ARTICLE |
CORRESPONDENCE Balbino Alarcón: Balarcon{at}cbm.uam.es
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Multimeric plasma membrane protein complexes typically assemble early in the secretory pathway, in parallel with the synthesis and folding of individual subunits in the ER. Exit from the ER is an important checkpoint for many such complexes, as only fully assembled complexes are allowed to pass this control step (1). ER retention/retrieval signals (herein referred to as ER retention signals) are present in some receptor subunits, and the assembly of complexes is thought to mask the ER retention signal, permitting targeting to the membrane of the fully assembled complexes (2). The TCR is an especially complex model because it is formed by six different subunits that all contain ER retention determinants.
In the TCR, the TCR and TCRß subunits (or TCR
and TCR
in
T cells) are responsible for the recognition of the MHC/antigen ligand. These are noncovalently bound to the signal-transducing subunits CD3
, CD3
, CD3
, and CD3
(the CD3
subunit is CD247). During assembly, CD3
first dimerizes with either CD3
or CD3
, and the resulting
and
dimers associate with the TCR
and TCRß subunits (35). The resulting
ß
or
ß
complexes are either retained in the ER or degraded in lysosomes (6). Only when CD3
is incorporated into the complex is the TCR transported to the plasma membrane (68). In this way, expression of signal-transducing subunits independent of ligand-binding subunits or vice versa is tightly controlled. Indeed, in T cell mutants and knockout mice lacking TCRß, CD3
, or CD3
, TCR expression is severely impaired (912). In addition, T cell precursors in RAG-deficient and SCID mice that are unable to express the TCR gene subunits have a very low level of CD3
and
dimer expression at the cell surface (13, 14), and their development is arrested at the most immature CD4 CD8 stage.
Removal of the ER retention signal in the cytoplasmic tail of CD3 permits this subunit to reach the cell surface by itself (15, 16). This signal consists of an elongated
helix followed by a ßI' turn and contains three important, closely spaced residues: tyrosine, leucine, and arginine. The presence of arginine in the ER retention signal is characteristic of type II proteins despite the fact that CD3
is a type I membrane protein (1719). Other ER retention signals in the TCR have not been analyzed in detail, although TCR
contains an ER retention signal in its transmembrane region (20) and TCRß in both its extracellular and transmembrane domains (21). With regard to the other CD3 subunits, CD3
has a conserved arginine residue in position 3 from the COOH terminus, and CD3
has either an arginine or a lysine residue at the same position. Their removal from Tac
and Tac
chimeras disrupts ER retention (22).
In addition to ER retention signals, binding of incompletely folded subunits and complexes to chaperonins such as calnexin can also influence ER retention (23). Moreover, endocytosis signals in several subunits of the TCR complex offer a further level of regulation (22, 24, 25). The CD3 subunit contains an important double leucine signal for endocytosis that is hidden in the complete TCR complex but unmasked by PKC-mediated phosphorylation of an upstream serine (26). In partial complexes, this double leucine signal is constitutively exposed and only masked upon integration of CD3
into the TCR complex (27, 28).
During assembly, all individual ER retention determinants in TCR subunits must be annulled before the TCR complex can be transported to the plasma membrane. The ER retention determinants may become progressively overridden as the TCR complex assembles or alternatively, all determinants might become inoperative at once, when all the TCR subunits are assembled. To study this process, we have characterized the ER retention signals in CD3 and analyzed the predominance of CD3
and CD3
signals in the
dimer. All the determinants in CD3
are overridden when it assembles with CD3
. However, the single ER retention signal in CD3
remains active in the
dimer and only becomes inoperative upon completion of the last assembly step, i.e., the incorporation of CD3
. These results support a model of sequential inactivation of ER retention signals during stepwise assembly.
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Results |
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The fact that some 44 chimera was detected at the plasma membrane might be due to excessive protein expression in transfected COS cells, thereby overriding the ER retention machinery. Hence, we studied the surface expression of the 44
chimera in stable transfectants of the human NK cell line YT. Several cell clones with each construct were studied to exclude clonal effects. Only the mutant 44
5 was expressed at the cell surface at a level that matched that of wild-type CD4 (Fig. 1 C). There was only a slight increase in surface expression of chimeras 44
1 through 44
3 and a marginal reduction of 44
4.
We determined how the rate of exit from the ER was affected in these 44 mutants. Human CD4 has two N-glycosylation sites, one of which is converted to the complex type in the mature protein (30). Hence, we assessed the acquisition of partial endo-H resistance of the 44
mutants after metabolic labeling of the COS cells. Export from the ER of the 44
1 and 44
2 chimeras was accelerated twofold. These mutants required 50 min to acquire 50% endo-H resistance (t1/2 = 50) compared with the 95 min for wild-type 44
(Fig. 1, D and E). Deletion of further amino acids reduced the exit rate to t1/2 = 115 min for 44
3, whereas the ER export of 44
4 and 44
5 was dramatically reduced (t1/2 >> 120 min). Thus, it seemed clear that signals other than those for ER retention also regulate the level of 44
chimera surface expression.
The cytoplasmic tail of CD3 contains a di-leucine endocytotic motif (Fig. 1 A; reference 25) and a putative DxE ER export signal (31). The combination of ER retention, export, and endocytosis is ultimately responsible for the surface expression of the 44
mutants. Deletion of the ER retention signal in the COOH-terminal end of the chimera might explain the accelerated rate of ER export for 44
1 and 44
2, whereas removal of the putative DxE ER export signal would explain the decrease in ER export of 44
4 and 44
5. The diminished ER exit of 44
3 compared with 44
2 might be due to a positional effect on the DxE signal. Nevertheless, it seems that the di-leucine endocytotic signal is mainly responsible for regulating the surface levels of the 44
chimera. This could explain why only 44
5 is highly expressed at the surface despite the reduced rate of ER export (Fig. 1, B, C, and E).
CD3 contains ER retention determinants in its extracellular, transmembrane, and cytoplasmic domains
To further characterize this ER retention signal and to determine the impact this signal has on 44 chimera surface expression, point mutations of the last three amino acids were introduced. To avoid interference from the di-leucine internalization signal, leucine 131 was replaced by alanine. Expression of the double mutants at the cell surface was analyzed in transfected COS cells and in stable YT transfectants. In both cell types, mutation of the di-leucine motif alone (44
L131A mutant) caused a two- (COS cells) to sixfold (YT cells) increase in surface expression of the 44
chimera (Fig. 2 A). Replacement of arginine 158 with alanine (44
L131A/R158A mutant) resulted in an additional two- (COS cells) to fourfold (YT cells) increase in surface expression. In contrast, mutation of the other two COOH-terminal amino acids (44
L131A/N160A and 44
L131A/R159A mutants) did not have a major impact on cell surface expression. The effect of arginine 158 mutation was also reflected in the cellular redistribution of this 44
mutant (Fig. S2, available at http://www.jem.org/cgi/content/full/jem.20041133/DC1). Thus, of the three amino acid residues deleted in mutant 44
1, only arginine 158 seems to be important for ER retention of 44
.
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Expression of the CD4/CD3 chimeras and of the CD3
point mutants was also evaluated by flow cytometry. Surface expression of the single di-leucine motif mutant of CD3
(
L131A) was slightly higher than the wild-type CD3
but lower than the mutant in the cytoplasmic ER retention signal (Fig. 2 E,
R158A). The double mutation of the cytoplasmic ER and endocytotic signals (
L131A/R158A) acted synergistically to increase the surface expression of CD3
. Nevertheless, all CD4-CD3
chimeras and CD3
mutants were expressed at lower levels than CD4 (Fig. 2 E), further indicating that ER retention determinants reside not only in the cytoplasmic tail of CD3
, but also in the transmembrane and extracellular domains. These extracellular and transmembrane retention determinants in CD3
could represent distinct sequence motifs or an unfolded state of the protein. In any case, it appears that in contrast to CD3
, which contains a single ER retention signal (15), retention of free CD3
is regulated by multiple signals. It therefore seems that the expression of CD3
on the cell surface is tightly regulated.
Dimerization with CD3 abolishes all ER retention determinants in CD3
One of the earliest steps in TCR assembly is the dimerization of CD3 with either CD3
or CD3
(3, 4, 6). The resulting
and
dimers are retained in the ER unless they assemble with TCR
, TCRß, and CD3
. Because both CD3
and CD3
contain ER retention signals, we examined the relative contribution of each signal to dimer retention. COS cells were cotransfected with the CD3
mutants and CD4 chimeras (refer to Fig. 2) and either wild-type CD3
or a deletion mutant of CD3
lacking its single ER retention signal (
mut; reference 15). The 4
4 chimera was excluded from this study because it lacks the extracellular domain of CD3
necessary for assembly with CD3
(32). Cellular distribution of the dimers was distinguished from that of the single chains by immunostaining with a CD3 dimer-specific antibody, UCHT1 (33). When associated with wild-type CD3
, all CD3
chimeras and mutants were located in the ER, independent of the presence of CD3
ER retention and endocytotic signals (Fig. 3 A). However, transfection of
mut resulted in export of the
dimers from the ER and targeting to the plasma membrane (Fig. 3, A and B). The same effect was seen when wild-type CD3
and
mut were expressed in COS cells (not depicted).
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These results suggest that the only functional ER retention signal in the dimer is that in CD3
, and all ER retention determinants in CD3
are abrogated upon dimerization with CD3
. Therefore, the ER retention determinants in CD3
do not seem to play a major role in regulating
expression at the cell surface. However, CD3
does contribute to this task by mediating the endocytosis of the
dimer via its di-leucine signal.
The single ER retention signal of CD3 is only overridden during the last assembly step
Once (or
) dimers are assembled in the ER, they associate with the TCR
and TCRß chains to form
ß
and
ß
complexes. These incomplete TCRCD3 complexes remain in the ER or are degraded in lysosomes. The TCR complex can only reach the plasma membrane when the
subunit is incorporated (68, 34). Indeed, reconstruction of the TCR complex in HeLa cells showed that transfection of the
subunit was sufficient to drive transport of
ß
and
ß
complexes to the cell surface (35). Bearing this in mind, we studied whether the CD3
ER retention mutant promoted surface expression of the
ß
complex or whether assembly of
was still required. COS cells were transfected with plasmids encoding these subunits, but the
L131A mutant was used to avoid internalization of the
ß
complex. When transfected with the CD3
ER retention mutant, both the CD3
dimer and TCRß were transported to the plasma membrane, even in the absence of
(Fig. 4 A). Two-color flow cytometry with anti-TCRß and anti-CD3
antibodies was used to quantify the
ß
complex on the cell surface. This showed that the
ß
complex is expressed at a high level independent of
(Fig. 4 B). In contrast, when wild-type CD3
was transfected, TCRß and the CD3
dimer were predominantly found in the ER and were consequently practically undetectable at the cell surface (Fig. 4, A and B). Similar results were obtained when wild-type CD3
instead of the di-leucine mutant was used, although surface expression of the
ß
complex reached lower levels (Fig. S3, available at http://www.jem.org/cgi/content/full/jem.20041133/DC1), probably because the
ß
complex was being endocytosed. These results show that mutation of the CD3
ER retention signal is sufficient for transport and expression of the incomplete
ß
complex to the cell surface. Interestingly, the presence of
did not induce a major increase in the surface expression of complexes containing wild-type CD3
, possibly due to the inefficient assembly of CD3
into the
ß
complex.
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Masking of the CD3 ER retention signal upon CD3
assembly is position dependent
A prediction of the stepwise model of ER retention signal annulment is that silencing of a given ER retention signal must be position specific; e.g., if CD3 assembly overrides the ER retention signal in CD3
, this must occur in the context of the specific topological position of CD3
in the TCR complex. To evaluate this hypothesis, we constructed two new CD3
chimeras. In one of the chimeras, the cytoplasmic tail of CD3
was substituted by the tail of CD3
(Fig. 6 A,
chimera). The other chimera was generated by appending the ER retention signal of CD3
at the COOH-terminal end of CD3
(Fig. 6 A,
ret). Next, we examined whether assembly of the CD3
chimeras with the ER retention mutant of CD3
resulted in expression of the
dimer at the cell surface. As previously demonstrated (Fig. 3), assembly of wild-type CD3
with
mut but not with wild-type CD3
allowed transport of the
dimer to the cell surface (Fig. 6 B). In contrast, assembly of the CD3
chimeras
and
ret with
mut prevented export of the
dimer to the plasma membrane (Fig. 6 B) and retained the dimer in the ER (Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20041133/DC1). These results therefore show that abrogation of the ER retention signals during assembly of the
dimer is dependent on both the sequence of the cytoplasmic tail and the position of the ER retention signals within the dimer.
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Discussion |
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Dual regulation through ER retention signals and proteasome-dependent degradation from the ER prevents TCR subunits from progressing through the secretory pathway (38, 39). ER retention signals have been described in CD3, TCR
, TCRß, CD3
, and CD3
(15, 2022, and this study), and it could be considered that all ER retention signals are abrogated simultaneously when the full TCR complex is assembled. However, our findings indicate that there is a hierarchy among retention signals such that they become overridden progressively as the TCR complex assembles. Hence, we propose a model for the export of the TCR complex (Fig. 7) in which the ER retention signals of CD3
, CD3
, TCR
, and TCRß become progressively inoperative as they assemble with CD3
. The ER retention signal in CD3
remains dominant in these complexes.
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What then is the mechanism that overrides CD3's ER retention signal upon assembly of
? It has been proposed that the cytoplasmic tail of CD3
hides the otherwise exposed di-leucine endocytosis motif in CD3
by steric hindrance (28). Indeed, in
-deficient cells it has recently been demonstrated that the TCR is more rapidly internalized and that expression of CD3
, or a CD3
chimera with its cytoplasmic tail partially replaced by a foreign sequence, restores normal TCR internalization (27). Steric masking of the dilysine ER retention motif in the
chain of the heterodimeric high affinity receptor for immunoglobulin E (Fc
RI) upon assembly with the
chain has been proposed to regulate plasma membrane targeting (2). Similarly, steric hindrance by the cytoplasmic tail of CD3
could be responsible for annulling the CD3
ER retention signal, although other mechanisms involving CD3
-dependent rearrangements of the TCR complex cannot be excluded. Interestingly, the
chain of Fc
RI and CD3
are structural and functionally related, and indeed, the Fc
RI
chain can take over the role of CD3
in TCR assembly in
/ mice (4244). These results suggest a common mechanism underlying ER retention of immune receptor complexes by components of the CD3
family. In any case, masking of the CD3
ER retention signal by CD3
assembly is position dependent. Thus, either replacement of the cytoplasmic tail of CD3
by CD3
or apposition of an extra CD3
ER retention signal to the COOH-terminal end of CD3
prevents surface expression of the full TCR complex (Fig. 6).
We have shown that the di-leucine endocytosis signal of CD3, together with the ER retention signal in CD3
, is also important to reduce the expression of the
dimer on the cell surface. Thus, high level expression of the
(and by extension
) dimer on the cell surface is prevented by impairing its export from the ERGolgi and by stimulating the rapid endocytosis of dimers from the cell surface. This regulation of CD3 dimer and free CD3 subunit expression is required to prevent ligand-independent triggering of signaling cascades. It has been shown that small amounts of
and
dimers expressed on the surface of immature thymocytes, also known as clonotypic-independent complexes, can promote thymic differentiation and proliferation upon cross-linking with anti-CD3 antibodies (45). One might ask what would be the consequence of augmenting the expression of CD3 dimers at the cell surface on thymic maturation. Studies in which a TCRß transgene that lacks the variable region (46), or even all extracellular domains (47), was expressed in MHC class I and IIdeficient mice indicate that the pre-TCR function is independent of ligand recognition. The pre-TCR could therefore serve merely as a platform to express sufficiently high levels of the CD3 dimers at the cell surface to initiate ligand-independent signaling. Assembly of the CD3 dimers with TCRß, pT
, and CD3
must override the CD3
ER retention signal as well as those in TCRß (21) and pT
(48). In this regard, it should be noted that the addition of an extra ER retention signal to TCRß abolishes pre-TCR function (49). These results suggest that the pre-TCR must be expressed on the cell surface to carry out its signaling role. Furthermore, unlike the natural ER retention signals present in the TCR and CD3 subunits, the artificial signal introduced in TCRß does not appear to be annulled during assembly.
We have made an initial attempt to characterize the signals that regulate the intracellular retention of the CD3 chain. In accordance with previous observations (22), we found that the cytoplasmic tail of CD3
contains an ER retention signal at the COOH terminus. Although this sequence is reminiscent of the double arginine ER retention signal in type II membrane proteins (18, 19), we have discovered that only one of the two arginines in CD3
is important for ER retention. Thus, the cytoplasmic CD3
ER retention signal better resembles that of CD3
, which contains only one important basic residue (arginine 3; references 15 and 16). In addition to the ER retention signal, the cytoplasmic tail of CD3
contains a putative ER export sequence of the DxE type (31) and a di-leucine endocytosis signal (22, 50). However, CD3
also contains ER retention determinants in its extracellular and transmembrane domains that have yet to be characterized. These extracellular and transmembrane retention determinants in CD3
could represent distinct sequence motifs or an unfolded state of the protein. In any case, it appears that in contrast to CD3
, the retention of free CD3
is regulated by multiple signals.
In conclusion, the results presented here suggest that the TCRCD3 complex is endowed with a complicated system of intracellular retention signals that become overridden in a stepwise fashion as assembly proceeds. Assembly is regulated in such a way that all intermediates have at least one functional retention signal. This system guarantees that only a full signaling-competent TCRCD3 complex is expressed at the cell surface.
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MATERIALS AND METHODS |
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Constructs.
All constructs were generated by PCR using human cDNAs as templates. PCR products were cloned into the pSR or pSR
-HA (unpublished data) vectors. The 44
chimera is composed of the extracellular and the transmembrane domains of CD4 (finishing in position V395 of the mature polypeptide) fused to the complete intracellular domain of CD3
(from position A115 of the mature polypeptide). 44
1 to 44
5 constructs have a stop codon at positions 157, 152, 148, 141, and 121, respectively, of the mature human CD3
protein. Truncated CD3
(
tr) contains only the first two amino acids of the cytoplasmic tail. Wild-type human CD3
and the CD3
mutant lacking the last five COOH-terminal amino acids (
mut) have been described (15, 51). Point mutants were generated by introducing the mutation encoding for alanine in the positions 131, 158, 159, and 160 of the mature human CD3
protein using 44
(44
L131A, 44
L131A/N160A, 44
L131A/R159A, and 44
L131A/R158A) or CD3
(
L131A,
R158A, and
L131A/R158A) as template. The 4
4,
44, and
constructs were created as isolated fragments by PCR, cloned into the intermediate vector pGEM-7Zf(+), and the chimeric cDNAs were subcloned into the pSR
-HA vector. The
ret construct was created by PCR using a 3' primer encoding for the last COOH-terminal 15 amino acids of human CD3
(KGQRDLYSGLNQRRI) appended to the last amino acid of CD3
, and the PCR product was cloned into the pSR
-HA vector.
Antibodies
The mAb antihuman SP34 that recognizes the CD3 extracellular domain, the mAb antihuman CD3 UCHT1, the mAb antihuman CD4 HP2/6, and the mAb antiCß Jovi.1 were provided by C. Terhorst (Beth Israel Deaconess Hospital, Boston, MA), P. Beverley (The Edward Jenner Institute for Vaccine Research, Berkshire, UK), F. Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain), and M. Owen (CRUK, London, UK), respectively. The anti-CD3
antiserum 448 has been described (5). The following mAbs were purchased as indicated: anti-HA epitope 12CA5 (Roche Diagnostics), anti-murine TCRß H57-597 and antihuman CD3 Leu4 (BD Biosciences), and antihuman CD3
SK7 (StemCell Technologies Inc.). All secondary fluorochrome-labeled antibodies were purchased from BD Biosciences.
Cell transfections
COS cells were transiently transfected as described previously (32), and stable transfectants of YT, 2B4, MA5.8, and R3.25 cells were generated by electroporation and selection in geneticin.
Flow cytometry
24 h after transfection, COS cells were detached from the plate with 0.02% EDTA in PBS and divided into two aliquots for surface and intracellular staining. For intracellular staining, cells were first fixed with 2% paraformaldehyde in PBS for 20 min at 4°C and then permeabilized with 0.1% saponin in PBS at 4°C for 1 h. Permeabilized and nonpermeabilized cells were incubated with 4 µg/ml of the appropriate mAb for 30 min at 4°C and then with a secondary FITC- or PE-labeled antibody. For two-color staining, directly labeled antibodies were used. Surface expression is indicated as a percentage, as mean fluorescence intensity (MFI), or by multiplying both parameters.
Confocal microscopy
Upon transfection, COS cells were plated on glass coverslips, fixed in paraformaldehyde at room temperature 24 h later, and then permeabilized with saponin as described above. The coverslips were mounted in Mowiol and examined with a confocal microscope (Radiance 2000; BioRad Laboratories).
Cell labeling and immunoprecipitation
48 h after transfection, COS cells were labeled with 35S-methionine for 15 min and then chased for different times in standard medium before lysing with 1% NP-40 lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.8, 10 mM iodoacetamide, 1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin). Postnuclear lysates were immunoprecipitated with an anti-CD4 antibody, and the immunoprecipitates were resuspended in endo-H buffer (50 mM sodium citrate, pH 5.5, 0.1% SDS, 1 mM PMSF, 1 µg/ml aprotinin, and 1 µg/ml leupeptin) before digesting half of the sample with endo-H. The samples were resolved by SDS-PAGE and analyzed by autoradiography.
For surface biotinylation, 50 x 106 MA5.8 and 2B4 were incubated with 0.5 mg/ml sulfo-NHS-biotin (Pierce Chemical Co.) in PBS supplemented with 0.1 mM CaCl2 and 1 mM MgCl2 for 45 min on ice. After washing, surface complexes were recovered by incubating the labeled intact cells with human anti-CD3 antibody Leu4 before lysis. Protein G was added and immunoprecipitates were subjected to two-dimensional SDS-PAGE (first dimension under nonreducing and second dimension under reducing conditions), immunotransferred to a nitrocellulose membrane, hybridized with streptavidin horseradish peroxidase (Southern Biotechnology Associates, Inc.), and developed by ECL (Bio-Rad Laboratories).
Online supplemental material
Fig. S1 shows intracellular distribution of 44 deletional mutants. Fig. S2 shows intracellular distribution of the 44
point mutants. In Fig. S3, the ER retention signal in CD3
and the di-leucine endocytosis signal in CD3
both regulate surface expression of the incomplete
ß
complex. Fig. S4 illustrates the intracellular distribution of
dimers. Figs. S1S4 are available at http://www.jem.org/cgi/content/full/jem.20041133/DC1.
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Acknowledgments |
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This work was supported by grants SAF2002-03589 from CICYT, 08.3/0030.1/2001 from the Comunidad de Madrid, and by funds from the Fundación Ramón Areces to the Centro de Biología Molecular.
The authors have no conflicting financial interests.
Submitted: 7 June 2004
Accepted: 23 December 2004
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References |
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