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IMMUNOBIOLOGY
From the Division of Immunology, Beth Israel Deaconess
Medical Center, Harvard Medical School, Boston, Massachusetts;
Departament de Ciencias Experimentals i de la Salut Laboratori de
Immunologia, Universitat Pompeu Fabra Barcelona, Spain; and Instituto
de Parasitología y Biomedicina, Consejo Superior de
Investigaciones Científicas, Granada, Spain.
CD150 signaling lymphocytic activation molecule (SLAM), a
T/B/dendritic cell surface glycoprotein, is a costimulatory receptor involved in T-cell activation and is also a receptor for measles virus.
CD150-induced signal transduction is controlled by
SAP/SH2D1A, the gene that is aberrant in X-linked
lymphoproliferative disease and familial hemophagocytic
lymphohistiocytosis. This report shows that CD150 colocalizes with the
T-cell receptor (TCR) following CD3 triggering in human peripheral
blood T cells and is rapidly and reversibly tyrosine phosphorylated on
TCR cross-linking. The Src-like kinases Lck and Fyn phosphorylate
tyrosine residues in the cytoplasmic tail of CD150. The results
demonstrate that the SAP protein has 2 modes of binding to CD150.
Binding to the motif Thr-Ile-Tyr281Ala-Gln-Val occurs in a
phosphotyrosine-independent fashion and to the motif
Thr-Val-Tyr327Ala-Ser-Val in a phosphotyrosine-dependent manner. Within
both SAP binding motifs the threonine residue at position CD150 signaling lymphocytic activation molecule
(SLAM) is a cell surface glycoprotein of relative mass 70 kd found on
activated/memory T cells, B cells, and dendritic cells.1,2
It is a member of the immunoglobulin superfamily and shares homology
with CD84, CD229, CD244, CD48, CD2, 19A, and Ly108.3 CD150
was recently shown to be the second receptor for measles virus in
addition to CD46.4-7 CD150 is a self-ligand8,9
with diverse immunologic functions including T/B-cell
costimulation,1,10,11 induction of interferon Although crystal structure and biacore data have shed light on the mode
of SAP binding to CD150, many fundamental questions remain to be
addressed regarding CD150/SAP binding and the role of CD150/SAP binding
in T-cell activation. It is unclear whether SAP binds to the motifs on
CD150 surrounding Tyr307 and Tyr327 in T cells. It is also
unclear which tyrosine kinases are able to phosphorylate CD150,
how stringent the SAP binding motif requirement is for SAP/CD150
interaction in T cells, or the exact mode of binding of SHP-2 to CD150.
Additionally, the requirement for CD150/SAP interaction in
CD150 lateral mobility and homophilic interactions between cells is
unknown. These questions are addressed in the present study.
Cells and antibodies
CD150 was immunoprecipitated using mouse monoclonal anti-hCD150
(2E7). Cell surface staining of CD150 was performed using mouse
monoclonal anti-hCD150 (A12). Horseradish peroxidase (HRP)-conjugated antiphosphotyrosine monoclonal antibody cocktail (PY-7E1, PY-1B2, PY20)
and HRP-conjugated streptavidin were from Zymed (San Francisco, CA).
Rabbit polyclonal anti-SHP2, anti-Fyn, and anti-Lck antibodies were
obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-FLAG
monoclonal M5 was obtained from Sigma Chemical (St Louis, MO).
Anti-mouse IgG1, IgG2a antisera conjugated to Texas red or fluorescein
isothiocyanate (FITC) were obtained from Southern Biotechnology
(Birmingham, AL).
Polymerase chain reaction-mediated mutagenesis
The sequences of the PCR primers are listed in Table
1.
Plasmid construction For expression in COS-7 and Jurkat-TAg cells, CD150 or CD150 tail mutants were cloned into the multiple cloning sites of pCDNA3 (Invitrogen), pCI (Promega), or enhanced green fluorescent protein (pEGFP; Clontech), and sequenced to confirm their identity. CD150 mutants encompassing the 77-amino acid tail of CD150 were also cloned into the EcoR1 and BamH1 sites of the yeast Gal-4 activation domain vector pGAD424 (Clontech). Human SAP was cloned into the first multiple cloning site of the yeast Gal4 binding domain vector pBRIDGE (Clontech) and also into pCDNA3 and pCMV-FLAG.Yeast 2-hybrid system and  -galactosidase activity (yeast protocols handbook,
Clontech). For phosphotyrosine-dependent binding analysis an altered
Fyn construct was inserted into the second multiple cloning
site of pBRIDGE as previously described.25
Immunoprecipitation and Western blotting COS-7 cells (107) were transfected by the dimethylaminoethyl (DEAE)-dextran method and biotinylated with sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) as described previously.16 Lysis was carried out with 1% Triton X-100 as described before.16 Cell lysates were clarified by centrifugation at 14 000g for 15 minutes at 4°C and the crude lysate was precleared using 30 µL protein G-agarose beads (Gibco) and 3 µg pooled mouse IgG for 1 hour. Immunoprecipitations were carried out using 3 µg of the indicated antibody and 30 µL protein G-agarose beads for 3 hours at 4°C. Beads were then washed 3 times with decreasing concentrations of Triton X (1%, 0.1%, 0%). Crude lysates and immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride filters (Millipore, Bedford, MA). Filters were blocked for 1 hour with 5% skim milk (or 3% bovine serum albumin) and then probed with the indicated antibodies. Bound antibody was revealed using HRP-conjugated secondary antibodies using enhanced chemiluminescence (Supersignal, Pierce). For antiphosphotyrosine blotting we used a directly HRP-conjugated antibody cocktail (Pierce).Immunofluorescent staining Peripheral blood was collected from healthy volunteers and PBMCs were isolated by centrifugation over a Ficoll gradient (Amersham Pharmacia Biotech, Piscataway, NJ). PBMCs were activated for 24 hours to up-regulate cell surface CD150 expression (data not shown) in RPMI supplemented with 10% fetal calf serum (FCS) with phytohemagglutinin (PHA) at 5 µg/mL plus interleukin-2 (IL-2) at 100 U/mL. Cells were then washed twice in ice-cold phosphate-buffered saline (PBS) prior to CD150 cross-linking experiments. CD150 or CD3 were cross-linked using 10 µg anti-CD150 A12 (mouse IgG1) or anti-CD3 OKT3 (mouse IgG2a) respectively, incubated on ice for 20 minutes followed by addition of FITC-conjugated anti-IgG1 or IgG2a with incubation at 37°C for 10 minutes. Cross-linking was terminated with 3 washes of ice-cold PBS before the cells were mounted on coverslips for microscopy. In some experiments cells were permeabilized with methanol and nuclei were stained with 1 mM Hoescht dye. Immunofluorescence was visualized with a Nikon Optiphot II microscope connected to a SPOT (Diagnostic Instruments, Sterling Heights, MI) digital camera.Transient transfection in Jurkat-TAg cells Jurkat TAg cells (20 × 106) were transfected by electroporation using a Biorad Genepulser apparatus using the settings 250 V, 960 µF, 200 , and 100 µg DNA resuspended in RPMI
with 100 mM HEPES in 0.4 cm gap cuvettes.
CD150 is rapidly and reversibly tyrosine phosphorylated following TCR cross-linking Because CD150 has previously been shown to have T-cell costimulation function1 in experiments using monoclonal antibodies, we investigated whether this effect could in part be due to an ability of CD150 to cluster in close proximity with the TCR at the T-cell plasma membrane. Human T cells were preactivated for 48 hours with PHA and IL-2 to induce high levels of surface CD150 expression (data not shown). After a "rest" period of 24 hours, anti-CD3 (OKT3) or anti-CD150 (A12) with an isotype-specific secondary antibody was used to cross-link one of the cell surface receptors. Next, the cells were stained for the second receptor (CD150 or CD3). Cross-linking of either CD150 or CD3 results in partial colocalization of both molecules on activated T cells; colocalization of CD3 and CD150 is more apparent when CD150 is cross-linked first (Figure 1A, lower panel). Cross-recognition of the first antibodies by the second antibodies was unlikely because isotype-specific reagents were used (A12 is IgG1, OKT3 is IgG2a). Moreover, FITC-labeled CD150+ B cells did not label with Texas red anti-IgG2a. Thus, CD150 appears to cluster in close proximity to the TCR/CD3 complex on the T-cell plasma membrane. Antibody-induced lateral mobility of CD150 may be analogous to CD150 movement into the interface of 2 Jurkat T cells transfected with CD150 shown below.
Triggering of the TCR with antibodies directed at CD3 partially mimics activation induced by major histocompatibility complex-peptide, resulting in phosphorylation of CD3 ITAMS and recruitment and activation of a variety of tyrosine kinases including the src-like tyrosine kinases Lck and Fyn [AI11] (see Clements et al26 for review). Because of the inducible proximity of CD150 and TCR/CD3, we next studied the effect of anti-CD3 triggering on CD150 phosphorylation. To this end a Jurkat transfectant was used. Following anti-CD3 treatment of Jurkat T cells stably transfected with CD150, we indeed observed rapid transient tyrosine phosphorylation of CD150 (Figure 1B). The intensity of phosphorylation peaked between 30 seconds and 1 minute and returned to basal levels after 5 minutes. These data suggest that proximal tyrosine kinases activated by TCR stimulation also function to phosphorylate tyrosine residues in the cytoplasmic tail of CD150. Tyrosine phosphorylation of CD150 did not occur in response to cross-linking of CD150 alone indicating that CD150 capping, and concomitant TCR cocapping (Figure 1A), is insufficient in itself to activate CD150-phosphorylating kinases consistent with the role of CD150 as a costimulatory molecule. All 3 tyrosines of CD150 (Tyr281, Tyr307, Tyr327) are phosphorylated by the src kinase Fyn To identify possible TCR-proximal tyrosine kinases that might be responsible for the rapid phosphorylation of CD150 following CD3 cross-linking, we focused on the 2 prominent kinases in TCR signaling Fyn and Lck. We have previously shown that the src kinase Fyn is able to phosphorylate CD150 in COS-7 cells.16 Chimeric constructs (Figure 2D) encoding the ectodomain of CD8 fused to the truncated cytoplasmic tail of CD150 encompassing the first SAP motif and Tyr307 (CD8-SLAM4 del1) or the first SAP binding motif alone (CD8-SLAM3) were therefore coexpressed with Fyn in COS-7 cells. COS-7 cells were used to avoid interference by endogenous Fyn. Immunoprecipitation of CD8 followed by Western blotting against phosphotyrosine demonstrates that Fyn is able to phosphorylate both CD8-SLAM3 (which contains the first SAP motif, Tyr281 only) and CD8-SLAM4 del1 (which contains Tyr281 and Tyr307 but not Tyr327) indicating that it phosphorylates Tyr281 and Tyr307 (Figure 1C). Coexpression of Lck with the chimeric constructs results in phosphorylation of CD8-SLAM4 del1 but not CD8 SLAM3, thus indicating that Lck phosphorylates Tyr307 but not Tyr281 (Figure 1C). Because Fyn phosphorylates both CD8-SLAM3 and CD8-SLAM4 del1, which contain Tyr281 or Tyr281 plus Tyr307, respectively, we further mapped CD150 phosphorylation using a series of CD150 constructs incorporating tyrosine-phenylalanine mutations (Figure 2B shows the nomenclature of these constructs). It is clear that Fyn is able to phosphorylate CD150 at position Tyr281 and Tyr327, but phosphorylates Tyr307 to a slightly lesser extent (Figure 3A, second panel).
SAP binds to amino acid residues on CD150 embedding Tyr281 or Tyr327 SAP binding to the membrane-proximal Tyr281 motif of CD150 has been described previously.16 As shown in Figure 2A, CD150 also has a perfect SAP binding motif surrounding Tyr327, but it is unclear whether SAP can indeed bind to this motif in a physiologic manner. We decided to use a point mutational mapping approach to address this question. CD150 constructs encoding various combinations of tyrosine-phenylalanine mutations in the cytoplasmic tail were generated for expression in mammalian cells (Figure 2B has details).Coexpression of CD150 with SAP in COS-7 cells leads to constitutive weak tyrosine phosphorylation of CD150 on all 3 SAP binding sites (Figure 3A, left panel). A large increase of phosphorylation of each construct was observed following cotransfection of Fyn. Predictably the single tyrosine mutants (Tyr1Phe, Tyr3Phe) were phosphorylated to a greater extent than the double mutants (Tyr12Phe, Tyr13Phe, Tyr23Phe; Figure 3A, right panel). Following immunoprecipitation of CD150, coimmunoprecipitation of SAP was observed on all the mutants except Tyr13Phe, which has Tyr281 and Tyr327 mutated to phenylalanine. This result demonstrates that SAP binds to Tyr281 and, following phosphorylation, Tyr327. SAP cannot bind to Tyr307 under these conditions. Because expression of CD150 and SAP in COS-7 cells leads to
constitutive weak tyrosine phosphorylation, dissection of the phosphotyrosine-independent binding of SAP to CD150 was performed using
the yeast 2-hybrid system. Yeast cells cannot accomplish tyrosine
phosphorylation unless they are transfected with tyrosine-kinase DNA.
They therefore provide a suitable expression system for
nonphosphotyrosine mapping of the SAP/CD150 interaction.25
CD150 Gal-4 activation domain and SAP Gal-4 binding domain
chimeric constructs (see "Materials and methods") were transformed
into the yeast strain Y190. SAP/CD150 interaction was then
measured with a liquid SHP-2 binding to CD150 involves both SAP binding consensus sites at Tyr281 and Tyr327 One major proposed function of the SH2D1A gene product SAP is that of blocking recruitment of other signal transduction molecules to CD150 and related molecules (CD84, CD229, CD244).22 One such signaling molecule, SHP-2 (a ubiquitous tyrosine phosphatase with tandem SH2 domains), is known to bind CD150, which event can be blocked by SAP.16 Because SAP can bind to the motifs surrounding Tyr281 and Tyr327 of CD150 (Figure 3), we further investigated the binding requirements for SHP-2 to CD150. SHP-2 exhibits a requirement for both tyrosines, Tyr281 and Tyr327, to bind CD150 (Figure 4). Mutation of Tyr281 (Tyr1Phe) or Tyr327 (Tyr3Phe) alone or in combination results in abrogation of SHP-2 binding (Figure 4, third panel). This result suggests that SAP binding to nonphosphorylated Tyr281 alone would be sufficient to block the SHP-2/CD150 interaction.
The threonine residue at position  2 is in fact critical for SAP binding independently of tyrosine phosphorylation on the motifs surrounding Tyr281 and Tyr327. Introduction of Thr>Ala point mutations in the SAP motifs completely abrogates binding to CD150, expressed in
COS-7 cells (Figure 5A). The majority of
SAP binding occurs on motif number 1 although in the presence of
fyn some SAP binding is observed at motif 3 (Figure 5A,
right panel, Thr1Ala mutation). In the absence of fyn the
CD150 constructs are constitutively tyrosine phosphorylated at low
levels (Figure 5A, left and right panels). To measure SAP binding to
nonphosphorylated CD150 incorporating threonine mutations we used the
yeast 2-hybrid system (Figure 5B). Mutation of the threonine residue in
either the first SAP motif (Thr1Ala), or the first and third motif in
combination (Thr13Ala) completely abrogates SAP binding (Figure 5B),
whereas mutation of Thr305 (Thr2Ala) or Thr325 (Thr3Ala) alone results
in strong -galactosidase activity (Figure 5B). These results show
that without tyrosine phosphorylation, SAP binding to Tyr281 is
dependent on the threonine residue at position 2. This confirms the
observations in COS-7 cells, that following tyrosine phosphorylation by
fyn the threonine residue stabilizes binding of SAP to
motifs surrounding TyrY281 and Tyr327. Thus, the threonine in the SAP
motif T(I/V)YxxV is essential for binding CD150 at each
motif.
The nonphosphotyrosine interaction of SAP with Tyr281 is partially stabilized by Leu278 of CD150 The presence of a threonine residue at position2 to the central
tyrosine in the SAP binding motif of CD150 is critical for stabilizing
the interaction in the presence or absence of tyrosine phosphorylation.
However, this does not explain the unique affinity of SAP for
nonphosphorylated Tyr281. For example, Tyr327 of CD150 is preceded by a
threonine residue Thr325, but SAP does not bind CD150 at this position
in the absence of tyrosine phosphorylation (Figures 3 and 4).We sought
to identify candidate amino acid residues N-terminal to the Try281 SAP
motif that might contribute to the nonphospho binding. Amino acid
residues surrounding the Tyr281 motif of human and mouse CD150 were
compared with those surrounding all the other SAP motifs of the human
and mouse CD150 family members (Table 2).
CD84, CD229, and CD244 are known to require tyrosine phosphorylation
for SAP binding.25,28 The plasma membrane-proximal SAP
consensus regions of human, mouse, and monkey CD150 are unique in
having a lysine residue at position 5, and a leucine residue at
position 3 to the central tyrosine (Table 2, residues denoted with an
asterisk, and Figure 6). Lys276
and Leu278 were mutated to the corresponding residues found at the
third SAP consensus motif N-terminal to Tyr327, which binds SAP only
following tyrosine phosphorylation. Yeast 2-hybrid analysis was
performed with CD150 mutants encoding point mutations of Lys276Asn and
Leu278Ile (Figure 7). Mutation of
Lys276Asn had no effect on the ability of SAP to bind nonphosphorylated
CD150 (Figure 7A). In contrast mutation of Leu278Ile resulted in a
marked reduction in nonphosphotyrosine binding of SAP to CD150 (Figure
7A). On introduction of fyn into the yeast this reduction in
binding was reversed (Figure 7B), showing that Leu278 contributes to
the phosphotyrosine-independent binding of SAP with CD150. Therefore
binding of SAP to CD150 is dependent on a number of residues N-terminal
and C-terminal to the central tyrosine in the motifs (Table 2 and
Figure 6). The unique blocking function of SAP binding to
nonphosphorylated motif number 1 is partially explained by a
stabilizing effect of Leu278.
CD150 exhibits lateral mobility in Jurkat T cells, forming adhesion plaques between neighboring cells; SAP is recruited to these contacts, but SAP-CD150 interaction is not required for their formation CD150 is proposed to function as a T-cell costimulatory receptor and we have observed that CD150 and CD3 colocalize on CD150 or CD3 triggering. We asked whether one mechanism of CD150 costimulation might be to facilitate the formation of homophilic adhesion contacts between adjacent cells.As a first approach to investigating the ability of CD150 to migrate to
cell contacts, we used Jurkat cells transiently transfected with
CD150-EGFP. The use of the EGFP tag on the C-terminus of CD150 allows
visualization of its location either within the cell or on the plasma
membrane with fluorescence microscopy. Following transfection of
Jurkat-TAg T cells with CD150-EGFP we observed aggregation of CD150 at
the plasma membrane contacts between neighboring cells (Figure
8A). Clustering of CD150 at the cellular
junctions results in recruitment of SAP to the same region (Figure 8C)
as visualized with an anti-SAP-Cy3 monoclonal antibody. We conclude that CD150 has a lateral mobility in the plasma membrane and forms adhesion contacts between cells, presumably due to homophilic interaction. Within these adhesion contacts there is a marked accumulation of SAP.
We next asked whether SAP/CD150 interaction was necessary for the
formation of the CD150 adhesion contacts. We transfected Jurkat-TAg T
cells with CD150-Tyr13Phe and Thr13Ala, both of which are
impaired in their ability to bind SAP, and used fluorescence microscopy
to visualize the cellular location of CD150. On transient transfection, 30% to 45% cell surface expression of CD150 was obtained (Figure 9A). SAP was able to
bind only to the intact CD150 construct (Figure 9B) as predicted from
the experiments in COS-7 cells and yeast. SAP binding to the mutant
CD150 constructs Tyr13Phe and Thr13Ala was undetectable in the presence
or absence of tyrosine phosphorylation of the CD150 tail, induced with
1 mM sodium pervanadate treatment (Figure 9B). Cell clusters of Jurkat-TAg expressing these CD150 constructs show clear CD150 concentration on the plasma membrane at the interface between neighboring CD150+ cells, with no differences between
wild-type CD150 or mutated CD150 (Figure 9C). This result demonstrates
that CD150 lateral mobility and formation of intercellular contacts on
T cells is not dependent on SAP binding.
CD150 was the first member of a growing family of lymphocyte cell surface receptors (CD84, CD229, CD244) that have been shown to bind to the signaling molecule SAP. This receptor/signaling molecule pair has recently acquired increased significance with the discovery that CD150 is the most commonly used receptor for measles virus entry into lymphocytes. Additionally, SAP is mutated in 3 immune diseases: X-linked lymphoproliferative disease, combined variable immunodeficiency disease, and familial hemophagocytic lymphohistiocytosis. CD150 has been reported to function as a costimulatory receptor on activated T cells, although the mechanism for this is unknown. In this study, we observed close physical proximity of the TCR with CD150 following cross-linking of either molecule. In addition, following triggering of CD3, CD150 was rapidly and transiently tyrosine phosphorylated. These results are consistent with a major signaling function of CD150 during costimulation. It is likely that tyrosine phosphorylation of CD150 in the immune synapse functions to recruit SH2 domain-containing signaling molecules, including SAP to the supramolecular activation cluster (SMAC). Indeed, in preliminary experiments we observe CD150 in the "immune synapse" formed between antigen-specific cytotoxic lymphocyte (CTL) clones and B cells pulsed with their cognate antigen (data not shown). CD150 is unique among its homologues in the immunoglobulin superfamily in that it is able to bind SAP, a floating SH2 domain, in the absence of tyrosine phosphorylation. In this study, using a detailed mutagenesis mapping approach we have shown that SAP binding to CD150 is in fact bimodal. Prior to tyrosine phosphorylation, SAP binds the membrane-proximal motif surrounding Tyr281. Following tyrosine phosphorylation by tyrosine kinases such as Fyn, SAP binds additionally to the distal motif surrounding Tyr327. SAP has been proposed to have 2 nonmutually exclusive functions. First, it functions to block recruitment to CD150 of SH2 domain-containing signaling molecules, the prototypic example being SHP-2.16 Second, it has been demonstrated to act as an adapter molecule, binding the guanine exchange factor p62dok1.23 It is reasonable to predict that immediately following TCR engagement, and subsequent CD150 phosphorylation, SAP is recruited to both SAP motifs at Tyr281 and Tyr327, to block or adapt at these positions. Physicochemical analyses of SAP binding to peptides spanning
Tyr281 of CD150 have suggested that amino acid residues
N-terminal to the central tyrosine of the SAP binding site
strengthen the interaction.24,27 Our findings extend
these observations to binding analysis in cells. The threonine residue
at position A major proposed function of adhesion molecule pairs such as leukocyte function-associated antigen 1 (LFA-1; CD11a/CD18)/intercellular adhesion molecule (ICAM-1) is to strengthen the interaction between T cells and antigen-presenting cells (APCs). Additionally, these interactions help to sequester TCR into the immune synapse. Here we demonstrate that CD150 has lateral mobility in the plasma membrane and readily clusters at the interface between 2 transfected cells. SAP is recruited into these CD150-rich regions, although SAP/CD150 interaction is not required for their formation. It is therefore likely that an important function of CD150 is to increase the affinity of memory T cells (which are CD150+) with APCs during the anamnestic response. In support of this concept, professional APCs (dendritic cells and activated macrophages) are highly positive for CD150 surface expression.
The authors would like to thank Drs Bruce and Barbara Furie for the use of their microscopy equipment, and Glenn Merrill-Skoloff for his expert assistance with digital microscopy. The authors are also indebted to Dr Charles Gullo, Dr Massimo Morra, and Dr William Faubion for critical reading of the manuscript and valuable discussions.
Submitted July 17, 2001; accepted September 14, 2001.
Supported by a grant from the National Foundation March of Dimes. D.H. is supported by a fellowship from the Leukemia and Lymphoma Society. M.S. is supported by a fellowship from Ministerio de Educacion y Ciencia.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Duncan Howie, Division of Immunology, RE-204, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Boston, MA 02215; e-mail: dhowie{at}caregroup.harvard.edu or terhorst{at}caregroup.harvard.edu.
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Top
Abstract
Introduction
(IFN-
constructs encoding the
ectodomain of CD8 and the cytoplasmic and membrane domains of
CD150.
, +SAP; 
, +pBridge.
Quantitation of 
