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C Terhorst

Publications and source records attributed to C Terhorst.

At least 91 records · Page 5Linked to original sources
PubMed1993

Covalent binding of guanine nucleotides to the CD3-gamma chain of the T cell receptor/CD3 complex.

In a search for proteins involved in signal transduction through the T cell receptor (TcR/CD3 complex), a recently developed highly efficient method for labeling of nucleotide binding proteins in permeabilized cells was applied. Here, we report that human CD3-gamma could be labeled by periodate-oxidized [alpha-32P] GTP (GTPoxi). In contrast to GTPoxi labeling of CD3-zeta (Peter, M. E., Hall, C., Rühlmann, A., Sancho, J. and Terhorst, C., EMBO J. 1992. 11:933), GTP-specific labeling of CD3-gamma reached a maximum when nucleotides were added 60 min prior to the cross-linking reaction. As CD3-gamma did not contain a known consensus sequence for nucleotide binding and since labeling kinetics of CD3-gamma coincided with those of cytosolic GTP-binding proteins, labeling may have been caused by a GTP-binding protein. This putative protein was not T cell specific because labeling of CD3-gamma could also be achieved when expressed in the endoplasmic reticulum of Chinese hamster ovary (CHO) cells. In CHO cells, labeling by GTPoxi took place only when CD3-gamma was associated with CD3-epsilon, whereas labeling could not be established upon association of CD3-gamma with CD3-delta or TcR alpha. The observation that CD3-gamma was labeled without leaving the endoplasmic reticulum led to the hypothesis that the association of CD3-gamma with a GTP-binding protein might be involved in an early step of the TcR/CD3 complex formation or transport.

Animals↗
PubMed1993

The T cell receptor associated CD3-epsilon protein is phosphorylated upon T cell activation in the two tyrosine residues of a conserved signal transduction motif.

Signal transduction through the T cell receptor for antigen, the TcR/CD3 complex, involves phosphorylation of tyrosine residues in the CD3-zeta chain. Since both CD3-epsilon and the zeta chain contain a tyrosine-based signaling motif, we examine phosphorylation of CD3-epsilon in human T cells. Engagement of the TcR/CD3 complex induced tyrosine phosphorylation of CD3-epsilon in vivo. Induction of CD3-epsilon phosphorylation followed similar kinetics to that of the zeta chain phosphorylation. In contrast to zeta, CD3-epsilon phosphorylation was strictly dependent upon cell surface expression of this member of the TcR/CD3 complex. Chemical and proteolytic cleavage combined with peptide-specific Western blotting established that CD3-epsilon phosphorylation occurred in the two tyrosine residues located in the signal transduction motif in the C-terminal portion of the molecule. Taken together, these data indicated that phosphorylation of CD3-epsilon by tyrosine protein kinases may serve to couple the TcR/CD3 complex to other effector molecules in the signaling cascade.

CD3 Complex↗
PubMed1993

Labeling of adenine and guanine nucleotide-binding proteins in permeabilized cells with in situ periodate-oxidized nucleotides.

A novel approach for identification of adenine and guanine nucleotide-binding proteins in permeabilized cells is described. Cells were incubated for various periods with alpha-32P-labeled nucleotides and then briefly treated with periodate. Condensation products formed in situ between the protein bound alpha-32P-labeled oxidized nucleotide (NTPoxi) and a lysine residue near the nucleotide-binding sites were rapidly stabilized by the addition of cyanoborohydride. Analysis by two-dimensional isoelectric focusing/sodium dodecylsulfate-poly-acrylamide gel electrophoresis showed that in the human leukemic T-cell line Jurkat a number of distinct intracellular proteins could be labeled with ATPoxi (M(r) 40,000-200,000) or GTPoxi (M(r) 19,000-80,000). Competition with deoxyribonucleotides confirmed the selectivity of these affinity labeling reactions. To test this method two classical GTP-binding proteins were further examined. First the alpha-subunits of the Gs and Gi-2 proteins were specifically labeled with [alpha-32P]GTPoxi but not with [alpha-32P]ATPoxi. Second, p21ras was crosslinked specifically to [alpha-32P]GTPoxi or to its bound endogenous ligand. Surprisingly, under optimized conditions 60% of the ras protein was specifically modified, demonstrating the high efficiency and sensitivity of the method. As a first step toward isolation of hitherto unidentified nucleotide-binding proteins, rabbit antisera specific for the modified amino acid residues were raised. The presented labeling method can be applied for identification of nucleotide-binding proteins in all eukaryotic cells.

Adenine Nucleotides↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1992

CD3-zeta surface expression is required for CD4-p56lck-mediated upregulation of T cell antigen receptor-CD3 signaling in T cells.

It has been proposed that during T cell receptor antigen recognition, CD4- or CD8-p56lck molecules interact with the T cell antigen receptor-CD3 complex (TCR-CD3) to phosphorylate various undefined substrates, which then initiate signal transduction through the TCR-CD3 complex. The ability of CD4 to modulate the TCR-CD3-induced increase in intracellular Ca2+, [Ca2+]i, and substrate tyrosine phosphorylation was studied in mutants of the human leukemic T cell line HPB-ALL characterized by their low expression of the TCR-CD3 complex on the cell surface. In TCR-CD3low cells, in which CD3-zeta was found to be associated with the TCR-CD3 complex, cross-linking CD3 with CD4 resulted in a profile of calcium mobilization, CD3-zeta, and phospholipase C-gamma 1 tyrosine phosphorylation similar to that observed in HPB-ALL cells, although the magnitude of generalized substrate tyrosine phosphorylation appeared to be smaller, as compared with wild-type cells. Responses were weak or absent when CD3 was cross-linked alone. In contrast, in a mutant in which association of CD3-zeta 2 with the TCR-CD3 was defective, cross-linking of CD3 with CD4 had a weaker effect on any of the activation parameters tested. These experiments showed that the presence of CD3-zeta 2 in the TCR-CD3 complex is of critical importance for the ability of CD4 to enhance early transducing signals inside the cell. The data also suggest that CD4-associated protein tyrosine kinase p56lck could up-regulate defective CD3-mediated induction of phospholipase C activity by increasing tyrosine phosphorylation of phospholipase C-gamma 1.

Amino Acid Sequence↗
PubMed1992

Developmental regulation of transmembrane signaling via the T cell antigen receptor/CD3 complex in human T lymphocytes.

We have examined transmembrane signaling events via the TCR/CD3 complex (TCR/CD3) at various stages of T cell development for evidence of developmental regulation. Engagement of TCR/CD3 induced defective activation of phospholipase C (PLC) in thymocytes relative to peripheral blood T lymphocytes. The defect in PLC activation via TCR/CD3 was restricted to immature thymocytes (CD3low, CD4+CD8+). Mature thymocytes (CD3high, CD4+CD8-/CD8+CD4-) were similar to PBL in signaling via TCR/CD3. Both immature and mature thymocytes expressed a similar profile of PLC isoenzyme mRNA species, indicating that the defect in signaling in immature thymocytes was not due to altered expression of PLC isoenzymes. Activation of tyrosine phosphorylation pathways implicated in the coupling of TCR/CD3 to PLC was impaired in immature thymocytes, as evidenced by depressed phosphorylation of CD3 zeta subunit after stimulation with anti TCR/CD3 mAb. This was associated with lower levels of p59fyn tyrosine kinase and minimal or undetectable stimulus-induced kinase activation in immature thymocytes relative to mature thymocytes. We conclude that the capacity to signal via TCR/CD3 is regulated during T cell development by mechanisms acting at the level of TCR/CD3-associated tyrosine phosphorylation pathways.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

Cloning and sequencing of the cDNA encoding the human homologue of the murine immunoglobulin-associated protein B29.

Membrane-bound immunoglobulins (Ig) on the surface of murine B cells are noncovalently associated with a heterodimeric protein complex of MB-1 and B29 (also called Ig-alpha and Ig-beta). The Ig-associated proteins are predicted to regulate the assembly and transport of the Ig complex to the cell surface and to couple membrane-bound Ig to intracellular signal transduction pathways. We have isolated and sequenced a full-length cDNA clone encoding the human homologue of the B29 protein. The predicted amino acid sequence was compared to its murine counterpart, to MB-1 and to the human T cell receptor (TcR)-associated CD3 proteins. The alignment of the human B29 protein with its murine counterpart revealed 90% homology in the C-terminal portion comprising the cytoplasmic tails, the transmembrane regions and the adjacent 26 amino acids of the extracellular regions. Only 59% homology was found in the rest of the Ig-like extracellular domains. The high degree of conservation observed for the C-terminal amino acids suggested that these domains of the proteins play important functional roles for the Ig complex. Indicative of this was the conservation of the antigen receptor tail motif D-(X)7-E/D-(X)2-Y-(X)2-L-(X)7-Y-(X)2-L/I which is thought to be a component of signal transduction pathways. This motif is also found in the human and murine MB-1 proteins as well as in the TcR-associated CD3 molecules. Further regions of homology between B29, MB-1 and the CD3 proteins included extracellular residues which were predicted to maintain the Ig-like structure, and hydrophilic residues within the transmembrane regions which may be utilized during the intracellular assembly and transport of the oligomeric Ig/MB-1/B29 or TcR/CD3 complexes. Thus the similarities found between B29, MB-1 and the CD3 proteins suggest conserved functions for both the Ig- and TcR-associated proteins.

Amino Acid Sequence↗
PubMed1992

The T-cell receptor zeta chain contains a GTP/GDP binding site.

In a search for nucleotide binding proteins associated with the T-cell receptor (TCR)-CD3 complex, a novel labeling technique involving introduction of [alpha-32P]GTP or [alpha-32P]ATP into permeabilized cells followed by in situ periodate oxidation was developed. To test the method we first demonstrated that p21ras and other classical GTP binding proteins could be labeled in a GTP-specific manner. In human T lymphocytes the TCR zeta chain was found to be specifically labeled by GTPoxi but not by ATPoxi or CTPoxi. Labeling kinetics and competition experiments demonstrated that zeta had a capacity to bind GTP and GDP but not GMP or ATP. Proteolytic cleavage experiments identified lysine 128 as the GTP crosslinking site. This result was confirmed by studies using oligonucleotide-directed mutagenesis. Lysine residues 128, 135 and 149 were each replaced by arginine and glycine 134 by valine and mutated proteins were expressed in CHO cells. Labeling of mutants K128R and G134V was abrogated whereas mutant proteins K135R and K148R could still be specifically crosslinked to GTP. We conclude that Lys128 and Gly134 are part of a GTP/GDP binding site suggesting that zeta is a unique GTP/GDP binding structure.

3T3 Cells↗
PubMed1991

Expression of a nonpolymorphic MHC class I-like molecule, CD1D, by human intestinal epithelial cells.

The human CD1 locus encodes three nonpolymorphic MHC class I-like cell surface glycoproteins, CD1a-c, which are expressed primarily by immature thymocytes. A mAb and antipeptide antiserum were utilized to determine the tissue distribution of a fourth CD1 molecule, CD1d. Within the lymphoid lineage, CD1d was expressed on B cells but not on thymocytes. Immunoperoxidase staining of fresh frozen intestinal tissues demonstrated that the majority of intestinal epithelial cells, with the exception of cells at the base of some crypts, expressed CD1d. The CD1d staining was observed in the cytoplasm and along the basolateral membranes of the epithelial cells. The intestinal epithelial cell expression of CD1d was confirmed by immunoblotting with a CD1d antipeptide antiserum. Further immunoperoxidase studies indicated that CD1d, unlike murine CD1, was also expressed by nonlymphoid tissues outside of the gastrointestinal tract. The expression of CD1d outside the lymphoid and myeloid lineages clearly distinguishes this molecule from CD1a-c and suggests that it may serve a distinct function. The prominent expression of CD1d by intestinal epithelial cells suggests that this molecule may be an important ligand for T lymphocytes within the gut-associated lymphoid tissue.

Animals↗
PubMed1991

Expression and function of a variant T cell receptor complex lacking CD3-gamma.

A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five-fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface-radioiodinated DIL2 cells contained TCR-alpha, TCR-beta, CD3-delta, CD3-epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma.

Antibodies, Monoclonal↗
PubMed1991

Depletion of cellular calcium accelerates protein degradation in the endoplasmic reticulum.

In this study the effects of A23187 and thapsigargin on the degradation of T-cell antigen receptor-beta (TCR-beta) and CD3-delta in the endoplasmic reticulum have been studied. Preliminary experiments showed that these drugs had different effects on the secretory pathway. Depletion of cellular calcium pools by incubation of cells with A23187 in calcium-free medium blocked transport between the endoplasmic reticulum and the Golgi apparatus whereas thapsigargin caused a modest increase in transport. When added to cells transfected with TCR-beta or CD3-delta the drugs caused an immediate stimulation of proteolysis of presynthesized protein and at maximum effective concentrations caused a 3-fold increase in the rate of degradation. They did not affect the lag period of 1 h which precedes degradation of newly synthesized proteins. Chelation of cytosolic calcium also accelerated degradation, suggesting that depletion of calcium from the endoplasmic reticulum was the main stimulus of proteolysis and that increased degradation was not caused by a transient increase in cytosolic calcium levels. The selectivity of degradation in the endoplasmic reticulum was maintained. A23187 had no effect on the stability of CD3-gamma nor co-transfected epsilon-beta dimers. Calcium depletion increased the overall rate of degradation in the endoplasmic reticulum and increased the rate of proteolysis of an "anchor minus" beta chain. The results suggested that proteolysis within the endoplasmic reticulum may be regulated by the high concentrations of Ca2+ which are stored in the organelle. Ca2+ may be required for protein folding. Calcium depletion may have caused the beta and delta chains to adopt a conformation that was more susceptible to proteolysis. Alternatively, calcium depletion may have disrupted the lumenal content of the endoplasmic reticulum and increased the access of proteases to potential substrates.

Animals↗
PubMed1991

Isolation and expression of cDNA encoding the murine homologues of CD1.

The cDNA encoding the murine CD1.1 and CD1.2 gene products were isolated and their complete nucleotide sequence was determined. The nucleotide sequence and genomic organization of these molecules were similar to human CD1. The sequences in the alpha 1- alpha 3 domains were almost identical to previously reported genomic clones from a different strain, indicating limited polymorphism among these molecules. The predicted amino acid sequence in the transmembrane region and in the cytoplasmic tail was identical for CD1.1 and CD1.2. The two cDNA were also homologous in the 5' untranslated region but diverged in the 3' untranslated region. In contrast to human CD1, which is expressed at high levels in thymus, the expression of CD1 message in murine thymus was not detected in either thymus leukemia Ag positive or negative strains. Cell expressing murine CD1.1 were generated after transfer of the CD1.1 cDNA into murine cell lines. Immunoprecipitation with a rat anti-mouse CD1.1 mAb showed that the transfected CD1 was expressed on the cell surface as a beta 2-microglobulin-linked heterodimer. These results demonstrate that the murine and human CD1 genes, although encoding homologous transmembrane glycoproteins, are expressed in distinct tissues and may serve different functions.

Amino Acid Sequence↗
PubMed1991

Genetic reconstitution of the T cell receptor (TcR) alpha/beta heterodimer restores the association of CD3 zeta 2 with the TcR/CD3 complex.

The cell surface expression of the T cell receptor (TcR)/CD3 complex and, consequently, the functional competence of the cell is partly dependent on CD3 zeta. In its absence, a pentameric complex (TcR alpha/beta/CD3 gamma delta epsilon) is formed which is inefficiently transported to the cell surface. Reconstitution of CD3 zeta by transfection, in turn, restores the cell surface expression and function of the complex. Through the use of transfection experiments, we here provide direct evidence that the association of CD3 zeta 2 with the TcR/CD3 complex is dependent on the presence of both the TcR alpha and beta polypeptide chains. Despite wild-type levels of the CD3 zeta protein in a TcR alpha-negative mutant human T cell line, a complex was formed intracellularly which lacked CD3 zeta 2 and consisted of beta gamma delta epsilon and beta 2 gamma delta epsilon. Upon transfection of the mutant with a TcR alpha cDNA, a TcR/CD3 complex which contained CD3 zeta 2 was observed intracellularly. In contrast to the partial subcomplex on the cell surface of the untransfected cell line, the TcR/CD3 complex on the transfectant was functional as demonstrated by its ability to mobilize intracellular calcium after stimulation with a mitogenic CD3 epsilon-specific monoclonal antibody. Transient transfection studies performed in COS cell fibroblasts indicated that CD3 zeta 2 was not interacting with the TcR alpha protein alone, implying that a conformation provided by either the TcR alpha/beta heterodimer or the TcR alpha/beta/CD3 gamma delta epsilon complex was necessary for the association of CD3 zeta 2. Transfection studies performed in a TcR alpha/beta-negative murine T-T hybridoma confirmed the requirement of both the TcR alpha and beta proteins in CD3 zeta 2 binding. We conclude that the TcR alpha and beta chains harbor polypeptide sequences essential for the association of CD3 zeta 2 with the TcR/CD3 complex.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.

The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were incubated with a panel of protease inhibitors, and the rates of degradation of the transfected proteins were followed using chain-specific enzyme-linked immunosorbent assays (ELISAs). Of the protease inhibitors tested, degradation of both chains was highly sensitive to sulfhydryl reagents and peptidyl inhibitors of cysteine proteases. Concentrations of inhibitors that produced near complete inhibition of degradation in the endoplasmic reticulum did not cause gross changes in cellular ATP levels nor did they significantly slow constitutive secretion from CHO cells. The inhibitors did not affect the ability of CHO cells to synthesize and assemble disulphide-linked TCR zeta dimers. We conclude that the protease inhibitors were not toxic to cells and did not affect the biosynthetic activity of the endoplasmic reticulum. Furthermore, they did not alter the ability of the endoplasmic reticulum to deliver its content to the Golgi apparatus. Taken together, these results suggest that the cysteine protease inhibitors slow degradation in the endoplasmic reticulum through an action on cysteine proteases. The results imply that the endoplasmic reticulum contains cysteine proteases that can be used to remove retained proteins.

Adenosine Triphosphate↗
PubMed1991

The CD45 protein tyrosine phosphatase is required for the completion of the activation program leading to lymphokine production in the Jurkat human T cell line.

Stimulation of the T cell antigen receptor, TCR-CD3, induces tyrosine phosphorylation of specific cellular proteins through activation of a tyrosine kinase. The possible regulatory role of the CD45 protein tyrosine phosphatase in this process was explored by studying the functional properties of cellular variants of the Jurkat T cell line which have been selected to have normal levels of the TCR-CD3 complex, but low or negative expression of CD45. These variants had less than 20% of the normal membrane tyrosine phosphatase activity. Triggering the TCR-CD3 receptor on the CD45 variants with anti-CD3 mAb induced the activation of a tyrosine kinase. Tyrosine phosphorylation of cellular substrates as well as of the CD3 zeta chain was qualitatively comparable to normal cells although the extent of stimulation was lower. No differences were observed between the variants and the normal cells in the duration of the tyrosine phosphorylation signal. The increase in intracellular calcium concentration following receptor stimulation was also less efficient, suggesting that CD45 is necessary for optimal generation of the second messengers of the activation. The CD45 deficient cells secreted highly reduced levels of lymphokines (IL-2, IL-3 or GM-CSF) after activation by anti-CD3 mAb combined with the phorbol ester TPA. This impaired lymphokines production is related to the absence of CD45 since a CD45+ revertant subclone, isolated from one CD45- clone, produced normal levels of cytokines upon activation via CD3, while CD45- subclones were unable to secrete cytokines following activation via CD3. However, upon activation with Ca2+ ionophore and PMA, all CD45- (sub)clones secreted cytokines at levels comparable to those produced by CD45+ cells. These results show that CD45 is required for cytokine production after activation via the TCR-CD3 complex.

Antigens, CD↗
PubMed1991

Requirements for cell surface expression of the human TCR/CD3 complex in non-T cells.

The T-cell antigen receptor (TCR) consists of a glycoprotein heterodimer (alpha/beta or gamma/delta) which is non-covalently associated with at least four or five invariant polypeptides (CD3 gamma, delta, epsilon, zeta and eta). In T-cell variants lacking TCR alpha, beta or zeta, it has been shown that incomplete TCR/CD3 complexes are retained within the cell. To examine requirements for cell surface expression of TCR/CD3, we transfected COS monkey kidney cells with cDNAs encoding TCR alpha, beta and CD3 gamma, delta, epsilon and zeta. We report that cell surface appearance of TCR/CD3 on COS cells requires coordinate expression of all six proteins. In the absence of the zeta chain, subcomplexes comprising from two to five chains were readily demonstrable in COS cells, but they failed to reach the cell surface or to acquire N-linked oligosaccharide side chains indicating failure to reach the medial Golgi. Pulse-chase metabolic labelling of transfected COS cells showed that three chains (CD3 gamma, CD3 epsilon, and zeta) were stable while three (TCR alpha, TCR beta and CD3 delta) were rapidly degraded. In two- and three-chain co-transfections specific intracellular subcomplexes were formed between TCR alpha and CD3 gamma, TCR alpha and CD3 delta, or TCR beta and CD3 epsilon. Binary subcomplexes having at least one stable chain (CD3 epsilon - TCR beta) were stable while one formed by two unstable chains (TCR alpha - CD3 delta) was still degraded. Assembly of the TCR/CD3 complex in COS cells thus appears centered around the metabolically stable CD3 gamma and CD3 epsilon proteins. Site-specific mutations of the negatively-charged transmembrane amino acid of residues of the CD3 chains to alanines served to either abolish (for TCR alpha - CD3 delta and TCR beta - CD3 epsilon) or diminish (for TCR alpha -CD3 gamma) these TCR-CD3 interactions. These mutations had no effect, however, on CD3-CD3 interactions or upon synthesis, metabolism, or intracellular distributions of the CD3 proteins. The transmembrane domains of CD3 gamma, delta, and epsilon thus appear to play a major role in associations of CD3 with TCR chains.

Animals↗