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 pl ace by pairwise interactions. Thus, CD3-epsilon interacts with either CD3-gamma or CD3-delta, and these dimers then associate with the TCR h eterodimer (alpha.beta or gamma.delta) and the CD3-zeta homodimer to c onstitute a full complex. We have now mapped the site in CD3-E respons ible 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 res ponsible for the interaction. Thus, deletion of this 16-amino acid ext racellular sequence resulted in the inhibition of up to 95% of the CD3 -epsilon/gamma interaction. A highly conserved sequence is also presen t in both CD3-gamma and CD3-delta, suggesting that the domain in these two chains may reciprocally be involved in the interaction with CD3-E , Indeed, an immobilized synthetic peptide corresponding to the CD3-ga mma sequence specifically associated to a bacterially expressed CD3-ep silon protein, suggesting the le-amino acid domain is sufficient to pr omote 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 int eractions. Indeed, it is shown that it mediates the formation of disul fide-linked homodimers and that the formation of homo-and heterodimers are mutually excluded. Finally, this domain contains a Cys-X-X-Cys se quence that resembles that of p56(lck), which is responsible for the i nteraction with the cytoplasmic tails of CD4 and CD8. Since the replac ement of the two cysteines (Cys(97) and Cys(100)) in CD3-epsilon by al anines strongly inhibited pair formation, the existence of a Cys-X-X-C ys motif involved in protein-protein interactions is suggested.