Fig. 1. The human EAT-2 gene. (A) Alignment of the human and mouse EAT-2 nucleotide sequences. The coding region sequences of the human (hEAT-2) and mouse (mEAT-2) EAT-2 cDNAs are compared. Exon boundaries are indicated (bold font, identity of nucleotides; regular font, difference of nucleotides). (B) Genomic organization of the human EAT-2 gene. The human EAT-2 gene consists of four exons that present an overall organization similar to that of the SH2D1A gene. The putative exon IIIA represents part of exon III (see text).

Fig. 2. EAT-2 is expressed in B lymphocytes and macrophages. RT–PCR and TaqMan analysis of murine EAT-2 and SH2D1A expression. (A) EAT-2 and SH2D1A expression in spleen and lymph nodes (LFN) of wild-type mice and T and NK cell-deficient tgε26 mice (Wang et al., 1994). (B) Expression of the mouse EAT-2 transcript in the murine B leukemia M12 and K46, but not in the T-cell line EL-4. (C) Purified B lymphocytes (B220⁺ cells) and macrophages (CD11b⁺ cells) from wild-type mice are positive for EAT-2 transcripts by TaqMan analysis (see Materials and methods).

Fig. 3. Structure of mouse EAT-2–CD150 phosphopeptide complex. (A) Structure-based sequence comparison of EAT-2 with other SH2 domains. Upper panel: the human and mouse EAT-2 and SH2D1A protein sequences are compared. Yellow areas, identical residues; green areas, blocks of similarity; blue areas, conserved positions. Exon boundaries are indicated (Ex, exon). Elements of secondary structure are indicated at the top and labeled using the standard SH2 domain nomeclature (Eck et al., 1993). Key residues for the peptide–SH2 domain interactions are indicated by the symbol ‘+’. Amino acid substitutions found in XLP patients are indicated at the bottom. An arrow indicates Cys15 of EAT-2 and Gly16 of SH2D1A. Lower panel: the mouse EAT-2 SH2 domain is compared with the SH2 domain of the human inositol polyphosphate-5-phosphatase (h SHIP), viral Abelson leukemia oncogene (v abl), Rous sarcoma virus oncogene (v src), human tyrosine kinase lck (h lck) and human tyrosine phosphatase SHP-2 (h SHP-2). Blocks of color indicate similarity or identity as indicated in the above panel. (B) Ribbon diagram showing the EAT-2 SH2 domain in complex with the CD150 phosphopeptide. The bound phosphopeptide is shown in a stick representation (yellow). Selected EAT-2 residues that form the binding site are shown in blue. The N-terminal residues of the peptide make a parallel β-sheet interaction with strand βD; the side chains of these residues make hydrophobic contacts with Leu49 and Tyr51 in strand βD (see text, and D). Interestingly, R12 (at position αA2), which is conserved in most SH2 domains and generally contributes to phosphotyrosine coordination, does not participate in phosphate binding in the EAT-2 complex. Instead, Arg54 (βD6) hydrogen-bonds with the phosphate group. Similar coordination was described for the SH2D1A SH2 domain–CD150 phosphotyrosine peptide complex (Poy et al., 1999). Interactions C-terminal to the phosphotyrosine are dominated by Val +3pY, which binds in a hydrophobic cleft. (C) Surface representation of the EAT-2 SH2 domain with the bound CD150 pTyr281 peptide. Hydrophobic residues at the –1 and –3 positions of the peptide (in a stick representation) intercalate with hydrophobic and aromatic residues on the surface of the SH2 domain. Thr2 (Thr 279 of SLAM) hydrogen-bonds with Glu16. C-terminal to the phosphotyrosine, Val +3 is buried in a mostly hydrophobic groove. Key residues for the SH2 domain–peptide interaction are represented in light green. (D) Stereo view showing the details of CD150 coordination. Residues 278–286 of the CD150 phosphopeptide are shown in green; EAT-2 residues surrounding the bound peptide are colored yellow. White lines indicate hydrogen bond interactions; red spheres represent ordered water molecules that bridge between the peptide and the SH2 domain. EAT-2 residues are labeled in white; N and C indicate the respective termini of the CD150 peptide. Green arrows indicate the phosphotyrosine and ‘+3’ binding pockets. Note the β-sheet hydrogen bonding pattern between the main chain of residues 49–53 of EAT-2 and the N-terminal residues of the CD150 phosphopeptide. The peptide essentially forms an additional strand in the central β-sheet of EAT-2. (E) Superimposition of the mouse EAT-2 and human SH2D1A structures bound to the CD150 pTyr281 peptide. Mouse EAT-2 (gray) and human SH2D1A (blue) α-carbon traces are superimposed. Note that the peptides are bound in essentially identical conformations. (F) Stereo view of the electron density map for the CD150 phosphopeptide bound to the EAT-2 SH2 domain. The 2F_o – F_c annealed omit map was calculated at 2.15 Å resolution and contoured at 1.2σ.

Fig. 4. EAT-2 binds exclusively to a phosphorylated peptide (pY281) derived from the cytoplasmic tail of CD150. (A) Fluorescence polarization analysis of the EAT-2 binding to a phosphorylated pY281 peptide. Different concentrations of GST–mouse EAT-2 (or GST–human SH2D1A) and an 11mer synthetic peptide identical to amino acid residues 276–287 of human CD150 (Sayos et al., 1998), tyrosine phosphorylated or not, were used. Top panel: binding of GST–mouse EAT-2 to the pY281 (filled triangles and continuous line) or the Y281 peptide (open squares and dashed line). Bottom panel: binding of GST–human SH2D1A to the pY281 (filled triangles and continuous line) or the Y281 peptide (open squares and dashed line). x-axis: protein concentration (nM); y-axis: polarization units (mP). The table summarizes the apparent dissociation constant (kD). (B) Hybrid system analysis of the interaction between EAT-2 and the cytoplasmic tail of CD150 in the presence or absence of fyn. Dashed bars indicate the interaction between the EAT-2 (or SH2D1A) full-length protein fused to a GAL4 DNA-binding domain and the GAL4 DNA activation domain fused to the cytoplasmic tail of the CD150 receptor. An empty pGAD424 vector was used as a control (solid bars). The test was conducted in either the presence or absence of fyn_420,531Y–F. y-axis = β-galactosidase (U/ml).

Fig. 5. EAT-2 binds only to the phosphorylated cytoplasmic tail of CD150, CD84, CD229 and CD244 in a modified yeast hybrid system. (A) Interactions between EAT-2 and CD150, CD244, CD229 or CD84 require the presence of the tyrosine kinase fyn. Yeast cells (strain Y187) were co-transformed with full-length mouse EAT-2 and mutant fyn_420,531Y–F in pBRIDGE, and with vector pGAD424 containing the cytoplasmic tail of CD150, CD244, CD229 or CD84. An empty pGAD424 vector was used as a control. Protein interactions were detected by measuring activation of β-galactosidase (U/ml) in the yeast lysate. Interactions in the presence of the protein tyrosine kinase fyn_420,531Y–F are indicated by dashed bars. In control experiments with the same transformed yeast cells, fyn_420,531Y–F expression was repressed specifically by adding l-methionine to the culture medium (solid bars). (B) Schematic representation of the putative EAT-2-binding motifs in the cytoplasmic tail of the CD150 family members. The motifs in the cytoplasmic tail of CD150 and CD229 that have been shown to bind SH2D1A by mutational analyses are indicated by an asterisk (Sayos et al., 2001; Howie et al., 2001).

Fig. 6. In vivo binding of EAT-2 to CD150 family members. Interactions were examined after co-transfection of combinations of mouse EAT-2 (in pCMV-FLAG) with CD150, CD244, CD229 or CD84, and fyn into COS-7 cells (plasmid combinations are indicated above each set of panels). All cells were surface biotinylated prior to being subjected to detergent lysis. Immunoprecipitates (I.P.) made with an antibody againsT cD150-related receptors (panels on the left of each figure) are analyzed by western blotting. Anti-phosphotyrosine = W.B. α-PY; avidin = W.B. AV-HRP; EAT-2 = W.B. α-FLAG; SHP-2 = W.B. α-SHP-2. In the panels on the right of each figure, lysates of the same transfections are analyzed by western blotting. (A) EAT-2 and CD150. (B) EAT-2 and CD244. (C) EAT-2 and CD84. (D) EAT-2 and CD229.

Fig. 7. Analysis of EAT-2 binding to Y–F CD150 and CD229 mutant receptors. (A) EAT-2 binding to CD150 Y–F mutants in a direct interaction hybrid system. Hybrid system direct interaction analysis of the binding between EAT-2 in pBRIDGE-Fyn_420,531Y–F (dashed bars) and different cD150 Y–F mutations in pGAD424. As control, vector pBRIDGE was used (solid bars). In particular, mutants CD150 Y1F (Tyr281 to phenylalanine), Y3F (Tyr327 to phenylalanine) or Y13F (Tyr281 and Tyr327 to phenylalanine) were used in this study. The lower part of the figures depicts the mutations in the CD150 cytoplasmic tail. (B) EAT-2 binding to CD229 Y–F mutants in a direct interaction hybrid system. Hybrid system direct interaction analysis of the binding between EAT-2 in pBRIDGE-Fyn_420,531Y–F (dashed bars) and different cD229 Y–F mutations in pGAD424. As control, vector pBRIDGE was used (solid bars). In particular, mutants CD229 Y2F (Tyr558 to phenylalanine), Y3F (Tyr581 to phenylalanine) and Y23F (Tyr558 and Tyr581 to phenylalanine) were used in this study. The lower part of the figure depicts the mutations in the CD229 cytoplasmic tail. (C) EAT-2 binding to CD150 Y–F mutants in an in vivo system. Full-length wild-type CD150, CD150 Y1F (Tyr281 to phenylalanine), CD150 Y2F (Tyr307 to phenylalanine), CD150 Y3F (Tyr327 to phenylalanine) and CD150 Y123F (Tyr281, Tyr307 and Tyr327 to phenylalanine) were co-transfected with EAT-2 and fyn in a COS-7 cell system. Mutant cD150 cDNAs used in the transfections are indicated above each set of panels. Immunoprecipitates (I.P.) were made with an anti-CD150 antibody and analyzed by western blotting. Anti-phosphotyrosine = W.B. α-PY; avidin = W.B. AV-HRP; EAT-2 = W.B. α-FLAG. The bottom panel shows the lysates of the same cell extracts used for the precipitation.

Fig. 8. A possible model for the interactions between EAT-2, SH2D1A and the four cell surface receptors at the interface of B lymphocytes, macrophages (Mφ) and T cells. The receptor–ligand interactions of CD48/CD244 (Brown et al., 1998) and CD150/CD150 (Punnonen et al., 1997; Mavaddat et al., 2000) have been reported.