Structural basis for the interaction of the free SH2 domain EAT-2 with SLAM receptors in hematopoietic cells
2001; Springer Nature; Volume: 20; Issue: 21 Linguagem: Inglês
10.1093/emboj/20.21.5840
ISSN1460-2075
Autores Tópico(s)Autoimmune and Inflammatory Disorders Research
ResumoArticle1 November 2001free access Structural basis for the interaction of the free SH2 domain EAT-2 with SLAM receptors in hematopoietic cells Massimo Morra Corresponding Author Massimo Morra Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Jun Lu Jun Lu Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Search for more papers by this author Florence Poy Florence Poy Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Search for more papers by this author Margarita Martin Margarita Martin Department of Cellular Biology and Pathology, Faculty of Medicine, University of Barcelona, Spain Search for more papers by this author Joan Sayos Joan Sayos Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Silvia Calpe Silvia Calpe Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Charles Gullo Charles Gullo Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Duncan Howie Duncan Howie Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Svend Rietdijk Svend Rietdijk Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Andrew Thompson Andrew Thompson Molecular Biology Institute, University of California, Los Angeles, CA, 90095 USA Search for more papers by this author Anthony J. Coyle Anthony J. Coyle Millennium Pharmaceuticals Inc., Inflammation Division, Cambridge, MA, 02139 USA Search for more papers by this author Christopher Denny Christopher Denny Molecular Biology Institute, University of California, Los Angeles, CA, 90095 USA Search for more papers by this author Michael B. Yaffe Michael B. Yaffe Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 USA Search for more papers by this author Pablo Engel Pablo Engel Department of Cellular Biology and Pathology, Faculty of Medicine, University of Barcelona, Spain Search for more papers by this author Michael J. Eck Michael J. Eck Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Search for more papers by this author Cox Terhorst Corresponding Author Cox Terhorst Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Massimo Morra Corresponding Author Massimo Morra Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Jun Lu Jun Lu Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Search for more papers by this author Florence Poy Florence Poy Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Search for more papers by this author Margarita Martin Margarita Martin Department of Cellular Biology and Pathology, Faculty of Medicine, University of Barcelona, Spain Search for more papers by this author Joan Sayos Joan Sayos Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Silvia Calpe Silvia Calpe Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Charles Gullo Charles Gullo Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Duncan Howie Duncan Howie Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Svend Rietdijk Svend Rietdijk Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Andrew Thompson Andrew Thompson Molecular Biology Institute, University of California, Los Angeles, CA, 90095 USA Search for more papers by this author Anthony J. Coyle Anthony J. Coyle Millennium Pharmaceuticals Inc., Inflammation Division, Cambridge, MA, 02139 USA Search for more papers by this author Christopher Denny Christopher Denny Molecular Biology Institute, University of California, Los Angeles, CA, 90095 USA Search for more papers by this author Michael B. Yaffe Michael B. Yaffe Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 USA Search for more papers by this author Pablo Engel Pablo Engel Department of Cellular Biology and Pathology, Faculty of Medicine, University of Barcelona, Spain Search for more papers by this author Michael J. Eck Michael J. Eck Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA Search for more papers by this author Cox Terhorst Corresponding Author Cox Terhorst Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Author Information Massimo Morra 1, Jun Lu2, Florence Poy2, Margarita Martin3, Joan Sayos1, Silvia Calpe1, Charles Gullo1, Duncan Howie1, Svend Rietdijk1, Andrew Thompson4, Anthony J. Coyle5, Christopher Denny4, Michael B. Yaffe6, Pablo Engel3, Michael J. Eck2 and Cox Terhorst 1 1Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 02115 USA 2Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02115 USA 3Department of Cellular Biology and Pathology, Faculty of Medicine, University of Barcelona, Spain 4Molecular Biology Institute, University of California, Los Angeles, CA, 90095 USA 5Millennium Pharmaceuticals Inc., Inflammation Division, Cambridge, MA, 02139 USA 6Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, 02139 USA *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2001)20:5840-5852https://doi.org/10.1093/emboj/20.21.5840 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The T and natural killer (NK) cell-specific gene SAP (SH2D1A) encodes a 'free SH2 domain' that binds a specific tyrosine motif in the cytoplasmic tail of SLAM (CD150) and related cell surface proteins. Mutations in SH2D1A cause the X-linked lymphoproliferative disease, a primary immunodeficiency. Here we report that a second gene encoding a free SH2 domain, EAT-2, is expressed in macrophages and B lympho cytes. The EAT-2 structure in complex with a phosphotyrosine peptide containing a sequence motif with Tyr281 of the cytoplasmic tail of CD150 is very similar to the structure of SH2D1A complexed with the same peptide. This explains the high affinity of EAT-2 for the pTyr motif in the cytoplasmic tail of CD150 but, unlike SH2D1A, EAT-2 does not bind to non-phosphorylated CD150. EAT-2 binds to the phosphorylated receptors CD84, CD150, CD229 and CD244, and acts as a natural inhibitor, which interferes with the recruitment of the tyrosine phosphatase SHP-2. We conclude that EAT-2 plays a role in controlling signal transduction through at least four receptors expressed on the surface of professional antigen-presenting cells. Introduction The SH2D1A (or SAP) gene encodes a 15 kDa protein whose absence or mutation causes X-lymphoproliferative (XLP) primary immunodeficiency (Coffey et al., 1998; Nichols et al., 1998; Sayos et al., 1998), a disease characterized by an extreme sensitivity to infection with Epstein–Barr virus (EBV) (Purtilo et al., 1975; Hamilton et al., 1980; Seemayer et al., 1995; Sullivan, 1999; Howie et al., 2000; Morra et al., 2001a). Both T and natural killer (NK) cell dysfunctions have been observed in XLP patients (Sullivan et al., 1980; Lanier, 1998; Benoit et al., 2000; Parolini et al., 2000). Uniquely, the SH2D1A protein comprises only a single SH2 domain with a 26 C-terminal amino acid tail (Coffey et al., 1998; Nichols et al., 1998; Sayos et al., 1998). SH2D1A, which is expressed in T and NK cells (Nagy et al., 2000), binds to a motif [TIpYxx(V/I)] in the cytoplasmic tail of SLAM (CD150) (Sayos et al., 1998), 2B4 (CD244) (Lanier, 1998; Tangye et al., 1999; Parolini et al., 2000; Sayos et al., 2000), Ly-9 (CD229) and CD84 (Sayos et al., 2001) via its SH2 domain. Classically, SH2 domain binding depends upon phosphorylation of the tyrosine in the ligand and requires additional contacts C-terminal to the pTyr, usually at the +3 position. Characteristically, SH2D1A uses a 'three-pronged' modality of binding to the Tyr281 motif of CD150 (Sayos et al., 1998; Li et al., 1999; Poy et al., 1999), where residues N-terminal to the phosphotyrosine, Ile (−1) and Thr (−2), interact in a specific manner with the β-pleated sheet βD and with the tyrosine pocket of SH2D1A, respectively (see Figure 3A and B for SH2 domain nomenclature). SH2D1A can bind to the unphosphorylated cytoplasmic tail of CD150 (Sayos et al., 1998), and it blocks recruitment of the SHP-2 phosphatase to the tail of phosphorylated CD150 (Sayos et al., 1998), CD244 (Tangye et al., 1999; Sayos et al., 2000), CD84 and CD229 (Sayos et al., 2001). Recently, SH2D1A has been shown to bind to a 62 kDa phosphoprotein adaptor (p62dok) (Sylla et al., 2000). Figure 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). Download figure Download PowerPoint Figure 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). Download figure Download PowerPoint Figure 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 2Fo − Fc annealed omit map was calculated at 2.15 Å resolution and contoured at 1.2σ. Download figure Download PowerPoint CD150 is found not only on cells of T and NK lineage, but also on resting B cells, dendritic cells and macrophages (Sidorenko and Clark et al., 1993; Cocks et al., 1995; Wang et al., 2001). Because CD150 is a self-ligand, it is involved bi-directionally in antigen-presenting cell (APC)–T cell interactions (Punnonen et al., 1997; Sayos et al., 1998; Mavaddat et al., 2000). CD244 is expressed on APCs such as macrophages or monocytes, and on NK cells and a subset of CD8+ T cells (Nakajima and Colonna, 2000). CD229 and CD84 are expressed on myeloid cells, macrophages, B cells and cells of the T lineage (Sandrin et al., 1992; De la Fuente et al., 1997). Thus, all four receptors, which interact with SH2D1A, are expressed on the surface of professional APCs, where SH2D1A is absent. Because of the importance of SH2D1A in T and NK cell signaling, we reasoned that APCs must contain a regulator with properties similar to SH2D1A. We focused on a previously reported cDNA, termed EAT-2, which encodes a 132 amino acid single SH2 domain protein with unknown functions (Thompson et al., 1996). Here we show that EAT-2 is the SH2D1A equivalent in B lymphocytes and macrophages as it binds to CD84, CD150, CD244 and CD229 through its SH2 domain. The structure of a complex of EAT-2 with a phosphotyrosine peptide (pTyr281) derived from the CD150 cytoplasmic tail is very similar to that of SH2D1A with the same peptide. Thus, EAT-2 and SH2D1A are free SH2 domains that define a new class of proteins that play a role either in T cells or in APCs. Results The human EAT-2 gene A cDNA library made with RNA from human splenocytes was used to clone a cDNA encoding human EAT-2. The human EAT-2 cDNA has a coding region of 399 nucleotides (DDBJ/EMBL/GenBank accession No. AF256653) (Figure 1A). Its nucleotide sequence is 83% identical to the mouse cDNA (Figure 1A). The complete genomic organization of human EAT-2 was obtained using BLAST analysis (Altschul et al., 1990) of the High Throughput Genomic (HTG) database and the human EAT-2 cDNA sequence. Using seven different GenBank sequences of pBACs containing the EAT-2 exons, but in particular pBAC AL359699 and AC068536, the EAT-2 gene was shown to have an exon–intron organization similar to that of mouse and human SH2D1A (Coffey et al., 1998; Wu et al., 2000) (Figure 1B). Like the SH2D1A gene, EAT-2 consists of four exons spanning ∼14 kb. The coding region of the first and second exons is highly conserved between human and mouse (87 and 90% identity, respectively), while exon 3 is slightly less conserved (81%) (Figure 1A). Two additional sequences highly homologous to the first and third exon are located in the same chromosomal area (exon IA, which is located ∼30 kbp upstream of the first exon, and exon IIIA) (Figure 1B). Interestingly, the sequence of the coding region of the fourth exon is extremely conserved between species (Figure 1A). Exon 1, exon 2 and approximately the first two-thirds of exon 3 code for the EAT-2 SH2 domain (Figure 3A), while the terminal portion of the third exon and exon 4 account for the EAT-2 tail (Figure 3A). A human expressed sequence tag (EST) sequence that is 99% identical to human EAT-2 and derived from a lung cDNA library has been found recently (#BG569733). The nucleotide region 5′ to the ATG contains a canonical TATA box at 335 nucleotides upstream of the ATG. The length of the 3′-untranslated region (UTR) was determined by comparing the genomic DNA sequence downstream of the stop codon with three ESTs (#BE896279, #BF375549 and #AW613569). We therefore predict that the major human EAT-2 mRNA will be ∼2400 nucleotides, similar to a major 2.5 kb cDNA encoding murine EAT-2 (Thompson et al., 1996). The 3′-UTR of EAT-2 contains three ARE recognition sites, which indicates that EAT-2 mRNA levels may be controlled post-transcriptionally by triggering cell surface receptors, as is the case with SH2D1A (Wu et al., 2000). EAT-2 is expressed in B lymphocytes and macrophages SH2D1A is expressed mostly in T lymphocytes and NK cells (Nagy et al., 2000). To establish whether EAT-2 was expressed in cells of the immune system that are SH2D1A-negative, several populations of immunocytes were tested. EAT-2 is highly expressed in organs such as spleen, lymph nodes, lung and small intestine (Thompson et al., 1996; M.Morra, data not shown). To enrich for B cells and other APCs, splenocytes from an immunodeficient mouse, tgϵ26 (Wang et al., 1994), which lacks NK and T cells, were used. EAT-2 but not SH2D1A is expressed in tgϵ26 spleen and lymph nodes (Figure 2A). No murine EAT-2 transcript was detected in the thymus (Figure 2B). The murine B-cell leukemia lines K46 and M12 tested positive for the EAT-2 transcript, while the T-leukemia line EL-4 is negative (Figure 2B). Human EAT-2 nucleotide sequences were amplified using RNA from five out of the six B-lymphoma or lymphoblastoid cell lines tested (Cess, Daudi, Namalwa, Raji and RPMI1888) (M.Morra, data not shown). Expression of mouse EAT-2 in highly purified cell-sorted B lymphocytes (B220+ cells) and macrophages (CD11b+ cells) was confirmed by TaqMan analysis (Figure 2C). Peritoneal exudate macrophages isolated from RAG-2 null mice that lack T and B cells also tested positive for EAT-2 expression (M.Morra, data not shown). Taken together, these results show that within the hematopoietic cell lineage, EAT-2 is expressed in APCs such as B lymphocytes and macrophages. The structure of EAT-2 is similar to that of SH2D1A The amino acid sequences of the SH2 domains of human and mouse EAT-2 share sequence homologies with all other SH2 domains (Figure 3A, lower panel). The EAT-2 SH2 domain is homologous to the SH2 domain of Grb2 (35%), Csk (30%), Lck (30%) and Syk (30%). However, the highest homology of the mouse and human EAT-2 SH2 domain is with the SH2 domain of mouse and human SH2D1A (47 and 40%, respectively) (Figure 3A, upper panel). To enable a comparison of the structures of EAT-2 and SH2D1A, an attempt was made to grow crystals of mouse EAT-2 with a short (14mer) peptide segment of the cytoplasmic tail of CD150 including Tyr281. Although both the unphosphorylated (Tyr281) and the phosphorylated (pTyr281) peptide had co-crystallized with human SH2D1A (Li et al., 1999; Poy et al., 1999), only a crystal of the mouse EAT-2 SH2 domain complexed with the pTyr281 peptide was obtained. We expressed and purified a fragment of EAT-2 (residues 1–103) lacking the C-terminal tail and crystallized it in complex with a 14 residue CD150 phosphopeptide (residues 273–286, with the Tyr281 phosphorylated). The structure was solved by molecular replacement with the SH2D1A SH2 domain and refined to an R-value of 21.6% at 2.15 Å resolution (see Materials and methods) (Figure 3F). The final model included all 103 residues of EAT-2, residues 277–286 of CD150 and 86 water molecules. EAT-2 has a characteristic SH2 fold (Kuriyan and Cowburn, 1997), which includes a central β-sheet with α-helices packed against either side (Figure 3B). Canonical SH2 domains bind phosphopeptides in a 'two-pronged' fashion: the phosphotyrosine residue binds in a pocket on one side of the central sheet, and the three to five residues C-terminal to it bind in a pocket on the opposite side (Kuriyan and Cowburn, 1997). The CD150 phosphotyrosine peptide retains these general binding features in the complex with EAT-2 (Figure 3B). Further, EAT-2, like SH2D1A (Poy et al., 1999), forms additional interactions with the three amino acids N-terminal to CD150 Tyr281 (Ile −1, Thr −2 and Leu −3; Figure 3B–D). The CD150 peptide makes a parallel β-sheet interaction with the βD strand of the domain, and the side chains of residues Leu −3 and Ile −1 of the CD150 peptide pack with Leu49 and Tyr51 in strand βD of EAT-2 (Figure 3B and D). Thr −2 (Thr279 of CD150) hydrogen-bonds with Glu16 of EAT-2, and with a buried water molecule that is also coordinated by Arg31 (Figure 3D). Corresponding interactions are also observed in SH2D1A–CD150 complexes (Poy et al., 1999). The phosphorylated Tyr281 is coordinated in a manner similar to that observed in other SH2 domain complexes; the conserved Arg31 forms the expected bi-dentate hydrogen bonds with phosphate oxygens (Figure 3D). The five residues following Tyr281 are coordinated by interactions with the EF and BG loops, and the βD strand of the central sheet (Figure 3D). These C-terminal interactions are similar to those seen in other SH2 domain–phosphopeptide complexes. Val284 (pY +3) inserts into a hydrophobic cleft in a manner analogous to that of an isoleucine at this position in Src family SH2 complexes (Eck et al., 1993; Waksman et al., 1993) (Figure 3D). We conclude that the interactions between the CD150 peptide and EAT-2 are very similar to those of SH2D1A with the same peptide and utilize the same unusual three-pronged binding mode (Li et al., 1999; Poy et al., 1999). Although similar to other SH2 domains in their phosphotyrosine and C-terminal (+3) recognition, they are different in their ability to recognize specifically residues N-terminal to the phosphotyrosine. Thus, SH2D1A and EAT-2 represent a distinct class of SH2 domains. These results validate previous data obtained by screening of a library of random peptides with the EAT-2 SH2 domain (Poy et al., 1999). In spite of only 40% identical residues between mouse EAT-2 and human SH2D1A, their structures are very similar. This point is illustrated clearly by superimposing the EAT-2 SH2 domain onto the SH2D1A SH2 domain. The two structures superimpose with an r.m.s. deviation of 0.69 Å for Cα atoms (Figure 3E). Moreover, amino acid residues that are substituted in the SH2D1A protein of XLP patients, and which severely affect the functions of SH2D1A (Morra et al., 2001b), are conserved in mouse and human EAT-2 as well as in mouse and human SH2D1A (Figure 3A, upper panel). These residues are located mostly in positions that are key for the interaction between the EAT-2 SH2 domain and the CD150 phosphopeptide (indicated by the symbol '+' in Figure 3A, upper panel), or in positions critical for the stability of the SH2D1A protein (Morra et al., 2001b). EAT-2 binds to the phosphorylated cytoplasmic tail of CD84, CD150, CD244 and CD229 The structural analysis revealed that EAT-2 binds to a phosphorylated peptide derived from the cytoplasmic tail of CD150 in a three-pronged fashion. To measure the affinity of binding between EAT-2 and the CD150 Tyr281 peptide, a fluorescence polarization assay was used. An 11mer peptide encompassing the CD150 cytoplasmic region 276–286 was labeled with fluorescein isothiocyanate (FITC) in its α-amino group. The affinity of binding of this peptide, either with or without phosphorylation of Tyr281, was determined in a polarimeter using varying concentrations of GST–EAT-2 and GST–SH2D1A. GST–EAT-2 (Figure 4, upper panel) binds the phosphorylated pTyr281 peptide with an affinity comparable with GST–SH2D1A (Figure 4, middle and lower panel) (Kd = 131 nM for EAT-2/pTyr281; Kd = 127 nM for SH2D1A/pTyr281). However, in contrast to SH2D1A, EAT-2 fails to bind the Tyr281 peptide in the absence of phosphorylation (Figure 4, upper and lower panel). Taken together, these in vitro binding studies distinguish between the SH2 domains of EAT-2 and SH2D1A in that only SH2D1A can bind to the peptide in the absence of phosphorylation. Figure 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 fyn420,531Y–F. y-axis = β-galactosidase (U/ml). Download figure Download PowerPoint Next, we determined whether EAT-2 binds to the complete cytoplasmic tails of CD150 and of the related receptors CD84, CD229 and CD244, as does SH2D1A (Sayos et al., 2000, 2001). An altered yeast two-hybrid system was used. We chose the yeast two-hybrid system because there is no tyrosine phosphorylation in yeast cells. Moreover, we previously had developed an altered yeast two-hybrid system in which phosphorylation of tyrosine could be established without interfering with the read-out of the assay (Sayos et al., 2001). Briefly, yeast cells were co-transformed with two vectors: (i) the first is a bi-cistronic vector, containing coding sequences for either the EAT-2 sequence alone (two-hybrid system) or EAT-2 and mutant tyrosine kinase fyn420,531Y–F (modified two-hybrid system); and (ii) the second vector contains the coding sequences for the cytoplasmic tail of human CD84, CD150, CD229 or mouse CD244. In line with the fluorescence polarization results, EAT-2 interacted with CD150 only in the presence of fyn, while no reporter activity was detected without fyn (Figure 4B). SH2D1A was used as a control for its ability to interact with CD150 in the absence of fyn (Sayos et al., 1998) (Figure 4B). Next, we expanded the analysis to the CD150-related receptors CD84, CD229 and CD244. As in the case of CD150, a reporter activity was evident only in the presence of fyn (Figure 5). Thus, this assay demonstrated the ability of EAT-2 to interact with CD150, CD244, CD229 or CD84. This interaction requires the presence of fyn420,531Y–F (Figure 5) and is dependent upon tyrosine phosphorylation of the receptors. Figure 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 fyn420,531Y–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 fyn420,531Y–F are indicated by
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