CD150 Association with Either the SH2-Containing InositolPhosphatase or the SH2-Containing Protein TyrosinePhosphatase Is Regulated by the Adaptor Protein SH2D1A1

Larysa M. Shlapatska,* Svitlana V. Mikhalap,* Anna G. Berdova,* Oleksander M. Zelensky,*Theodore J. Yun,† Kim E. Nichols,‡ Edward A. Clark, 2† and Svetlana P. Sidorenko*

CD150 (SLAM/IPO-3) is a cell surface receptor that, like the B cell receptor, CD40, and CD95, can transmit positive or negativesignals. CD150 can associate with the SH2-containing inositol phosphatase (SHIP), the SH2-containing protein tyrosine phospha-tase (SHP-2), and the adaptor protein SH2 domain protein 1A (SH2D1A/DSHP/SAP, also called Duncan’s disease SH2-protein(DSHP) or SLAM-associated protein (SAP)). Mutations in SH2D1A are found in X-linked lymphoproliferative syndrome andnon-Hodgkin’s lymphomas. Here we report that SH2D1A is expressed in tonsillar B cells and in some B lymphoblastoid cell lines,where CD150 coprecipitates with SH2D1A and SHIP. However, in SH2D1A-negative B cell lines, including B cell lines fromX-linked lymphoproliferative syndrome patients, CD150 associates only with SHP-2. SH2D1A protein levels are up-regulated byCD40 cross-linking and down-regulated by B cell receptor ligation. Using GST-fusion proteins with single replacements of tyrosineat Y269F, Y281F, Y307F, or Y327F in the CD150 cytoplasmic tail, we found that the same phosphorylated Y281 and Y327 areessential for both SHP-2 and SHIP binding. The presence of SH2D1A facilitates binding of SHIP to CD150. Apparently, SH2D1Amay function as a regulator of alternative interactions of CD150 with SHP-2 or SHIP via a novel TxYxxV/I motif (immunoreceptortyrosine-based switch motif (ITSM)). Multiple sequence alignments revealed the presence of this TxYxxV/I motif not only in CD2subfamily members but also in the cytoplasmic domains of the members of the SHP-2 substrate 1, sialic acid-binding Ig-like lectin,carcinoembryonic Ag, and leukocyte-inhibitory receptor families. The Journal of Immunology,2001, 166: 5480–5487.

T he B cell receptor (BCR),3 CD40 or CD95/Fas each canplay a dual role in the regulation of the B cell fate. Theoutcome after ligation of any of these receptors depends

on the stage of B cell differentiation, the combination and sequenceof signals delivered via these receptors, and the involvement ofother molecules such as CD80, CD86, and IL-4R (1, 2). However,little is known about cell surface receptors that may modulate Bcell fate at later stages of differentiation.

A possible candidate for a regulator of mature B cells is CD150(signaling lymphocytic activation molecule/IPO-3) (3, 4). Ligationof CD150 on resting B lymphocytes with CD150 mAb induces arapid elevation of intracellular free calcium ([Ca21]i) and aug-

ments proliferation induced by CD40 mAb and IL-4 (3). On theother hand, CD150-induced signals can synergize with and aug-ment CD95-mediated apoptosis (5). Engaging CD150 with mAbpromotes IL-2- and CD28-independent but cyclosporin A-sensi-tive proliferation of T cells (6). Furthermore, ligation of CD150also induces IFN production by CD41 T cell clones and Ig pro-duction by activated B cells (4, 6, 7). Given that in Th1 cellsCD150 is expressed at 7- to 25-fold higher levels than in Th2 cells(8), CD150 may be involved in expanding Th0/Th1 immune re-sponses (9). How CD150 mediates these effects in lymphocytes isnot known.

CD150 in T cells associates with the small SH2-containingadaptor protein 1A (SH2D1A), also called Duncan’s disease SH2-protein (DSHP) or SLAM-associated protein (SAP) (10, 11). Mu-tations in theSH2D1Agene lead to X-linked lymphoproliferativesyndrome (XLP), an immunodeficiency associated with dysregu-lated proliferation of T and B lymphocytes in the setting of pri-mary EBV infection (10, 12–14). SH2D1A binds to a sequencesurrounding Y281 in the cytoplasmic tail of CD150 in a tyrosine-independent manner (15, 16). One possibility is that SH2D1Afunctions as a signaling inhibitor by blocking and/or regulatingbinding of signal transducing molecules to SH2 docking sites (10,13). Indeed SH2D1A may block recruitment of the protein tyrosinephosphatase SHP-2 to CD150 in T cells and the 2B4 receptor inNK cells (10, 11, 17). This block may lead to selectiveimpairmentof 2B4-mediated NK cell activation and possibly T cell function inXLP patients (10, 18, 19). However, defects in T and NK signalingmay not be completely responsible for such phenotypic manifesta-tions of XLP as dysgammoglobulinemia and B cell non-Hodgkinlymphomas (20, 21).

Recently, we found that in B cells CD150 can bind not onlySHP-2 but also SH2-containing inositol phosphatase (SHIP) (5).

*Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NationalAcademy of Sciences of the Ukraine, Kiev, Ukraine;†Department of Microbiology,University of Washington, Seattle, WA 98195; and‡Pediatric Oncology, Children’sHospital of Philadelphia, Philadelphia, PA 19104

Received for publication June 2, 2000. Accepted for publication February 23, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Howard Hughes Medical Institute Grant 76195-548101, and INTAS Grant 96-1493 (to S.P.S.), U.S. Civilian Research and Devel-opment foundation Grants UN2-437 (to S.P.S.) and USB-383 (to L.M.S.), and Na-tional Institutes of Health Grant GM37905 (to E.A.C.).2 Address correspondence and reprint requests to Dr. Edward A. Clark, Department ofMicrobiology, Box 357242, University of Washington, Seattle, WA 98195. E-mailaddress: eclark@bart.rprc.washington.edu3 Abbreviations used in this paper: BCR, B cell antigen receptor; B-LCL, B lymphoblas-toid cell line; BL, Burkitt’s lymphoma cell line; [Ca21]i, intracellular free calcium;CD150ct, cytoplasmic tail of CD150; PY, phosphotyrosine; SH2D1A, SH2 domain pro-tein 1A; SHIP, SH2-containing inositol phosphatase; SHP-2, SH2-containing protein ty-rosine phosphatase; XLP; X-linked lymphoproliferative syndrome; ITSM, immunorecep-tor tyrosine-based switch motif; SHPS, SHP-2 substrate 1; CEA, carcinoembryonic Ag;NP-40, Nonidet P-40; HMM, hidden Markov model; Siglec, sialic acid-binding Ig-likelectin; ITIM, immunoreceptor tyrosine-based inhibitory motif; SIT, SHP-2-interactingtransmembrane adaptor protein; LIR, leukocyte-inhibitory receptors.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

However, whether SH2D1A can compete with SHP-2 and/or SHIPin B cells was unclear. Here we report that SH2D1A is expressedin tonsillar B cells and in some B lymphoblastoid cell lines, whereit associates with CD150. In these cell lines, CD150 coprecipitateswith SHIP and SH2D1A, but in SH2D1A-negative cell linesCD150 associated with SHP-2. Using mutational analysis, wefound that both Y281 and Y327 in the CD150 cytoplasmic tail(CD150ct) are essential for binding of SHP-2 as well as SHIP.Apparently, SH2D1A regulates SHIP vs SHP-2 binding to aTxYxxV/I motif (immunoreceptor tyrosine-based switch motif(ITSM)) in CD150. Multiple sequence alignments revealed that theITSM motif is not only in cytoplasmic tails of CD2 subfamilymembers (CD150, CD84, Ly-9/CD220, and 2B4/CD244), but alsoin the SHPS (SHP-2 substrate 1), sialic acid-binding Ig-like lectin(Siglec), carcinoembryonic Ag (CEA), and leukocyte-inhibitoryreceptor (LIR) families, underscoring the functional importance ofthis motif.

Materials and MethodsAbs and reagents

Rabbit antisera against SHIP, SHP-2, and p38 mitogen-activated proteinkinase; were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).Glutathione-agarose was purchased from Sigma (St. Louis, MO, protein A-and protein G-Sepharose were from Pharmacia (Piscataway, NJ). Forcross-linking of surface receptors, we used the following mAbs: IPO-3anti-CD150 (IgG1); IPO-4 anti-CD95 (IgM) (22); and G28-5 anti-CD40(IgG1) (23). F(ab9)2 of goat anti-human IgM (Jackson ImmunoResearch,West Grove, PA) were used for IgM cross-linking on human B cell lines.To generate a polyclonal Ab recognizing SH2D1A (DSHP), one of us(K.E.N.) immunized rabbits with a 26-aa peptide conjugated to keyholelimpet hemocyanin (peptide sequence: EKKSSARSTQGTTGIREDPDVCLKAP). After three injections, serum was collected and the anti-SH2D1Atiter was determined using an ELISA with free peptide bound in the solidphase (Research Genetics, Huntsville, AL).

Plasmid constructs

The GST-fusion protein construct of the cytoplasmic tail of CD150 (GST-CD150ct) was prepared as described (5). Forward and reverse primers withthe appropriate restriction sites for in-frame cloning into the pGEX-2Tplasmid were used to amplify the cDNA fragments usingpfu polymerase(Stratagene, La Jolla, CA). Using PCR-based site-directed mutagenesis(24), we made constructs of GST-CD150ct fusion proteins with phenylal-anine (F) replacements at tyrosines Y269, Y281, Y307, and Y327. Plas-mids with the correct nucleotide sequences were transformed into the bac-terial strain XLI-BlueMRF9 (Stratagene) for fusion protein production.Plasmids containing GST-CD150ct were also transformed into theEsche-richia coli strain TKX1 (Stratagene) for production of tyrosine-phospho-rylated fusion proteins. Tyrosine phosphorylation of these fusion proteinsapparently was restricted only to the corresponding cytoplasmic tails, be-cause GST was not tyrosine phosphorylated when expressed alone in thesame bacteria. Expression and purification of GST fusion proteins wereperformed as described (25).

SH2D1A sequence analysis

DNA was extracted from EBV-immortalized cell lines according to stan-dard protocols. The SH2D1A coding sequence was PCR amplified usingExpand Taq polymerase (Boehringer Mannheim, Indianapolis, IN) andprimers flanking each of the four SH2D1A exons as described (13).

Cell lines and stimulation

The pre-B cell lines REH and Namalwa; Burkitt’s lymphoma cell linesRamos, BJAB, and Raji; the B lymphoma line B104; the B lymphoblastoidcell lines (B-LCL) CESS, MP-1, T5-1, 6.16, and RPMI-1788; and theJurkat T cell line were maintained as described (26). B-LCL from XLPpatients included: IARC 739 (interstitial deletion of SH2D1A), XLP-D(C3T mutation at the position 462), XLP-8005 (C3T mutation at theposition 471), XLP-8002 (no mutations in SH2D1A) (13). T cell-depletedtonsillar cells were prepared as described (3, 25).

Cell staining

For determination of cell surface phenotype and cytoplasmic expression ofSH2D1A, cells were surface stained with biotin-labeled anti-IgD, anti-

CD150, or anti-CD95, followed by streptavidin-PerCP (Becton Dickinson,Mountain View, CA) and PE-labeled anti-CD38 (PharMingen, San Diego,CA), anti-CD3, or anti-CD20 (Becton Dickinson). Cells were fixed in 1%paraformaldehyde for 20 min and permeabilized with 0.2% Tween 20 for15 min. Then rabbit anti-SH2D1A serum (affinity-purified using SH2D1Apeptide) was added followed by goat F(ab9)2 anti-rabbit IgG. Cells werewashed twice with PBS containing 2.5% FCS and 0.2% Tween 20 and thenanalyzed on a FACScan flow cytometer (Becton Dickinson).

Biochemical methods

Cell lysis, immunoprecipitation, SDS-PAGE, in vitro kinase assays, andsubcellular fractionations were performed as described (3, 27, 28). Westernblotting was performed with an ECL kit (Amersham, Arlington Heights,IL). For evaluation of kinase activities, immunoprecipitates were washedwith Nonidet P-40 (NP-40) lysis buffer or with NP-40 lysis buffer contain-ing 0.5 M NaCl, twice with high salt buffer (0.5 M LiCl), and once withNP-40 lysis buffer and were subjected to in vitro kinase assays.

Modeling of the CD150 cytoplasmic tail

Preliminary CD150 homologue searches were performed with BLAST andPSI-BLAST over various protein databases including TrEMBL, Swiss-Prot, Kabad, and PDB. CD150 homologues were aligned with the ClustalXprogram. Program alignment output was slightly modified manually withJalView and GeneDoc alignment viewers to align TxYxxV/I-containingregions. We used HMMER package tools (29–31) to build hidden Markovmodel (HMM) profiles for the multiple alignment of CD150, 2B4, CD84,and Ly9 and to search TrEMBL, Kabat, and Swiss-Prot databases usingthis profile as a query. As the number of identified homologues grew, newsequences were added to the multiple alignments, and new HMM profileswere built. Identification of CD150ct structural homologues was done byboth sequence (BLAST, Fasta, CPHmodels, UCSC HMM) and structurealignment H3P2 at UCLA-Department of Energy (32, 33). CD150 se-quence fitting to the recognized fold was performed in a Swiss PDB viewer(34, 35) and submitted for modeling to the Swiss-Model server (http://www.expasy.ch/swissmod/SWISS-MODEL.html). The quality of the mod-eled structure was assessed by ERRAT (36) and Verify3D (37) programs.We used PovRay ray tracing software to prepare the presented picture.

Results and DiscussionSH2D1A is expressed in B cells where it associates with CD150

CD150 is expressed on the surface of B cells and is up-regulatedafter activation (3). Using immunohistology and a microarray anal-ysis, CD150 was found in diffuse large B cell lymphoma (22, 38).Because it was shown that in T cells CD150 associates withSH2D1A, we tested whether SH2D1A is expressed in B cells andwhether it associates with CD150 in B cells. We assess the ex-pression of SH2D1A protein in B lineage cells in a panel of B celllines representing different stages of maturation. All studied B-LCL, including cell lines from patients with XLP (IARC 739,XLP-D, XLP-8002, XLP-8005), expressed high levels of CD150peak fluorescence intensity,.10.0). The BL lines Raji, Namalwa(EBV1), and BJAB (EBV2) expressed CD150 at a moderate level(peak fluorescence intentisy 7.0); all other B cell lines tested(REH, Ramos, B104, RPMI-8226) were CD150 negative. Westernblot analysis of whole cell lysates revealed that SH2D1A was ex-pressed in only two of the B lymphoblastoid cell lines studied,MP-1 and CESS (Fig. 1A). Flow cytometry also showed intracel-lular expression of SH2D1A in MP-1, but not in the BJAB cell line(Fig. 1B).

The surface phenotype of MP-1 cells resembles early germinalcenter cells: IgM1IgD2CD381CD39lowCD951CD1501. TheCESS cell line has a surface phenotype similar to memory B cells;e.g., it expresses membrane IgG, but not IgM, and is CD39high. Inagreement with prior studies (13), immunohistochemical analysisrevealed SH2D1A expression in frozen sections from tonsils,lymph nodes, and diffuse large B cell lymphoma. SH2D1A waspresent within the cytoplasm of scattered large cells localized to Bcell areas (data not shown). To evaluate the SH2D1A expression insubsets of B cells in vivo, we performed three-color analysis of Tcell-depleted tonsillar cells (2.86 0.3% of CD31 lymphocytes).

5481The Journal of Immunology

These studies revealed SH2D1A in 11.5–18.0% of CD201 tonsil-lar B cells. Low levels of SH2D1A expression were found in naiveIgD1CD382B cells (mean, 85.4). SH2D1A was detected in 32.0%of germinal center IgD2CD381 (9% of total B cells; mean, 94.4;peak, 112.0). The highest level of SH2D1A protein was detected in46.1% of CD381CD1501 cells (mean, 101.5; peak, 119.0) (Fig.1C). At the same time IgD2CD382 cells, which include memoryB cells, were SH2D1A negative (mean, 70.3) (Fig. 1C). These dataagree with immunohistochemical studies of frozen tonsillar sec-tions that did not reveal preferential localization of SH2D1A inany B cell zone, but did detect the highest level of SH2D1A ex-pression within large cells with cleaved nuclei localized in germi-nal centers (data not shown).

Association of CD150 with SHIP, SH2D1A, or SHP-2 in B cells

Because our data implied that CD150 and SH2D1A are coex-pressed in B cells, we tested whether these molecules associate inB cells. In both SH2D1A1 cell lines (MP-1 and CESS), SH2D1Acoprecipitated with CD150. As expected, SH2D1A was not de-tected with CD150 in SH2D1A2 lines T5-1, IARC 739, XLP-8005, or BJAB (Fig. 2A). In some B cell lines, CD150 coprecipi-tated with SHIP, and a tyrosine-phosphorylated fusion protein ofthe CD150ct can bind SHP-2 (5). Also in COS-7 and mouse Tcells, CD150 binds SHP-2, and this association can be blocked bySH2D1A (10, 11). Immunoprecipitation experiments followed byWestern blot analysis clearly showed that in the SH2D1A-express-ing cell lines MP-1 and CESS, CD150 coprecipitated with SHIP,and not SHP-2 (Fig. 2A). In contrast, in all SH2D1A-negative cell

lines tested, CD150 associated only with SHP-2 (Fig. 2A). Traceamounts of SHP-2 were detected together with CD150 in theCESS cell line that has a much lower level of SH2D1A expressionthan the MP-1 line. Apparently, this differential binding of SHIPwith SH2D1A vs SHP-2 did not depend on CD150 tyrosine phos-phorylation, since CD150 is constitutively phosphorylated on ty-rosine in both the SH2D1A1 and SH2D1A2 cell lines studied(Fig. 2B). To evaluate a possible role for differential phosphory-lation of any of four tyrosine residues in CD150ct, we used ty-rosine-phosphorylated GST-fusion proteins of CD150ct (5). In theabsence of SH2D1A in cell lysates (cell line BJAB), this fusionprotein precipitated SHP-2, but in the presence of SH2D1A (celllysates from the MP-1 cell line), GST-CD150ctPY bound not onlySHP-2 but also SHIP (Fig. 2C). Thus, the presence of SH2D1A-facilitated binding of SHIP to CD150. GST-CD150ctPY copre-cipitation with both SHIP and SHP-2, apparently depends on thelevel of SH2D1A association with CD150. Preferential SHIP bind-ing to the native CD150 molecules in SH2D1A1 cell lines mayreflect associations in the context of intracellular localization.

Short term ligation of IgM on SH2D1A1 MP-1 cells did notchange the level of SH2D1A and SHP-2 coprecipitated withCD150 but did reduce the level of SHIP associated with CD150(Fig. 3A). At the same time, the level of precipitable CD150 re-mained constant (Fig. 3A). Under the same conditions, SHIP andSH2D1A levels in the cytosolic and particulate cell fractions re-mained constant (Fig. 3B). We did not detect BCR-induced relo-calization of either SHIP or SH2D1A to detergent-insoluble gly-colipid-enriched domains (Fig. 3B). In contrast, IgM cross-linkinghad no effect on the level of either SHIP or SHP-2 association withCD150 in the SH2D1A2 cell line BJAB (Fig. 3A, lower panels) orXLP B-LCLs (data not shown). This suggests that BCR ligationcan alter association of CD150 with SHIP, but the amount of SHIPassociated with CD150 does not depend on SH2D1A levels. Thus,SH2D1A does not simply compete with SHIP for binding toCD150.

Because CD150 expression is up-regulated on naive B cells byeither CD40 or BCR cross-linking (3), we tested whether SH2D1Aexpression in B cells could be induced and/or up-regulated. Up to48 h after ligation of CD40, IgM, or CD150, SH2D1A expressionwas not induced in cell lines BJAB, Raji, or XLP-8002. In theSH2D1A1 cell line, CESS, ligation of CD40 or CD150 did notchange the level of SH2D1A expression (data not shown). How-ever, 48 h of stimulation via CD40 up-regulated SH2D1A expres-sion in MP-1 cells (Fig. 3C). Cross-linking of CD150 on thesecells did not affect SH2D1A levels, whereas IgM ligation down-regulated SH2D1A expression (Fig. 3C).

Tyr281 and Tyr327 in the CD150ct are essential for both SHIPand SHP-2 binding

To clarify the molecular basis of SHIP vs SHP-2 binding toCD150, we constructed GST-fusion proteins of the CD150ct withsingle replacements of tyrosine at Y269F, Y281F, Y307F, orY327F (Fig. 4A). Since SHIP and SHP-2 binding to CD150ct isphosphotyrosine dependent (5), all fusion proteins were expressedin both tyrosine-phosphorylated (PY) and nonphosphorylatedforms. The major 145-kDa protein coprecipitated with GST-CD150ct-PY and phosphorylated in the in vitro kinase assay pre-viously was identified as SHIP (Ref. 5; Fig. 2C). Mutations in anyone of the tyrosines did not affect binding of SH2D1A to CD150ct(Fig. 4B). However, SHIP bound to GST-CD150ct-PY, M1-PY(Y269F), and M3-PY(Y307F) in a phosphotyrosine-dependentmanner (Fig. 4B and data not shown). At the same time, we did not

FIGURE 1. Expression of SH2D1A in B cells.A, The presence ofSH2D1A in B-LCL lines MP-1 and CESS was revealed by Western blot-ting with an anti-SH2D1A serum. NP-40 lysates of 53 105 cells/well wereresolved in 15% SDS-PAGE. Positive control was the T cell line Jurkat.One of four experiments.B, Expression of SH2D1A in B cell lines BJABand MP-1. Rabbit IgG served as a negative control for purified anti-SH2D1A rabbit Ab.C, Expression of SH2D1A in tonsillar B cells. Cellswere stained for IgD, CD38, or CD150 surface expression and cytoplasmicSH2D1A using three-color flow cytometry. Rabbit IgG was a negativecontrol for purified anti-SH2D1A Ab. Naive IgD1CD382 B cells; mean,85.4. Germinal center IgD2CD381 B cells, SH2D1A1 cells, 32.0%; mean,94.4; peak channel (PKchan), 112.0. CD381CD1501 cells, SH2D1A1

cells, 46.14%; mean, 101.5; PKchan, 119.0. IgD2CD382 cells, mean 70.3.One of four experiments.

5482 SH2D1A REGULATES CD150 ASSOCIATION WITH SHIP AND SHP-2

FIGURE 3. A, BCR ligation down-regulates association of SHIP with CD150 but does not affect CD150 association with SH2D1A and SHP-2. Westernblot of CD150 immunoprecipitates. After activation of cells with anti-IgM sera, immunoprecipitations were performed as in Fig. 2. As a control for CD150levels after BCR ligation, we included immunoprecipitation of surface-biotinylated CD150 followed by Western blot with streptavidin peroxidase.One offive experiments.B, At the same time BCR ligation did not change the level of SHIP and SH2D1A in particulate or cytosolic fractions or detergent-insolubleglycolipid-enriched domains (DIGs). The presence of Lyn served as a control for detergent-insoluble glycolipid-enriched domains preparation (lowerpanel). Western blot of subcellular fractions. One of three experiments.C, Regulation of SH2D1A expression via BCR and CD40. BCR ligation down-regulated and CD40 engagement up-regulated SH2D1A expression in MP-1 cells after 48 h of stimulation. Western blot analysis with anti-SH2D1A serumon NP-40 lysates. Anti-p38 mitogen-activated protein kinase (p38) blots controlled equal loading. One of three experiments.

FIGURE 2. A, Coprecipitation of CD150 with SHP-2 vs SHIP and SH2D1A. CD150 was immunoprecipitated with mAb IPO-3 directly coupled toSepharose, and MOPC 21 myeloma protein directly coupled to Sepharose was used as a negative control; 503 106 cells/immunoprecipitation. Westernblot analysis with Abs against SHIP, SHP-2, and SH2D1A was performed using ECL. Whole cell lysates served as a positive control. All cell linesexpressed CD150 at comparable levels. One of four experiments.B, Tyrosine phosphorylation of CD150 revealed in Western blot with mAb 4G10. CD150was immunoprecipitated with mAb IPO-3 as described inA. C, Presence of SH2D1A-facilitated SHIP association with CD150. Nonphosphorylated andtyrosine-phosphorylated GST-fusion proteins of CD150ct were used for precipitations from MP-1 cells followed by Western blot with anti-SHIP, anti-SHP-2, and anti-SH2D1A serum. Tyrosine phosphorylation of fusion proteins was controlled with mAb 4G10 (Anti-PY).

5483The Journal of Immunology

detect SHIP in the precipitates with M2-PY(Y281F) and M4-PY(Y327F) (Fig. 4B). SHP-2 was also precipitated with GST-CD150ct-PY, M1-PY(Y269F), and M3-PY(Y307F) fusion pro-teins, and again both M2-PY(Y281F) and M4-PY(Y327F) failed tobind SHP-2 in cell lysates from SH2D1A1 cells (Fig. 4,C andD).On the other hand, in the absence of SH2D1A (BJAB cell lysates)both M2-PY and M4-PY were able to bind SHP-2 (Fig. 4D), in-dicating that SH2D1A and SHP-2 are competing not only for Y281but also for Y327. The fact that M2-PY and M4-PY bind moreSH2D1A than SHP-2-binding mutants also may reflect competi-tion for the same binding sites. These results suggest that the sametyrosines within TxYxxV/I motif in CD150ct (Y281 and Y327) arerequired for SHIP and SHP-2 association with CD150.

This study provides evidence of SH2D1A expression in humanB cells. Using in vivo and in vitro approaches, we showed thatboth SHIP and SHP-2 are able to bind CD150 in B cells. Pointmutations of tyrosines in GST-fusion proteins of the CD150 cy-toplasmic tail revealed that tyrosine phosphorylation of the sameresidues Y281 and Y327 are essential for SHIP as well as SHP-2binding. Replacements at residues Y269 and Y307 did not affecteither SHIP or SHP-2 association with CD150. How then is SHIPand SHP-2 binding to the same sites regulated? Our data indicatethat the adaptor protein SH2D1A is involved in this regulation.Despite the reported absence of SH2D1A expression in murine Bcells (11), SH2D1A mRNA (12, 13) and protein are expressed inhuman B cells, including the B cell lines MP-1 and CESS and

tonsillar B cells (Fig. 1). As in T cells, CD150 coprecipitates withSH2D1A in B cells, and neither phosphorylation nor mutations oftyrosines in CD150 affect this association (Figs. 2 and 4). The SH2domain of SH2D1A binds the sequence surrounding Y281 devoidof tyrosine (15, 16) and also binds in a phosphotyrosine-dependentmanner to the 2B4 receptor (17). Furthermore, SH2D1A can com-pete with SHP-2 for binding to the cytoplasmic tail of CD150 and2B4 (10, 17). Here we found that in SH2D1A-expressing B celllines, CD150 coprecipitates with SHIP; however, in SH2D1A2

cell lines, CD150 associates only with SHP-2, and the presence ofSH2D1A facilitates SHIP binding to CD150ct (Fig. 2C). Probably,the regulation of CD150 association with SHIP vs SHP-2 bySH2D1A may be under control of BCR and CD40 signaling since:1) short term signal via BCR decreases SHIP association withCD150 (Fig. 3A); and 2) long term (48 h) BCR ligation reducesand CD40 cross-linking up-regulates the level of SH2D1A expres-sion (Fig. 3C).

Modeling of the CD150ct

The cytoplasmic tail of CD150 has the paired tyrosine-based motifTxYxxV/I, which we propose to be designated as a “switch” motif(ITSM). This motif with the help of the adaptor protein SH2D1Amay control binding of tyrosine vs inositolphosphatases to recep-tors. This motif is different from the well-defined immunoreceptortyrosine-based activation motifs D/ExxYxxL/I(x)6-8YxxL/I in BCR

FIGURE 4. A, Replacements of tyrosine by phenylalanine in GST-fusion protein constructs of the CD150ct.B, Y281 and Y327 in CD150ct are involvedin SHIP recruitment. Nonphosphorylated and tyrosine-phosphorylated GST fusion proteins of CD150ct were used for precipitations from MP-1 cellsfollowed by in vitro kinase assays on precipitates (SHIP) and Western blot with anti-SHIP (data not shown) and anti-SH2D1A serum. The tyrosine-phosphorylated fusion proteins GST-M2-PY (Y281F) and GST-M4-PY (Y327F) did not precipitate SHIP. One of five experiments.C, D, Both Y281 andY327 in the CD150ct are involved in SHP-2 recruitment. Western blot analysis of fusion proteins precipitates with anti-SHP-2 and anti-SH2D1A Abs.Tyrosine-phosphorylated fusion proteins were used for precipitations from MP-1 (C andD, upperandmiddle panels) and BJAB (D, lower panel) cell lines.The tyrosine-phosphorylated fusion proteins GST-M2-PY (Y281F) and GST-M4-PY (Y327F) did not precipitate SHP-2 from the MP-1 cell line in thepresence of SH2D1A. At the same time, mutations in Y269 and Y307 (fusion proteins GST-M1-PY and GST-M3-PY did not affect binding of SHP-2 toCD150ct (upper panelsof C andD). Recruitment of SH2D1A to CD150ct was revealed by Western blot with anti-SH2D1A serum. In the absence ofSH2D1A (cell line BJAB), GST-M2-PY (Y281F) and GST-M4-PY (Y327F) were able to recruit SHP-2(D). Tyrosine phosphorylation of fusion proteinswere controlled with anti-PY mAb 4G10 (D, lower panel). One of four experiments.

5484 SH2D1A REGULATES CD150 ASSOCIATION WITH SHIP AND SHP-2

and TCR complexes, which on phosphorylation recruit protein ty-rosine kinases such as Syk and ZAP-70 (39, 40). However, theCD150/2B4 motif has some similarities with immunoreceptor ty-rosine-based inhibitory motifs (ITIMs) I/VxYxxL/V(x)26-31I/VxYxxL/V found within cytoplasmic domains of “inhibitory receptorsuperfamily” members such as FcRIIb, CD22, CD72, killer Ig-relatedreceptor, paired Ig-like receptors, p49B, Ig-like transcript (ILT), andleukocyte-associated Ig-like receptor (41). These ITIMs inhibit acti-vation receptors by recruiting SH2-containing tyrosine phosphatasesSHP-1 and SHP-2, and also SHIP (1, 41, 42).

We performed a series of protein sequence databases searches tobroaden the list of ITSM-containing molecules. Most of the pre-viously reported CD150 homologues, 2B4 (CD244), CD48, CD84,and Ly9 (CD220), belong to CD2 subfamily of the Ig superfamily.Multiple alignments of CD2 subfamily members with the highestlevel of homology to CD150 (2B4, Ly9, CD84) were used to buildHMM profiles for position-specific matching searches against aSwiss-Prot database with a HMMER program package. Becausewe consider the ITSM motif the main functional unit within theCD150ct, the key criterion for sequence selection was the presenceof tyrosine-based motifs that fit the ITSM consensus (Fig. 5A). Thecommon feature for members of the CD2 subfamily is a pairedITSM and the existence of differentially spliced truncated formsleading to only a single ITSM for CD150, 2B4, and Ly-9 (Fig. 5,A andB).

Search results for CD150, 2B4, CD84, and Ly9 alignment revealedhomology to several members of SHPS-1 (SHP-2 substrate 1) family:SHPS-1, BIT, and MYD. These sequences are highly homologous toeach other and contain two ITSM-like motifs alternated with ITIMconsensus motifs (Fig. 5,A andB). The cytoplasmic tail of SHPS-1,like CD150, is able to bind SHP-2 phosphatase.

The third mostrepresented group of sequences belongs to theSiglec/CD33 family. The cytoplasmic domains of CD31/platelet en-dothelial cell adhesion molecule-1, CD33, Siglec-5 (OB/BP2), andSiglec-9 all have a similar tyrosine-based motif distribution pattern.A ITSM-like motif is situated 3–7 residues from the C terminusand is preceded by a conventional ITIM motif. Interestingly, simi-lar to CD150ct, a GST platelet endothelial cell adhesion molecule/CD31 cytoplasmic tail can bind both SHP-2 and SHIP, and SHIPinteracts predominantly with the ITSM motif in CD31 (43).

CEA superfamily members were also widely represented in theretrieved sequences with ITSM motifs. Bgp-1, Bgp-2, C-CAM 105ecto-ATPase, and related molecules contain Y-based motifs thesequences and positions of which in the cytoplasmic domain arehighly similar to the positions and sequences ofITSMs: tyrosinesat the C terminus followed by a group of positively charged residues.Similar to CD150, which recently was shown to serve as alternativemeasles virus receptor (44), both Bgp-1 and Bgp-2 are receptors formouse hepatitis virus and also have truncated forms (45).

Unlike sialoadhesin and CEA family members, other ITSM andITIM-containing molecules retrieved by the HMM search demon-strate experimentally confirmed inhibitory activity. CD150showed a weak sequence homology to several members of therecently established monocyte-inhibitory receptor/LIR/ILT family,like paired Ig-like receptors B and programmed death-1 (PD-1)receptor. However, ITIMs rather than ITSMs are presented morewidely in the cytoplasmic domains of this group, and motif ho-mology to CD150 ITSMs is weaker than in the case of Siglecs orCEAs (Fig. 5,A and B). Nevertheless, the structural similaritybetween these sequences and CD150 creates a potentially impor-tant bridge between these two groups of molecules. ITSM-con-taining molecules also include the catalytically inactive tyrosine

FIGURE 5. A, Multiple alignments of CD150 TxYxxV/I motifs (ITSM) and the tyrosine-containing motifs from the sequences found by HMM searches.B, Schematic chart representing the distribution of ITIM and ITSM-like motifs in the cytoplasmic domains of the molecules with homology to CD150.h,ITIM; ■, ITSM. PD-1, programmed death-1 receptor.C, Ribbon diagram illustrating the three-dimensional structure of CD150ct. The diagram was preparedusing a Swiss PDB Viewer.

5485The Journal of Immunology

kinase human receptor related to tyrosine kinase and the mem-brane adaptor protein called SHP-2-interacting transmembraneadaptor protein (SIT) (46) (Fig. 5,A andB). The effector moleculesthat bind the ITIM motif in SIT have not been identified, but it islikely that SIT regulates TCR-mediated induction of IL-2 genetranscription via this motif (47).

The distribution of ITSM-like tyrosine-containing motifs inCD150 homologues confirms that these motifs represent an im-portant and structurally unique but poorly studied group of ty-rosine-containing regulatory motifs. Multiple alignment of ITSM-like motifs from distant homologues of CD150 indicate theconservation pattern of this kind of motif: 1) in most of the motifspresented on Fig. 5A, at least one additional conserved position isevident: a threonine or serine in position24 with respect to theITSM tyrosine. This pattern is present in all ITSMs (Y281 andY327 in CD150, 2B4, CD31) that have been shown to be func-tionally significant; 2) in members of CD2, Siglec, and CEA fam-ilies, one ITSM is positioned 3–5 residues upstream of the C ter-minus; 3) CD150, Ly9, 2B4, and murine Bgp-2 have alternativelyspliced forms, and shorter isoforms possess only a single ITSM; 4)in several families (SHPS, Siglec, and LIR, both ITIM and ITSMconsensus motifs are present.

We applied computational biology methods to explore possiblemechanisms of CD150ct interactions with associated moleculesand to explain the available experimental data. No sequences withsignificant homology to CD150 were found in Protein Data Bank(PDB) by either search algorithm we used, including the position-specific scoring methods (30, 48); therefore, homology modelingwas inapplicable. Threading of the CD150ct sequence by the H3P2method predicted an Ig-likeb sandwich fold for CD150ct. Onestructure (IgG1 H chain, PDB access code 1tet, residues 113–213)with the bestz score and alignment to CD150 was selected formodeling. The model we built represents a Greek key fold, typicalfor Ig-like domains, with two parallelb sheets that contain two andfour strands (Fig. 5C). Checking the quality of the resulting struc-ture by ERRAT (36) and Verify3D (37) programs indicated thatthe modeled structure might be natural. Realizing the limitations ofthe used modeling method, nevertheless, we can use this model foranalysis of the experimental data from a structural perspective andmake functional predictions.

Since Y281 and Y327 are essential for SHP-2 binding toCD150, it is possible that both SH2 domains of SHP-2 mediatethisassociation. Using our model and SHP-2 structure revealed by crys-tallography (49), we predicted that simultaneous binding of Y281 andY327 by two SHP-2 SH2 domains is unlikely, because the distancebetween tyrosine-binding pockets of N- and C-terminal domains ofSHP-2 is 40 Å, compared with 8.18 Å, between Y281 and Y327residues in CD150ct model. Moreover, it was shown that only theN-SH2, but not the C-SH2 domain of SHP-2 phosphopeptides bindsto both ITIM- and ITSM-like motifs of CD31 (43).

Apparently, displacement of SHP-2 by SHD1A (10) makesY281 and Y327 available for binding by SHIP, which also requiresY281 and Y327 (Fig. 4B). This possibility is supported by in vivodata demonstrating differential coprecipitation of CD150 withSHIP in SH2D1A1 cell lines or with SHP-2 in SH2D1A2 celllines (Fig. 2). Although SHIP possesses only one SH2 domain,both Y281 and Y327 in CD150 are important for association withSHIP (Fig. 4). Similarly, the association of SHIP with FcgRIIBalso requires two tyrosines; not only Y279 within the ITIM of theFcgRIIB is required, but also Y296 is necessary for stable asso-ciation of SHIP with FcgRIIB (50). Interestingly, that SH2 domainof SHIP has a much higher affinity to immobilized phosphopeptideof CD31 ITSM-like motif, than N-SH2 domain of SHP-2 (43).However, using only surface plasmon resonance with evaluation of

dissociation constants for SH2 domains and their affinity to ITSMsin CD150, we will be able to build a kinetic model of their com-petitive binding.

Why should SH2D1A binding to the sequence surroundingY281 in CD150 prevent its association with SHP-2 but not com-pete with SHIP? Amino-terminal residues adjacent to Y281 in thecytoplasmic tail of CD150 are critical for high affinity binding ofSH2D1A to CD150 (16). At the same time, mutational analysis ofthe FcgRIIB tail revealed that the residue Y279 within the ITIMmotif is required for SHP-2 but not SHIP binding to the cytoplas-mic tail of FcgRIIB (50, 51). Therefore, high affinity binding ofSH2D1A to residues amino-terminal to Y281 in CD150 may com-pete with SHP-2 but not affect association of SHIP with CD150.Taken together, SH2D1A may play a role as a molecular “switch”that regulates SHIP vs SHP-2 association with CD150. This modelis consistent with the reported involvement of SHIP in regulationof B cell development as well as immune responses to antigenicchallenge (52–54). In other words, CD150 may transmit SHIP-dependent or SHP-2-mediated signals at distinct stages of B cellmaturation, and the adaptor protein SH2D1A may regulate thisswitch. Using DT40 sublines, we are currently investigating whatCD150-mediated signal transduction pathways are regulated bySH2D1A. After this paper was submitted, Nagy et al. (55) reportedthe expression of SH2D1A in B cell lines derived from EBV-positive Burkitt’s lymphomas.

AcknowledgmentsWe thank Dario Magaletti for expert technical assistance, Dr. O. V. Yurchenkofor immunohistochemical analysis, and Kate Elias for editorial assistance.

References1. Healy, J. I., and C. C. Goodnow. 1998. Positive versus negative signaling by

lymphocyte antigen receptors.Annu. Rev. Immunol. 16:645.2. Craxton, A., K. L. Otipoby, A. Jiang, and E. A. Clark. 1999. Signal transduction

pathways that regulate the fate of B lymphocytes.Adv. Immunol. 73:79.3. Sidorenko, S. P., and E. A. Clark. 1993. Characterization of a cell surface gly-

coprotein IPO-3, expressed on activated human B and T lymphocytes.J. Immu-nol. 151:4614.

4. Cocks, B. G., C. C. Chang, J. M. Carballido, H. Yssel, J. E. de Vries, andG. Aversa. 1995. A novel receptor involved in T-cell activation.Nature 376:260.

5. Mikhalap, S. V., L. M. Shlapatska, A. G. Berdova, C. L. Law, E. A. Clark, andS. P. Sidorenko. 1999. CDw150 associates with Src-homology 2-containing ino-sitol phosphatase and modulates CD95-mediated apoptosis.J. Immunol.162:5719.

6. Punnonen, J., B. G. Cocks, J. M. Carballido, B. Bennett, D. Peterson, G. Aversa,and J. E. de Vries. 1997. Soluble and membrane-bound forms of signaling lym-phocytic activation molecule (SLAM) induce proliferation and Ig synthesis byactivated human B lymphocytes.J. Exp. Med. 185:993.

7. Aversa, G., C. C. Chang, J. M. Carballido, B. G. Cocks, and J. E. de Vries. 1997.Engagement of the signaling lymphocytic activation molecule (SLAM) on acti-vated T cells results in IL-2-independent, cyclosporin A-sensitive T cell prolif-eration and IFN-g production.J. Immunol. 158:4036.

8. Hamalainen, H., S. Meissner, and R. Lahesmaa. 2000. Signaling lymphocyticactivation molecule (SLAM) is differentially expressed in human Th1 and Th2cells.J. Immunol. Methods 242:9.

9. Aversa, G., J. Carballido, J. Punnonen, C. C. Chang, T. Hauser, B. G. Cocks, andJ. E. De Vries. 1997. SLAM and its role in T cell activation and Th cell responses.Immunol. Cell. Biol. 75:202.

10. Sayos, J., C. Wu, M. Morra, N. Wang, X. Zhang, D. Allen, S. van Schaik,L. Notarangelo, R. Geha, M. G. Roncarolo, H. Oettgen, J. E. De Vries, G. Aversa,and C. Terhorst. 1998. The X-linked lymphoproliferative-disease gene productSAP regulates signals induced through the co-receptor SLAM.Nature 395:462.

11. Castro, A. G., T. M. Hauser, B. G. Cocks, J. Abrams, S. Zurawski, T. Churakova,F. Zonin, D. Robinson, S. G. Tangye, G. Aversa, et al. 1999. Molecular andfunctional characterization of mouse signaling lymphocytic activation molecule(SLAM): differential expression and responsiveness in Th1 and Th2 cells.J. Im-munol. 163:5860.

12. Coffey, A. J., R. A. Brooksbank, O. Brandau, T. Oohashi, G. R. Howell,J. M. Bye, A. P. Cahn, J. Durham, P. Heath, P. Wray, et al. 1998. Host responseto EBV infection in X-linked lymphoproliferative disease results from mutationsin an SH2-domain encoding gene.Nat. Genet. 20:129.

13. Nichols, K. E., D. P. Harkin, S. Levitz, M. Krainer, K. A. Kolquist, C. Genovese,A. Bernard, M. Ferguson, L. Zuo, E. Snyder, et al. 1998. Inactivating mutationsin an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome.Proc. Natl. Acad. Sci. USA 95:13765.

5486 SH2D1A REGULATES CD150 ASSOCIATION WITH SHIP AND SHP-2

14. Sullivan, J. L. 1999. The abnormal gene in X-linked lymphoproliferative syn-drome.Curr. Opin. Immunol. 11:431.

15. Poy, F., M. B. Yaffe, J. Sayos, K. Saxena, M. Morra, J. Sumegi, L. C. Cantley,C. Terhorst, and M. J. Eck. 1999. Crystal structures of the XLP protein SAPreveal a class of SH2 domains with extended, phosphotyrosine-independent se-quence recognition.Mol. Cell 4:555.

16. Li, S. C., G. Gish, D. Yang, A. J. Coffey, J. D. Forman-Kay, I. Ernberg,L. E. Kay, and T. Pawson. 1999. Novel mode of ligand binding by the SH2domain of the human XLP disease gene product SAP/SH2D1A.Curr. Biol.9:1355.

17. Tangye, S. G., S. Lazetic, E. Woollatt, G. R. Sutherland, L. L. Lanier, andJ. H. Phillips. 1999. Cutting edge: human 2B4, an activating NK cell receptor,recruits the protein tyrosine phosphatase SHP-2 and the adaptor signaling proteinSAP.J. Immunol. 162:6981.

18. Tangye, S. G., J. H. Phillips, L. L. Lanier, and K. E. Nichols. 2000. Functionalrequirement for SAP in 2B4-mediated activation of human natural killer cells asrevealed by the X-linked lymphoproliferative syndrome.J. Immunol. 165:2932.

19. Parolini, S., C. Bottino, M. Falco, R. Augugliaro, S. Giliani, R. Franceschini,H. D. Ochs, H. Wolf, J. Y. Bonnefoy, R. Biassoni, et al. 2000. X-linked lym-phoproliferative disease: 2B4 molecules displaying inhibitory rather than acti-vating function are responsible for the inability of natural killer cells to killEpstein-Barr virus-infected cells.J. Exp. Med. 192:337.

20. Brandau, O., V. Schuster, M. Weiss, H. Hellebrand, F. M. Fink, A. Kreczy,W. Friedrich, B. Strahm, C. Niemeyer, B. H. Belohradsky, and A. Meindl. 1999.Epstein-Barr virus-negative boys with non-Hodgkin lymphoma are mutated in theSH2D1A gene, as are patients with X-linked lymphoproliferative disease (XLP).Hum. Mol. Genet. 8:2407.

21. Strahm, B., K. Rittweiler, U. Duffner, O. Brandau, M. Orlowska-Volk,M. A. Karajannis, U. Stadt, M. Tiemann, A. Reiter, M. Brandis, et al. 2000.Recurrent B-cell non-Hodgkin’s lymphoma in two brothers with X-linked lym-phoproliferative disease without evidence for Epstein-Barr virus infection.Br. J. Haematol. 108:377.

22. Sidorenko, S. P., E. P. Vetrova, O. V. Yurchenko, A. G. Berdova,L. N. Shlapatskaya, and D. F. Gluzman. 1992. Monoclonal antibodies of IPOseries against B cell differentiation antigens in leukemia and lymphoma immu-nophenotyping.Neoplasma 39:3.

23. Clark, E. A., and J. A. Ledbetter. 1986. Activation of human B cells mediatedthrough two distinct cell surface differentiation antigens, Bp35 and Bp50.Proc.Natl. Acad. Sci. USA 83:4494.

24. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989.Site-directed mutagenesis by overlap extension using the polymerase chain re-action.Gene 77:51.

25. Law, C. L., K. A. Chandran, S. P. Sidorenko, and E. A. Clark. 1996. Phospho-lipase C-g1 interacts with conserved phosphotyrosyl residues in the linker regionof Syk and is a substrate for Syk.Mol. Cell. Biol. 16:1305.

26. Law, C. L., S. P. Sidorenko, K. A. Chandran, K. E. Draves, A. C. Chan, A. Weiss,S. Edelhoff, C. M. Disteche, and E. A. Clark. 1994. Molecular cloning of humanSyk: A B cell protein-tyrosine kinase associated with the surface immunoglobulinM-B cell receptor complex.J. Biol. Chem. 269:12310.

27. Sidorenko, S. P., C. L. Law, S. J. Klaus, K. A. Chandran, M. Takata, T. Kurosaki,and E. A. Clark. 1996. Protein kinase Cm (PKCm) associates with the B cellantigen receptor complex and regulates lymphocyte signaling.Immunity 5:353.

28. Law, C. L., S. P. Sidorenko, K. A. Chandran, Z. Zhao, S. H. Shen, E. H. Fischer,and E. A. Clark. 1996. CD22 associates with protein tyrosine phosphatase 1C,Syk, and phospholipase C-g(1) upon B cell activation.J. Exp. Med. 183:547.

29. Eddy, S. R. 1995. Multiple alignment using hidden Markov models.Proc. Int.Conf. Intell. Syst. Mol. Biol. 3:114.

30. Eddy, S. R. 1998. Profile hidden Markov models.Bioinformatics 14:755.31. McClure, M. A., C. Smith, and P. Elton. 1996. Parameterization studies for the

SAM and HMMER methods of hidden Markov model generation.Proc. Int.Conf. Intell. Syst. Mol. Biol. 4:155.

32. Karplus, K., K. Sjolander, C. Barrett, M. Cline, D. Haussler, R. Hughey, L. Holm,and C. Sander. 1997. Predicting protein structure using hidden Markov models.Proteins Suppl. 1:134.

33. Karplus, K., C. Barrett, and R. Hughey. 1998. Hidden Markov models for de-tecting remote protein homologies.Bioinformatics 14:846.

34. Guex, N., A. Diemand, and M. C. Peitsch. 1999. Protein modelling for all.TrendsBiochem. Sci. 24:364.

35. Guex, N., and M. C. Peitsch. 1997. SWISS-MODEL and the Swiss-PdbViewer:an environment for comparative protein modeling.Electrophoresis 18:2714.

36. Colovos, C., and T. O. Yeates. 1993. Verification of protein structures: patternsof nonbonded atomic interactions.Protein Sci. 2:1511.

37. Luthy, R., J. U. Bowie, and D. Eisenberg. 1992. Assessment of protein modelswith three-dimensional profiles.Nature 356:83.

38. Alizadeh, A. A., M. B. Eisen, R. E. Davis, C. Ma, I. S. Lossos, A. Rosenwald,J. C. Boldrick, H. Sabet, T. Tran, X. Yu et al. 2000. Distinct types of diffuse largeB-cell lymphoma identified by gene expression profiling.Nature 403:503.

39. Campbell, K. S. 1999. Signal transduction from the B cell antigen-receptor.Curr.Opin. Immunol. 11:256.

40. van Leeuwen, J. E., and L. E. Samelson. 1999. T cell antigen-receptor signaltransduction.Curr. Opin. Immunol. 11:242.

41. Long, E. O. 1999. Regulation of immune responses through inhibitory receptors.Annu. Rev. Immunol. 17:875.

42. Tsubata, T. 1999. Co-receptors on B lymphocytes.Curr. Opin. Immunol. 11:249.43. Pumphrey, N. J., V. Taylor, S. Freeman, M. R. Douglas, P. F. Bradfield,

S. P. Young, J. M. Lord, M. J. Wakelam, I. N. Bird, M. Salmon, andC. D. Buckley. 1999. Differential association of cytoplasmic signalling moleculesSHP-1, SHP-2, SHIP and phospholipase C-g1 with PECAM-1/CD31.FEBS Lett.450:77.

44. Tatsuo, H., N. Ono, K. Tanaka, and Y. Yanagi. 2000. SLAM (CDw150) is acellular receptor for measles virus.Nature 406:893.

45. Nedellec, P., G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile,K. V. Holmes, and N. Beauchemin. 1994.Bgp2,a new member of the carcino-embryonic antigen-related gene family, encodes an alternative receptor for mousehepatitis viruses.J. Virol. 68:4525.

46. Marie-Cardine, A., H. Kirchgessner, E. Bruyns, A. Shevchenko, M. Mann,F. Autschbach, S. Ratnofsky, S. Meuer, and B. Schraven. 1999. SHP2-interactingtransmembrane adaptor protein (SIT), a novel disulfide-linked dimer regulatinghuman T cell activation.J. Exp. Med. 189:1181.

47. Schraven, B., A. Marie-Cardine, H. C, E. Bruyns, and I. Ding. 1999. Integrationof receptor-mediated signals in T cells by transmembrane adaptor proteins.Im-munol. Today 20:431.

48. Barrett, C., R. Hughey, and K. Karplus. 1997. Scoring hidden Markov models.Comput. Appl. Biosci. 13:191.

49. Hof, P., S. Pluskey, S. Dhe-Paganon, M. J. Eck, and S. E. Shoelson. 1998. Crystalstructure of the tyrosine phosphatase SHP-2.Cell 92:441.

50. Cambier, J. C., D. Fong, and I. Tamir. 1999. The unexpected complexity ofFcgRIIB signal transduction.Curr. Top. Microbiol. Immunol. 244:43.

51. Famiglietti, S. J., K. Nakamura, and J. C. Cambier. 1999. Unique features ofSHIP, SHP-1 and SHP-2 binding to FcgRIIb revealed by surface plasmon reso-nance analysis.Immunol. Lett. 68:35.

52. Ono, M., H. Okada, S. Bolland, S. Yanagi, T. Kurosaki, and J. V. Ravetch. 1997.Deletion of SHIP or SHP-1 reveals two distinct pathways for inhibitory signaling.Cell 90:293.

53. Liu, Q., A. J. Oliveira-Dos-Santos, S. Mariathasan, D. Bouchard, J. Jones,R. Sarao, I. Kozieradzki, P. S. Ohashi, J. M. Penninger, and D. J. Dumont. 1998.The inositol polyphosphate 5-phosphatase ship is a crucial negative regulator ofB cell antigen receptor signaling.J. Exp. Med. 188:1333.

54. Helgason, C. D., C. P. Kalberer, J. E. Damen, S. M. Chappel, N. Pineault,G. Krystal, and R. K. Humphries. 2000. A dual role for Src homology 2 domain-containing inositol-5-phosphatase (SHIP) in immunity: aberrant development andenhanced function of B lymphocytes in ship(2/2) mice.J. Exp. Med. 191:781.

55. Nagy, N., C. Cerboni, K. Mattsson, A. Maeda, P. Gogolak, J. Sumegi, A. Lanyi,L. Szekely, E. Carbone, G. Klein, and E. Klein. 2000. SH2D1A and SLAMprotein expression in human lymphocytes and derived cell lines.Int. J. Cancer88:439.

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