SAP (or SH2D1A), an adaptor-like molecule expressed in immune cells, is composed almost exclusively of a Src homology 2 (SH2) domain. In humans, SAP is mutated and either absent or non-functional in X-linked lymphoproliferative (XLP) syndrome, a disease characterized by an inappropriate response to Epstein-Barr virus (EBV) infection. Through its SH2 domain, SAP associates with tyrosines in the cytoplasmic domain of the SLAM family of immune cell receptors, and is absolutely required for the function of these receptors. This property results from the ability of SAP to promote the selective recruitment and activation of FynT, a cytoplasmic Src-related protein tyrosine kinase (PTK). Here, we demonstrate that SAP operates in this pathway by binding to the SH3 domain of FynT, through a second region in the SAP SH2 domain distinct from the phosphotyrosine-binding motif. We demonstrate that this interaction is essential for SAP-mediated signalling in T cells, and for the capacity of SAP to modulate immune cell function. These observations characterize a biologically important signalling mechanism in which an adaptor molecule composed only of an SH2 domain links a receptor devoid of intrinsic catalytic activity to the kinase required for its function.

The adaptors SAP, EAT-2 and ERT are specific to cells of the immune system and belong to the SAP family. All three are expressed in natural killer (NK) cells. Here we examined the global function of the SAP family using mice lacking SAP, EAT-2 and ERT. These adaptors acted together in a mechanism that was essential for the elimination of hematopoietic but not nonhematopoietic cells by NK cells. This function was mediated by many receptors of the SLAM family on NK cells that were engaged by ligands found solely on hematopoietic cells. In the absence of SAP-related adaptors, SLAM receptors lost their activating function and became inhibitory receptors that repressed other activating receptors, such as NKG2D. Hence, the SAP family is essential for the elimination of unwanted hematopoietic cells by NK cells.

EAT-2 is an adaptor expressed in innate immune cells, including natural killer (NK) cells. It is closely related to the adaptor SAP, which regulates signaling lymphocyte activation molecule (SLAM)-related receptors by recruiting the kinase FynT to the receptors. Here we have studied the function of EAT-2 in NK cells by creating mice lacking or overexpressing EAT-2. Like SAP, EAT-2 was associated with the SLAM-related receptor 2B4 in NK cells. However, unlike SAP, EAT-2 was an inhibitor of NK cell function. EAT-2 repressed natural cytotoxicity and interferon-gamma secretion by a mechanism involving tyrosine phosphorylation of its C terminus. We have demonstrated a similar function for the adaptor ERT, a newly identified SAP family member expressed in mouse NK cells. These data identify a previously unknown mechanism of NK cell inhibition. Moreover, they indicate that EAT-2 and SAP have distinct and at times opposing functions in natural immunity.

2B4 is a SLAM-related receptor expressed on natural killer (NK) cells and cytotoxic T cells. It can regulate killing and gamma interferon secretion by NK cells, as well as T-cell-mediated cytotoxicity.There are conflicting data regarding the mechanism of action of 2B4. In these studies, we attempted to understand better the nature and basis of 2B4 signaling. Our studies showed that engagement of 2B4 on NK cells triggered a tyrosine phosphorylation signal implicating 2B4, Vav-1, and, to a lesser extent, SHIP-1 and c-Cbl. Structure-function analyses demonstrated that this response was defined by a series of tyrosine-based motifs in the cytoplasmic region of 2B4 and was not influenced by the extracellular or transmembrane segment of 2B4. In addition, the 2B4-induced signal was absolutely dependent on coexpression of SAP, a Src homology 2 (SH2) domaincontaining adaptor associating with SLAM-related receptors and mutated in X-linked lymphoproliferative disease. It was also observed that 2B4 was detectably associated with the Src-related protein tyrosine kinase FynT in an immortalized NK cell line. Mutation of arginine 78 of SAP, a residue critical for binding of SAP to FynT, eliminated 2B4-mediated protein tyrosine phosphorylation, implying that SAP promotes 2B4 signaling most probably by recruiting FynT. Finally, despite the similarities in the signaling modalities of 2B4 and its relative SLAM, the natures of the tyrosine phosphorylation signals induced by these two receptors were found to be different. These differences were not caused by variations in the extent of binding to SAP but rather were dictated by the tyrosine-based sequences in the cytoplasmic domain of the receptors. Taken together, these data lead to a better understanding of 2B4 signaling. Furthermore, they provide firm evidence that the signals transduced by the various SLAM-related receptors are unique and that the specificity of these signals is defined by the distinctive arrays of intracytoplasmic tyrosines in the receptors.2B4, also named CD244, is a member of the SLAM family of immune cell-specific receptors that also includes SLAM, CD84, Ly-9, NTB-A/Ly-108, and CRACC (12,23,32,34). It is expressed in natural killer (NK) cells, cytotoxic CD8ϩ T cells, ␥␦ T cells, and mast cells. Like its relatives, 2B4 possesses immunoglobulin (Ig)-like domains in its extracellular region, a single transmembrane domain, and an intracellular segment bearing several tyrosine-based motifs. The ligand for 2B4 is CD48, a glycosylphosphatidylinositol-linked receptor expressed on diverse types of immune cells (5, 16). While an analysis of mice lacking 2B4 has yet to be reported, engagement of 2B4 by anti-2B4 antibodies or CD48 was shown to promote cytotoxicity and gamma interferon (IFN-␥) secretion by NK cells (24). Furthermore, it augments antigen-induced IFN-␥ secretion by activated CD8 ϩ T cells (14, 21). Like most other members of the SLAM family, 2B4 interacts through its cytoplasmic domain with SAP (or SH2D1A), a small intracellular Src homology 2 (SH2) domain-c...