The adaptor protein 3BP2 binds human CD244 and links this receptor to Vav signaling, ERK activation, and NK cell killing.

Adaptor proteins, molecules that mediate intermolecular interactions, are crucial for cellular activation. The adaptor 3BP2 has been shown to positively regulate NK cell-mediated cytotoxicity. In this study we present evidence for a physical interaction between 3BP2 and the CD244 receptor. CD244, a member of the CD150 family, is a cell surface protein expressed on NK, CD8+ T, and myeloid cells. CD244 interacts via its Src homology 2 domain with the X-linked lymphoproliferative disease gene product signaling lymphocytic activation molecule-associated protein (SAP)/SH2 domain protein 1A. 3BP2 interacts with human but not murine CD244. CD244-3BP2 interaction was direct and regulated by phosphorylation, as shown by a three-hybrid analysis in yeast and NK cells. Tyr337 on CD244, part of a consensus motif for SAP/SH2 domain protein 1A binding, was critical for the 3BP2 interaction. Although mutation of Tyr337 to phenylalanine abrogated human 3BP2 binding, we still observed SAP association, indicating that this motif is not essential for SAP recruitment. CD244 ligation induced 3BP2 phosphorylation and Vav-1 recruitment. Overexpression of 3BP2 led to an increase in the magnitude and duration of ERK activation, after CD244 triggering. This enhancement was concomitant with an increase in cytotoxicity due to CD244 ligation. However, no differences in IFN-gamma secretion were found when normal and 3BP2-transfected cells were compared. These results indicate that CD244-3BP2 association regulates cytolytic function but not IFN-gamma release, reinforcing the hypothesis that, in humans, CD244-mediated cytotoxicity and IFN-gamma release involve distinct NK pathways.

Identification of Grb2 as a novel binding partner of the signaling lymphocytic activation molecule-associated protein binding receptor CD229.

Ag recognition by the TCR determines the subsequent fate of the T cell and is regulated by the involvement of other cell surface molecules, termed coreceptors. CD229 is a lymphocyte cell surface molecule that belongs to the CD150 family of receptors. Upon tyrosine phosphorylation, CD229 recruits various signaling molecules to the membrane. One of these molecules is the signaling lymphocytic activation molecule-associated protein, of which a deficiency leads to the X-linked lymphoproliferative syndrome. We report that CD229 interacts in a phosphorylation-dependent manner with Grb2. We mapped this interaction showing that the Src homology 2 domain of Grb2 and the tyrosine residue Y606 in CD229 are required for CD229-Grb2 complex formation. The Grb2 motif in the cytoplasmic tail of CD229 is distinct and independent from the two tyrosines required for efficient signaling lymphocytic activation molecule-associated protein recruitment. CD229, but not other members of the CD150 family, directly bound Grb2. We also demonstrate that CD229 precipitates with Grb2 in T lymphocytes after pervanadate treatment, as well as CD229 or TCR ligation. Interestingly, the CD229 mutant lacking the Grb2 binding site is not internalized after CD229 engagement with specific Abs. Moreover, a dominant negative form of Grb2 (containing only Src homology 2 domain) impaired CD229 endocytosis. Unexpectedly, Erk phosphorylation was partially inhibited after activation of CD229 plus CD3. Consistent with this, CD229 ligation partially inhibited TCR signaling in peripheral blood cells and CD229-Jurkat cells transfected with the 3XNFAT-luciferase reporter construct. Altogether, the data suggest a model whereby CD229 ligation attenuates TCR signaling and Grb2 recruitment to CD229 controls its rate of internalization.

The cell surface expression of SAP-binding receptor CD229 is regulated via its interaction with clathrin-associated adaptor complex 2 (AP-2).

CD229 (Ly9) is a cell surface receptor selectively expressed on T and B lymphocytes, and it belongs to the CD150 receptor family. Like other receptors of this family, CD229 interacts with SAP/SH2D1a protein, mutation of which is responsible for the fatal X-linked lymphoproliferative disease. Receptors of the CD150 family function as costimulatory molecules, regulating cytokine production and cytotoxicity. Thus, their signaling and regulation in lymphocytes may be critical to an understanding of the pathogenesis of the X-linked lymphoproliferative disease. Here we show that CD229 interacts with the mu(2) chain of the AP-2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD229 was the only member of the CD150 family associated with AP-2. We also show that the mu(2) chain interacts with the Y(470)EKL motif of CD229. The integrity of this site was necessary for CD229 internalization, but it was not involved in SAP recruitment. Moreover, CD229 binds to the AP-2 complex in T and B cell lines, and it is internalized rapidly from the cell surface on T cells after antibody ligation. In contrast, cross-linking of CD229 receptors with intact antibody inhibited CD229 internalization on B cells. However, when F(ab')(2) antibodies were used, CD229 internalization was similar on T and B cells, suggesting that Fcgamma receptors control CD229 cell surface expression. Furthermore, CD229 was regulated by T cell receptor and B cell receptor signaling because coligation with antibodies against anti-CD3 and anti-IgM increased the rate of CD229 endocytosis. These data suggest that CD229 cell surface expression on lymphocytes surface is strongly and differentially regulated within the CD150 family members.