Manolios N, Kemp O, Li Z G
Rheumatology Department, Royal North Shore Hospital, St. Leonards, Sydney, NSW, Australia.
Eur J Immunol. 1994 Jan;24(1):84-92. doi: 10.1002/eji.1830240114.
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.
最近已证实CD3链与克隆型T细胞抗原受体（TCR）的α、β链之间存在选择性的两两相互作用。在本研究中，对参与组装的克隆型链与CD3链之间的相互作用区域进行了检测。为确定蛋白质相互作用位点，构建了多种基因改造的TCR链。这些包括：缺失跨膜和/或胞质结构域的截短蛋白；嵌合蛋白，其中细胞外、跨膜或胞质结构域被源自Tac抗原或CD4的相似结构域所取代；以及点突变的TCR链。用cDNA转染COS-1细胞，进行代谢标记，并用非平衡pH凝胶电泳（NEPHGE）-SDS/PAGE分析免疫沉淀产物。结果表明，TCR-α链与TCR-β链之间的组装发生在细胞外水平。TCR-α链与CD3-δ和CD3-ε的组装定位于TCR-α跨膜结构域内的一个八氨基酸基序。该基序内TCR-α带电残基（精氨酸、赖氨酸）的位点特异性突变为亮氨酸，以及跨膜CD3-ε和CD3-δ电荷基团的类似点突变导致组装被废除。相反，TCR-β与CD3-ε二元复合物通过其细胞外结构域相互作用。与TCR-α类似，TCR-β与CD3-δ的组装位点在跨膜区域。尽管对CD3-γ和ζ进行了多种基因操作，但这些蛋白未能与TCR-α组装。同样，TCR-β与ζ之间也没有相互作用。这些发现与关于两两相互作用和高阶亚复合物形成的信息相结合，扩展了我们对TCR复合物结构的模型。
