Genetic reconstitution of the T cell receptor (TcR) α/β heterodimer restores the association of CD3 ζ₂ with the TcR/CD3 complex

Abstract

The cell surface expression of the T cell receptor (TcR)/CD3 complex and, consequently, the functional competence of the cell is partly dependent on CD3 zeta. In its absence, a pentameric complex (TcR alpha/beta/CD3 gamma delta epsilon) is formed which is inefficiently transported to the cell surface. Reconstitution of CD3 zeta by transfection, in turn, restores the cell surface expression and function of the complex. Through the use of transfection experiments, we here provide direct evidence that the association of CD3 zeta 2 with the TcR/CD3 complex is dependent on the presence of both the TcR alpha and beta polypeptide chains. Despite wild-type levels of the CD3 zeta protein in a TcR alpha-negative mutant human T cell line, a complex was formed intracellularly which lacked CD3 zeta 2 and consisted of beta gamma delta epsilon and beta 2 gamma delta epsilon. Upon transfection of the mutant with a TcR alpha cDNA, a TcR/CD3 complex which contained CD3 zeta 2 was observed intracellularly. In contrast to the partial subcomplex on the cell surface of the untransfected cell line, the TcR/CD3 complex on the transfectant was functional as demonstrated by its ability to mobilize intracellular calcium after stimulation with a mitogenic CD3 epsilon-specific monoclonal antibody. Transient transfection studies performed in COS cell fibroblasts indicated that CD3 zeta 2 was not interacting with the TcR alpha protein alone, implying that a conformation provided by either the TcR alpha/beta heterodimer or the TcR alpha/beta/CD3 gamma delta epsilon complex was necessary for the association of CD3 zeta 2. Transfection studies performed in a TcR alpha/beta-negative murine T-T hybridoma confirmed the requirement of both the TcR alpha and beta proteins in CD3 zeta 2 binding. We conclude that the TcR alpha and beta chains harbor polypeptide sequences essential for the association of CD3 zeta 2 with the TcR/CD3 complex.

Apoptosis but Not Other Activation Events Is Inhibited by a Mutation in the Transmembrane Domain of T Cell Receptor β That Impairs CD3ζ Association

“…The hampered transport of the TCR⅐CD3 complex from the ER in MUTC2 is compatible with a deficit in the association of CD3 chain which has been suggested to be the last step in the assembly of the TCR⅐CD3 complex (9,20). Indeed, although some CD3 homodimer was detected in metabolic labelings of WTB7 cells (Fig.…”

“…Thus, transient transfection studies performed in COS cells indicated that CD3 does not interact directly with TCR or CD3 chains alone, but it requires the previous association of both TCR␣ and -␤ to the other CD3 chains in order to provide adequate conformation for binding of CD3 (8,20).…”

Characterization of immature thymocyte lines derived from T-cell receptor or recombination activating gene 1 and p53 double mutant mice.

“…The surface expression of CD3-X in cells that are unable to express TCR-a chains is in contradiction to a number of studies implicating a requirement for the formation of a TCR-a,B heterodimer prior to the integration of CD3-; into the TCR-CD3 complex, which is generally regarded as the last step in CD3-TCR assembly (3,16,20,31). It should be taken into account, however, that those data were obtained by using mature T-cell lines, from which mutants were derived that were then rescued by transfection of CD3 or TCR components, or by transfecting CD3 and TCR genes into nonlymphoid cells.…”

“…Stable pairwise assembly has previously been observed between single TCR-a or TCR-13 chains and CD3 chains (18). In contrast, the CD3-C dimer assembles more efficiently with the TCR chains when all of the other CD3 components are present (16,(18)(19)(20) the CD3-; chain was associated with the TCR-f3 chain in the TCR-a mutant cell lines, cells were solubilized in a buffer containing 1% Brij-35, a detergent which is sufficiently weak to maintain the integrity of the TCR-CD3 complex. Cell lysates were immunoprecipitated with H57-597, a mAb recognizing all TCR-f3 chains.…”

Cloning and sequencing of the cDNA encoding the human homologue of the murine immunoglobulin‐associated protein B29

“…Conversely, the TcR transmembrane segments harbor positive charges which have been suggested to stabilize interactions between TcR and CD3 chains by mediating ion or hydrogen bonds [20,211. Removing both of the positive charges from the TcR cx transmembrane segment was shown to abolishTcR alp as well as subsequent TcR/CD3 interactions [22]. Likewise, removing the negative charges from CD3 proteins prevented the formation of TcRaICD36 and TcRP/CD3s subcomplexes [23].…”