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

Using a newly developed in situ affinity-labeling method of nucleotide-binding proteins (NTP^oxi technique) we discovered that the human T-cell receptor-associated CD3-ζ protein might bind GTP/GDP. To further characterize GTP/GDP binding to CD3-ζ, murine T-cell lines expressing ζζ homodimers or CD3-ζ/FcεR1γ heterodimers were used. Specific GTP^oxi labeling of CD3-ζ was found in all murine T cells in which a complete CD3-ζ polypeptide chain was expressed, including cells in which CD3-ζ was disulfide bridged to the FcεR1γ chain. In murine T cells the kinetics of labeling of CD3-ζ was similar to that of small G-proteins. Upon activation of murine T cells a slight but significant increase in GTP^oxi labeling of CD3-ζ was detected. Whether all 3 so-called 'Reth motifs' (ζ_A, ζ_B and/or ζ_C) were necessary for the binding of GTP/GDP was addressed by using cells expressing truncated CD3-ζ molecules. Whereas truncated CD3-ζ, in which ζ_A and part of ζ_B were deleted, was still able to bind GTP, upon deletion of all 3 Reth motifs cross-linking by the GTP^oxi method became impossible. Regardless of whether this implies a direct or indirect binding of GTP/GDP to CD3-ζ, these nucleotides and their hydrolysis must play an important role in T-cell activation through the TCR/CD3 complex.