The high affinity Fc epsilon receptor gamma subunit (Fc epsilon RI gamma) facilitates T cell receptor expression and antigen/major histocompatibility complex-driven signaling in the absence of CD3 zeta and CD3 eta.

The T cell receptor (TCR) is a molecular complex formed by at least seven transmembrane proteins: the antigen/major histocompatibility complex recognition unit (Ti alpha-beta heterodimer) and the invariant CD3 chains (gamma, delta, epsilon, zeta, and eta). In addition to targeting partially assembled Ti alpha-beta CD3 gamma delta epsilon TCR complexes to the cell surface, CD3 zeta appears to be essential for interleukin-2 production after TCR stimulation with antigen/major histocompatibility complex. The gamma chain of the high affinity Fc receptor for IgE (Fc epsilon RI gamma) has significant structural homology to CD3 zeta and the related CD3 eta subunit. To identify the functional significance of sequence homologies between CD3 zeta and Fc epsilon RI gamma in T cells, we have transfected a Fc epsilon RI gamma cDNA into a T cell hybridoma lacking CD3 zeta and CD3 eta proteins. Herein we show that a Fc epsilon RI gamma-gamma homodimer associates with TCR components to up-regulate TCR surface expression. A TCR composed of Ti alpha-beta CD3 gamma delta epsilon Fc epsilon RI gamma-gamma is sufficient to restore the coupling of TCR antigen recognition to the interleukin-2 induction pathway, demonstrating the functional significance of structural homology between the above receptor subunits. These results, in conjunction with the recent finding that CD3 zeta, CD3 eta, and Fc epsilon RI gamma are coexpressed in certain T cells as subunits of an unusual TCR isoform, suggest that Fc epsilon RI gamma is likely to play a role in T cell lineage function.     

Assembly, intracellular processing, and expression at the cell surface of the human alpha beta T cell receptor/CD3 complex. Function of the CD3-zeta chain

The TCR/CD3 complex is a multimeric protein complex composed of a minimum of seven transmembrane chains (TCR alpha beta-CD3 gamma delta epsilon zeta 2). Whereas earlier studies have demonstrated that both the TCR-alpha and -beta chains are required for the cell surface expression of the TCR/CD3 complex, the role of the CD3 chains for the TCR/CD3 expression have not been experimentally addressed in human T cells. In this study the function of the CD3-zeta chain for the assembly, intracellular processing, and expression of the TCR/CD3 complex in the human leukemic T cell line Jurkat was investigated. The results indicate that: 1) CD3-zeta is required for the cell surface expression of the TCR/CD3 complex; 2) the pentameric form (TCR alpha beta-CD3 gamma delta epsilon) of the TCR/CD3 complex and single TCR chains associated with CD3 (TCR alpha-CD3 gamma delta epsilon and TCR beta-CD3 gamma delta epsilon) are produced in the endoplasmic reticulum in the absence of CD3-zeta; 3) the CD3-zeta does not associate with TCR alpha-CD3 gamma delta epsilon or TCR beta-CD3 gamma delta epsilon complexes; 4) CD3-zeta associate with the pentameric form of the TCR/CD3 complex in the endoplasmic reticulum to form the heptameric complex (TCR alpha beta-CD3 gamma delta epsilon----TCR alpha beta-CD3 gamma delta epsilon 2); and 5) CD3-zeta is required for the export of the TCR/CD3 complex from the endoplasmic reticulum to the Golgi apparatus for subsequent processing.     

Biochemical evidence for the presence of a single CD3delta and CD3gamma chain in the surface T cell receptor/CD3 complex

The T cell antigen receptor (TCR) consists of an alphabeta heterodimer and associated invariant CD3gamma, delta, epsilon, and zeta chains (TCR/CD3 complex). The general stoichiometry of the receptor complex, which is believed to be one molecule each of TCRalpha, TCRbeta, CD3gamma, and CD3delta and two molecules each of CD3epsilon and CD3zeta, is not clearly understood. Although it has been shown that there are two chains of CD3epsilon and CD3zeta, the stoichiometry of CD3gamma or CD3delta chains in the surface antigen receptor complex has not been determined. In the present study, transgenic mice expressing an altered form of mouse CD3delta and CD3gamma were employed to show that the surface TCR complexes contain one molecule each of CD3delta and CD3gamma. Thymocytes from wild type and CD3 chain transgenic mice on the appropriate knockout background were surface-biotinylated and immunoprecipitated using a specific antibody. The immunoprecipitates were resolved in two dimensions under nonreducing/reducing conditions to determine the stoichiometry of CD3delta and CD3gamma in the surface antigen receptor complex. Our data clearly show the presence of one molecule each of CD3delta and CD3gamma in the surface TCR/CD3 complex.     

The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules.

Recent studies have demonstrated that the CD3-zeta subunit of the T cell antigen receptor (TCR) complex is involved in signal transduction. However, the function of the remaining invariant subunits, CD3-gamma, -delta, and epsilon, is still poorly understood. To examine their role in TCR function, we have constructed TCR/CD3 complexes devoid of functional zeta subunit and showed that they are still able to trigger the production of interleukin-2 in response to antigen or superantigen. These data, together with previous results, indicate that the TCR/CD3 complex is composed of at least two parallel transducing units, made of the gamma delta epsilon and zeta chains, respectively. Furthermore, the analysis of partially truncated zeta chains has led us to individualize a functional domain that may have constituted the building block of most of the transducing subunits associated with antigen receptors and some Fc receptors.     

Masking of the CD3 gamma di-leucine-based motif by zeta is required for efficient T-cell receptor expression

The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.     

Intracellular redistribution of nucleolin upon interaction with the CD3epsilon chain of the T cell receptor complex

T cell activation through the antigen receptor (TCR) involves the cytoplasmic tails of the CD3 subunits CD3gamma, CD3delta, CD3epsilon, and CD3zeta. Whereas the biological significance of the cytoplasmic tails of these molecules is suggested, in part, by their evolutionarily conserved sequences, their interactions with signal transduction molecules are not completely understood. We used affinity chromatography columns of glutathione S-transferase fused to the CD3epsilon cytoplasmic tail to isolate proteins that specifically interact with this subunit. In this way, we identified the shuttling protein nucleolin as a specific CD3epsilon-interacting molecule. Using competition studies and affinity chromatography on peptide columns, we were able to identify a central proline-rich sequence as the nucleolin-interacting sequence in CD3epsilon. Transfection in COS cells of wild type CD3epsilon, but not of nonbinding mutants of CD3epsilon, resulted in redistribution of nucleolin from the nucleus and nucleoli to the cytoplasm. This property was transferred to a CD8 protein chimera by appending the cytoplasmic tail of CD3epsilon. We also found that nucleolin associated with the TCR complex. This association was increased upon TCR engagement, suggesting that the CD3epsilon/nucleolin interaction may have a role in T cell activation.     

Assembly and function of the T cell antigen receptor. Requirement of either the lysine or arginine residues in the transmembrane region of the alpha chain

The T cell receptor (TCR) for antigen consists, on the majority of peripheral lymphocytes, of an immunoglobulin-like, disulfide-linked heterodimeric glycoprotein: the alpha and beta chain. These proteins are noncovalently linked to at least four nonvariant proteins which comprise the CD3 complex: CD3 gamma, delta, epsilon, and zeta. Whereas the TCR alpha and beta proteins have positively charged residues in the transmembrane region, all the CD3 proteins have similarly placed negatively charged amino acid residues. It has been suggested that these basic and acidic amino acid residues may play an important role in TCR.CD3 complex assembly and/or function. In this paper, the structural and functional role of the lysine and arginine residues of the TCR alpha chain was addressed using oligonucleotide mediated site directed mutagenesis. The Arg256 and Lys261 residues of the TCR alpha cDNA of the HPB-ALL cell line were mutated to either Gly256 and/or Ile261. The altered cDNAs were transfected into a TCR alpha negative recipient mutant cell line of REX, clone 20A. Metabolic labeling of the T cell transfectants showed that mutation of either the Arg256 or Lys261 amino acid residues had no effect on the ability of the TCR alpha chain to form either a heterodimer with the TCR beta chain or a complex with the CD3 gamma, delta, and epsilon proteins. Consequently, the Arg256 to Gly256 and Lys261 to Ile261 mutations did not prevent the formation of a mature, functional TCR.CD3 complex on the cell surface as determined by immunofluorescence, cell surface radioiodination, and the ability of the transfectants to mobilize intracellular calcium after stimulation with a mitogenic anti-CD3 epsilon monoclonal antibody. In contrast, a mutant cDNA in which both the Arg256 and Lys261 residues were mutated to Gly256 and Ile261, respectively, failed to reconstitute the cell surface expression of the TCR.CD3 complex and, consequently, the ability to respond to mitogenic stimuli. In the absence of both the Arg256 and Lys261 residues, TCR alpha beta heterodimer formation was not observed. Cotransfection studies in COS cells showed that the failure of assembly of a heterodimer was likely due to an inability of the mutated TCR alpha chain to form a subcomplex with either the CD3 gamma, delta, epsilon, or zeta proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

T cell receptor (TCR) beta chain homodimers on the surface of immature but not mature alpha, gamma, delta chain deficient T cell lines.

Transfected T cell receptor (TCR) beta chain genes are expressed as homodimers on the surface of immature (Sci/ET27F) but not on mature (58 alpha-beta-) T cell lines which lack TCR alpha, gamma and delta chains. The homodimer on Sci/ET27F cells is tightly bound to CD3 delta and CD3 epsilon while the association with CD3 gamma and CD3 zeta proteins is rather weak. Crosslinking of the TCR beta homodimers resulted in a strong and rapid calcium flux. In 58 alpha-beta- T cells the beta TCR chain could be easily visualized intracellularly but was not transported to the cell surface. The Scid cell lines considerably facilitate the molecular analysis of early differentiation events in the thymus which are likely to be regulated by the beta TCR homodimer.     

Developmental and functional impairment of T cells in mice lacking CD3 zeta chains.

CD3 zeta is a component of the T cell antigen receptor (TCR) complex and is important for signal transduction. We have established mice selectively lacking CD3 zeta but able to express CD3 eta, a polypeptide produced from the same locus through alternative splicing, using the method of gene targeting in embryonic stem cells. In homozygous mutant mice, the numbers of thymocytes and peripheral T cells were greatly reduced and the expression levels of TCR on these cells were 5-fold lower than those on wild-type cells. By contrast, TCR gamma delta+ intestinal intraepithelial lymphocytes were not obviously affected by the mutation. T cells from homozygous mutants exhibited an impaired proliferative response. These results imply that CD3 zeta has a critical role in the development and signal transduction of T cells in vivo.     

The gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors

The T cell receptor for antigen (TCR) is composed of six different transmembrane proteins. T cells carefully control the intracellular transport of the receptor and allow only complete receptors to reach the plasma membrane. In an attempt to understand how T cells regulate this process, we used c-DNA transfection and subunit-specific antibodies to follow the intracellular transport of five subunits (alpha beta gamma delta epsilon) of the receptor. In particular, we assessed the intracellular stability of each chain. Our results showed that the chains were markedly different in their susceptibility to intracellular degradation. TCR alpha and beta and CD3 delta were degraded rapidly, whereas CD3 gamma and epsilon were stable. An analysis of the N-linked oligosaccharides of the glycoprotein subunits suggested that the chains were unable to reach the medial Golgi during the metabolic chase. This was supported by immunofluorescence micrographs that showed both the stable CD3 gamma and unstable CD3 delta chain localized in the endoplasmic reticulum. To study the effects of subunit associations on intracellular transport we used cotransfection to reconstitute precise combinations of subunits. Associations between stable and unstable subunits expressed in the same cell led to the formation of stable complexes. These complexes were retained in or close to the endoplasmic reticulum. The results suggested that the intracellular transport of the T cell receptor could be regulated by two mechanisms. The TCR alpha and beta and CD3 delta subunits were degraded rapidly and as a consequence failed to reach the plasma membrane. CD3 gamma or epsilon were stable but were retained inside the cell. The results also demonstrated that there was an interplay between the two pathways such that the CD3 gamma and epsilon subunits were able to protect labile chains from rapid intracellular degradation. In this way, they could seed subunit assembly in or close to the endoplasmic reticulum and allow a stable receptor to form before its transport to the plasma membrane.     

A redundant role of the CD3 gamma-immunoreceptor tyrosine-based activation motif in mature T cell function

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3gamma, CD3delta, and CD3epsilon) or three (CD3zeta) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3zeta chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-gammadeltaepsilon module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3gamma-, delta-, and epsilon-ITAMs regulate TCR signaling. We here report on the role of the CD3gamma chain and the CD3gamma-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3gamma null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3gamma. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3gamma-ITAM. Loss of integrity of the CD3gamma-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3gamma-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3gamma-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3delta and/or the CD3epsilon chains endow the TCR with qualitatively unique signaling functions.     

Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER

The T cell antigen receptor (TCR) is an oligomeric protein complex made from at least six different integral membrane proteins (alpha beta gamma delta epsilon and zeta). The TCR is assembled in the ER of T cells, and correct assembly is required for transport to the cell surface. Single subunits and partial receptor complexes are retained in the ER where TCR alpha, beta, and CD3 delta chains are degraded selectively. The information required for the ER degradation of the TCR beta chain is confined to the membrane anchor of the protein (Wileman et al., 1990c; Bonifacino et al., 1990b). In this study we show that the rapid degradation of the TCR beta chain is inhibited when it assembles with single CD3 gamma, delta, or epsilon subunits in the ER, and have started to define the role played by transmembrane anchors, and receptor ectodomains, in the masking proteolytic targeting information. Acidic residues within the membrane spanning domains of CD3 subunits were essential for binding to the TCR beta chain. TCR beta chains and CD3 subunits therefore interact via transmembrane domains. However, when sites of binding were restricted to the membrane anchor of the TCR beta chain, stabilization by CD3 subunits was markedly reduced. Interactions between membrane spanning domains were not, therefore, sufficient for the protection of the beta chain from ER proteolysis. The presence of the C beta domain, containing the first 150 amino acids of the TCR ectodomain, greatly increased the stability of complexes formed in the ER. For assembly with CD3 epsilon, stability was further enhanced by the V beta amino acids. The results showed that the efficient neutralization of transmembrane proteolytic targeting information required associations between membrane spanning domains and the presence of receptor ectodomains. Interactions between receptor ectodomains may slow the dissociation of CD3 subunits from the beta chain and prolong the masking of transmembrane targeting information. In addition, the close proximity of TCR and CD3 ectodomains within the ER may provide steric protection from the action of proteases within the ER lumen.     

The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.     

Role of CD3 gamma in T cell receptor assembly

The T cell receptor (TCR) consists of the Ti alpha beta heterodimer and the associated CD3 gamma delta epsilon and zeta 2 chains. The structural relationships between the subunits of the TCR complex are still not fully known. In this study we examined the role of the extracellular (EC), transmembrane (TM), and cytoplasmic (CY) domain of CD3 gamma in assembly and cell surface expression of the complete TCR in human T cells. A computer model indicated that the EC domain of CD3 gamma folds as an Ig domain. Based on this model and on alignment studies, two potential interaction sites were predicted in the EC domain of CD3 gamma. Site-directed mutagenesis demonstrated that these sites play a crucial role in TCR assembly probably by binding to CD3 epsilon. Mutagenesis of N-linked glycosylation sites showed that glycosylation of CD3 gamma is not required for TCR assembly and expression. In contrast, treatment of T cells with tunicamycin suggested that N-linked glycosylation of CD3 delta is required for TCR assembly. Site-directed mutagenesis of the acidic amino acid in the TM domain of CD3 gamma demonstrated that this residue is involved in TCR assembly probably by binding to Ti beta. Deletion of the entire CY domain of CD3 gamma did not prevent assembly and expression of the TCR. In conclusion, this study demonstrated that specific TCR interaction sites exist in both the EC and TM domain of CD3 gamma. Furthermore, the study indicated that, in contrast to CD3 gamma, glycosylation of CD3 delta is required for TCR assembly and expression.     

CD3.TCR1, a human CD3 epitope expressed on viable gamma/delta lymphocytes exclusively.

T lymphocytes express either the alpha/beta or the gamma/delta receptor (TCR) in a mutually exclusive fashion. Both structures are associated on the cell membrane with the CD3 proteins which are thought to transduce signals resulting from antigen recognition. The CD3 complex is present in both alpha/beta and gamma/delta cells and includes at least five proteins (designated gamma, delta, epsilon, zeta and eta). We have developed here a novel mAb, anti-CD3.TCR1, which immunoprecipitates the CD3 molecules from both alpha/beta and gamma/delta cells lysates following solubilization with Triton X-100. While the SDS-PAGE migration profile of the material recognized by either anti-CD3.TCR1 or anti-OKT3 are superimposable in both cell types, this mAb recognizes viable untreated gamma/delta T lymphocytes exclusively. These findings further support the view that molecular interactions within the TCR/CD3 protein complex are distinct in the two T lymphocyte populations.     

Failure to synthesize the CD3-gamma chain. Consequences for T cell antigen receptor assembly, processing, and expression.

The TCR consists of the Ti alpha beta heterodimer and the associated CD3 chains, CD3 gamma delta epsilon zeta 2 or zeta eta. The structural relationships between the subunits of the Ti/CD3 complex are still not fully understood. To explore the roles of the individual CD3 chains for the assembly, intracellular processing, and expression of the TCR, mutants of the T cell line Jurkat were isolated. One variant, JGN, was found to produce all the Ti/CD3 components with the exception of CD3-gamma. The results indicate that: 1) the tetrameric form (Ti alpha beta-CD3 delta epsilon) of the Ti/CD3 complex is produced in the endoplasmic reticulum in the absence of CD3-gamma; 2) CD3-zeta does not associate with the Ti alpha beta-CD3 delta epsilon complex; 3) the Ti alpha beta-CD3 delta epsilon complex is not exported from the endoplasmic reticulum to the Golgi apparatus; and 4) CD3-gamma is required for cell surface expression of the Ti/CD3 complex. Transfection of the wild-type CD3-gamma gene into JGN reconstituted expression of functional Ti/CD3 complexes, and analysis of T cell lines producing different amounts of CD3-gamma indicated that CD3-gamma and CD3-delta competed for the binding to CD3-epsilon.     

T cell antigen receptor--structure, expression and function

T cell receptor complex is composed of at least 7 different polypeptides and is one of the most sophisticated receptor. There are two types of T cell receptor (TCR); alpha beta and gamma delta, both of which are composed of a heterodimer and associated with invariant CD3 complexes on the cell surface. T cells expressing alpha beta dimer recognize antigen-peptides in the context of self-MHC molecules, whereas the specificity and function of gamma delta T cells are largely unknown. Gene organization of alpha beta and gamma delta indicates the difference of mechanism to generate diversity. Whereas alpha and beta genes have a large number of V genes, those of gamma and delta genes are limited. However, especially for delta gene, the repertoire is largely produced by junctional diversity. There are increasing data showing new TCR heterodimers; such as beta delta heterodimer in human, beta homodimer in mouse and unknown new heterodimer in chicken, which are expressed on the cell surface in the association with CD3 complex. The characterization of these new receptor dimers and the function of cells expressing these receptors have to be determined. Among CD3 complex, zeta and eta chains are most important for signal transduction after antigen-recognition by TCR. eta gene is recently cloned and now found to be produced by an alternative splicing of a common gene with zeta chains gene. Tyrosine++ phosphorylation of zeta chain seems to be one of the earliest events of T cell activation. Since fyn, one of src oncogene family possessing tyrosine++ kinase function, is co-precipitated with TCR-CD3 complex, fyn seems to be involved in early phosphorylation for T cell activation. Positive and negative selection of thymocytes has been shown to occur via TCR using TCR-transgenic mice model. Molecular mechanism of the selection should be determined.