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.
Franco
R
R
Division of Immunology, Beth Israel Hospital, Harvard Medical School, Boston, MA 02215.
Peter
M E
ME
Choi
M S
MS
Malissen
B
B
Sancho
J
J
Terhorst
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017252:CD3 Complex / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D017455:Receptors, IgE / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism
D017252:CD3 Complex; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D017455:Receptors, IgE; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins