Structural and functional elucidation of the primary transducer module of the B cell antigen receptor

Interaction of an antigen with the B cell antigen receptor (BCR) initiates signaling cascades leading to B cell activation, proliferation and differentiation. Signal transduction downstream of the BCR depends on the adaptor protein SLP65 (SH2 domain-containing leukocyte adaptor protein of 65 kDa). Upon BCR activation, SLP65 translocates from the cytosol to the plasma membrane via a poorly understood mechanism, and recruits the enzymes Btk (Bruton’s tyrosine kinase) and PLCγ2 (Phospholipase C-γ2), necessary for the release of the key secondary messenger Ca2+. As recently shown by our group, the pre-formed complex of SLP65 with CIN85 (Cbl-interacting protein of 85 kDa) is indispensable for efficient BCR signaling initiation (Oellerich et al., 2011). For my dissertation, I investigated the plasma membrane targeting process of this so called “primary BCR transducer module”. Using imaging and biochemical assays, my colleagues and I showed that signaling-competent SLP65 is distributed between the cytosol and intracellular exosome-like vesicles in resting DT40 cells. Vesicular localization of SLP65 depended on its positively charged amino (N)-terminal region, encompassing 50 amino acids. I found that the N terminus of SLP65 is a direct lipid-binding module, which preferentially binds to negatively charged and curved membranes in vitro. In vivo, the N terminus of SLP65 could be functionally substituted by the curvature-sensitive lipid-binding N-BAR domain of Amphiphysin. However, lipid binding by the N terminus or the BAR domain was insufficient to target SLP65 to vesicles in the absence of CIN85 binding, indicating that the N terminus and CIN85 cooperate for precise subcellular targeting of SLP65. The finding that pre-assembly of signaling adaptors on vesicles is required for B cell activation indicates that these signaling complexes may be delivered to the BCR via vesicular transport. Analysis of the secondary structure of SLP65 by Nuclear Magnetic Resonance (NMR) spectroscopy, in collaboration with the group of Prof. Christian Griesinger, showed that the N terminus of SLP65 is intrinsically disordered in solution, as well as upon lipid binding. Large regions of intrinsic disorder were also observed in the central part of SLP65. I investigated the functional role of intrinsic disorder in this region by substituting it with a flexible GS linker, or by rearranging the Btk- and PLCγ2- binding motifs. My results indicate that secondary structure in this region is dispensable for the assembly of the Ca2+ initiation complex, but might be required for the membrane targeting of SLP65 via the vesicular route. Altogether, my studies contribute to the understanding of the membrane targeting process of the adaptor protein SLP65 and the BCR signaling initiation