The TCR/CD3 complex is composed of six subunits which are expressed on the cell surface in a coordinate fashion after assembly in the endoplasmic reticulum (ER). The TCR/CD3 complex is assembled after a series of pairwise interactions involving the formation of dimers of CD3 epsilon with either CD3 gamma or CD3 delta. These dimers assemble with TCR alpha and TCR beta chains, and finally, the CD3 zeta homodimer is added to allow export of the full complex from the ER. A model has been proposed suggesting that during assembly the CD3 epsilon/CD3 gamma dimer interacts exclusively with TCR beta and the CD3 epsilon/CD3 delta dimer with TCR alpha to form a complex with a single TCR alpha/beta heterodimer. We show in this study, by immunoprecipitation and two-dimensional gel electrophoresis, that in the human T cell line Jurkat as well as in total human thymocytes, this preferential interaction does not occur and instead, the CD3 epsilon/CD3 gamma and CD3 epsilon/CD3 delta dimers associate with both TCR chains simultaneously and indistinctly. These data are confirmed by the analysis of the TCR alpha-negative T cell line MOLT-4 in which TCR beta is found separately associated with CD3 epsilon/CD3 gamma and with CD3 epsilon/CD3 delta dimers. Indirectly, our results support a model of stoichiometry in which two TCR alpha/beta heterodimers are present in a TCR/CD3 complex. Furthermore, immunoprecipitation with anti-CD3 gamma and anti-CD3 delta antibodies from 1% NP40 and 1% Brij96 cell lysates showed that these subunits form independent partial complexes which are cross-linked through the CD3 zeta homodimer. This suggests that CD3 zeta mediates the interaction between both TCR alpha/beta heterodimers contained in the double TCR complex. Further proof for this hypothesis is obtained after analysis of a Jurkat cell transfectant containing a point mutation in the transmembrane domain of TCR beta that impairs the association of CD3 zeta. In this mutant cell line, unlike a control line with wild-type TCR beta, the CD3 gamma- and CD3 delta-containing complexes were found completely independent. Altogether, these results support a model of TCR/CD3 assembly and stoichiometry in which two TCR-alpha/beta heterodimers form two hemicomplexes containing either CD3 epsilon/gamma or CD3 epsilon/delta dimers which become associated via the CD3 zeta homodimer.

The transmembrane domain of T cell receptor (TCR) ␤ contains a conserved immunoreceptor tyrosine-based activation-like motif consisting of a duplicated YXXL sequence. The motif is also present in TCR␥, the equivalent chain to TCR␤ in ␥␦ T lymphocytes but is absent in TCR␣ and TCR␦. To determine the putative role of this sequence in TCR⅐CD3 complex assembly and signal transduction, a TCR␤ chain cDNA was mutated in the C-terminal tyrosine of the motif, cloned in an expression vector, and transfected into TCR␤-negative Jurkat cells. Transfectants of the mutated chain (MUT) expressed, on average, much less TCR⅐CD3 complex on the membrane than wild type TCR␤ transfectants. Radiolabeling experiments suggested that the mutation caused a loose association of the CD3 chain resulting in a defective assembly. However, stimulation of high TCR⅐CD3 expressing wild type and MUT clones with monoclonal antibodies and Staphylococcus aureus enterotoxin B resulted in similar levels of CD25 and CD69 expression, interleukin-2 secretion, and TCR⅐CD3 complex downregulation. By contrast, MUT cells were clearly resistant to activation-induced cell death, and they did not express CD95-ligand upon activation. These results suggest a differentiated intracellular signaling pathway leading to apoptosis in which Tyr-TM11 of the immunoreceptor tyrosine-based activation motif-like motif and CD3 appear to be involved. The T cell antigen receptor⅐CD3 complex (TCR⅐CD3)1 is formed by a clonotypic heterodimer (␣␤ or ␥␦), which provides ligand specificity, non-covalently linked to at least four invariant chains (CD3⑀, -␥, -␦, and -) (for review see Refs. 1-3). Assembly occurs by pairwise interactions (4), and as a result, the TCR⅐CD3 complex is formed by four dimeric components as follows: (a) clonotypic TCR␣ and TCR␤ chains that are covalently linked via a single extracellular disulfide bond; (b) the non-covalent CD3␥⑀ dimer; (c) the non-covalent CD3␦⑀ dimer; and (d) a disulfide-linked family dimer consisting of any of the five defined members of this family, , , , , and the ␥ chain of the high affinity Fc⑀ receptor (5). In addition to these interactions, stable pairwise associations can also be observed between single clonotypic and CD3 chains (4, 6, 7). Nevertheless, the CD3 dimer will only assemble in the complex if all the other subunits are present, thus explaining why CD3 is the last component to be integrated into the TCR⅐CD3 complex during assembly (8 -10). The stoichiometry, as well as the possible formation of alternative TCR⅐CD3 complexes, is still a question of debate.The ability of antigen receptors to transduce signals to multiple biochemical cascades is the central event of immune cell activation (11). Engagement of the multicomponent TCR⅐CD3 complex with its antigen/MHC ligand, agonist mAbs, or superantigens results in several biochemical processes critical for the functional activation of T lymphocytes, including cellular proliferation, cell differentiation, and programmed cell death. Using chimeric molecules and reconstituted recept...

We have previously shown that a tyrosine to leucine replacement in the transmembrane region of T cell receptor (TCR)-β results in a deficient induction of CD95-L and apoptosis upon TCR triggering in a transfected T cell line. By contrast, interleukin (IL)-2 production and the expression of CD25 and CD69 were normally induced. Since the mutation in TCR-β also resulted in impaired association of CD3-ζ, it was proposed that this chain is specifically required for the induction of apoptosis. We now show that the deficient induction of CD95-L and apoptosis does not derive from a general lower production of second messengers, since intracellular Ca2+ fluxes and tyrosine phosphorylation of total proteins were elicited at wild-type levels. Unlike in T cell clones stimulated with partial agonists, both p21 and p18 forms of tyrosine-phosphorylated CD3-ζ were detected, although the overall level of tyrosine-phosphorylated CD3-ζ was low. More strikingly, inducible association of ZAP70 to CD3-ζ was strongly inhibited, despite a normal induction of ZAP70 tyrosine phosphorylation. Finally, ZAP70 was not concentrated near the plasma membrane in the apoptosis-deficient cells. These results suggest that CD3-ζ is necessary for engagement of a specific signaling pathway leading to CD95-L expression that also needs the recruitment of ZAP70.