Molecular Dissection of 2B4 Signaling: Implications for Signal Transduction by SLAM-Related Receptors

Abstract

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...

SAP Facilitates Recruitment and Activation of LCK at NTB-A Receptors during Restimulation-Induced Cell Death

“…FYN is considered the key Src-family kinase required for SLAM-R signaling, based on its SAP-dependent association with several other SLAM-Rs (13)(14)(15)(16)(17)(18)(19)(20). Although LCK was previously implicated in phosphorylation of both SLAM and CD84 (17,24), no direct association between LCK and a SLAM-R had been detected to date.…”

“…Previous studies either relied on overexpression constructs for biochemical analysis of NTB-A signaling (19) or focused on thymocytes, NK cells, or NK cell lines (13)(14)(15)(16)(17)(18)(19)(20). In the latter case, it is possible that cell type-based differences in the expression of FYN, LCK, SAP, or the SLAM family receptor examined could account for differences in the associations detected.…”

“…Two Srcfamily kinases are primarily involved in initiating TCR signaling: LCK and FYN (12). FYN is the major Src-family kinase shown to mediate signal transduction of SLAM-Rs (including NTB-A) in thymocytes and NK cells (13)(14)(15)(16)(17)(18)(19)(20). To examine the role of both FYN and LCK in RICD, we first used highly selective inhibitors for FYN (SU6656) (21) or LCK [LCK inhibitor I (LCKi)] (22).…”

Modulation of 2B4 (CD244) activity and regulated SAP expression in human NK cells

“…SAP can function as a natural blocker of SH2 domain-mediated interactions and might therefore prevent the interactions between SHP-2 and possibly SHP-1 with 2B4 [3,7,14], explaining why 2B4 can mediate inhibitory signals in the absence of SAP. SAP is also involved in the positive signaling by 2B4 and other SRR by recruiting and activating the Src family kinase Fyn through an unusual SH2-SH3 domain interaction [15,16].…”

The X-linked lymphoproliferative disease gene product SAP associates with PAK-interacting exchange factor and participates in T cell activation

“…Recent genetic and biochemical evidence demonstrates that the SAP plays an essential role in coupling a group of immune receptors, including SLAM and 2B4, to mediate proper immune activation (11,(22)(23)(24)(25). Although a good deal of information has been accumulated regarding the SAP recruitment of Fyn to SLAM-family receptors and the importance of the SAP-Fyn interaction in immune cell functions (12,18,26,27), little is known about the mechanism by which SAP initiates various signaling processes.…”