The Impact of Single Amino Acid Substitutions in CD3γ on the CD3ε{lunate}γ Interaction and T-Cell Receptor-CD3 Complex Formation

The human T-cell receptor-CD3 complex consists of at least eight polypeptide chains; CD3γε{lunate}- and δε{lunate}-dimers associate with the disulfide linked αβ- and ζζ-dimers to form a functional receptor complex. The exact structure of this complex is still unknown. We now have examined the interaction between CD3γ and CD3ε{lunate} in human T-cells. For this purpose, we have generated site-directed mutants of CD3γ that were introduced in human T-cells defective in CD3γ expression. Cell-surface and intracellular expression of the introduced CD3γ chains was determined, as was the association with CD3δ, CD3ε{lunate}, and the T-cell receptor. Although the introduction of wild type CD3γ and CD3γ (78Y-F) fully restored T-cell receptor assembly and expression, the introduction of CD3γ (82C-S), CD3γ (85C-S), and CD3γ (76Q-E) all resulted in an impaired association between CD3γ and CD3ε{lunate} and a lack of cell-surface expressed CD3γ. Finally, the introduction of CD3γ (76Q-L) and CD3γ (78Y-A) restored the expression of TCR-CD3δε{lunate}γε{lunate}ζ₂ complexes, although the association between CD3γ and CD3ε{lunate} was impaired. These results indicate that several amino acids in CD3γ are essential for an optimal association between CD3γ and CD3ε{lunate} and the assembly of a cell-surface expressed TCR-CD3δε{lunate}γε{lunate}ζ₂ complex.