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The Journal of Immunology, 1999, 162: 878-885.
Copyright © 1999 by The American Association of Immunologists

The Extracellular Domain of the {zeta}-Chain Is Essential for TCR Function

Britt Johansson, Ed Palmer and Luca Bolliger1

Basel Institute for Immunology, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The {zeta}-chain homodimer is a key component in the TCR complex and exerts its function through its cytoplasmic immunoreceptor-tyrosine activation motif (1). The {zeta}-chain extracellular (EC) domain is highly conserved; however, its functional and structural contributions to the TCR signaling have not been elucidated. We show that the EC domain of the {zeta} homodimer is essential for TCR surface expression. To gain a more detailed structural and functional information about the {zeta}-chain EC domain, we applied a cysteine scanning mutagenesis to conserved amino acids of the short domain. The results showed that the interchain disulfide bridge can be displaced by seven or eight amino acids along the EC domain. The TCR signaling efficacy was dramatically reduced during peptide/MHC engagement in the {zeta} mutants containing the displaced disulfide bond. These signaling defective {zeta} mutants produced an unconventional early tyrosine phosphorylation pattern. While the tyrosine phosphorylated forms of {zeta} (p21 and p23) could be observed during Ag stimulation, downstream signaling events such as the generation of phospho-p36, higher m.w. forms of phospho-{zeta}, and phospho-{zeta}/ZAP-70 complexes were impaired. Together these results suggest an important function of the phylogenetically conserved {zeta}-EC domain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The TCR/CD3 complex is a multimeric complex composed of at least six different integral membrane proteins (2). While the recognition of MHC/peptide complexes is mediated by the disulfide-linked clonotypical TCR {alpha}- and ß-chains, the noncovalently associated invariant CD3 chains, {gamma}, {delta}, {epsilon}, and the homodimeric disulfide-linked {zeta}, are needed for its surface expression and function (reviewed in 3 . The biogenesis of the TCR complex proceeds through the formation of partial complexes between the different TCR components in the endoplasmic reticulum (ER)2, before the TCR{alpha}ß{delta}{gamma}{epsilon}2 and {zeta}-{zeta} homodimer assemble, to reach the cell surface (4, 5). The association of the {zeta}-chain to the partial TCR complex is rate-limiting for the transport of complete TCR complexes to the surface (reviewed in 6 .

The molecular mechanism of how the TCR complex mediates the translocation of the signal to the cytoplasm across the plasma membrane has been the subject of intense study. It is known that the immunoreceptor-tyrosine activation motifs (ITAMs) present in one copy in each of the cytoplasmic tails of the CD3 components, and in three copies in the {zeta}-chain, actively participate in the cytoplasmic signal transduction. These signaling modules are required to link the TCR complex components to the soluble cellular transduction machinery (7, 8), which directs the signal transduction pathway(s) and diversifies the array of physiological responses (3).

Recent studies have suggested a possible role of the extracellular (EC) domain of the {zeta}-chain in TCR function. Besides being highly conserved among different species, the accessibility of the {zeta}-EC domain to chemical modification varies during TCR engagement (9, 10). Furthermore, the Fc{epsilon}RI{gamma} subunit, which has a shorter EC domain compared with the {zeta}-chain and carries only one ITAM, can replace the {zeta}-chain in the {alpha}ß T cell subset (11, 12, 13) but not in the {gamma}{delta} T cell subset (14). Finally, our previous work showed that mutations of the unique positive charge present in the {zeta}-EC domain influences the antigenic response mediated by the engagement of the TCR (15). Together, these studies suggest that the EC domain of {zeta} may play a significant role in TCR function.

In this study, we produced a {zeta}-chain with a deleted portion of the EC domain and showed that it was not able to sustain the surface expression of the TCR complex. To gain more detailed functional and structural information about the essential {zeta}-EC domain, we utilized a cysteine scanning mutagenesis. This method has been successfully used to directly determine interacting amino acids within neighboring transmembrane (TM) {alpha} helices of the bacterial aspartate receptor (16, 17). Individual amino acid exchanges to cysteine at conserved positions were evaluated for their effect on TCR expression and function in the presence or absence of the normal disulfide bridge. We reasoned that the formation of a displaced disulfide bridge could physically lock the structure of the {zeta} homodimer, and thereby disturb the function of the TCR. The experiments described here show that the {zeta}-chain EC domain is essential for TCR surface expression and is intimately involved in the signal transduction cascade of a TCR engaged with MHC-peptide complexes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and peptide

The 2B4 derivative MA5.8, lacking endogenous {zeta} expression, was reconstituted with the {zeta} mutants described in this study. P13.9, an L cell derivative expressing I-Ek, B7.1, and ICAM were used for phosphorylation experiments. LK35.2 (18) and the B cell line expressing human MHC class II DR1, DAP.3, were used as Ag-presenting cell lines. The ecotropic packaging cell line Bosc23 was purchased from American Type Culture Collection (Manassas, VA). All cells were grown in Iscove’s modified Dulbecco’s medium supplemented with 5% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 50 µM 2-ME. The antigenic peptide of pigeon apocytochrom c from amino acid 90 to 104 (ERADLIAYLKQATAK) was synthesized at the Basel Institute for Immunology (Basel, Switzerland) using FastMoc chemistry on a 430A peptide synthesizer (Applied Biosystems, Foster City, CA).

Mutations, transfections, infections, and FACS analysis

The strategy was outlined in Bolliger et al. (15). Mutations were introduced with PCR with oligonucleotides including the desired changes and were cloned in pGEM3Z (19). Sequencing was performed on an automated sequencing machine (Applied Biosystems). The correct constructs were cloned into a retroviral vector carrying the puromycin resistance gene, LXSP (20, 21, 22). Large DNA preparations were performed according to standard procedures. Bosc23 cells were transfected using a calcium-phosphate method, and MA5.8 cells were infected as previously described (23), except that DEAE-dextran (40 µg/ml) was used. MA5.8 or transfectants were stained with fluorescein isothiocyanate-labeled anti-CD3{epsilon} subunit (145-2C11) (24), or anti-TCRß (H57-597), V{alpha}11, and Vß3 directly conjugated Abs (PharMingen, San Diego, CA). Acquisition was performed using a FACScan flow cytometer and analysis with Cellquest software (Becton Dickinson, San Diego, CA).

Cell surface biotinylation, phosphorylation, immunoprecipitation (IP), SDS-PAGE analysis, and Western blot analysis

Methods have been described by Bolliger et al. (15). In short, cells were biotinylated bicarbonate buffer (20 mM NaHCO3 and 150 mM NaCl) with sulfo-NHS-biotin (100 µg/ml; Pierce, Rockford, IL) at 107 cells/ml. The reaction was blocked by the addition of PBS with 3% v/v FCS and 50 mM lysine (25). Cell lysis was conducted in 1% digitonin or TX-100 (Sigma, Buochs, Switzerland), 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM PMSF, 10 µg/ml of aprotinine, and 10 µg/ml of leupeptine at a cell concentration of 108 cells/ml for 20 min on ice. Cleared lysates were immunoprecipitated for 2 h at 4°C with 2 µg of 145-2C11 (anti-CD3{epsilon}) or H146-968 mAbs bound to 15 µl of equilibrated protein G Sepharose (Pharmacia, Uppsala, Sweden) per 5 x 107 cells. The pellets were resuspended in nonreducing or reducing sample buffer, boiled for 2 min at 95°C, loaded on a 12% SDS-PAGE, and transferred onto a nitrocellulose membrane (Bio-Rad, Mountainview, CA). The {zeta}-chain was detected with the mAb H146-968 (26).

Phosphorylation assay. The cell line P13.9 expressing I-Ek, B7.1, and ICAM was loaded with 100 µM peptide overnight in a 24-well plate (0.8 x 106 cells/well in 500 µl of 10% FCS Iscove’s modified Dulbecco’s medium) and used as an Ag-presenting line as described. (15, 27). Shortly, 107 responders were added in each well to 0.75 x 106 APC and incubated at 37°C according to described times. Cells were harvested, and cell pellets were lysed for 20 min in 500 µl of 1% TX-100, 10 mM Tris-HCl (pH 7.4), and 150 mM NaCl. The lysis buffer was supplemented with 1 mM orthovanadate, 100 µM pervanadate from a fresh stock solution of 10 mM orthovanadate and 15 mM H2O2, 10 mM NaF, 1 mM PMSF, 10 µg/ml of aprotinine, and 10 µg/ml of leupeptine. IP with H146-968 or an antiserum specific for ZAP-70 (1222, kindly donated by Dr. L. Samelson), SDS-PAGE, and Western blot analysis followed as previously described.

Western blot analysis. After the transfer of proteins onto nitrocellulose, the membrane was blocked with 5% low-fat milk-PBS-Tween (0.2% v/v Tween 20 in PBS) for 20 min at room temperature (RT). Blots were probed with 200 ng/ml of H146-968, 100ng/ml of 4G10 (Upstate Biotechnology, Lake Placid, NY), or 500 ng/ml of ZAP-70 (Transduction Laboratories, Lexington, KY) for 1 h at RT in PBS or Tris-buffered saline (TBS)-Tween. H146-968 mAbs were visualized with a polyclonal peroxidase-conjugated goat anti-hamster Ig conjugate (Southern Biotechnology Associates, Birmingham, AL). Biotinylated proteins or secondary biotinylated mAb were detected using streptavidin-peroxidase (Southern Biotechnology Associates). Complexes were visualized with an enhanced chemiluminescence system (Pierce). For the detection of phosphorylated tyrosines, 5% BSA (BSA stock solution; Pierce) in TBS was used as a blocker. BSA solution (1%) was used during every incubation. To detect phosphotyrosines, the mAb 4G10 was used at 1 µg/ml in conjunction with a goat anti-mouse antiserum which was conjugated to horseradish peroxidase as a secondary reagent (Bio-Rad).

Stimulation assays

Ab stimulation. As described by Bolliger et al. (15), 145-2C11 and H57-597 mAbs were bound to a 96-well flat-bottom plate, and 50 µl of transfected cells at 106 cells/ml were added to each well. Cells were incubated for 20 h at 37°C in 7.5% CO2. Culture supernatants were harvested and frozen before measuring the amount of IL-2 present.

Superantigen (SAg) stimulation. DAP.3 cells were incubated with the indicated amounts of SAg (Staphylococcus aureus A toxin, Sigma) at 5 x 105 cells/ml. A total of 50 µl/well were distributed on flat-bottom 96-well plates. Finally, 50 µl of transfected cells at 106 cells/ml were added in each well and incubated for 20 h at 37°C in 7.5% CO2. Supernatants were treated as previously described.

Ag stimulation. Apocytochrom c peptide was dissolved in water and used as a 50 mg/ml stock solution. LK35.2 cells were incubated with the indicated peptide concentrations at 5 x 105 cells/ml and plated out in flat-bottom 96-well plates. A total of 50 µl of responders at 106 cells/ml were added, and culture supernatants were assayed for IL-2 concentration as described. All experiments were done in triplicates, and every experiment was repeated at least three times. The amount of IL-2 present in the supernatants was quantified using the IL-2-dependent cell line HT-2 in a standard IL-2 assay. Alamar blue substrate (Alamar Biosciences, Sacramento, Ca) was used to assess viability of the IL-2 dependent cell line, HT-2, according to the manufacturer’s instructions. Plates were read in a Molecular Devices SpectoMAX 340 machine, and results were analyzed with SOFTmax pro software (Molecular Devices, Sunnyvale, Ca). Murine rIL-2 was used as a standard (PharMingen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The extracellular domain of the {zeta}-chain is essential for TCR surface expression.

The sequence and functional homology of the Fc{epsilon}RI{gamma} to the {zeta}-chain has suggested that the length and composition of the {zeta}-EC domain can influence {alpha}ß T cell function (11, 12, 13). We examined the structural requirements of the {zeta}-EC domain by deleting the nine amino acids covering the domain (EX-) (Fig. 1Go). In this construct the TM of the {zeta}-chain was directly used as a noncleavable ER-targeting signal sequence to overcome the inefficient cleavage by the signal peptidase in the proximity of the membrane. The dipole determined by the charge difference of the TM proximal domains still respected the requirements for a type I integral membrane protein, thus allowing its correct membrane orientation (28). Transfection of the {zeta}-chain-deficient T cell hybridoma MA5.8 (29) with EC-negative {zeta}-chain cDNA (EX-) did not rescue TCR surface expression, in contrast to the wild-type (WT) {zeta}-chain as demonstrated by the FACS analysis of bulk transfectants (Fig. 2GoA). This result suggested that EC-negative {zeta}-chain cannot associate with the TCR complex. To show the correct location of the {zeta}-(EX-) mutant, we took advantage of the fact that the {zeta}-chain covalently dimerizes via an interchain disulfide bridge only after having reached the oxidizing environment of the ER, in contrast to the reducing conditions of the cytoplasm. IP of lysates containing the WT {zeta} or {zeta}-(EX-) mutant were performed with specific Abs to the {zeta}-chain or CD3{epsilon} (Fig. 2GoB). In the WT {zeta}-transfectant, the covalently linked {zeta}-chain homodimer could be recovered with both anti-CD3{epsilon} and anti-{zeta} Abs, demonstrating its association to the TCR complex (WT in lanes 1, 2, and 5, 6 under reducing conditions, Fig. 2GoB). In contrast, the EX- covalently linked {zeta}-homodimer could be immunoprecipitated only with anti-{zeta} Abs, indicating that {zeta}-(EX-) mutant had reached the ER but was unable to associate with the TCR/CD3 complex (EX- in lanes 3, 4, and 7, 8 under reducing conditions, Fig. 2GoB). Interestingly, the {zeta}-(EX-) mutant produced some of the monomeric form under nonreducing conditions, suggesting a possible inefficient translocation to the ER. However, this monomeric form was not wrongly oriented in the membrane, since a mAb directed to the very C term of cytoplasmic tail of the {zeta}-homodimer failed to stain the transfectants (data not shown).



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  		FIGURE 1. Primary sequence comparison of the EC and part of the TM domain of Fc{epsilon}R{gamma} subunit and the {zeta}-chain mutants used in this study. The numbering used all along this study is highlighted on top of the sequences. The {zeta}-chain mutant missing the EC domain is displayed as well (EX-).

 


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  		FIGURE 2. A, TCR expression pattern of the truncated EC domain of the {zeta}-chain. MA5.8 is the recipient cell line stained with anti-CD3{epsilon} FITC-labeled Abs. WT and EX- are the profiles of bulk MA5.8 infected with WT {zeta}-chain and the truncated form of the {zeta}-chain, respectively. B, Western blot of WT and EX- transfectant lysates immunoprecipitated with anti-CD3{epsilon} or {zeta}-specific Abs probed with mAb specific for the {zeta}-chain. The same samples were equally divided and run on a 12% SDS-PAGE either under nonreducing or reducing conditions. {zeta} dimers ({zeta}-{zeta}) and monomers ({zeta}) are highlighted.

 
In summary, these results showed that {zeta}-chain lacking the EC domain cannot associate with the TCR complex to restore TCR surface expression, and further suggest that the EC domain mediates an essential step in the biogenesis of the TCR/CD3 complex.

Cysteine scanning mutagenesis along the EC domain

To extend our analysis of the {zeta}-EC domain in TCR-mediated functions, we performed a cysteine scanning mutagenesis by exchanging highly conserved amino acids to cysteine. The advantages of cysteine over the classical amino acid of choice, alanine (alanine scanning mutagenesis), are given by the possibility to form covalent bonds between protein domains. The formation of new covalent bonds can constrain the mobility and the conformation, and, therefore, directly affect the function of the protein, in this case, of the {zeta}-chain. We also produced double mutants to directly evaluate the formation of a displaced disulfide bridge by replacing the cysteine which is involved in {zeta}-chain covalent homodimerization with a glycine (cysteine to glycine at position 11, 11CG; see Fig. 1Go for the nomenclature used). The mutants generated are listed in Fig. 1Go.

The 11CG exchange produced a noncovalently-linked {zeta} homodimer, but by itself did not significantly influence TCR function (compare WT and 11CG in Fig. 4Go, and in agreement with previous data (30)), even if its ability to sustain TCR surface expression was reduced by 25–30% (compare mean channel fluorescence intensity in Fig. 3Go, WT vs 11CG). Single-point mutations introduced at position 7 (aspartic acid to cysteine, 7DC) and 5 (leucine to cysteine, 5LC) in the EC domain of the {zeta}-chain clearly impaired TCR surface expression, suggesting that those residues may be important for interactions with a yet undefined partner of the TCR complex. Interestingly, the absence of a cysteine at position 11 in those two mutants completely blocked TCR surface expression (compare profiles of 7DC-11CG and 5LC-11CG to the WT in Fig. 3Go) suggesting that an incorrectly placed disulfide bridge produced a {zeta} homodimer not able to assemble into the TCR complex.



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  		FIGURE 4. Biochemical analysis of cell surface biotinylated {zeta}-chain mutants. After cell surface biotinylation cells were lysed and cleared, lysates were subjected to IP (anti-CD3{epsilon} or anti-{zeta}), nonreducing SDS-PAGE, and Western blot analysis. A, Biotinylated proteins recovered after IP were highlighted with streptavidin-HRP. TCR{alpha}ß, the CD3 complex ({gamma}{delta}{epsilon}), and the {zeta} dimer ({zeta}-{zeta}) or ({zeta}) are indicated. B, The same Western blot was probed with Abs specific for the {zeta}-chain. The monomeric and dimeric forms of {zeta} are indicated.

 


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  		FIGURE 3. TCR surface expression profiles of bulk {zeta} transfectants. The TCR surface expression of the recipient cell line MA5.8 infected with the different mutants (see text) was quantified with an anti-CD3{epsilon} FITC-conjugated mAb. Mean channel fluorescence values are indicated within the icon top right.

 
In contrast, the exchange to a cysteine at position 4 (glycine to cysteine) supported TCR surface expression, even in the absence of the normal disulfide bridge (compare 4GC and 4GC-11CG in Fig. 3Go). Therefore, a {zeta}-chain containing a possible single-shifted disulfide bridge could still lead to TCR expression. Similarly, the exchange of phenylalanine at position 3 to a cysteine efficiently rescued TCR surface expression to 11CG levels, both in the presence or absence of the normal disulfide bridge at position 11 (compare 3FC and 3FC-11CCG in Fig. 3Go).

These experiments showed that the exchange of conserved amino acids in the short EC domain of the {zeta}-chain to cysteine modulated the level of TCR surface expression and suggest an interaction with other components of the TCR complex. Moreover, they emphasized the sensitivity of TCR surface expression to improperly "homodimerized" {zeta} homodimer.

The natural disulfide bridge of the {zeta}-chain can be shifted by seven and eight amino acids along the EC domain

To analyze the composition of the TCR complexes and to test the presence of the disulfide bridge in the mutant {zeta}-chains at the cell surface, we subjected these {zeta}-chain mutants to surface biotinylation, IP, SDS-PAGE analysis under nonreducing conditions, and Western blot analysis (Fig. 4Go). IP of WT {zeta} transfectants with anti-CD3{epsilon} Abs yielded the following surface labeled proteins: an 87-kDa TCR{alpha}ß heterodimer, a 32-kDa {zeta}-homodimer, CD3{delta} and a doublet of CD3{gamma} and {epsilon} (28 kDa and 25 kDa, respectively), as shown in Fig. 4GoA. The same blot was probed with a specific Ab to the {zeta}-chain and revealed a 32-kDa band corresponding to the mobility corresponding to the {zeta}-{zeta} homodimer described in Fig. 4GoA (Fig. 4GoB, indicated as {zeta}-{zeta}). Mutant cell lines 7DC, 4GC, 3FC, and 3FC-11CG produced the same pattern of surface-labeled TCR complex subunits as the WT transfectant, suggesting that all of the expected components of the TCR complex were associated and that all of the {zeta}-chain mutants were indeed disulfide-linked (Fig. 4GoA for surface labeling, and Fig. 4GoB for {zeta}-specific probe). Thus, 3FC-11CG contained a single interchain disulfide bridge that was displaced from its normal position by eight amino acids. As expected, 11CG, the {zeta}-chain without cysteines required for the covalent dimerization, migrated as a 16-kDa surface-labeled monomer (Fig. 4GoA)and was specifically detected with an anti-{zeta} Ab (Fig. 4GoB, indicated with {zeta}). However, this result did not allow us to draw any conclusions about the presence of 11CG in the TCR complex in a monomeric form. The 4GC-11CG mutant {zeta}-chain was not recovered upon IP with anti-CD3{epsilon} Abs (Fig. 4Go, A and B, respectively), but the surface-labeled {zeta}-chain could be recovered in IPs as a covalently linked homodimer using a specific mAb directed to {zeta} (compare IP CD3{epsilon} to IP {zeta} of 4GC-11CG in Fig. 4Go, A and B, respectively). This demonstrated its presence at the cell surface as a covalently linked homodimer that was loosely associated to the TCR/CD3 complex.

The displaced interchain disulfide bridge was clearly formed at positions 4 and 3 along the EC domain of the 4GC-11CG and 3FC-11CG {zeta} mutants, and both mutants were clearly able to support TCR complex surface expression (Fig. 3Go). That these two mutants could be biochemically distinguished in their association to the TCR/CD3 complex suggested that the position of a disulfide bridge could influence the stability of the {zeta}-chain in the TCR complex. Interestingly, the 4GC and 4GC-11CG {zeta} mutants could also be similarly distinguished in their TCR complex association. The fact that 4GC and 4GC-11CG differed only by the presence or absence of the cysteine forming the native disulfide bridge suggested that the presence of the normal disulfide bridge might stabilize the {zeta} association to the TCR/CD3 complex.

The shifted disulfide bridge impairs T cell activation by Ag

To determine the functional effects of a shifted disulfide bridge within the {zeta}-chain homodimer, mutants were subjected to stimulation with mAbs, SAg, and the canonical peptide. Stimulation with plate-bound bivalent anti-TCR or anti-Thy-1 (31) mAb similarly induced the production of IL-2 in all of the mutants shown (Fig. 5GoA, anti-CD3{epsilon}; and Fig 5GoB, anti-TCRß; anti-Thy-1, data not shown). The SAg stimulation of the mutants occurred similarly to the WT (Fig. 5GoC), though the dose-response curves of hybridomas expressing mutant {zeta}-chains were slightly reduced compared with the WT or 11CG cell lines. Unexpectedly, stimulation with the canonical peptide Ag segregated the mutants from the WT and 11CG transfectants (Fig. 5GoD). In fact, while the WT and 11CG were comparable in their dose-response curves, the {zeta} mutants 4GC, 4GC-11CG, 3FC, and 3FC-11CG clearly demonstrated a 25-fold shift in the IL-2 dose-response curve (Fig. 5GoD). Despite the heterogeneity of TCR expression and the stability of the {zeta} mutant homodimer within the complex, all mutants demonstrated a similarly reduced IL-2 response. Therefore, these data suggested that the presence of the shifted disulfide bridge was causing the defect during the peptide stimulation.



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  		FIGURE 5. Functional analysis of the {zeta}-chain mutants. The amount of IL-2 produced is quantified on the y-axis, and the dose of Ag is on the x-axis. A, Stimulation with plate-bound mAb anti-CD3{epsilon} and B, anti-TCRß-chain. C, Stimulation with SAg (SEA) presented by DAP3 cells, and D, nominal peptide (pigeon apocytochrom c 90–104) presented by LK35.2.

 
These results showed that displaced disulfide bridges at positions 3 and 4 in the EC domain of the {zeta}-chain lead to clear defects in TCR signaling. Furthermore, they suggest that the requirement of the EC domain was altered during the engagement of the TCR either with mAb or SAg and MHC-peptide.

{zeta} Mutants generate different phosphorylation patterns upon MHC-peptide Ag stimulation

Tyrosine phosphorylation of ancillary TCR components upon engagement is a very sensitive measure of productive TCR engagement (7, 32). We tested the 4GC, 4GC-11CG, 3FC, and 3FC-11CG {zeta} mutants, which were defective in the production of IL-2 upon Ag stimulation (Fig. 5GoD), in a tyrosine phosphorylation assay to identify possible alterations in the phosphorylation pattern.

Peptide Ag stimulation of WT {zeta} transfectants yielded the expected tyrosine-phosphorylated bands, revealed by IP with anti-{zeta}- or anti-ZAP-70-specific Abs. The phosphorylated forms of {zeta} (p21 and p23), phospho-CD3{epsilon}, phopho-ZAP-70, phospho-p36, and a group of discrete bands that may represent ubiquitinated forms of phospho-{zeta} can be seen in lysates of Ag-stimulated hybridomas expressing the WT {zeta}-chain (compare lane 1 with lanes 2 and 3 in Fig. 6Go) (33). In contrast to the IP with anti-{zeta} mAb, where the p21 form of phosphorylated {zeta} was dominant, the ZAP-70 IP produced equimolar ratios of both p21 and p23. Unphosphorylated ZAP-70 could only be detected in the IP specific for ZAP-70 (Fig. 6Go, lower panel), suggesting that only a minor fraction of tyrosine phosphorylated ZAP-70 was associated to the {zeta}-chain. Both p36 and the discrete bands above were similarly recovered from both IPs, suggesting that they belong to the same complex (34). The different ratio of p21 and p23 in the two IPs may result from the associated ZAP-70 masking the {zeta}-chain epitope recognized by the anti-{zeta} mAb.



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  		FIGURE 6. Phosphorylation patterns upon nominal peptide stimulation. Bulk {zeta} transfectants were stimulated with peptide presented by the P13.9 cell line. After IP, probes were resolved on a 12% SDS-PAGE and Western blotted. Blots were probed with antiphosphotyrosine mAb (upper panel) and anti-ZAP-70 mAb (lower panel). Significant phosphorylated bands are indicated, and higher phospho-{zeta}-intermediates are shown with a bracket.

 
The two {zeta}-chain mutants with a displaced disulfide bridge shown in Fig. 6Go (4GC-11CG and 3FC-11CG, and 4GC and 3FC, data not shown) produced a clearly different tyrosine phosphorylation pattern compared with the WT. The p21 and p23 {zeta} forms were efficiently induced and recovered in both mutants. However, neither p36 nor any of the other discrete phosphotyrosine-containing bands around 40 kDa could be immunoprecipitated from the 4GC-11CG and 3FC-11CG lysates using anti-{zeta} or anti-ZAP Abs. Furthermore, phospho-ZAP-70 could be recovered from WT lysates by IP with either anti-{zeta} or anti-ZAP-70 Abs, whereas, it could only be recovered from the mutant lysates with an anti-ZAP-70 antiserum but not with the anti-{zeta} mAb. Similar results were also obtained with selected clones expressing comparable levels of WT and 4GC-11CG {zeta}-chain mutant (see next paragraph).

These biochemical alterations likely explain the decrease in IL-2 production reported in this study.

Low TCR expression does not account for the phenotype of hybridomas expressing mutant {zeta}-chain

It was possible that differences in TCR surface expression between the WT and mutant {zeta} transfectants could account for the inefficient TCR stimulation with MHC/peptide. To address this issue, we produced T cell clones from the bulk transfectants (WT and 4GC-11CG) by limiting dilution, and isolated matched pairs of WT and mutant clones with similar TCR surface expression (low and high, as described in Fig. 7GoA). We did not isolate clones from 3FC or 3FC-11CG {zeta}-chain mutants because their surface expression was similar to 11CG, which behaved like WT {zeta}-chain (see Fig. 3Go and 5Go).



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  		FIGURE 7. Analysis of two matched groups of WT and 4GC-11CG clones. A, FACS quantification of TCR complex surface expression with anti-CD3{epsilon} mAb. B, Quantification of IL-2 production upon peptide/MHC stimulation. C, Western blot analysis of tyrosine-phosphorylated proteins of WT and 4GC-11CG clones after peptide/MHC stimulation and anti-{zeta} mAb IP. Significant phosphorylated bands are indicated, and higher phospho-{zeta}-intermediates are shown with a bracket.

 
The biochemical analysis of the 4GC-11CG mutants (low and high TCR expressors) revealed its loose association into the TCR complex (data not shown, and in agreement with Fig. 4Go). The stimulation of the respective pairs of hybridomas demonstrated that the 4GC-11CG clones were clearly defective in the response to antigenic peptide. This was particularly evident in the pair of clones expressing high amounts of surface TCR (Fig. 7GoB). Furthermore, MHC/peptide stimulation and subsequent phosphotyrosine analysis was conducted with these TCR-matched pairs of clones (Fig. 7GoC). Similar to what is shown in Fig. 6Go, the production of p36 and high molecular tyrosine-phosphorylated proteins was defective in the 4GC-11CG mutant clones. In the low-expressing clone pair the amount of p21 and p23 forms of phospho-{zeta} in the mutant were even reduced compared with the WT.

These results clearly showed that the difference in the TCR surface expression was not the primary cause of the impairment of peptide/MHC-induced signals, but rather the presence of a displaced interchain disulfide bridge was responsible for the defect.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Most studies regarding the role of the TCR components during T cell activation have focused on site-directed mutagenesis of distinct motifs present in the cytoplasmic or TM domains (e.g., TM charged residues, ITAMs, and dileucine internalization motifs) (35, 36, 37, 38). In the present study, the EC domain of the {zeta}-chain was examined because recently published results highlighted its potential involvement in TCR signaling (9, 10, 15).

In the present set of experiments, we first showed that the truncation of the EC domain of the {zeta}-chain EX- completely abolished TCR complex surface expression. These results show that the EC domain is not needed for the homodimerization of {zeta} but is required for an early step during the biogenesis of the TCR complex. In this respect, the EC domain may allow a stable association with the TCR complex, and once assembled into the complex, the {zeta}-chain may mask the degradation signals of the CD3 subunits with its bulky cytoplasmic tail. Alternatively, the {zeta}-EC domain may be involved in TCR superdimerization (39, 40, 41).

We subsequently performed a cysteine scanning mutagenesis to gain an insight into the contribution of the {zeta}-EC domain to TCR signaling. The experiments presented in this study show that single-point mutations in the EC domain of {zeta}-chain at positions 3, 4, and, in particular, 5 and 7, influenced TCR surface expression, suggesting that this part of the EC domain interacts with a component of the TCR/CD3 complex. Strikingly, {zeta}-chains harboring only the displaced cysteine at positions 5 or 7 (5LC-11CG and 7DC-11CG) were not able to rescue TCR surface expression. Taken together, these results emphasize that a correct conformation of the {zeta}-{zeta} homodimer, determined by the placement of the disulfide bond, is essential to permit the {zeta}-chain to assemble into the TCR complex. Although, a single-point mutation at position 3 or 4 also influenced TCR surface expression (4GC, 4GC-11CG, 3FC, and 3FC-11CG {zeta} mutants), the presence or the absence of the normal cysteine at position 11 did not have any further effect on expression. In these {zeta} mutants, the shifted disulfide bridge was clearly formed, as shown in Fig. 4Go, and produced a TCR/CD3 complex containing a disulfide-linked {zeta}-homodimer. However, this does not produce an optimal conformation of the {zeta}-chain EC domain since the TCR surface expression was altered compared with the WT. Thus, it is possible to shift the normal disulfide bridge along the EC domain of the {zeta}-chain, but only a limited number of positions produce TCR compatible {zeta}-homodimers. In fact, it is conceivable that the formation of a shifted disulfide bridge in the EC domain may influence the orientation of downstream domains (TM and cytoplasmic) and block the assembly into the TCR/CD3 complex. That the normal {zeta}-disulfide bridge could be shifted by seven and eight amino acids in an N-terminal direction along the EC domain, respects the requirements for an {alpha}-helical structure. Since one {alpha}-helical turn covers 3.6 amino acids, the shift of the disulfide bond by seven or eight amino acids speculatively suggests that the {zeta}-EC domain is comprised of two {alpha}-helical turns.

The observation that, in contrast to 4GC, the 4GC-11CG {zeta}-chain was not recovered in the TCR complex during IP with anti-CD3 mAb, suggested that, in this specific case, the presence of a second covalent linkage was advantageous for {zeta}-chain stability within the complex without affecting TCR surface expression. However, a more stably associated {zeta}-homodimer (i.e., 4GC, 3FC, and 3FC-11GC) did not give any obvious advantage during MHC/peptide stimulation, as these four mutants were equally deficient in their IL-2 production when compared with the WT.

In general, the {zeta} mutants described here displayed a segregation of responses triggered by the same TCR complex. It is interesting that responses to anti-TCR mAbs and to SAg were only modestly affected in the mutants, while the responses to peptide/MHC ligands were dramatically affected (Fig. 5GoD and Fig. 7GoB). Our experiments clearly show that recognition of a MHC-peptide ligand relies on an intact {zeta}-EC domain to generate a signal. Importantly, the EC domain of the {zeta}-chain does not contribute to Ag binding, and given its EC location, it cannot directly influence ITAM function. However, the {zeta}-EC domain may couple the Ag-binding {alpha}ß heterodimer to the cytoplasmic domains.

A potential criticism of our interpretation of the results is that the mutants express less TCR at the cell surface compared with the WT, and this might be the only reason for a reduced signaling efficacy. Three lines of evidence argue against this being the cause of the phenotypes described here. First, the dose-response curve during TCR engagement with SAg of the mutants was minimally reduced compared with the WT or the 11CG {zeta}-chains. As SAg relies on the same TCR{alpha}ß requirements as MHC/peptide engagement, it is unlikely that only the difference in surface expression explains the phenotype, but rather suggests that intrinsic differences in TCR triggering can be accounted for this difference. Second, the 11GC {zeta} mutant has a reduced TCR surface expression compared with the WT, nevertheless, a similar TCR surface expression to 3FC and 3FC-11CG; however, functionally, it behaved indistinguishably from the WT. Third, and most importantly, we tested clones derived from the 4GC-11CG bulk transfectants and compared them to WT clones expressing a comparable amount of surface TCR complex (Fig. 7Go).

Although the mutant {zeta}-chains were tyrosine phosphorylated to produce the p21 and p23 species, the production of other tyrosine phosphoproteins was clearly defective. In lysates of Ag-stimulated hybridomas, phosphorylated ZAP-70 was not associated with the mutant {zeta}-chains. Furthermore, the phosphorylated form of p36 failed to be coimmunoprecipitated with the mutant {zeta}-chains, as well. {zeta}-Chain phosphorylation per se is not obviously blocked in hybridomas expressing these mutants, but rather the block seems to occur further downstream. In fact, the signaling blockade involves the failure to recruit and/or phosphorylate ZAP-70 and p36. The lack of p36 phosphorylation in the mutant hybridomas is consistent with an early block in the signaling cascade. The fact that a {zeta}-chain with a mutant EC domain allows its phosphorylation but not subsequent steps in the signal transduction implies that the shifted disulfide bond might alter the conformation of the {zeta} cytoplasmic tails and impede proper docking of ZAP-70 (42) and/or p36 during peptide/MHC engagement. It is likely that the p36 protein corresponds to the LAT protein that was recently cloned and is pivotal during TCR signal transduction (43).

We would like to propose that the short {zeta}-EC domain is directly involved in the reorganization of the TCR complex that initiates the signaling cascade (9, 10, 44). In our mutants, this activation step might have a higher threshold because the shifted disulfide bridge constrains the mobility of the {zeta} homodimer, and, therefore, disturbs the TCR complex reorganization during engagement. Since the stimulation with mAb and, to some extent SAg (45), induces a cross-linking of TCR complexes, the rate-limiting step for MHC/peptide-induced activation might be thus physically bypassed. For this reason, the {zeta} mutants did not essentially differ from the WT in stimulation assays with mAb and SAg. A recent report supports our finding that mAb and SAg may have different requirements for signaling compared with MHC/peptide. It has been shown that, in contrast to MHC/peptide stimulation, SAg stimulation is independent of lck (46). Alternatively, the defect in the phosphorylation could reflect quantitative, rather than "mechanical," effects that are produced by the difference in the strength of the stimuli used: strong with cross-linking Abs or SAg, and weak with peptide/MHC. In this case, the EC domain of the {zeta}-chain could be involved in setting the threshold for activation.

That ITAM-deficient {zeta}- and Fc{epsilon}RI{gamma}-chains are signaling defective has been reported in hybridomas and reconstituted {zeta}-/- mice (12, 13, 47, 48). It is interesting that the {zeta} mutants reported in this study display a similarly impaired phenotype upon Ag stimulation, even though all of the three {zeta} ITAMs are still present. These two experimental results are not necessarily contradictory because the two types of mutation are structurally different. In fact, the accessibility of the CD3{gamma}, {delta}, and {epsilon} ITAMs in the TCR complexes harboring the tail-less {zeta} homodimer might be increased, and the disadvantage of having fewer ITAMs might be overcome with a less bulky {zeta} cytoplasmic tail, as recently reported (49). The different location of the disulfide bridge along the {zeta}-EC domain, accounts for the defective behavior of the mutant {zeta}-homodimers. A structurally locked {zeta}-EC domain could impair the normal tyrosine phosphorylation events, and possibly hinder an optimal interaction with downstream signaling partners such as ZAP-70 and p36. As the activation of T cells from the point of view of the {zeta}-chain is an ordered and sequential process in terms of its lck-dependent phosphorylation and ubiquitination (33, 50, 51, 52), a missing link would have important effects on the all-signaling cascade. By analogy to the signaling scaffold proposed for the B-cell receptor (53), it is therefore possible to assume that the recently proposed reorganization of the TCR complex upon Ag stimulation (54) may be defective in {zeta}-chain mutants described in this manuscript.


   Acknowledgments
 
We thank S. Stotz, Drs. T. Göbel, H. Jacobs, and J. Bluestone for critically reading the manuscript; B. Pfeiffer, H. Spalinger, and H. P. Stahlberger for photography and artwork; and Drs. L. Samelson and R. Germain for reagents. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche LTD, Basel, Switzerland.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Luca Bolliger, Basel Institute for Immunology, Grenzacherstrasse 487, 4005 Basel, Switzerland. E-mail address: Back

2 Abbreviations used in this paper: ER, endoplasmic reticulum; ITAM, immunoreceptor-tyrosine activation motif; EC, extracellular; TM, transmembrane; WT, wild type; SAg, superantigen; IP, immunoprecipitation. Back

Received for publication July 21, 1998. Accepted for publication September 29, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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