Functional and structural requirements for the internalization of distinct BCR‐ligand complexes

Abstract

Antigen (Ag) binding to the BCR rapidly initiates two important events: a phosphorylation cascade that results in the production of secondary signaling intermediaries and the internalization of Ag-BCR complexes. Previous studies using anti-BCR antibodies (Ab) have suggested that BCR signaling is an essential requirement for BCR endocytosis and have further implicated lipid rafts as essential platforms for both BCR functions. However, published data from our laboratory indicate that lipid rafts and consequently raft-mediated signaling are dispensable for BCR-mediated internalization of Ag-specific BCR. Therefore, we investigated the relationship between BCR signaling and endocytosis by defining the role of early kinase signaling in the BCRmediated internalization of a model Ag (haptenated protein). The results demonstrate that Src kinases and Syk-mediated BCR signaling are not essential for BCR-mediated Ag internalization. Moreover, by comparing Ag and Ab, it was determined that while both localize to clathrin-coated pits, the internalization of Ab-BCR complexes is more susceptible to inhibition of signaling and highly sensitive to disruption of lipid rafts and the actin cytoskeleton compared to Ag-BCR complexes. Thus, these results demonstrate that the nature of the ligand ultimately determines the functional requirements and relative contribution of lipid rafts and other membrane structures to the internalization of BCR-ligand complexes.

The Syk-binding Ubiquitin Ligase c-Cbl Mediates Signaling-dependent B Cell Receptor Ubiquitination and B Cell Receptor-mediated Antigen Processing and Presentation

“…Reagents-The following reagents were used for this study: hen egg lysozyme (HEL, catalog no. L-6876, Sigma), HEL BCR Signaling-Intracellular calcium measurements were performed as described previously (18,27).…”

“…BCR Internalization-The kinetics of antigen-BCR endocytosis were determined as described previously (18,27).…”

“…BCR Ubiquitination-Analysis of BCR ubiquitination by ubiquitin pull-down and SDS-PAGE Western blot analysis was done as described previously (18,27).…”

“…BCR Processing-Analysis of BCR processing by Western blot analysis was done as described previously (18,27). For BCR stimulation, biotinylated rabbit anti-human IgM was used at 10 g/ml, and the cells were pulsed with ligand for the indicated times.…”

“…Flow Cytometric Antigen Processing and Presentation AssayFlow cytometric analysis of HEL 46 -61 -I-A k expression was done as reported previously (16,18,(27)(28)(29), except for the following modifications. Splenocytes from either MD4.B10.Br or B10.Br mice were pretreated with inhibitors (4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo [3,4- Cells were infected with lentiviral particles at 37°C with constant inversion for 30 min and returned to culture in complete media to allow viral integration for 48 h. After 48 h, flow cytometric analysis confirmed a transfection efficiency of 8 -15% on the basis of GFP expression.…”