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.
Manolios
N
N
Rheumatology Department, Royal North Shore Hospital, St. Leonards, Sydney, NSW, Australia.
Kemp
O
O
Li
Z G
ZG
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