Physical and Functional Association between Thymic Shared Antigen-1/Stem Cell Antigen-2 and the T Cell Receptor Complex*

Atsushi KosugiDagger §, Shin-ichiroh Saitoh, Satoshi Noda, Kensuke Miyakeparallel , Yoshio Yamashitaparallel , Masao Kimotoparallel , Masato Ogata, and Toshiyuki Hamaoka

From the Dagger  School of Allied Health Sciences, Faculty of Medicine, Osaka University, Osaka 565,  Biomedical Research Center, Osaka University Medical School, Osaka 565, and parallel  Department of Immunology, Saga Medical School, Saga 849, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Thymic shared antigen-1 (TSA-1)/stem cell Ag-2 (Sca-2) is a glycosylphosphatidylinositol (GPI)-anchored antigen expressed on lymphocytes. We have previously demonstrated that a signal via TSA-1/Sca-2 inhibits T cell receptor (TCR)-mediated T cell activation and apoptosis. To elucidate a molecular mechanism for TSA-1-mediated modulation of the TCR-signaling pathway, we examined whether TSA-1 is physically coupled to the TCR in the present study. TSA-1 was clearly associated with CD3zeta chains in T cell hybridomas, activated T cells, and COS-7 cells transfected with TSA-1 and CD3zeta cDNA. The physical association was confirmed on the surface of T cells in immunoprecipitation and confocal microscopy. The analysis using stable and transient transfectants expressing a transmembrane form of TSA-1 revealed that the association of CD3zeta did not require the GPI anchor of TSA-1. Finally, tyrosine phosphorylation of CD3zeta chains was induced after stimulation with anti-TSA-1, suggesting that a functional association between these two molecules also exists. These results imply that the physical association to CD3zeta underlies a regulatory role of TSA-1/Sca-2 in the TCR-signaling pathway.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Thymic shared antigen-1 (TSA-1)1/stem cell antigen-2 (Sca-2) is a Ly-6-related differentiation antigen expressed on immature thymocytes and thymic epithelial cells (1-4). Recently, cDNA encoding human TSA-1 has been isolated, and it was shown that TSA-1 mRNA is expressed in human lymphoid tissues as well as various nonlymphoid tissues (5). Although TSA-1/Sca-2 is a useful marker in early T cell development and T cell activation and seems to play a regulatory role in thymocyte differentiation (6-8), functions of TSA-1/Sca-2 remain largely obscure.

In a previous study, we have analyzed a role of TSA-1 in mature T cells and demonstrated that it functions as a modulator of T cell receptor (TCR)-signaling pathway (6, 9, 10). Anti-TSA-1 mAb inhibited tyrosine phosphorylation of CD3zeta chains and IL-2 production induced by anti-CD3 stimulation in T cell hybridomas (9), suggesting that a signal via TSA-1 regulates early and late events in TCR signaling. The findings observed in this in vitro study were further strengthened by the fact that in vivo injection of anti-TSA-1 mAb completely blocked anti-TCR/CD3-mediated apoptosis of thymocytes (10). Thus, TSA-1/Sca-2 seems to be an important cell surface molecule regulating T cell differentiation and activation by virtue of its ability for modulating TCR-mediated signal transduction. However, since TSA-1 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein and thus does not have its transmembrane and cytoplasmic regions, it is not known how TSA-1 transmit signals into the cytoplasm of the cell.

In the present study, we addressed the above question by analyzing the molecular interaction between TSA-1 and the TCR. The data clearly demonstrated that TSA-1 is physically and functionally associated with CD3zeta chains of the TCR complex, and strongly suggested that the regulatory role of TSA-1 on TCR signaling is based on this intermolecular association.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
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References

Cell Lines and Hybridomas-- 2B4 is a murine T cell hybridoma that is specific for pigeon cytochrome c plus I-Ek (11). LK35.2 is a B cell hybridoma and used as accessory cells (12). Jurkat-derived transfectants expressing either GPI-anchored or a transmembrane form of TSA-1 have been established by us as described previously (9). All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37 °C in a humidified atmosphere of 5% CO2. For maintaining Jurkat-derived transfectants, G418 (Life Technologies, Inc.) was added at a concentration of 1 mg/ml.

mAb and Reagents-- The following monoclonal antibodies were used: 145-2C11 (13) and HMT3-1 (14), anti-CD3epsilon ; H146-968 (15), anti-CD3zeta ; A2B4 (11), anti-clonotypic antibody recognizing TCR-alpha of 2B4; M17/5.2 (16), anti-leukocyte function-associated antigen-1 (LFA-1); M1/42 (obtained from American Type Culture Collection, Rockville, MD), anti-major histocompatibility complex class I; D7 (17), anti-Ly-6A/E; 6C3 (18), anti-Ly-6C; G7 (19), anti-Thy-1.2; and PRST1 (6) and GR12, anti-TSA-1. GR12 is a rat mAb against TSA-1 that has been newly established by us, and the specificity of this mAb has been defined using Jurkat-derived transfectants expressing mouse TSA-1 (data not shown). No. 387, polyclonal antiserum against CD3zeta , was generously provided by Dr. Allan M. Weissman. Normal rat and hamster IgG were purchased from Cappel (Durham, NC).

Cell Preparations-- T cells were enriched from spleen cells of C57BL/6 mice by immunomagnetic negative selection as described previously (9).

DNA Transfection-- A total of 1 × 107 COS-7 cells was washed with Hepes-buffered saline and resuspended in 1 ml of ice-cold Hepes-buffered saline. Fifteen micrograms of plasmid DNA were added to the cell suspension in a cuvette (Gene Pulser Cuvette, Bio-Rad), and the electric pulse (250 V, 960 µF) was applied by a Gene Pulser (Bio-Rad). After 2 days of culture in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, cells were harvested, washed twice with phosphate-buffered saline, pelleted by centrifugation, and frozen at -20 °C before analysis by immunoblotting. The samples were electrophoresed followed by immunoblotting. The expression vectors encoding GPI-anchored TSA-1 or a transmembrane form of TSA-1 were constructed as described previously (9).

Surface Biotinylation, Immunoprecipitation, and Immunoblotting Analysis-- Cell surface biotinylation, immunoprecipitation, and immunoblotting analysis were performed as described previously (20, 21). Cells were solubilized with ice-cold lysis buffer (1% digitonin, 20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 5 mM iodoacetamide, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). After preclearing with normal rat IgG prebound to protein G-Sepharose (Pharmacia Biotech Inc.), the lysate was immunoprecipitated with various mAbs prebound to protein G-Sepharose.

Immunofluorescence Staining and Confocal Microscopy-- Cells were incubated at 4 °C for 30 min with FITC antibody, washed twice, incubated at 4 °C for 30 min with biotinylated antibody, washed twice, and incubated at 4 °C for 10 min with Texas Red streptavidin (Molecular Probes, Eugene, OR). Negative control staining was performed using FITC-conjugated Leu-4 mAb or Texas Red streptavidin without biotinylated antibody. Confocal microscopy was performed on Zeiss LSM410 model confocal microscopes. Green fluorescence was detected following excitation at 488 nM, and red fluorescence, following excitation at 543 nM. No immunofluorescence signal was detected in cells stained with negative control reagents (data not shown).

Phosphorylation-- 2B4 cells (1.5 × 107) were stimulated with various mAb (10 µg/ml) in the presence of LK cells (7.5 × 106) for 30 min at 37 °C. Immunoprecipitation and detection of tyrosine phosphorylation of CD3zeta were performed as described previously (9).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Physical Association between TSA-1/Sca-2 and the CD3zeta -- To elucidate a molecular mechanism for TSA-1/Sca-2-mediated modulation of the TCR signaling pathway, we examined whether TSA-1 could be physically associated with the TCR subunits. When 2B4, a T cell hybridoma which constitutively expressed TSA-1, was solubilized with Triton X-100 lysis buffer, immunoprecipitated with anti-TSA-1 mAb, and immunoblotted with anti-CD3zeta mAb, no physical association between TSA-1 and CD3zeta was observed (Fig. 1B). However, when 2B4 cells were solubilized with digitonin lysis buffer, a clear band migrating at 16 kDa was observed in anti-TSA-1 immunoprecipitates (Fig. 1A). Although the amount of CD3zeta associated with TSA-1 was low, the result was very reproducible; we have observed the association in at least 10 independent experiments. The immunoprecipitation with M17/5.2, a mAb against mouse LFA-1, did not co-precipitate any CD3zeta , indicating that the association between TSA-1 and CD3zeta is specific.


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Fig. 1.   Association between TSA-1/Sca-2 and CD3zeta chains in T cell hybridoma. 2B4 cells were solubilized either in digitonin (A) or in Triton X-100 (B) lysis buffer as indicated. Postnuclear supernatants were immunoprecipitated with M17/5.2 (anti-LFA-1 mAb) as a negative control, PRST1 (anti-TSA-1 mAb), or 2C11 (anti-CD3epsilon mAb). 1 × 107 cell equivalent/lane was immunoprecipitated with M17/5.2 or PRST1, and 5 × 106 cell equivalent/lane with 2C11. Immunoprecipitates were subjected to 14% SDS-PAGE under reducing condition, and immunoblotted with anti-CD3zeta mAb, H146-968, and horseradish peroxidase-conjugated protein A. CD3zeta is indicated by an arrowhead. The bands migrating at approximately 70 and 60 kDa are likely to be the heavy chain of the mAbs used for immunoprecipitation. Molecular sizes (kDa) are shown on the left.

We further examined whether the association of CD3zeta is detected in other GPI-anchored proteins. 2B4 cells were solubilized with digitonin lysis buffer, immunoprecipitated with various mAbs against other GPI-anchored and transmembrane surface proteins, and immunoblotted with anti-CD3zeta (Fig. 2). Again, the association of CD3zeta with TSA-1 was observed and this was confirmed using another rat mAb against TSA-1, GR12. However, none of mAbs against other GPI-anchored proteins such as Thy-1, Ly-6A/E, and Ly-6C, nor mAbs against transmembrane proteins such as LFA-1 and class I co-precipitated CD3zeta . Since these control mAbs were able to immunoprecipitate their corresponding surface Ags efficiently (data not shown), the result demonstrated a selectivity of CD3zeta chains for the ability to associate with TSA-1 among GPI-anchored molecules.


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Fig. 2.   Co-precipitation of CD3zeta chains using various mAbs. 2B4 cells were solubilized in digitonin lysis buffer. The lysates were immunoprecipitated with various mAbs as indicated, electrophoresed in 14% SDS-PAGE under reducing condition, and immunoblotted with anti-CD3zeta mAb. Antibodies used are M17/5.2, anti-LFA-1; M1/42, anti-class I major histocompatibility complex; G7, anti-Thy-1; GR12 and PRST1, anti-TSA-1; D7, anti-Ly-6A/E; 6C3, anti-Ly-6C; 2C11, anti-CD3epsilon ; and NRIgG, normal rat IgG. 1 × 107 cell equivalent/lane was analyzed. CD3zeta is indicated by an arrowhead. Molecular sizes (kDa) are shown on the left.

We next asked whether other chains of the TCR complex could be associated with TSA-1. The lysates of 2B4 cells were immunoprecipitated with anti-TSA-1, subjected to two-dimensional electrophoresis, and immunoblotted either with anti-CD3zeta or with anti-CD3epsilon . The result of Fig. 3 clearly demonstrated that TSA-1 was associated with CD3epsilon as well as CDzeta . Since CD3epsilon was not bound to TSA-1 in the 2B4 mutant that lacks expression of CD3zeta , the association between TSA-1 and CD3epsilon seemed to be dependent on the existence of CD3zeta chains (data not shown).


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Fig. 3.   Association between TSA-1/Sca-2 and CD3epsilon chains in T cell hybridoma. The lysates of 2B4 cells were immunoprecipitated with GR12 (A and C, anti-TSA-1 mAb) or with 2C11 (B and D, anti-CD3epsilon mAb), subjected to two-dimensional SDS-PAGE, and immunoblotted with anti-CD3zeta mAb (A and B) or with anti-CD3epsilon mAb (C and D). 2 × 107 cell equivalent/sample was immunoprecipitated with GR12, and 5 × 106 cell equivalent/sample with 2C11. Positions of CD3zeta or -epsilon chains are indicated. The diagonal of the gel is indicated by a broken line.

To investigate the direct association of TSA-1 to CD3zeta , COS-7 cells were transiently transfected with TSA-1 cDNA, CD3zeta cDNA, or both, and analyzed for the association (Fig. 4). A band migrating at 16 kDa was detected in COS-7 cells transfected with CD3zeta (Fig. 4B), confirming the band as CD3zeta . When COS-7 cells were transfected with TSA-1 in combination with CD3zeta , CD3zeta was clearly co-precipitated in anti-TSA-1 immunoprecipitates (Fig. 4C), demonstrating that the association between TSA-1 and CD3zeta can be produced in the absence of other chains of the TCR complex.


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Fig. 4.   Association between TSA-1/Sca-2 and CD3zeta chains in COS-7 cells. COS-7 cells were transfected with GPI-anchored TSA-1 (A), CD3zeta (B), GPI-anchored TSA-1 plus CD3zeta (C), or a transmembrane TSA-1 plus CD3zeta (D). Transfected COS-7 cells were solubilized in digitonin lysis buffer, and postnuclear supernatants were immunoprecipitated with NRIgG, GR12 (anti-TSA-1 mAb), or H146 (anti-CD3zeta mAb). 8 × 106 cell equivalent/lane was immunoprecipitated with NRIgG or GR12, and 1 × 106 cell equivalent/lane with H146. Immunoprecipitates were subjected to 14% SDS-PAGE under reducing conditions and immunoblotted with anti-CD3zeta mAb, H146-968. CD3zeta is indicated by an arrowhead. Molecular sizes (kDa) are shown on the left.

Association between TSA-1/Sca-2 and the TCR on the Cell Surface-- To determine whether TSA-1/Sca-2 was associated with the TCR on the cell surface, 2B4 cells were surface-labeled with biotin followed by immunoprecipitation with anti-TSA-1 mAb or with control mAbs (Fig. 5A). Although a mAb against LFA-1 and that against Ly-6C were able to immunoprecipitate their corresponding surface antigens, neither of these mAbs co-precipitated surface TCR chains. In contrast, in addition to TSA-1 per se, anti-TSA-1 mAb immunoprecipitated 46- and 26-kDa proteins, which seemed to correspond to TCR-alpha , and CD3delta and -epsilon chains, respectively. The bands migrating at 21 kDa in anti-TSA-1 immunoprecipitates might correspond to CD3gamma , but the mobility of the bands was lower than that of CD3gamma in anti-CD3epsilon immunoprecipitates. The co-precipitation of surface TCR chains in anti-TSA-1 immunoprecipitates was further analyzed by two-dimensional SDS-PAGE (Fig. 5B). Although the amount of each TCR chain associated with TSA-1 was relatively small compared with the total amount on the cell surface, every component of the TCR complex, TCR-alpha beta heterodimers and CD3delta , -epsilon , -gamma and -zeta chains, seemed to be co-precipitated with TSA-1.


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Fig. 5.   Association between TSA-1/Sca-2 and the TCR on the cell surface. A, 2B4 cells were surface biotinylated and solubilized in digitonin lysis buffer. The lysates were immunoprecipitated with various mAbs as indicated, and analyzed by 14% SDS-PAGE. 1 × 107 cell equivalent/lane was immunoprecipitated with NRIgG, GR12 (anti-TSA-1), M17/5.2 (anti-LFA-1), or 6C3 (anti-Ly-6C), and 4 × 106 cell equivalent/lane with 2C11 (anti-CD3epsilon ). Closed arrowheads indicate TSA-1 (12 kDa), alpha  (180 kDa), and beta  (95 kDa) chains of LFA-1, and Ly-6C (16 kDa). Open arrowheads indicate TCR chains. B, the lysates of surface-biotinylated 2B4 cells were analyzed by two-dimensional SDS-PAGE. 2 × 107 cell equivalent/sample was immunoprecipitated with GR12, and 1 × 107 cell equivalent/sample with 2C11. Positions of TCR chains and TSA-1 are indicated.

The surface association between TSA-1 and the TCR in the biochemical analysis was confirmed in immunofluorescence confocal microscopy. As shown in Fig. 6, A and B, the TCR complex and TSA-1 were stained with FITC-conjugated anti-CD3epsilon mAb and biotinylated anti-TSA-1 mAb plus Texas Red streptavidin, respectively. When the relative localization of both markers was determined by merging the two images, TSA-1 (red) was found to co-localize extensively with the TCR (green), yielding a yellow fluorescence signal (Fig. 6C). Taken together, these results demonstrated that the TCR/TSA-1 complex is expressed on the surface of 2B4 T cell hybridomas.


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Fig. 6.   Co-localization of TSA-1/Sca-2 and the TCR on the cell surface by immunofluorescence confocal microscopy. 2B4 cells were stained with FITC-conjugated anti-CD3epsilon mAb (2C11) (A), biotinylated anti-TSA-1 mAb (PRST1) plus Texas Red streptavidin (B), or FITC-conjugated anti-CD3epsilon mAb followed by biotinylated anti-TSA-1 mAb plus Texas Red streptavidin (C). Optically merged images are shown such that coincident staining appears yellow. Although the staining patterns for CD3epsilon and TSA-1/Sca-2 were not identical, extensive colocalization was observed.

Activation-dependent Association between TSA-1/Sca-2 and the CD3zeta in Normal T Cells-- We next examined whether TSA-1 can also be associated with CD3zeta in normal T cells. Since mRNA and protein expression of TSA-1 cannot be detected in resting T cells (6), no association was observed when we used freshly isolated T cells for immunoprecipitation analysis (data not shown). However, a clear co-precipitation of CD3zeta was detected in anti-TSA-1 immunoprecipitates from concanavalin A-activated T cells (Fig. 7). The association seemed to be specific, since the immunoprecipitation with anti-LFA-1 mAb did not co-precipitate any CD3zeta (Fig. 7).


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Fig. 7.   Association between TSA-1/Sca-2 and CD3zeta chains in normal activated T cells. Splenic T cells were incubated with concanavalin A for 3 days. Cells were harvested and solubilized in lysis buffer. The lysates were analyzed by immunoprecipitation and immunoblotting as described in the legend of Fig. 1. 3 × 107 cell equivalent/lane was immunoprecipitated with NRIgG, GR12, or M17/5.2, and 3 × 106 cell equivalent/lane with 2C11. CD3zeta is indicated by an arrowhead. Molecular sizes (kDa) are shown on the left.

Physical Association between TSA-1/Sca-2 and the CD3zeta in Jurkat-derived Transfectants Expressing a Transmembrane Form of TSA-1-- We had established three types of Jurkat-derived transfectants in our previous study (9). J2A11 cells were transfected with the expression vector alone, J6C4 cells with wild-type GPI-anchored TSA-1, and J4B1 cells with a transmembrane form of TSA-1, which consisted of the transmembrane and cytoplasmic portion of class I Db fused to the extracellular portion of TSA-1. The immunoprecipitation and immunoblotting analysis was performed using these transfectants to determine whether the attachment to the plasma membrane via the GPI anchor is required for the physical association between TSA-1 and CD3zeta . As shown in Fig. 8, CD3zeta was bound to TSA-1 in J6C4 cells but not in J2A11 cells, showing the ability of murine GPI-anchored TSA-1 to associate with human CD3zeta . In J4B1 cells, although the amount of CD3zeta bound to TSA-1 was small compared with that observed in J6C4 cells, the association of CD3zeta with a transmembrane TSA-1 was apparently identified. Moreover, in COS-7 cells transfected with a transmembrane TSA-1 together with CD3zeta , the association between these two molecules was clearly observed (Fig. 4D). Thus, the GPI anchor does not seem to be critical for the interaction between TSA-1 and CD3zeta , which is concordant with our previous data regarding the functional role of TSA-1 in TCR signaling.


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Fig. 8.   Association between TSA-1/Sca-2 and CD3zeta chains in Jurkat-derived transfectants. Jurkat cells transfected with the vector encoding GPI-anchored TSA-1 (J6C4) or a transmembrane TSA-1 (J4B1), or with vector alone (J2A11) were solubilized in digitonin lysis buffer. Postnuclear supernatants were immunoprecipitated with NRIgG (normal rat IgG), GR12 (anti-TSA-1 mAb), or H146 (anti-CD3zeta mAb). 3 × 107 cell equivalent/lane was immunoprecipitated with NRIgG or GR12, and 3 × 106 cell equivalent/lane with H146. Immunoprecipitates were subjected to 14% SDS-PAGE under reducing condition, and immunoblotted with anti-CD3zeta antiserum, no. 387. CD3zeta is indicated by an arrowhead. Molecular sizes (kDa) are shown on the left.

Functional Association between TSA-1/Sca-2 and the CD3zeta -- Finally, we assessed whether biochemical changes could be induced in CD3zeta by activating through TSA-1. To this end, we analyzed tyrosine phosphorylation of CD3zeta chains from 2B4 T cell hybridomas after stimulation with anti-TSA-1 in the presence with accessory cells. As shown in Fig. 9, stimulation of 2B4 cells with 2C11 resulted in tyrosine phosphorylation of CD3zeta . In contrast, stimulation with anti-LFA-1 or anti-class I did not induce any CD3zeta phosphorylation, indicating that engagement of any cell surface molecules by the mAb does not generally lead to the induction of CD3zeta phosphorylation. However, when 2B4 cells were stimulated through TSA-1 with GR12 or PRST1, the induction of tyrosine phosphorylation of CD3zeta was evident (Fig. 9). Interestingly, the amount of phosphorylated CD3zeta induced with PRST1 was much greater than that induced with GR12, although GR12 co-immunoprecipitated CD3zeta more efficiently than did PRST1 (see Fig. 2). The result suggested that the direct physical association of TSA-1 to CD3zeta leads to positive signaling events such as phosphorylation in CD3zeta chains when the signal was delivered to TSA-1 by cross-linking with the mAbs.


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Fig. 9.   Tyrosine phosphorylation of CD3zeta induced by cross-linking of TSA-1/Sca-2. 2B4 cells (1.5 × 107) were stimulated with the indicated antibodies in the presence of LK cells (7.5 × 106) for 30 min at 37 °C. Cells were pelleted by centrifugation, lysed, and immunoprecipitated with A2B4 (anti-TCR-alpha mAb). 90% of each immunoprecipitate was blotted with an anti-phosphotyrosine mAb, 4G10 (upper panel), whereas 10% was blotted with an anti-CD3zeta mAb, H146-968 (lower panel). Phophorylated CD3zeta is indicated by an arrowhead. The bands migrating at approximately 55 and 24 kDa in each lane are likely to be the heavy and light chains of the mAbs used for immunoprecipitation. Molecular sizes (kDa) are shown on the left.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Many rodent and human GPI-anchored proteins have been implicated in regulation of T cell activation, since mAbs against these GPI-anchored proteins induce T cell activation as monitored by interleukin-2 production and proliferation. T cell activation induced by a signal through GPI-anchored proteins is dependent upon expression of the TCR; anti-Thy-1 and anti-Ly-6 mAbs fail to stimulate a TCR- variant cell line, and the defect was able to be restored by TCR expression in these variant cell lines (22, 23). In addition to the positive regulation by GPI-anchored proteins in T cell activation, some GPI-anchored proteins transduce a negative signal that inhibits anti-CD3-mediated TCR signaling (24). We have previously demonstrated that a signal via TSA-1/Sca-2 inhibits TCR/CD3-mediated activation and apoptosis both in vitro and in vivo (6, 9, 10). Thus, the TCR seems to be an essential molecule in signaling pathway of GPI-anchored proteins at least in T cells.

A number of studies have indirectly suggested that there is a physical and/or functional association between some GPI-anchored proteins and the TCR. By using chemical cross-linkers, it was reported that CD45 is mutually associated with Thy-1 and the TCR, indicating that Thy-1 can physically interact with the TCR through CD45 (25). In another study, a T cell clone was stably transfected with antisense Ly-6A RNA (26). Cell surface expression of Ly-6A was markedly suppressed in this transfectant, but surprisingly surface expression of the TCR was greatly inhibited as well because of the reduction of TCR-beta mRNA. The Ly-6A antisense transfectant was then transfected with TCR-beta cDNA, and surface TCR expression was reconstituted without the expression of Ly-6A. However, TCR signaling was still impaired in this transfectant due to the absence of Ly-6A.

Despite these observations, it seems to be very difficult to demonstrate a direct association of the TCR to Thy-1, Ly-6, or other GPI-anchored proteins in immunoprecipitation analysis. Nonetheless, we are able to provide evidence that TSA-1/Sca-2 is physically associated with TCR in the present study. When TSA-1/Sca-2 expressed on the cell surface was stimulated with anti-TSA-1 mAbs, CD3zeta in the TCR complex was induced to be phosphorylated in its tyrosine residues (Fig. 9). This result indicates that a functional association also exists between these two molecules, and argues against the possibility that the interaction between TSA-1 and CD3zeta occurs merely during the process of solubilization and immunoprecipitation.

We do not know why we can successfully detect the physical association of the TCR to TSA-1 among many GPI-anchored proteins. Since none of mAbs against Thy-1, Ly-6A/E, and Ly-6C co-precipitated CD3zeta in an experiment in which both mAbs, PRST1 and GR12, against TSA-1 clearly co-precipitated CD3zeta (Fig. 2), the association between TSA-1 and CD3zeta is considered to be specific. Given that TSA-1 is a GPI-anchored protein and CD3zeta has a very short extracellular portion, the interaction between TSA-1 and CD3zeta could be mediated by an as yet undefined membrane protein, which could serve as a linker between these two proteins (27). This "linker" protein presumably functions not only in T cells but in COS-7 cells (Fig. 4). Moreover, the association between TSA-1 and the "linker" protein could not be dependent on the GPI anchor, but on primary sequence motifs of TSA-1. An effort should be made to identify the "linker" protein in biochemical analysis.

Alternatively, another possibility may account for the mechanism underlying the physical association between TSA-1 and CD3zeta . GPI-anchored proteins are known to be localized to caveolae, glycosphingolipid-rich areas in the cell membrane (28-30). Caveolae are also enriched in signal-transducing molecules, such as GTP-binding proteins, small G proteins, and nonreceptor-type tyrosine kinases (31). It has been proposed that caveolae could represent a specialized signaling compartment at the cell surface (32). Although lymphocytes do not have caveolae due to the lack of caveolin, there is the same membrane microdomain that are enriched in glycosphingolipids in lymphocytes (33). Thus, if the TCR complex or the CD3zeta may reside in this microdomain, GPI-anchored proteins could be associated with CD3zeta by lipid-protein interactions, thereby forming a signaling compartment at the surface of T cells. Stimulation of GPI-anchored proteins with mAbs results in the delivery of a signal through this signaling compartment. If this possibility is correct, TSA-1 is not special among other GPI-anchored proteins, but our mAbs against TSA-1 could be special among other mAbs against GPI-anchored proteins. Although we can not thus far explain whether and how a transmembrane TSA-1 could be localized in this signaling compartment, the above hypothesis is very attractive, given that most of GPI-anchored proteins have a signal-transducing ability when cross-linked with mAbs.

Although the mechanism is not fully understood, our previous finding that a signal through TSA-1 down-modulates the TCR signaling pathway could be explained by the physical association between TSA-1 and CD3zeta . Cross-linking of TSA-1 with the mAb induces the phosphorylation of tyrosine residues in CD3zeta chains (Fig. 9) through the activation of the Src family tyrosine kinases, which may subsequently cause recruitment of another protein tyrosine kinase, ZAP-70 (34). Thus, intracellular signal-transducing molecules could be sequestered from TCR-signaling pathways to the TSA-1/CD3zeta complex, resulting in down-modulation of TCR signaling. Studies are in progress to elucidate a molecular mechanism for the TSA-1-signaling pathway.

    ACKNOWLEDGEMENT

We are grateful to Dr. Yasuhiro Minami for helpful discussions.

    FOOTNOTES

* This work was supported by Grant-in-aid 08839014 from the Ministry of Education for Scientific Research and by a grant from Precursory Research for Embryonic Science and Technology, Research Development Corporation of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence and reprint requests should be addressed: School of Allied Health Science, Faculty of Medicine Osaka University, 1-7, Yamada-oka, Suita, Osaka 565, Japan. Tel./Fax: 81-6-879-2599; E-mail: kosugi{at}sahs.med.osaka-u.ac.jp.

1 The abbreviations used are: TSA-1, thymic shared Ag-1; Sca-2, stem cell Ag-2; TCR, T cell receptor; GPI, glycosylphosphatidylinositol; LFA-1, leukocyte function-associated antigen-1; FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; NRIgG, normal rat immunoglobulin G.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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