Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands.

In both infection and autoimmunity, the development of high-affinity Abs and memory requires B cells to efficiently capture and process Ags for presentation to cognate T cells. Although a great deal is known about how Ags are processed, the molecular mechanisms by which the BCR captures Ag for processing are still obscure. In this study, we demonstrate that the Igβ component of the BCR is diubiquitinylated and that this is dependent on the E3 ligase Itch. Itch−/− B lymphocytes manifest both a defect in ligand-induced BCR internalization and endocytic trafficking to late endosomal Ag-processing compartments. In contrast, analysis of ubiquitinylation-defective receptors demonstrated that the attachment of ubiquitins to Igβ is required for endosomal sorting and for the presentation of Ag to T cells, yet, ubiquitinylation is dispensable for receptor internalization. Membrane-bound Igμ was not detectably ubiquitinylated nor were the conserved lysines in the mu cytosolic tail required for trafficking to late endosomes. These results demonstrate that ubiquitinylation of a singular substrate, Igβ, is required for a specific receptor trafficking event. However, they also reveal that E3 ligases play a broader role in multiple processes that determine the fate of Ag-engaged BCR complexes.

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The weaver (wv) gene has been identified as a glycine to serine substitution at residue 156 in the H5 region of inwardly rectifying K(+) channel, GIRK2. The mutation is permissive for the expression of homotetrameric channels that are nonselective for cations and G-protein-independent. Coexpression of GIRK2wv with GIRK1, GIRK2, or GIRK3 in Xenopus oocytes along with expression of subunit combinations linked as dimers and tetramers was used to investigate the effects of the pore mutation on channel selectivity and gating as a function of relative subunit position and number within a heterotetrameric complex. GIRK1 formed functional, K(+) selective channels with GIRK2 and GIRK3. Coexpression of GIRK2wv with GIRK1 gave rise to a component of K(+)-selective, G-protein-dependent current. Currents resulting from coexpression of GIRK2wv with GIRK2 or GIRK3 were weaver-like. Current from dimers of GIRK1-GIRK2wv, GIRK2-GIRK2wv, and GIRK3-GIRK2wv was phenotypically similar to that obtained from coexpression of monomers. Linked tetramers containing GIRK1 and GIRK2wv in an alternating array gave rise to wild-type, K(+)-selective currents. When two mutant subunits were arranged adjacently in a tetramer, currents were weaver-like. These results support the hypothesis that in specific channel stoichiometries, GIRK1 rescues the weaver phenotype and suggests a basis for the selective neuronal vulnerability that is observed in the weaver mouse.

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