Requirements of SLP76 tyrosines in ITAM and integrin receptor signaling and in platelet function in vivo.

Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP76), an adaptor that plays a critical role in platelet activation in vitro, contains three N-terminal tyrosine residues that are essential for its function. We demonstrate that mice containing complementary tyrosine to phenylalanine mutations in Y145 (Y145F) and Y112 and Y128 (Y112/128F) differentially regulate integrin and collagen receptor signaling. We show that mutation of Y145 leads to severe impairment of glycoprotein VI (GPVI)-mediated responses while preserving outside-in integrin signaling. Platelets from Y112/128F mice, although having mild defects in GPVI signaling, exhibit defective actin reorganization after GPVI or alpha IIb beta 3 engagement. The in vivo consequences of these signaling defects correlate with the mild protection from thrombosis seen in Y112/128F mice and the near complete protection observed in Y145F mice. Using genetic complementation, we further demonstrate that all three phosphorylatable tyrosines are required within the same SLP76 molecule to support platelet activation by GPVI.

Roles of the proline-rich domain in SLP-76 subcellular localization and T cell function.

The adaptor protein Src homology (SH)2 domain-containing and leukocyte-specific phosphoprotein of 76 kDa (SLP-76) is critical for signal transduction in multiple hematopoietic lineages. It links proximal and distal T cell receptor signaling events through its function as a molecular scaffold in the assembly of multimolecular signaling complexes. Here we studied the functional roles of sub-domains within the SLP-76 proline-rich region, specifically the Gads binding domain and the recently defined P1 domain. To gain a further understanding of the functions mediated by this region, we used three complementary approaches as follows: reconstitution of SLP-76-deficient cells with functional domain deletion mutants, blocking molecular associations through the expression of a dominant negative protein fragment, and directed localization of SLP-76 to assess the role of the domains in SLP-76 recruitment. We find the Gads binding domain and the P1 domain are both necessary for optimal SLP-76 function, and in the absence of these two regions, SLP-76 is functionally inert. Furthermore, we provide direct evidence that SLP-76 localization and, in turn, function are dependent upon association with Gads.