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 CD3ζ. In its absence, a pentameric complex (TcR α/β/CD3γδϵ) is formed which is inefficiently transported to the cell surface. Reconstitution of CD3ζ 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 ζ₂ with the TcR/CD3 complex is dependent on the presence of both the TcR α and β polypeptide chains. Despite wild‐type levels of the CD3ζ protein in a TcR α‐negative mutant human T cell line, a complex was formed intracellularly which lacked CD3 ζ₂ and consisted of βγδϵ and β₂γδϵ. Upon transfection of the mutant with a TcR α cDNA, a TcR/CD3 complex which contained CD3 ζ₂ 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 ζ‐specific monoclonal antibody. Transient transfection studies performed in COS cell fibroblasts indicated that CD3 ζ₂ was not interacting with the TcR α protein alone, implying that a conformation provided by either the TcR α/β heterodimer or the TcR α/β/CD3 γδϵ complex was necessary for the association of CD3 ζ₂. Transfection studies performed in a TcR α/β‐negative murine T‐T hybridoma confirmed the requirement of both the TcR α and β proteins in CD3 ζ₂ binding. We conclude that the TcR α and β chains harbor polypeptide sequences essential for the association of CD3 ζ₂ with the TcR/CD3 complex.