Analysis of SLP-76 function in macrophages, thymocytes, and mature T cells

The hematopoietic-specific adapter protein, SH2-domain containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), contains protein-protein interacting domains that mediate associations with effectors important for T cell receptor (TCR) signal transduction. SLP-76 is critical for both T cell development and mature TCR signaling and is thought to serve as a molecular scaffold, recruiting specific effector molecules in a spatial/temporal manner following TCR engagement. The structural requirements of SLP-76 for mediating thymopoiesis and mature T cell function remain largely unknown. Additionally, SLP-76 plays critical functions in transducing certain protein tyrosine kinase-dependent pathways in other hematopoietic cell types. Although not required for macrophage development, the requirement for SLP-76 in macrophage FcγR function has not been investigated. To address the former question, transgenic mice were generated to examine the functional requirements for specific protein interacting domains of SLP-76 in thymocytes and peripheral T cells in vivo. Using the CD2 promoter, wild-type and mutant SLP-76 transgenes were expressed exclusively within the T lineage compartment. Whereas the wildtype SLP-76 transgene rescues T cell development and function in the absence of endogenous protein, a mutant of SLP-76 that cannot be tyrosine phosphorylated (Y3F) fails to support normal thymocyte development. A second mutant of SLP-76, which carries a deletion of amino acid residues 224–244 (Δ224–244) within the proline-rich domain, reveals a required role for this region in providing optimal SLP-76 function in both thymopoiesis and T cell function. In contrast, mice expressing a mutation in the SH2 domain of SLP-76 exhibit significantly less impaired thymopoiesis, but show significant defects in mature T cell function. These data demonstrate a differential requirement for SLP-76 domains in thymocytes and T cells. To assess the role of SLP-76 in macrophage FcγR-mediated functions and signaling events, bone marrow-derived macrophages (BMMs) from SLP-76−/− mice were assessed for FcγR-induced signaling events, phagocytosis, and reactive-oxygen intermediate production. This study demonstrates that although SLP-76 becomes rapidly tyrosine phosphorylated in response to FcγR stimulation of wildtype BMMs, FcγR-dependent functions and signaling events measured in these studies can occur independently of SLP-76. Collectively, these findings suggest that SLP-76 and its specific domains may play unique functions in distinct cell types