TCRζ Chain Research Articles

Hematopoietic Cell Phosphatase (HCP) Regulates p56LCKPhosphorylation and ZAP-70 Binding to T Cell Receptor ζ Chain

Ligation of the T cell receptor complex and CD4 leads to activation of the protein tyrosine kinases p56lckand p59fynresulting in phosphorylation of TcRζ chain and the recruitment of ZAP-70. In this study, we have reconstituted p56lckphosphorylation of TcRζ and ZAP-70 recruitment in heterologous cells and examined the role of the tyrosine phosphatase HCP in regulating the process. Both p56lckand p59fyninduce significant phosphorylation of TcRζ. However, under conditions of comparable p56lckand p59fynexpression, p56lckwas found to induce three to four fold greaterin vivophosphorylation of TcRζ. HCP dephosphorylated p56lck, ZAP-70 and the TcRζ chain. Further, dephosphorylation of the different TcRζ isoforms results in disruption of the interaction between TcRζ and ZAP-70. These results indicate that HCP acts to negatively regulate signal transduction pathways in T cells.

Cloning, sequencing and expression of the bovine CD3ε and TCR-ζ chains, two invariant components of the T-cell receptor complex

CD3ε and the ζ-chain of the bovine T-cell receptor (TCR) are two invariant molecules with an important role in signal transduction via the TCR/CD3 complex. The nucleotide sequence of a bovine CD3ε cDNA clone containing the complete coding sequence was determined and the deduced amino acid (aa) sequence compared to that of other species. The cytoplasmic domains of the different CD3ε clearly show a higher degree of conservation than the extracellular domains. Bovine CD3ε produced in Escherichia coli using different bacterial expression vectors was recognised by antibodies (Ab) directed against the intracytoplasmic domain of human CD3ε. A partial bovine TCRζ-chain cDNA was generated by the polymerase chain reaction (PCR) using primers that were based on sequences that are conserved between different species; 3′ and 5′ RACE-PCR were carried out to obtain the complete TCRζ-chain cDNA sequence. A comparison of the predicted TCRζ-chain aa sequence reveals that the GDP/GTP-binding motif, which is conserved in other species, shows marked differences in the bovine and ovine TCRζ-chains. In contrast to CD3ε, the short extracellular domain of the TCRζ-chain is 100% conserved between the different species and the transmembrane domain also shows a high degree of identity. Ab were raised against the TCRζ-chain, produced as a glutathione S-transferase fusion protein in E. coli, and were used in Western blot analysis to further characterise TCRζ-chain expression in T-cells. These reagents provide valuable tools for the study of signal transduction pathways in normal and transformed bovine T-cells

Insights into T cell development and signal transduction provided by TCR-zeta chain deficient mice.

The T cell antigen receptor (TCR) transduces signals that mediate different responses depending on the stage of development of the T cell and the nature of the ligand it engages. The presence of multiple signal transducing subunits (CD3-gamma-delta,-epsilon and zeta chain) suggests the potential to control these responses by altering the subunit composition of the TCR. zeta chain represents an especially important signalling molecule as it contains multiple signalling motifs within its cytoplasmic tail. The generation and analysis of zeta deficient (zeta-/-) and zeta-transgenic mice has provided insight into the role of zeta as well as the CD3 subunits in TCR surface expression, T cell activation and thymocyte development. Herein, we discuss the results from such experiments which suggest distinct roles for zeta chain and the CD3 components at different stages of T cell development.

Tyrosine-phosphorylated T cell receptor ζ chain associates with the actin cytoskeleton upon Activation of mature T lymphocytes

The multichain T cell antigen receptor (TCR) is composed of an antigen binding ( α β ) domain and associated signal-transducing complexes, the CD3 (γ, σ, and ϵ) and the ζ chains. The ζ chain (TCRζ) plays a key role in signal transduction. We show here that TCR ligation induces association of tyrosine-phosphorylated TCRζ with the detergent-insoluble cell fraction. The microfilament poison, cytochalasin D, disrupts this association and enhances the coprecipitation of actin with TCRζ after receptor ligation. This microfilament association is specific to TCR-associated polypeptides containing at least one intact immunoreceptor tyrosine-based activation motif (ITAM). Mapping studies using transfectants and chimeric TCRζ chain constructs suggest that the third ITAM is necessary and sufficient for association, if the distal tyrosine is intact. This cytoskeletal association is directly correlated with IL-2 production, and ligation of TCR on immature thymocytes does not induce TCRζ-cytoskeleton association. These data thus provide direct evidence of a developmentally regulated activation-dependent interaction between a lymphocyte antigen receptor and the actin cytoskeleton.

Isolation of immature and mature T cell receptor complexes by lectin affinity chromatography

The antigen-specific T cell receptor (TCR) is a multisubunit complex composed of at least six different polypeptide (α, β, γ, δ, ε, ζ), several of which are glycoproteins. Assembly of the TCR occurs in the endoplasmic reticulum (ER) and involves intermediary complexes of CD3-γ, δ, ε proteins and TCRα and -β molecules. Egress of TCR from the ER and transport through the Golgi apparatus is most often monitored by the sensitivity of TCR glycoproteins to endoglycosidase H (Endo H), an enzyme specific for immature oligosaccharides which have not yet been processed by Golgi glycosidases and glycosyltransferases. Because they are not glycosylated, the subcellular localization of CD3-ε and TCRζ chains cannot be directly determined by Endo H treatment, and therefore must be inferred by association with glycoprotein members of the TCR complex. Thus, when both immature and mature TCR glycoproteins are present within a given sample, this becomes extremely difficult. In this report, we describe a method for the physical separation of immature and mature murine TCR complexes based on processing of N-linked carbohydrate side chains. Specifically, we report the use of wheat germ agglutinin-affinity matrices to separate TCR complexes which have reached the trans Golgi compartment of the cell from those that have not. This technique is rapid, sensitive, and does not affect the integrity of assembled TCR complexes.