Abstract
Functionally mature human T lymphocytes express a cell-surface receptor for antigen (T cell receptor (TCR)-CD3) composed of at least six polypeptides (TCR-alpha and -beta; T3-gamma, -delta, -epsilon, and -zeta). Immature thymocytes and variants of T cell lines lacking one of the TCR.CD3 polypeptide chains fail to express surface receptor and accumulate the other chains intracellularly. Here we show that the assembly of the TCR.CD3 complex within the endoplasmic reticulum (ER) began with a core of CD3-gamma, -delta, and -epsilon to which TCR-alpha and -beta bound. A recently described intracellular protein, CD3-omega, participated in the assembly since it was found to be associated with the free TCR-alpha or -beta chains or with the CD3 chains. CD3-omega dissociated as TCR.CD3 complexes were formed in the ER. Association of non-disulfide-linked TCR-alpha and -beta chains with CD3 was detected before that of disulfide-bridged TCR-alpha/beta heterodimers. These data suggest that during assembly, the association of TCR-alpha and -beta chains with the CD3 complex precedes the formation of a TCR-alpha/beta dimer. The existence of intermediates consisting of CD3-gamma, -delta, and -epsilon chains and a single TCR-alpha or -beta chain was also confirmed by using a series of variant T cell lines lacking the TCR-beta or -alpha chain, respectively. Once the single TCR-alpha and -beta chains were associated with CD3, disulfide linkages were formed, and a 70-kDa form of the TCR was detected within the ER. This intracellular precursor of the TCR.CD3 complex was subsequently processed into the mature 90-kDa TCR as the TCR.CD3 complex passed through the Golgi apparatus. Assembly of the TCR.CD3 complex is a rather rapid process, whereas export from the ER occurs at a slow rate. After 1 h, 75% of the receptor complex remained within the ER.
Highlights
From the $Laboratory of Molecular Immunology and the I(Division of Tumor Immunology, Dana-Farber Cancer Institute, Haruard Medical School, Boston,Massachusetts 02115
The CD3 proteins disulfide-linked TCR-a and -B chains with CD3 was have been proposed to function in transductionof the signal detected beforethat of disulfide-bridged TCR-a/B het- induced by antigenlreceptorinteraction.Inadditionto a erodimers
Identification of the 42-kDa TCR-a and the36-kDa TCR-j3 chains was based on comparisons with immunoprecipitations with the anti-TCR-a reagent H36 (Fabbi et al, 1985) and the anti-TCR-j3 reagent j3F1 (Brenner et al.,1987b) (Fig. 1, B and C) and reprecipitation of proteins eluted from the SDS gel
Summary
TCR-aIp Heterodimers and Single Chains Associatewith the CD3 Proteins-In order to determine distinct stepsin the assembly of the TCR-a/P.CD3 complex, pulse-chase immunoprecipitation experiments were performed using the human leukemic T cell lines Jurkat, REX, HPB-ALL, PEER, and MOLT4. Of the Human TCR*CD3 Complex these experiments (Fig. 1, A and B ) , the CD3-o chain had dence that theEndo-H treatment was completeand resulted disappeared before the 70-kDa TCR-a/B heterodimer had in the polypeptide chain backbone of TCR-a and -B was been converted into the mature 90-kDa form.Taken together, provided by the observation that thesame digestion products these results strongly suggested that CD3-wwas associated were obtained by N-glycosidase F treatment. Quantitative measurements the one hand, and the TCR-a and -P forms, on the other, showed that 70% of the 70-kDa TCR heterodimer associated align vertically on the two-dimensional gel, this suggests that with CD3 is in the “immature” form 1 h after labeling and the only dimers present in untreated cells are either eoior 25% 6 h afterthe radioactive pulse was given (Fig.).
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