Kooperation funktioneller Domänen des Adapterproteins SLP-65 für die Ca2+-Antwort in B-Lymphocyten

Specific antigen recognition by B cells is mediated via their antigen receptor (BCR), resulting in several intracellular signaling events. The recruitment of the Ca^2+ initiation complex, consisting of SLP-65, PLC-[gamma]2 and Btk, to the plasma membrane is a key element of BCR signal transduction. The exact mechanism of this SLP-65 mediated localization remains obscure. In 2005 the N-terminus of SLP-65 was described as an important component and a leucine-zipper function was postulated. However, I could show by targeted inactivation of this motif, that a leucine-zipper is not responsible for the correct subcellular organisation of the Ca^2+ initiation complex. The function depended rather on the basic characteristics of this region, which I name basic effector domain (BED). Furthermore, I could identify Calmodulin as a new BED-dependent binding partner of SLP-65. Ca^2+ concentration-dependent interaction studies, as well as Ca^2+ mobilization kinetics of cells expressing SLP-65 variants that do not bind Calmodulin, suggest a function downstream of the Ca^2+ mobilization. However, the importance of BED for the membrane localization of SLP-65 could be attributed to a different aspect. I showed a BED-mediated interaction of SLP-65 directly with lipid components of cell membranes in vitro, namely phosphatidylinositol-phosphates (PIP). I confirmed the functional relevance in vivo showing that the defined PIP binding motif of the adaptor protein TIRAP can fully substitute BED. This suggests that a direct interaction of BED with PIP is involved in membrane localization of SLP-65 and thus of the Ca^2+ initiation complex. I also contributed to the functional understanding of the SH2 domain as the second essential component for membrane recruitment of SLP-65. By co-expression of SLP-65 variants lacking BED or SH2 domain, I could show an in cis cooperation of these domains. To characterize this in more detail chimeric SLP-65 variants were generated, in which BED was replaced by various membrane recruitment domains. Additional inactivation of the SH2 domain within these proteins allowed analysis of an individual function of the SH2 domain. The results demonstrated that simple membrane localization is not sufficient to substitute for both membrane anchors of SLP-65. Instead, the protein must localize specifically to BCR-proximal signalling compartments within membrane microdomains. This is mediated by the SH2 domain. The results show that membrane recruitment of SLP-65 underlies a complex mechanism requiring the concerted function of at least two domains. The model I propose is, that BED contributes to SLP-65 membrane recruitment by direct PIP-binding and together with the SH2 domain stabilizes SLP-65 at the plasma membrane by a two-armed anchoring. The SH2 domain additionally mediates a specific localization of the protein, and therefore the Ca^2+ initiation complex, in defined signalling platforms, the lipid rafts.