B cell antibody responses are triggered by the binding of antigen to the clonally distributed B cell antigen receptors (BCRs). Over the last several years a great deal has been learned about the biochemistry of the complex signal cascades triggered by BCR antigen engagement. Signaling is initiated by phosphorylation of the BCR by a membrane associated member of the Src family kinase, Lyn. At present, the initiating event in B cell activation that brings the antigen bound BCR into contact with Lyn is not known. It now appears that cholesterol- and sphingolipid-rich membrane microdomains, termed lipid rafts, serve as a platform for BCR signaling. Lipid rafts can be isolated from cells based on their relative insolubility in certain nonionic detergents at 4 degrees Celsius. Using detergent insolubility to isolate rafts we learned that in resting cells the BCR is excluded from rafts that concentrate Lyn but upon multivalent antigen binding, the BCR oligomerizes and associates with rafts where it is phosphorylated by Lyn and signaling is initiated. The translocation of the BCR into rafts does not require two of the earliest events in BCR signaling, namely the phosphorylation of the BCR by Lyn or association of the BCR with the actin cytoskeleton. However, the failure of the BCR to signal or to associate with the actin cytoskeleton results in only weak and transient association of the BCR with lipid rafts. The initiation of signaling in the rafts is followed by raft clustering and ultimately by the formation of a highly organized structure termed an immunological synapse from which BCR signaling may be prolonged. Over the last year we have applied the new technology of FRET imaging that allows us to view the interactions of proteins and lipids in living cells, to better characterize the earliest events in antigen-driven BCR signaling, namely, the oligomerization of the receptor and its association with lipid rafts. Using quantitative FRET imaging we showed that the BCR is a monomer of the surface of resting cells and that multivalent antigen binding resulted in the simultaneous phosphorylation of the receptor?s cytoplasmic domains, a change in the BCR?s cytoplasmic domains from a clustered to an open form and the rapid yet transient association with lipids that compose lipid rafts. These events precede the activation of downstream signaling events and require the continuous activity of Src-family kinases but not the binding of Syk. Thus, the initiation of BCR signaling is a remarkably dynamic process accompanied by reversible conformation changes and raft lipid associations induced by Src-family kinase activity. Efforts are under way to introduce appropriate FRET donor and acceptor pairs into transgenic mice to allow us to image signal events in normal B cell subsets. An exciting theme that emerged from our studies of the relationship of the BCR with rafts is one in which BCR raft association is regulated by a variety of factors that control the outcome of the B cell's encounter with antigen including the developmental state of the B cell, the engagement of coreceptors and viral infection. Determining how these factors influence BCR/raft association should add fundamentally to our understanding of how rafts function. Over the last year we have made progress in defining the mechanisms by which coreceptors function to regulate the association of the BCR with lipid rafts and as a consequence regulate signaling. The B cell coreceptors CD19/CD21 when coligated to the BCR through the binding of complement tagged antigens prolongs BCR residency in and signaling from rafts. We determined that the ability of the CD19/CD21 complex to function in rafts was dependent on a tetraspanin CD81 that is a component of the CD19/CD21 complex. Thus, in B cells from CD81-deficient mice and B cells expressing chimeric CD19 receptors that fail to associate with CD81, the CD19/CD21 complex when coligated to the BCR failed to stabilize the BCR in rafts. Many proteins that associate with lipid rafts do so by virtue of their acylation in particular by their palmitoylation, a reversible acylation event. We determined that CD81 becomes palmitoylated in the lipid rafts following crosslinking of the BCR and the CD19/CD21 complex. Palmitoylation appeared essential for the function of CD81 as blocking palmitoylation using the inhibitor 2-bromopalmitate blocked the ability of the CD19/CD21 complex to stabilize the BCR in lipid rafts when coligated to the BCR. Studies are in progress to determine the nature of the palmitoylating enzyme and the cysteines in the cytoplasmic domains of CD81 that are targets of palmitoylating enzymes. Progress was also made in determining how the FcgammaRIIB, a potent negative regulator of BCR signaling when coligated to the BCR, signals for apoptosis when crosslinked to itself. We learned that the FcgammaRIIB when crosslinked to itself becomes associated with lipid rafts and signals for apoptosis by a mechanism dependent on c-Abl but independent of both the phosphatase SHIP and the FcgammaRIIB?s ITIM motifs that are required for FcgammaRIIB?s inhibition of BCR signaling. Although the signaling pathways following homo-versus hetero-aggregation are distinct there appears to be a feedback mechanism by which once signaling is initiated in one pathway signaling is shut down in the opposing pathway. Studies are in progress to define the role of c-Abl and SHIP in this feedback mechanism.
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