B cell Ag receptor (BCR) signaling changes dramatically during B cell development, resulting in activation in mature B cells and apoptosis, receptor editing, or anergy in immature B cells. BCR signaling in mature B cells was shown to be initiated by the translocation of the BCR into cholesterol- and sphingolipid-enriched membrane microdomains that include the Src family kinase Lyn and exclude the phosphatase CD45. Subsequently the BCR is rapidly internalized into the cell. Here we show that the BCR in the immature B cell line, WEHI-231, does not translocate into lipid rafts following cross-linking nor is the BCR rapidly internalized. The immature BCR initiates signaling from outside lipid rafts as evidenced by the immediate induction of an array of phosphoproteins and subsequent apoptosis. The failure of the BCR in immature B cells to enter lipid rafts may contribute to the dramatic difference in the outcome of signaling in mature and immature B cells.

No abstract

Cholesterol‐ and sphingolipid‐rich membrane microdomains termed lipid rafts appear to play a central role in B cell activation. In mature B cells, signaling through the B cell antigen receptor(BCR) is initiated from within rafts and leads to activation. In immature B cells, the BCR is excluded from rafts and signaling leads to apoptosis. CD40, a member of the tumor necrosis receptor family, is expressed by B cells throughout development and has been shown to influence the results of the engagement of antigen by the BCR in both mature B and immature B cells. Here evidence is provided that CD40 is excluded from the lipid rafts of both mature and immature B cells and remains excluded from rafts even after cross‐linking. Nevertheless, in mature B cells CD40 signaling influences the association of the BCR with rafts resulting in an increase in the amount of BCR that translocates into rafts following ligand binding and a subsequent acceleration of the movement of the BCR from rafts. In immature B cells, the cross‐linked BCR remains excluded from rafts in the presence of CD40 signaling, conditions under which BCR‐induced apoptosis is blocked. These results indicate that CD40 functions outside lipid rafts to influence raft‐dependent events in mature B cells and raft‐independent events in immature B cells.

HLA-DM has been shown in vitro to catalyze the release of invariant chain (Ii) derived peptides from the peptide-binding groove of class II molecules, thereby facilitating the binding of antigenic peptides. Previous studies showed that at steady state, the majority of DM resides in the class II peptide-loading compartment (IIPLC) where Ii dissociates from class II molecules and antigenic peptides are bound. Here we characterize the expression of DM in vivo in subcellular fractions containing the IIPLC. Using quantitative immunoblotting, we show that in the cell as a whole, class II molecules are expressed in 23-fold molar excess of DM. However, DM is concentrated in the IIPLC, where it is present in a considerably higher concentration relative to the class II molecules, in a molar ratio of 5DR:1 DM. This molar ratio of DM to DR in the IIPLC in vivo is consistent with the catalytic function proposed for DM from studies in vitro. We also provide both biochemical and genetic evidence that DM associates with complexes which contain Ii fragments and class II molecules in the IIPLC. Such complexes are only observed in leupeptin-treated cells in which Ii fails to be completely degraded and complexes containing the leupeptin-induced fragment of Ii (LIP) and class II molecules accumulate in the IIPLC. This observation is consistent with LIP-class II complexes being a substrate for DM in vivo and suggests that interactions of DM and LIP-class II are extremely transient under normal conditions.