Structural mutations of C-domains in members of the Ig superfamily. Consequences for the interactions between the T cell antigen receptor and the zeta 2 homodimer.
Several molecules belonging to the Ig superfamily are expressed together with noncovalently associated subunits. This applies for membrane-bound IgM and IgD, some of the FcR, and the Ti dimers of the TCR. The interactions between members of the Ig superfamily and their associated subunits are still not fully understood. We locate critical amino acid residues for TCR assembly in the Ti-alpha and -beta extracellular C-domains. A point mutation (phenylalanine195----valine) in a highly conserved residue in the Ti-alpha chain of the Jurkat variant J79 was identified by DNA sequencing. This mutation did not prevent cytoplasmic association of Ti alpha beta and CD3 gamma delta epsilon, but abolished binding of the zeta 2 homodimer to the rest of the TCR. The consequences of this mutation for TCR assembly were confirmed by transfection of a site-directed mutagenized Ti-alpha chain into a Ti-alpha-deficient Jurkat variant. Computer model analysis showed that the Ti-alpha phenylalanine195 directly contributed to the beta-sheet facing away from the Ti-beta chain, indicating that it could be directly involved in the interactions between one or more of the CD3 chains or the zeta 2 dimer. Site-directed mutagenesis of the corresponding residue in the Ti-beta chain demonstrated that a phenylalanine216----valine substitution had similar effects on TCR assembly as the Ti-alpha mutation, whereas a phenylalanine216----histidine substitution allowed TCR assembly and expression. Whether the consequences for TCR assembly of the Ti-alpha and -beta mutations were due to any direct effects on the interaction between zeta and the Ti alpha beta dimer or to indirect effects are discussed.
Geisler
C
C
Department of Tumor Cell Biology, Fibiger Institute, Copenhagen, Denmark.
Rubin
B
B
Caspar-Bauguil
S
S
Champagne
E
E
Vangsted
A
A
Hou
X
X
Gajhede
M
M
D000595:Amino Acid Sequence; D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry* / Q000648:ultrastructure; D001483:Base Sequence; D017252:CD3 Complex; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000648:ultrastructure; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D013329:Structure-Activity Relationship
D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta