The 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 (Brodsky et al., 2001
). 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, lipid rafts, which play a crucial role in the initiation and organization of signaling cascades, have been implicated in the control of clathrin-mediated internalization of the BCR (Stoddart et al., 2002
). Here, we define rafts as cholesterolrich membrane microdomains, enriched in glycosphingolipids. These lipid microdomains vary in size depending on the aggregation of their contents, and they are domains with which acylated signaling molecules, such as SRC
-family kinases, can associate. We previously presented evidence that lipid rafts spatially organize signaling cascades with clathrin for regulation of BCR internalization. An analysis of several different cell lines showed that BCR uptake occurred only when clathrin heavy chain (CHC) was associated with lipid rafts and tyrosine phosphorylated. These observations suggested the existence of a clathrin-mediated uptake pathway that is associated with rafts and regulated by receptors signaling in rafts.
In addition to a cooperative effort between rafts and clathrin, lipid rafts on their own may drive internalization of the BCR. However, the extent of raft involvement has been debated. It was originally reported that antigen-BCR complexes resident within plasma membrane lipid rafts can be internalized and delivered to endocytic compartments (Cheng et al., 1999
). Whether this was direct or via raftassociated clathrin was not addressed. It was subsequently argued that lipid raft-mediated internalization cannot be a major pathway by which the bulk of antigen-BCR complexes gain access to the endocytic pathway, because the kinetics of BCR-mediated antigen internalization versus lipid raft-mediated cholera toxin B internalization are distinct (Putnam et al., 2003
). Whether a cell uses a clathrinor raft-mediated pathway, membrane deformation is required for invagination and subsequent internalization. The actin cytoskeleton has been implicated in this process for both endocytic pathways. Actin has been found in association with rafts and caveolae, and actin-binding proteins, such as Hip 1R, link the clathrin coat to the actin cytoskeleton (Cheng et al., 1999
; Chen and Brodsky, 2005
). The BCR rapidly associates with the actin cytoskeleton following cross-linking (Hartwig et al., 1995
), and actin polymerization appears necessary for scission and detachment of CCVs during BCR internalization (Brown and Song, 2001
). Thus the actin cytoskeleton is implicated in BCR internalization in addition to its reported role in BCR signaling (Jugloff and Jongstra-Bilen, 1997
Cross-linked cell surface receptors are ultimately degraded following uptake, so internalization eventually negatively regulates signaling. However, endocytosis can also play a positive role in receptor-mediated cellular activation. Some receptors continue to signal in endosomes, amplifying their impact after internalization and before degradation. For example, internalization of transforming growth factor-β, nerve growth factor, and epidermal growth factor receptors via the clathrin pathway is believed to trigger or amplify signaling from early endosomes (Grimes et al., 1996
; Vieira et al., 1996
; Howe et al., 2001
; Hayes et al., 2002
; Wang et al., 2002
; Miaczynska et al., 2004
). Thus, internalization can result in two different outcomes, depending on the surface receptors and context of their activation. Whether BCR internalization leads exclusively to inactivation of receptor signaling or is important for signal amplification has not been firmly established.
The recent development of a B cell line DKOS that is conditionally deficient in clathrin expression made it possible to investigate the relative contributions of different pathways to BCR internalization and their impact on BCR signaling. The hierarchy of internalization pathways was established by systematically studying the effects of raft and actin antagonists on clathrin-sufficient and -depleted B cells. The data suggest that the most extensive BCR internalization occurs when clathrin cooperates with rafts and actin. However, the BCR's promiscuous membrane associations enable it to exploit alternative, albeit less efficient, clathrin-independent routes of entry under certain cellular conditions. In cells where all pathways of BCR internalization were blocked, receptor activation, as measured by generalized tyrosine phosphorylation and ERK phosphorylation, was prolonged. This suggests that BCR endocytosis leads primarily to inactivation of receptor signaling.