The T cell antigen receptor alpha and beta chains interact via distinct regions with CD3 chains.
Selective pairwise interactions between CD3 chains and the clonotypic T cell antigen receptor (TCR)-alpha, -beta chains has recently been established. In this study, the region of interaction between clonotypic and CD3 chains involved with assembly was examined. To determine the site of protein interaction a variety of genetically altered TCR chains were constructed. These included: truncated proteins, lacking transmembrane and or cytosolic domains; chimeric proteins, in which extracellular, transmembrane or cytosolic domains were replaced with similar domains derived from either the Tac antigen or CD4; and point mutagenized TCR chains. COS-1 cells were transfected with cDNA, metabolically labeled, and immunoprecipitates analyzed using non-equilibrium pH gel electrophoresis (NEPHGE)-SDS/PAGE. The results demonstrated that assembly between TCR-alpha and TCR-beta chains occurred at the extracellular level. Assembly of the TCR-alpha chain with CD3-delta, and CD3-epsilon was localized to an eight-amino acid motif within the transmembrane domain of TCR-alpha. Site-specific mutations of the TCR-alpha charged residues within this motif (arginine, lysine) to leucine and similar point mutations of the transmembrane CD3-epsilon and CD3-delta charge groups resulted in the abrogation of assembly. In contrast, TCR-beta and CD3-epsilon binary complexes interacted via their extracellular domain. Analogous to TCR-alpha, the site of TCR-beta and CD3-delta assembly was at the transmembrane region. Despite multiple genetic manipulations on CD3-gamma and zeta these proteins failed to assemble with TCR-alpha. Similarly, there was no interaction between TCR-beta and zeta. These findings when coupled with the information on pairwise interactions and formation of higher order subcomplexes extend our model for the structure of the TCR complex.
Rheumatology Department, Royal North Shore Hospital, St. Leonards, Sydney, NSW, Australia.
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N
Manolios
O
O
Kemp
Z G
ZG
Li
D000818:Animals; D017252:CD3 Complex / Q000737:chemistry* / Q000378:metabolism; D004274:DNA, Recombinant; D015180:Electrophoresis, Gel, Two-Dimensional; D011233:Precipitin Tests; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000378:metabolism*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D017252:CD3 Complex; D004274:DNA, Recombinant; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins