Physical and Functional Association between Thymic Shared
Antigen-1/Stem Cell Antigen-2 and the T Cell Receptor Complex*
Atsushi
Kosugi
§,
Shin-ichiroh
Saitoh¶,
Satoshi
Noda¶,
Kensuke
Miyake
,
Yoshio
Yamashita
,
Masao
Kimoto
,
Masato
Ogata¶, and
Toshiyuki
Hamaoka¶
From the
School of Allied Health Sciences, Faculty of
Medicine, Osaka University, Osaka 565, ¶ Biomedical Research
Center, Osaka University Medical School, Osaka 565, and
Department of Immunology, Saga Medical School, Saga
849, Japan
 |
ABSTRACT |
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 CD3
chains in T cell hybridomas,
activated T cells, and COS-7 cells transfected with TSA-1 and CD3
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 CD3
did not require
the GPI anchor of TSA-1. Finally, tyrosine phosphorylation of CD3
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 CD3
underlies a
regulatory role of TSA-1/Sca-2 in the TCR-signaling pathway.
 |
INTRODUCTION |
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 CD3
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
CD3
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 |
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-CD3
; H146-968 (15),
anti-CD3
; A2B4 (11), anti-clonotypic antibody recognizing TCR-
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 CD3
, 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 CD3
were performed as described previously (9).
 |
RESULTS |
Physical Association between TSA-1/Sca-2 and the CD3
--
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-CD3
mAb, no physical association between
TSA-1 and CD3
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 CD3
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 CD3
,
indicating that the association between TSA-1 and CD3
is
specific.

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Fig. 1.
Association between TSA-1/Sca-2 and CD3
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-CD3 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-CD3 mAb, H146-968, and
horseradish peroxidase-conjugated protein A. CD3 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.
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We further examined whether the association of CD3
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-CD3
(Fig. 2). Again, the
association of CD3
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 CD3
. Since these control mAbs were able to
immunoprecipitate their corresponding surface Ags efficiently (data not
shown), the result demonstrated a selectivity of CD3
chains for the
ability to associate with TSA-1 among GPI-anchored molecules.

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Fig. 2.
Co-precipitation of CD3 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-CD3 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-CD3 ; and NRIgG, normal rat IgG. 1 × 107 cell equivalent/lane was analyzed. CD3 is indicated
by an arrowhead. Molecular sizes (kDa) are shown on the
left.
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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-CD3
or with anti-CD3
. The result
of Fig. 3 clearly demonstrated that TSA-1
was associated with CD3
as well as CD
. Since CD3
was not bound
to TSA-1 in the 2B4 mutant that lacks expression of CD3
, the
association between TSA-1 and CD3
seemed to be dependent on the
existence of CD3
chains (data not shown).

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Fig. 3.
Association between TSA-1/Sca-2 and CD3
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-CD3 mAb),
subjected to two-dimensional SDS-PAGE, and immunoblotted with
anti-CD3 mAb (A and B) or with anti-CD3 mAb
(C and D). 2 × 107 cell
equivalent/sample was immunoprecipitated with GR12, and 5 × 106 cell equivalent/sample with 2C11. Positions of CD3
or - chains are indicated. The diagonal of the gel is indicated by a
broken line.
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To investigate the direct association of TSA-1 to CD3
, COS-7 cells
were transiently transfected with TSA-1 cDNA, CD3
cDNA, or
both, and analyzed for the association (Fig.
4). A band migrating at 16 kDa was
detected in COS-7 cells transfected with CD3
(Fig. 4B),
confirming the band as CD3
. When COS-7 cells were transfected with
TSA-1 in combination with CD3
, CD3
was clearly co-precipitated in
anti-TSA-1 immunoprecipitates (Fig. 4C), demonstrating that the association between TSA-1 and CD3
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 CD3
chains in COS-7 cells. COS-7 cells were transfected with
GPI-anchored TSA-1 (A), CD3 (B), GPI-anchored
TSA-1 plus CD3 (C), or a transmembrane TSA-1 plus CD3
(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-CD3 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-CD3 mAb, H146-968. CD3 is
indicated by an arrowhead. Molecular sizes (kDa) are shown
on the left.
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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-
, and CD3
and -
chains, respectively. The bands migrating at 21 kDa in anti-TSA-1
immunoprecipitates might correspond to CD3
, but the mobility of the
bands was lower than that of CD3
in anti-CD3
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-
heterodimers and CD3
, -
, -
and -
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-CD3 ). Closed arrowheads indicate TSA-1
(12 kDa), (180 kDa), and (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.
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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-CD3
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-CD3 mAb (2C11)
(A), biotinylated anti-TSA-1 mAb (PRST1) plus Texas Red
streptavidin (B), or FITC-conjugated anti-CD3 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 CD3 and
TSA-1/Sca-2 were not identical, extensive colocalization was
observed.
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Activation-dependent Association between TSA-1/Sca-2
and the CD3
in Normal T Cells--
We next examined whether TSA-1
can also be associated with CD3
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 CD3
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 CD3
(Fig. 7).

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Fig. 7.
Association between TSA-1/Sca-2 and CD3
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. CD3 is indicated by an arrowhead. Molecular
sizes (kDa) are shown on the left.
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Physical Association between TSA-1/Sca-2 and the CD3
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 CD3
. As shown in Fig.
8, CD3
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 CD3
. In J4B1 cells, although the amount of CD3
bound to TSA-1 was small compared with
that observed in J6C4 cells, the association of CD3
with a
transmembrane TSA-1 was apparently identified. Moreover, in COS-7 cells
transfected with a transmembrane TSA-1 together with CD3
, 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 CD3
, 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 CD3
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-CD3 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-CD3 antiserum, no. 387. CD3 is indicated
by an arrowhead. Molecular sizes (kDa) are shown on the
left.
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Functional Association between TSA-1/Sca-2 and the
CD3
--
Finally, we assessed whether biochemical changes could be
induced in CD3
by activating through TSA-1. To this end, we analyzed tyrosine phosphorylation of CD3
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 CD3
. In
contrast, stimulation with anti-LFA-1 or anti-class I did not induce
any CD3
phosphorylation, indicating that engagement of any cell
surface molecules by the mAb does not generally lead to the induction
of CD3
phosphorylation. However, when 2B4 cells were stimulated
through TSA-1 with GR12 or PRST1, the induction of tyrosine
phosphorylation of CD3
was evident (Fig. 9). Interestingly, the
amount of phosphorylated CD3
induced with PRST1 was much greater
than that induced with GR12, although GR12 co-immunoprecipitated CD3
more efficiently than did PRST1 (see Fig. 2). The result suggested that
the direct physical association of TSA-1 to CD3
leads to positive
signaling events such as phosphorylation in CD3
chains when the
signal was delivered to TSA-1 by cross-linking with the mAbs.

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Fig. 9.
Tyrosine phosphorylation of CD3 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- mAb). 90% of each
immunoprecipitate was blotted with an anti-phosphotyrosine mAb, 4G10
(upper panel), whereas 10% was blotted with an anti-CD3
mAb, H146-968 (lower panel). Phophorylated CD3 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.
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 |
DISCUSSION |
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-
mRNA.
The Ly-6A antisense transfectant was then transfected with TCR-
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, CD3
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 CD3
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 CD3
in an
experiment in which both mAbs, PRST1 and GR12, against TSA-1 clearly
co-precipitated CD3
(Fig. 2), the association between TSA-1 and
CD3
is considered to be specific. Given that TSA-1 is a GPI-anchored
protein and CD3
has a very short extracellular portion, the
interaction between TSA-1 and CD3
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 CD3
. 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 CD3
may reside in
this microdomain, GPI-anchored proteins could be associated with CD3
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
CD3
. Cross-linking of TSA-1 with the mAb induces the phosphorylation
of tyrosine residues in CD3
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/CD3
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.
 |
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