent that internalization pathways are regulated by signaling cascades. For example, inhibitors or genetic deletions of protein tyrosine kinases prevent BCR internalization (Pure and Tardelli, 1992;Dykstra et al., 2001;Ma et al., 2001). Moreover, ...

Actin-Binding Protein 1 Regulates B Cell Receptor-Mediated Antigen Processing and Presentation in Response to B Cell Receptor Activation

“…Previously, we showed that BCR activation induced the recruitment of clathrin to the BCR and phosphorylation of clathrin within lipid rafts, both of which were required for BCR internalization (26). In this study, we found that BCR activation induced the recruitment of Abp1 to the plasma membrane and to the internalizing BCR, suggesting that BCR signaling regulates the function of Abp1.…”

Large‐scale proteomic analysis of tyrosine‐phosphorylation induced by T‐cell receptor or B‐cell receptor activation reveals new signaling pathways

“…The clathrin-dependent pathway has been shown to be the primary pathway for ligand-induced BCR endocytosis, and raft structures appear to be required for efficient phosphorylation of clathrin heavy chain [21,33]. Tyrosine-phosphorylation of clathrin heavy chain is induced by an SRC-type protein kinase in response to stimulation of various receptor types, and the level of phosphorylation correlates with the rate of receptor internalization [21,[34][35][36][37]. Our data support a requirement for phosphorylation of clathrin heavy chain as well as for that of other endocytosis-related factors in BCR internalization.…”

“…Receptors remaining at the cell surface were detected by flow cytometry with a FACScan instrument (Becton Dickinson) or by confocal laser-scanning microscopy [21]. The percentage of cells with surface receptors was calculated and normalized by the corresponding initial value.…”

Ubiquitinylation of Igβ Dictates the Endocytic Fate of the B Cell Antigen Receptor

“…Itch constitutively colocalizes with endophilin A1 and clathrin H chain (41). As internalization of the BCR is dependent upon clathrin (42), residence on these membrane-associated structures may be sufficient for Itch to ubiquitinylate the BCR.…”