Characterization of the region involved in CD3 pairwise interactions within the T cell receptor complex.
Assembly of the six-chain T cell antigen receptor-CD3 complex takes place by pairwise interactions. Thus, CD3-epsilon interacts with either CD3-gamma or CD3-delta, and these dimers then associate with the TCR heterodimer (alpha.beta or gamma.delta) and the CD3-zeta homodimer to constitute a full complex. We have now mapped the site in CD3-epsilon responsible for the interaction with CD3-gamma and CD3-delta by analysis of a series of deletional mutants encompassing the most conserved regions. We found that the highly conserved juxtamembrane domain is mainly responsible for the interaction. Thus, deletion of this 16-amino acid extracellular sequence resulted in the inhibition of up to 95% of the CD3-epsilon/gamma interaction. A highly conserved sequence is also present in both CD3-gamma and CD3-delta, suggesting that the domain in these two chains may reciprocally be involved in the interaction with CD3-epsilon. Indeed, an immobilized synthetic peptide corresponding to the CD3-gamma sequence specifically associated to a bacterially expressed CD3-epsilon protein, suggesting the 16-amino acid domain is sufficient to promote CD3-epsilon/CD3-gamma assembly. The conservation of the motif in the CD3 chains suggest that, in addition to CD3-epsilon/CD3-gamma and CD3-epsilon/CD3-delta interactions, it may also mediate homotypic interactions. Indeed, it is shown that it mediates the formation of disulfide-linked homodimers and that the formation of homo- and heterodimers are mutually excluded. Finally, this domain contains a Cys-X-X-Cys sequence that resembles that of p56(lck), which is responsible for the interaction with the cytoplasmic tails of CD4 and CD8. Since the replacement of the two cysteines (Cys97 and Cys100) in CD3-epsilon by alanines strongly inhibited pair formation, the existence of a Cys-X-X-Cys motif involved in protein-protein interactions is suggested.
Borroto
A
A
Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, Cantoblanco, Madrid 28049, Spain.
Mallabiabarrena
A
A
Albar
J P
JP
Martínez-A
C
C
Alarcón
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017252:CD3 Complex / Q000737:chemistry / Q000378:metabolism*; D019556:COS Cells; D017931:DNA Primers; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017252:CD3 Complex; D017931:DNA Primers; D017260:Receptor-CD3 Complex, Antigen, T-Cell