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.

Signaling via mitogen-activated protein kinase kinase (MEK1) is required for Golgi fragmentation during mitosis.

We have developed an assay using permeabilized cells to monitor fragmentation of the Golgi complex that occurs during mitosis. Golgi stacks, in permeabilized interphase normal rat kidney (NRK) cells, upon incubation with mitotic extracts undergo extensive fragmentation, and the fragmented Golgi membranes are dispersed throughout the cytoplasm. We find that the continued presence of p34cdc2, the mitosis initiation kinase, is not necessary for Golgi fragmentation. Instead, fragmentation depends on cytosolic mitogen-activated protein kinase kinase 1 (MEK1 or MAPKK1). However, the known cytoplasmic substrates for MEK1, ERK1, and ERK2 are not required for this process. Interestingly, we find a Golgi-associated ERK, which we propose as the likely target for MEK1 in Golgi fragmentation.

A tyrosine-containing motif mediates ER retention of CD3-epsilon and adopts a helix-turn structure.

The CD3-epsilon endoplasmic reticulum (ER) retention motif has been characterized by mutagenesis and NMR spectroscopy. Tyr177, Leu180 and Arg183 are involved in ER retention. The motif forms an elongated alpha-helix in which the tyrosine and leucine residues are closely apposed, followed by a beta I' turn that places Arg183 in the vicinity of Leu180. The structure formed by Tyr177 and the leucine in position +3 is reminiscent of the beta-turn structure adopted by tyrosine-containing endocytosis signals. Moreover, substitution of the transferrin receptor (TfR) internalization sequence by the CD3-epsilon motif still allowed the rapid internalization of the TfR and, conversely, the chimeric protein resulting from the substitution of the CD3-epsilon motif by the endocytosis signal of the low density lipoprotein receptor was ER located. These data support the idea of a functional homology between the two types of signal.

An endoplasmic reticulum retention signal in the CD3 epsilon chain of the T-cell receptor.

Isolated polypeptide chains of the T-cell antigen receptor complex are degraded or retained in the endoplasmic reticulum (ER). Assembly of the multisubunit complex allows the individual chains to escape retention in the ER and to be expressed on the cell surface. We engineered a series of deletions in the CD3 epsilon subunit of the human T-cell receptor in order to find the sequences responsible for its retention in the ER. Deletion of amino acids 171 to 180 in the cytosolic tail resulted in the cell-surface expression of the isolated chain. This sequence also promotes retention when it is appended to CD4, a plasma membrane protein. Mutagenesis of the 10-amino-acid CD3 epsilon sequence established that the tyrosine and serine residues are important for ER retention. This and other ER retention signals must be hidden when a complete T-cell receptor complex is assembled in order to allow its expression on the cell surface.