From the Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas, Facultad de Biología, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
Received for publication, May 20, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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Engagement of the During intrathymic development of However, the level of expression of a cell surface receptor is the
result of an equilibrium between the synthesis and transport of new
polypeptides and their internalization, recycling, and degradation
(reviewed in Ref. 11). Extracellular stimuli induce changes in one or
several of these processes and therefore modify the level of expression
of a given receptor. In the particular case of the TCR, receptor
engagement by its natural ligands results in down-regulation of TCR-CD3
cell surface expression (11-14), which is a critical event intimately
associated with TCR signaling and T-cell activation (14). Different
molecular mechanisms have been proposed to account for the
down-modulation of ligated TCR complexes. Most studies support the
position that TCR ligation results in a significant increase of
the TCR internalization rate followed by the degradation of the
internalized complex (15, 16). However, TCR-CD3 complexes are
continuously internalized and recycled back to the cell surface in
nonstimulated T cells (17, 18), suggesting that constitutive TCR
recycling on resting T cells has to be affected by TCR ligation. In
this regard, Wiest and co-workers (19) have recently proposed that TCR
engagement has little effect on the TCR internalization rate; rather,
it prevents TCR recycling back to the cell surface by inducing the intracellular retention of ligated complexes and their degradation by
lysosomes and proteasomes.
In sharp contrast to the TCR, pre-TCR signaling occurs apparently
without any need for ligation (5, 6). This concurs with the finding
that the pre-TCR spontaneously clusters and localizes on the cell
surface into lipid rafts (20, 21), in a manner similar to that found
for the Cell Lines and Transfectants--
The Internalization and Recycling Assays--
SUP-T1 cells and
Immunoprecipitation and Anti-TCR Surface Biotinylation, Immunoprecipitation, and
Immunoblotting--
Cells (107/ml) either untreated or
pretreated for 2 h with 50 µM chloroquine, 20 µM lactacystin, or 1 µM epoxomicin
(Calbiochem) were washed three times in complete 1× PBS at 4 °C and
labeled with 0.5 mg/ml Sulfo-NHS-biotin (Pierce) in complete 1× PBS
for 30 min on ice. Excess biotin was eliminated by washing the cells three times with 10 mM L-lysine (Sigma) in 1×
PBS. Then, cells were cultured (5-10 × 106/ml) in
the absence or presence of the indicated lysosome or proteasome inhibitors. At each time point, cells (20 × 106/immunoprecipitation) were washed with 1× PBS,
lysed with 1% Brij, and immunoprecipitated as described above with the
UCHT-1 or SP-34 (kindly provided by Dr. B. Alarcón) anti-CD3 Confocal Microscopy and Colocalization Analysis--
Confocal
microscopy was performed essentially as described previously (9). Cells
were adhered to poly-L-lysine (Sigma)-precoated coverslips,
fixed with 4% paraformaldehyde in PBS for 15 min at 4 °C, and
stained at the cell surface with an anti-CD4 mAb (BD Biosciences). For
intracellular staining, fixed cells were permeabilized with a
commercial lysing solution (BD Biosciences) and stained with the
anti-CD63 mAb TEA3/18 (VI International Leukocyte Typing Workshop),
kindly provided by Dr. F. Sánchez-Madrid (Hospital de la
Princesa, Madrid, Spain). Rhodamine Red X-coupled goat anti-mouse Igs
(Molecular Probes) was used as the second-step reagent for both surface
and cytoplasmic staining. Samples were examined under a confocal
microscope (Radiance 2000, Bio-Rad Laboratories) coupled to an Axiovert
S100TV inverted microscope (Zeiss). Serial optical sections were
recorded at 0.3-0.5 µm intervals with a 63× lens under an optimal
iris setup. Colocalization analyses were performed using
Metamorph, version 5.03, software (Universal Imaging).
Surface Pre-TCR-CD3 Complexes Are Continually Internalized but Do
Not Recycle Back to the Cell Surface--
To study the dynamics of
pre-TCR cell surface expression, we used a human pre-T cell line,
SUP-T1, which has been shown to display low pre-TCR surface levels as
found on primary human pre-T cells (Ref. 9 and Fig.
1A). The relative contribution
of synthesis and secretion of new chains as compared with
internalization and recycling of expressed ones to actual pre-TCR-CD3
surface levels was explored by comparative cycloheximide or brefeldin-
A (BFA) treatment, as described recently for the mature TCR-CD3 complex (19). Pre-TCR surface expression was measured with an anti-TCRV
In sharp contrast to the TCR, surface expression of the pre-TCR was
dramatically affected by cycloheximide, indicating that it is dependent
on newly synthesized complexes. Pre-TCR levels fell to less than 20%
of control levels after 6 h of treatment and remained essentially
undetectable after 12 h (Fig. 1C). BFA also induced a
rapid and marked decrease on pre-TCR surface expression (80% after
2 h of treatment), which resulted in the down-modulation of the
complex (Fig. 1, A and B). These data suggest
that pre-TCR-CD3 complexes are continually internalized from the
surface of SUP-T1 pre-T cells, with internalization rates that are
apparently higher than those for the TCR-CD3. Surprisingly, however,
endocytosed pre-TCR-CD3 complexes do not recycle back to the cell surface.
The pT TCR
Supporting this possibility, confocal microscopy analysis showed that
untreated
It has been shown that the primary mechanism mediating down-modulation
of the TCR upon ligand binding involves targeting of endocytosed
complexes for intracellular degradation, predominantly by lysosomes,
but also by proteasomes (16, 19). To investigate whether proteasome was
also involved in degradation of endocytosed pre-TCR or ChTCR complexes,
biochemical studies were performed aimed at analyzing the intracellular
fate of TCR The pT
To investigate the intracellular degradation pathway followed by the
pT Molecular Mechanisms Involved in Constitutive Pre-TCR
Down-regulation--
Phosphorylation of the cytoplasmic tail of CD3
An intriguing possibility is that protein tyrosine kinases (PTK) such
as Lck, which might play a key role in cell-autonomous signaling
through the pre-TCR (20) could participate in its down-regulation, as
shown for engaged TCR-CD3 complexes (29). To address this possibility,
we analyzed the effects of the src family (Lck/Fyn) PTK
inhibitor PP2 on surface pre-TCR expression. As shown in Fig.
6B, inhibition of src kinases resulted in
increased expression levels of the pre-TCR and the ChTCR in SUP-T1 and
In this study, comparative analyses on the dynamics of human
pre-TCR and TCR cell surface expression and down-modulation revealed striking differences in the behavior and intracellular fate of unligated TCR and pre-TCR complexes. We have shown that TCR-CD3 complexes expressed on SUP-T1 cells, upon transfection with TCR Ligand-independent activation of the pre-TCR has recently been proposed
to result from its constitutive co-localization in membrane rafts with
signaling molecules, such as Lck (20), which may trigger
cell-autonomous activation of proximal signaling including CD3 Alternatively, it can be proposed that constitutive internalization and
degradation of the pre-TCR depends on unique endocytosis and/or
degradation motifs. In this regard, the CD3 and TCR T cell receptor (TCR) by
its ligand results in the down-modulation of TCR cell surface
expression, which is thought to be a central event in T cell
activation. On the other hand, pre-TCR signaling is a key process in
T cell development, which appears to proceed in a constitutive
and ligand-independent manner. Here, comparative analyses on the
dynamics of pre-TCR and TCR cell surface expression show that unligated
pre-TCR complexes expressed on human pre-T cells behave as engaged TCR
complexes, i.e. they are rapidly internalized and degraded
in lysosomes and proteasomes but do not recycle back to the cell
surface. Thus, pre-TCR down-regulation takes place constitutively
without the need for extracellular ligation. By using TCR
/pT
chain chimeras, we demonstrate that prevention of recycling and
induction of degradation are unique pre-TCR properties conferred by the
cytoplasmic domain of the pT
chain. Finally, we show that pre-TCR
internalization is a protein kinase C-independent process that
involves the combination of src
kinase-dependent and -independent pathways. These data suggest that constitutive pre-TCR down-modulation regulates pre-TCR surface expression levels and hence the extent of
ligand-independent signaling through the pre-TCR.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
T cells, thymocytes that
have a successful rearrangement at the T cell receptor
(TCR
)1 locus express a
pre-TCR complex composed of the TCR
chain paired with the invariant
pre-TCR
(pT
) chain and associated with CD3 components (1-3).
Surface expression of this pre-TCR (4) triggers the selection,
expansion, and further differentiation of developing pre-T cells in a
ligand-independent manner (5, 6), finally resulting in the induction of
rearrangements at the TCR
locus. Upon productive TCR
gene
rearrangements, the TCR
chain pairs with TCR
and associates with
CD3
, -
, -
, and -
chains, and thymocytes undergo a second
step of selection upon binding of the TCR
to self-peptide-major
histocompatibility complex molecules (1-3). Despite experimental
evidence on the similar biochemical compositions of the pre-TCR and TCR
in terms of their associated CD3 subunits (7-9), current studies
support the theory that mechanisms regulating the assembly and
intracellular transport of these complexes may differ markedly, because
the pre-TCR is expressed only transiently and very inefficiently during
thymocyte development, at levels about 50-100-fold lower than those of
the TCR on mature T cells (3, 10). By using TCR
-pT
chain
chimeras, we have shown recently (9) that limited expression of
the human pre-TCR is pT
chain-dependent. Particularly,
the pT
cytoplasmic (Cy) domain was found to serve an endoplasmic
reticulum retention function that could contribute in part to the
regulation of pre-TCR assembly and surface expression (9).
TCR following ligand binding (22, 23). These findings
support the view that surface pre-TCR complexes are constitutively
activated without any need for ligation (20) and raise the question of
what is the intracellular fate of such "activated" pre-TCR
complexes. In this study, we have analyzed the dynamics of pre-TCR-CD3
cell surface expression and down-modulation in unstimulated pre-T cells
and show that, similar to ligated TCR complexes, surface pre-TCR
complexes are continually and rapidly endocytosed and degraded in the
absence of extracellular ligation but do not recycle back to the cell
surface. Moreover, we show that cell-autonomous pre-TCR down-modulation
depends on the pT
cytoplasmic tail. The possibility that
constitutive endocytosis and degradation of the pre-TCR is a self-safe
mechanism responsible for its limited expression on the cell surface is discussed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
wt and
/CypT
stable transfectants were derived as described elsewhere (9) from the
pre-T cell line SUP-T1, which expresses an endogenous TCR
(V
1)
chain and the pT
chain but lacks TCR
. Briefly, SUP-T1 cells were
transfected, respectively, either with full-length cDNAs encoding a
conventional TCR
(V
12.1) chain or with a TCR
/pT
chimeric
construct in which the Cy domain of TCR
was replaced by the
equivalent domain of pT
(CypT
). G418-selected transfectants were
grown in RPMI 1640 (BioWhittaker) supplemented with 10% fetal calf
serum (Invitrogen). Likewise, TCR
-GFP stable transfectants
were derived from SUP-T1 cells transfected with a plasmid encoding a
COOH-terminal fusion protein of the TCR
chain with the green
fluorescence protein (GFP). The TCR
-GFP fusion was performed by PCR
amplification of a complete TCR
chain cDNA (kindly provided by
Dr. B. Alarcón, Centro de Biología Molecular Severo
Ochoa, Madrid, Spain) with the sense 5'-CGC GCG CCC GGG ATG AAG TGG AAG
GCG CTT-3' and antisense 5'-GCG CGC CCG GGC CCC GCG AGG GGG CAG GGC-3'
primers followed by digestion and ligation into the SmaI
site of the pEGFP-N1 plasmid vector (Clontech). Pre-TCR surface expression levels analyzed on 48 selected TCR
-GFP clones were similar to those on parental SUP-T1 cells.
wt,
/CypT
, and TCR
-GFP stable transfectants were cultured
(5-8 × 105/ml) in 24-well plates (Costar) either in
the absence or presence of cycloheximide (40 µg/ml, Calbiochem),
brefeldin-A (1 µg/ml, Sigma), phorbol 12-myristate 13-acetate (10 ng/ml, Calbiochem), and/or PP2 (2 µM, Calbiochem). At
different time points, surface expression of the endogenous pre-TCR was
analyzed by flow cytometry (EPICS XL Coulter Corp.) with
phycoerythrin-labeled anti-human TCR
-V
1 (Endogen) or
anti-CD3
(BD Biosciences) mAbs. Surface levels of the conventional
TCR
-TCR
(TCR) or the chimeric TCR
/CypT
-TCR
(ChTCR)
receptors expressed respectively on
wt and
/CypT
transfectants was analyzed with the anti-TCR
mAb BMA031 (Behringwerke AG) plus
phycoerythrin-labeled F(ab')2 goat anti-mouse Igs
(Caltag Laboratories). The percentage of surface expression at the
indicated time points was determined from the mean fluorescence values
of treated cells using the untreated controls as reference. Background values were determined with isotype-matched irrelevant antibodies.
Immunoblotting--
Immunoprecipitation and immunoblotting were
performed as described previously (9). In brief, cells were cultured as
described above in the presence or absence of cycloheximide (40 µg/ml) with or without inhibitors of lysosomal degradation
(NH4Cl, 40 mM, Merck; or chloroquine, 50 µM, Sigma) or a proteasome inhibitor (lactacystin, 20 µM, Sigma) for 8 h. Then, cells (5 × 106/immunoprecipitation) were washed in 1× PBS,
lysed with 1% Brij 96-containing lysis buffer and immunoprecipitated
as described (9) with either anti-TCR
(
F1, Endogen) or
anti-CD3
(UCHT-1, ATCC AN 1009425) mAbs. Immunoprecipitated proteins
were resolved by 12% SDS-PAGE under nonreducing conditions and
transferred onto polyvinylidene difluoride membranes
(ImmobilonTM-P, Millipore). Blots were probed with the
anti-human TCR
rabbit antiserum 448 (kindly provided by Dr. B. Alarcón) plus horseradish peroxidase-labeled polyclonal donkey
anti-rabbit Igs (Amersham Biosciences) and then developed using ECL
Lumi-LightPLUS (Roche Molecular Biochemicals).
Densitometric analysis was performed with a Bioimaging BAS 1500 (Fujifilm).
mAb. The immunoprecipitates were resolved by 12% SDS-PAGE under
nonreducing conditions, and blots were probed with
streptavidin-horseradish peroxidase (Pierce) and revealed by the ECL
method. When indicated, blots were stripped and reprobed with the 448 anti-TCR
antiserum or an anti-
-tubulin mAb (Sigma) as above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 mAb
(9). The dynamics of TCR-CD3 surface expression was analyzed for
comparison in the same cellular environment, namely SUP-T1 cells stably
transfected with a TCR
chain (
wt transfectants), which
co-expressed the conventional TCR
-CD3 complex (>99%
TCR
+) together with the endogenous pre-TCR (Ref. 9,
and Fig. 1A). The results confirmed that, as described (19),
the mature surface TCR-CD3 was relatively synthesis-independent, that
is, surface TCR-CD3 expression levels remained essentially stable
within the studied 12-h time period (Fig. 1C). Therefore,
mature TCR complexes are long-lived on
wt transfectants. As reported
(19), the effect of BFA on TCR expression was very different from that
of cycloheximide and resulted in a partial reduction of TCR membrane
levels (Fig. 1A), which fell rapidly during the first 2 h of treatment (about 30% reduction) and remained low (50% of control
levels) for 8 h thereafter (Fig. 1B). Because
surface expression of the mature TCR was found independent of newly
synthesized complexes, this reduction in TCR surface levels cannot be
due to the reported BFA-induced blockade of the anterograde transport
from the endoplasmic reticulum to the Golgi complex (24, 25). Rather,
it may be caused by the documented capacity of BFA to induce tubulation and fusion of the trans-Golgi network with early endosomes, which, although previously reported to leave cycling between plasma membrane and endosomes of certain molecules such as transferrin unperturbed (24), has recently been shown to affect the endocytic transport of the
TCR (19, 26). Therefore, our data indicate that TCR-CD3 complexes
expressed on SUP-T1
wt transfectants are continually internalized
and recycled back to the cell surface and thus behave as conventional
TCR-CD3 complexes on resting T cells (19).
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Fig. 1.
Surface pre-TCR and chimeric
TCR-pT (ChTCR) complexes
containing the pT
cytoplasmic domain are
constitutively internalized but do not recycle back to the cell
surface. A, SUP-T1 untransfected cells or cells stably
transfected with a TCR
(
wt) or with a chimeric
/CypT
chain (
/CypT
) were treated for
6 h with cycloheximide (thin line) or brefeldin-A
(thick line) or were left untreated (shaded
histogram). Pre-TCR surface expression on SUP-T1 cells was
determined by flow cytometry with a phycoerythrin-labeled mAb against
the endogenous TCR
(V
1) chain. Surface expression of TCR and
ChTCR complexes on
wt and
/CypT
transfectants, respectively,
was assessed with the BMA031 mAb against monomorphic determinants of
human TCR
heterodimer. The effect of brefeldin-A (B)
and cycloheximide (C) on the expression levels of the
endogenous pre-TCR on SUP-T1 cells, the TCR
on
wt
transfectants, and the ChTCR on
/CypT
transfectants was analyzed
by flow cytometry at the indicated time points, as described in
A. The percentage of surface expression was determined from
the mean fluorescence values of treated cells, using the untreated
controls as reference. Results are representative of four independent
experiments.
Chain Cytoplasmic Domain Is Responsible for the Impaired
Recycling of the Pre-TCR-CD3 Complex--
Because the pre-TCR and TCR
are co-expressed on
wt transfectants, the differential intracellular
fate of the two complexes cannot be cell type-dependent but,
rather, is structure-dependent (i.e. the substitution of
pT
in the pre-TCR with TCR
in the TCR). To uncover the structural
properties of pT
that are responsible for the impaired recycling of
the endocytosed pre-TCR, TCR
/pT
chimeric constructs involving
distinct pT
and TCR
domains were stably transfected into SUP-T1
cells (9), and the surface expression dynamics of the resulting
ChTCR was analyzed by flow cytometry. Particularly, we focused
on
/CypT
chimeric constructs in which the Cy domain of TCR
was
replaced by the equivalent domain of pT
. As shown Fig.
1A, the effects of BFA and cycloheximide on surface
expression of the resulting
/CypT
-TCR
ChTCR, measured with an
anti-TCR
mAb, were equivalent to those observed on endogenous pre-TCR expression. BFA induced a significant reduction of ChTCR expression levels after 2 h, which was followed by the complete loss of the complex after 6 h of treatment (Fig. 1, A
and B), and cycloheximide treatment also resulted in ChTCR
down-modulation (Fig. 1C). These data indicate that the
pT
Cy domain actively mediates impaired recycling of the pre-TCR-CD3
complex to the cell surface without the need of ligand binding.
Chains Associated to Internalized pre-TCR and ChTCR
Complexes Are Degraded Intracellularly--
To investigate the
mechanisms that could account for the impaired return of endocytosed
pre-TCR (and ChTCR) complexes to the cell surface, we followed the
intracellular fate of pre-TCR-associated TCR
chains in SUP-T1 cells
stably transfected with a plasmid encoding a COOH-terminal fusion
protein of the TCR
chain with the green fluorescence protein
(
-GFP). As shown in Fig. 2A
for one representative
-GFP+ clone of four, flow
cytometry analysis allowed us to simultaneously measure the surface
levels of the pre-TCR complex and the total cellular content of the
-GFP protein. These studies revealed that, as shown in Fig. 1 for
the parental SUP-T1 pre-T cell line, pre-TCR complexes expressed on
-GFP transfectants are continually internalized from the cell
surface and become down-modulated after treatment with cycloheximide,
BFA, or both. Kinetic analysis showed that pre-TCR down-modulation
paralleled a gradual decrease of the total content of
-GFP when
protein synthesis was blocked, so that green fluorescence became barely
detectable (<20% of control expression levels) after 12 h of
treatment with cycloheximide (Fig. 2B), suggesting that the
-GFP chimeric chains, including those associated with the
internalized pre-TCR, were degraded intracellularly. In contrast,
pre-TCR down-modulation was coupled with a dramatic increase of green
fluorescence in BFA-treated cells, indicating that
-GFP protein
chimeras had accumulated in the cytoplasm. More importantly, blocking
of endosome to lysosome trafficking induced by BFA (24) counteracted
the effect of cycloheximide and prevented the disappearance of GFP
expression, which remained stable although partly reduced (
60% of
control levels) after 12 h (Fig. 2, A and
B). Therefore, loss of green fluorescence induced by
cycloheximide could be the result of selective degradation of
-GFP
fusion proteins in lysosomes.
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Fig. 2.
The pre-TCR-associated TCR
chain localize intracellularly to lysosomes.
A, Sup-T1 stable transfectants homogeneous for the
expression of a TCR
-GFP chimeric protein were cultured in the
absence (shaded histogram) or presence of cycloheximide
(CHX), BFA, or both (CHX+BFA) for 8 h.
Two-color flow cytometry analysis was performed to measure
simultaneously the surface levels of pre-TCR expression with a
phycoerythrin-coupled anti-CD3
mAb (red fluorescence) and
the total cellular content of TCR
-GFP chimeric chains (green
fluorescence). B, green fluorescence analyses
were performed at the indicated time points in
-GFP transfectants
cultured in the presence of either cycloheximide, BFA, or both. Results
are given relative to untreated cells. The results of confocal
microscopy and colocalization analyses of
-GFP transfectants, either
surface-stained with an anti-CD4 mAb (C) or permeabilized
and stained with a mAb against the lysosomal marker CD63
(D), are shown. Rhodamine Red X-coupled goat anti-mouse Ig
was used as second step reagent.
-GFP transfectants were treated
with either BFA (E) or BFA plus cycloheximide (F)
for 6 h and analyzed by confocal microscopy. Serial optical
sections were captured at 0.3-0.5 µm. The images in each panel show
a single 0.2 µm section of each cell (n = 50).
Results are representative of at least four experiments in one of four
different
-GFP clones.
-GFP transfectants had low levels of surface
-GFP that
colocalized with the membrane marker CD4 (Fig. 2C), whereas
a significant amount of green fluorescence was expressed intracellularly and accumulated in the cytoplasmic structures that
expressed the lysosomal marker CD63 (27). As shown in Fig. 2D a high proportion of
-GFP (55 ± 17%)
colocalized with CD63 on different 0.2 µm sections. According to flow
cytometry data,
-GFP expression was sensitive to BFA treatment, so
that a significant increase of green fluorescence was observed in
BFA-treated cells in which
-GFP accumulated intracellularly and
acquired a characteristic distribution in tubular structures typical of
BFA-treated cells (Fig. 2E). Expectedly, no
-GFP
expression was detected upon cycloheximide treatment (data not shown),
again supporting the possibility that pre-TCR-associated
intracellular TCR
was degraded. However, intracellular accumulation
of
-GFP was observed when cells were treated simultaneously with
cycloheximide and BFA (Fig. 2F), indicating that degradation of cytoplasmic
-GFP was blocked because of the impaired trafficking to lysosomes induced by BFA.
associated to internalized complexes in cells treated
with drugs that block lysosome (NH4Cl and chloroquine) or
proteasome (lactacystin) function. None of these drugs affected surface
receptor expression levels or cellular viability (data not shown).
However, as shown by immunoblot analysis (Fig.
3A), they induced a
significant accumulation of intracellular TCR
2
dimers that were immunoprecipitated associated to the pre-TCR or the
ChTCR from SUP-T1 cells or
/CypT
transfectants, respectively. In
contrast, these drugs did not significantly affect the levels of TCR
chain associated to the mature TCR complex in
wt transfectants,
which is consistent with an active and selective degradation of
TCR
2 dimers associated with the pre-TCR and the ChTCR
but not with the conventional TCR. Accordingly, degradation of
TCR
2 dimers associated to pre-TCR and ChTCR complexes
was observed when protein synthesis was blocked with cycloheximide (Fig. 3A), but TCR
chain degradation was inhibited by
NH4Cl and chloroquine, and to a lesser extent, by
lactacystin (see densitometric analysis in Fig. 3B). Taken
together, our data provide evidence that, as shown previously for
ligated TCR-CD3 complexes, constitutive internalization of unligated
pre-TCR-CD3 complexes is followed immediately by TCR
chain
degradation, mainly by lysosomes, but also by proteasomes, which
prevents recycling to the cell surface. Moreover, they suggest that the
pT
Cy tail is involved selectively in that process.
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Fig. 3.
TCR chains
associated to constitutively internalized pre-TCR and ChTCR complexes
are degraded by lysosomes and proteasomes. A, SUP-T1
cells,
wt, and
/CypT
transfectants were cultured in the
presence or absence of cycloheximide with or without NH4Cl,
chloroquine, or lactacystin. The total content of TCR
chain
associated either to the pre-TCR immunoprecipitated with an anti-CD3
mAb from SUP-T1 cells or to the ChTCR or the conventional TCR
immunoprecipitated with an anti-TCR
mAb from
wt or
/CypT
transfectants, respectively, was determined by Western blotting.
B, densitometric analysis of the bands obtained in the
absence (control) or presence of the indicated drugs.
CLQ, chloroquine; LCY, lactacystin. The
percentage of immunoprecipitated TCR
was determined from the band
density values of treated cells, using the untreated controls as
reference. Data are representative of three experiments.
Cy Domain Is Sufficient to Divert the TCR
-TCR
Heterodimer from a Recycling Pathway to Intracellular
Degradation--
To determine whether, as shown for
TCR
2 dimers, internalized pT
-TCR
heterodimers are
targeted for intracellular degradation, we next analyzed biochemically
the intracellular fate of biotin-labeled surface pre-TCR-CD3 complexes.
Immunoblot analysis with avidin-peroxidase of anti-CD3
immunoprecipitates confirmed that the pT
-TCR
heterodimer was
rapidly degraded in SUP-T1 pre-T cells. As shown in Fig.
4A, 40% of the biotinylated
heterodimers had disappeared after 90 min, and less than 30% of the
input pT
-TCR
complexes remained after 4 h, as assessed by
densitometric analysis (Fig. 4B). In contrast, no
degradation of the mature TCR
heterodimer was observed in
wt
transfectants, in which biotinylated complexes remained stable for
4 h. Strikingly, heterodimers composed of the TCR
chain bound
to the chimeric
/CypT
chain behaved essentially as pT
-TCR
heterodimers. Moreover, the simultaneous analysis of the ChTCR and the
endogenous pre-TCR coexpressed in
/CypT
transfectants revealed
that the total content of these two heterodimers decreased with
identical kinetics (Fig. 4B), demonstrating that the pT
Cy tail is sufficient to determine the degradation fate of the pre-TCR
components.
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Fig. 4.
Intracellular degradation of internalized
pT -TCR
and
/CypT
-TCR
heterodimers. A, the total content of
internalized biotin-labeled surface pre-TCR (pT
-
),
ChTCR (
/CypT
-
), and conventional TCR
(
) was determined by anti-CD3
immunoprecipitation
and Western blotting with streptavidin-horseradish peroxidase at the
indicated time points. Note that both the endogenous pre-TCR and the
ChTCR were co-expressed and simultaneously degraded in
/CypT
transfectants. The migration positions of the molecular size
standards (in kDa) are indicated on the left, and the
positions of the pT
-TCR
,
/CypT
-TCR
, and TCR
-TCR
heterodimers are shown on the right. B,
densitometric analysis of the bands obtained at the indicated time
points. The percentage of surface labeled-heterodimers was determined
from the band density values obtained after 1.5 and 4 h of culture
using the controls at time 0 as reference. Data represent the mean of
four experiments ± S.D.
-TCR
heterodimer, we next performed immunoblot analysis of
anti-CD3
immunoprecipitates from biotin-labeled SUP-T1 pre-T cells
treated with drugs that affect either the lysosome (cloroquine) or
proteasome (lactacystin and epoxomicin) function. Blotting with
anti-
-tubulin was used as an internal control of protein loading
(Fig. 5A). These studies
revealed that about 70% of the biotinylated pT
-TCR
heterodimers
was lost after a 6-h chase as assessed by densitometric analysis (Fig.
5B). Surprisingly, a complete inhibition of the degradation
of pT
-TCR
heterodimers was observed in cells treated with
lactacystin, an degradation was likewise sensitive to epoxomicin, two
specific and irreversible proteasome inhibitors, whereas a weak
inhibitory effect was observed after a 6-h chase in the presence of the
lysosome inhibitor chloroquine. Therefore, although these data can not
rule out the possibility that a proportion of the pT
-TCR
heterodimers is degraded in lysosomes, they are consistent with a
prominent role for the proteasome in the constitutive degradation of
the internalized pT
-TCR
heterodimers.
View larger version (38K):
[in a new window]
Fig. 5.
Proteasome-dependent degradation
of internalized pT -TCR
heterodimers. A, the total content of
internalized biotin-labeled surface pre-TCR (pT
-TCR
)
was determined by anti-CD3
immunoprecipitation and Western blotting
with streptavidin-horseradish peroxidase in SUP-T1 cells after a 6-h
chase in the presence or absence of either cloroquine (CLQ),
lactacystin (LCY), or epoxomicin (EPOX). Blots
were subsequently stripped and reprobed with an anti-
-tubulin mAb
for control loading. B, a relative densitometric analysis of
the immunoblot shown in A is displayed. Data are
representative of three experiments.
by PKC is the mechanism responsible for constitutive TCR
internalization in unstimulated T cells (28). Therefore, we analyzed
whether constitutive pre-TCR internalization in unstimulated pre-T
cells is also PKC-dependent. However, no
effects on surface pre-TCR levels were observed after treatment of SUP-T1 cells with doses of phorbol 12-myristate
13-acetate, which induced 60% down-regulation of the mature TCR in
wt transfectants. Neither was ChTCR down-modulation induced
by phorbol 12-myristate 13-acetate in
/CypT
transfectants even
after 2 h of treatment (Fig.
6A). These data, together with
the fact that bisindolylmaleimide and Ro-31-7549, two specific PKC
inhibitors, did not affect pre-TCR surface expression (data not shown),
suggest that PKC is not involved in the constitutive internalization of
the pre-TCR.
View larger version (25K):
[in a new window]
Fig. 6.
Down-modulation of surface pre-TCR and ChTCR
is independent of PKC but partly dependent on src
kinase activity. A, SUP-T1 cells, wt, and
/CypT
transfectants were cultured in the absence or presence of
phorbol 12-myristate 13-acetate (PMA) and analyzed by flow
cytometry for receptor surface expression at the indicated time points
as described in the legend for Fig. 1. B, cells were
cultured overnight in the absence (shaded histogram)
or presence of PP2 (thick line) and then analyzed for
receptor surface expression as described in A. Background
values (thin line) were determined by staining with
isotype-matched irrelevant Abs. C, SUP-T1 cells and
wt
transfectants were cultured in the presence of cycloheximide
(CHX) with or without PP2 and analyzed at the indicated time
points for receptor surface expression as described in A.
The percentage of surface expression was determined from the mean
fluorescence values of treated cells using the untreated controls as
reference. Results are representative of three independent
experiments.
/CypT
transfectants, respectively. In contrast, PP2 treatment had
no effect on the expression levels of the mature TCR in Jurkat T cells
(data not shown), although some increase in TCR expression was observed
in
wt transfectants. To assess whether the increase in surface
pre-TCR levels upon Lck/Fyn inhibition could represent a blockade in
both pre-TCR internalization and degradation, we next analyzed the
kinetics of pre-TCR down-regulation in cycloheximide-treated SUP-T1
pre-T cells, with or without PP2. As shown in Fig. 6C, inhibition of src kinases consistently delayed, but could
not block, the constitutive internalization and down-regulation of the
pre-TCR. Therefore, mechanisms involving phosphorylation by src kinases are partially, but not fully, responsible for
constitutive pre-TCR down-regulation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, are
constitutively internalized and recycled back to the cell surface in
the absence of ligand binding, and thus behave as conventional unligated TCR complexes on resting mature T cells (17-19). In
contrast, pre-TCR complexes expressed on unstimulated SUP-T1 pre-T
cells are continually and rapidly endocytosed but do not recycle back to the cell surface. As reported for TCR complexes internalized following antigenic stimulation (16, 19), we show here that intracellular degradation is the mechanism responsible for the impaired
recycling of pre-TCR in unstimulated pre-T cells. Strikingly, we found
that
chain dimers associated to the internalized pre-TCR are sorted
for degradation in lysosomes and proteasomes and thus follow the
intracellular fate of TCR-
chain complexes internalized following
antigenic stimulation (16, 19), whereas evidence is provided that the
proteasome plays a prominent role in the constitutive degradation of
the internalized pT
-TCR
heterodimers. In this regard, it is worth
noting that, as observed upon TCR ligation, internalized
chains
associated with the unligated pre-TCR are found mostly in a
phosphorylated state, a characteristic event associated with T cell
activation (30). Therefore, the human pre-TCR complex behaves
constitutively as an activated TCR without any need for ligand
binding. After submission of this manuscript, Panigada et
al. (31) provided evidence of constitutive pre-TCR internalization
and degradation in the mouse. Our results extend the peculiar behavior
of the pre-TCR to the human, and map it to the cytoplasmic tail of the
pT
chain by comparison with TCR
, which does not share this capacity.
and
Zap70 phosphorylation in a manner similar to that observed for the
ligated
TCR (22, 23). Such a particular membrane distribution
might depend on the unique biochemical structure of the pre-TCR.
Particularly, it was proposed that palmitoylation of the conserved
juxtamembraneous cysteine residue of the pT
Cy domain might be
required for the cell-autonomous raft localization of the pre-TCR (20).
However, a very recent study has proved that this is not an
essential component for endowing the murine pre-TCR with
cell-autonomous signaling capability (32). Although this finding might
support the current view that the Cy domain of the murine pT
molecule is dispensable for pre-TCR function, the same study provides
direct evidence that the COOH-terminal proline-rich domain of the
murine pT
Cy tail plays a crucial role in pre-TCR signaling and T
cell development (32). This finding supports our proposal that the Cy
tail of the human pT
molecule is an essential component of pre-TCR
function (9) and concurs with the present finding that the pT
Cy
tail is sufficient to confer constitutive internalization and
degradation properties to the conventional TCR. It is thus likely that
the same mechanisms involved in ligand-induced TCR signaling and
down-regulation could control the pre-TCR-CD3 intracellular fate.
Accordingly, phosphorylation of CD3
by PKC, which is currently
believed to control the internalization and recycling of unligated TCR
complexes but not ligand-induced TCR down-modulation (11, 28), does not
seem to play a role in pre-TCR down-modulation. Regarding the potential
role of PTKs involved in TCR signaling such as Lck and Fyn in TCR
down-regulation, the available data support the view that
down-regulation of engaged TCR complexes involve both
PTK-dependent and -independent mechanisms, which are most
likely controlled by the concentration of ligand and final receptor
occupancy (11, 13, 30). Because pre-TCR internalization was found to be
only partly dependent on src kinase activity, the situation
may be equivalent to that reported for the TCR at maximal receptor
occupancy (13).
components shared by pre-TCR and TCR display internalization/sorting motifs of
both the dileucine- and the tyrosine-based types, which could mediate
clathrin-dependent internalization and intracellular
sorting to degradation (reviewed in Ref. 11). Interestingly, a
consensus tyrosine-based motif ( ...
226YPTC229 ... ) exists within the Cy
domain of the human pT
molecule as well, which could become
cell-autonomously exposed in the activated pre-TCR conformation to
fulfill both the internalization and degradation functions. It is also
possible that association of the pre-TCR into lipid rafts could
regulate a constitutive clathrin-independent endocytic pathway similar
to that recently described for the interleukin-2 receptor (33).
Whatever the mechanism involved, we would suggest that constitutive
internalization and degradation of the pre-TCR is a key process that
controls surface receptor levels and provides the cell with a self-safe
mechanism to avoid sustained ligand-independent signaling through a
potent, potentially oncogenic, cell growth receptor (34).
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ACKNOWLEDGEMENTS |
---|
We thank Drs. R. Kurrle, B. Alarcón, M. A. Alonso, F. Sánchez-Madrid, and R. Bragado for the generous gifts of antibodies and reagents and Drs. B. Alarcón, M. A. Alonso, J. R. Regueiro, I. V. Sandoval, and E. Fernández for helpful discussions and for reading the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnología (SAF 97-0161 and SAF2001-1629), Dirección General de Enseñanza Superior (PB97-1194), Comunidad de Madrid (08.3/0021/2000), Fondo de Investigación Sanitaria (FIS 00/1044), and Fundación Eugenio Rodríguez Pascual and also by an institutional grant to the Centro de Biología Molecular "Severo Ochoa" from Fundación Ramón Areces.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.
Present address: Lymphocyte Interaction Laboratory, Cancer
Research, UK, 44 Lincoln's Inn Fields, P. O. Box 123, London WC2A 3PX, United Kingdom.
§ Both authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 34-91-397-8076; Fax: 34-91-397-8087; E-mail:mtoribio@cbm.uam.es.
Published, JBC Papers in Press, December 8, 2002, DOI 10.1074/jbc.M204944200
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ABBREVIATIONS |
---|
The abbreviations used are:
TCR, T cell
receptor;
pT, pre-TCR
;
Cy, cytoplasmic;
mAb, monoclonal antibody;
ChTCR, chimeric TCR;
BFA, brefeldin-A;
GFP, green fluorescence protein;
mAb, monoclonal antibody;
PKC, protein kinase C;
PTK, protein tyrosine
kinase.
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