Characterization of the GTP/GDP binding site in the murine CD3-zeta polypeptide chain

Using a newly developed in situ affinity-labeling method of nucleotide-binding proteins (NTPoxi technique) we discovered that the human T-cell receptor-associated CD3-zeta protein might bind GTP/GDP. To further characterize GTP/GDP binding to CD3-zeta, murine T-cell lines expressing zeta zeta homodimers or CD3-zeta/Fc epsilon R1 gamma heterodimers were used. Specific GTPoxi labeling of CD3-zeta was found in all murine T cells in which a complete CD3-zeta polypeptide chain was expressed, including cells in which CD3-zeta was disulfide bridged to the Fc epsilon R1 gamma chain. In murine T cells the kinetics of labeling of CD3-zeta was similar to that of small G-proteins. Upon activation of murine T cells a slight but significant increase in GTPoxi labeling of CD3-zeta was detected. Whether all 3 so-called 'Reth motifs' (zeta A, zeta B and/or zeta C) were necessary for the binding of GTP/GDP was addressed by using cells expressing truncated CD3-zeta molecules. Whereas truncated CD3-zeta, in which zeta A and part of zeta B were deleted, was still able to bind GTP, upon deletion of all 3 Reth motifs cross-linking by the GTPoxi method became impossible. Regardless of whether this implies a direct or indirect binding of GTP/GDP to CD3-zeta, these nucleotides and their hydrolysis must play an important role in T-cell activation through the TCR/CD3 complex.