Characterization of the Region Involved in CD3 Pairwise Interactions within the T Cell Receptor Complex

Borroto, Aldo; Mallabiabarrena, Arrate; Albar, Juan Pablo; Martı́nez-A, Carlos; Alarcón, Balbino

doi:10.1074/jbc.273.21.12807

Cited by 57 publications

(55 citation statements)

References 35 publications

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“…Unlike in the mouse system, the introduction of hCD3␥ C82S or hCD3␥ C85S failed to support association with CD3 or T cell surface expression (42). These data are consistent with earlier studies showing the striking effect of cysteine mutations of hCD3 stalk residues on both hCD3␥ and hCD3␦ in heterodimer formation (44). Although in the in vitro system, CD3 , CD3␥, or CD3␦ stalk cysteine mutations impaired assembly of CD3 heterodimers with the ␣␤ TCR, there was apparently little effect on hCD3 ␦ or hCD3 ␥ heterodimer formation per se.…”

Section: Discussionsupporting

confidence: 89%

Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

Touma

Sun

Clayton

et al. 2007

The Journal of Immunology

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CD3εγ and CD3εδ are noncovalent heterodimers; each consists of Ig-like extracellular domains associated side-to-side via paired terminal β-strands that are linked to individual subunit membrane proximal stalk segments. CD3ε, CD3γ, and CD3δ stalks contain the RxCxxCxE motif. To investigate the functional importance of a CD3 stalk and terminal β-strand, we created a CD3γ double mutant CD3γC82S/C85S and a CD3γ β-strand triple mutant CD3γQ76S/Y78A/Y79A for use in retroviral transduction of lymphoid progenitors for comparison with CD3γwt. Although both mutant CD3γ molecules reduced association with CD3ε in CD3εγ heterodimers, CD3γQ76S/Y78A/Y79A abrogated surface TCR expression whereas CD3γC82S/C85S did not. Furthermore, CD3γC82S/C85S rescued thymic development in CD3γ−/− fetal thymic organ culture. However, the numbers of double-positive and single-positive thymocytes after CD3γC82S/C85S transduction were significantly reduced despite surface pre-TCR and TCR expression comparable to that of CD3γ−/− thymocytes transduced in fetal thymic organ culture with a retrovirus harboring CD3γwt cDNA. Furthermore, double-negative thymocyte development was perturbed with attenuated double-negative 3/double-negative 4 maturation and altered surface-expressed CD3εγ, as evidenced by the loss of reactivity with CD3γ N terminus-specific antisera. Single histidine substitution of either CD3γ stalk cysteine failed to restore CD3εγ association and conformation in transient COS-7 cell transfection studies. Thus, CD3γC82 and CD3γC85 residues likely are either reduced or form a tight intrachain disulfide loop rather than contribute to a metal coordination site in conjunction with CD3εC80 and CD3εC83. The implications of these results for CD3εγ and TCR structure and signaling function are discussed.

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Section: Discussionsupporting

confidence: 89%

Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

Touma

Sun

Clayton

et al. 2007

The Journal of Immunology

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“…The APA1͞1 antibody recognizes the proline-rich sequence in the tails of human and murine CD3 (ref. 21 and data not shown) only after engagement of the TCR (16). We showed that the epitope recognized by APA1͞1 is displayed only in immunological synapses when the antigen-presenting cell is loaded with a full, but not a partial, agonist.…”

Section: Discussionmentioning

confidence: 57%

A conformational change senses the strength of T cell receptor–ligand interaction during thymic selection

Risueño

Santen

Alarcón³

2006

Proc. Natl. Acad. Sci. U.S.A.

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T cell antigen receptor (TCR) triggering determines the fate of immature thymocytes. The affinity of the TCR for its endogenous peptide͞MHC ligands serves as a signal for positive or negative selection through mechanisms that are still little understood. We have used a conformation-specific antibody to demonstrate that recognition of TCR ligands that lead to negative selection induces a conformational change in the TCR in situ. In contrast, this conformational change is elicited in only a small percentage of immature thymocytes during positive selection. Using a TUNEL assay, we demonstrate that the conformational change in the TCR is strongly linked to activation of programmed cell death in conditions leading to negative selection. Furthermore, the few conformational change-positive thymocytes detected in conditions that preferably lead to positive selection are also TUNELpositive. Thus, the conformational change in the TCR may underlie the discrimination of ligands leading to positive and negative selection.signal transduction ͉ thymic differentiation ͉ negative selection T he T cell antigen receptor (TCR) complex is composed in most T cells of the ligand-binding TCR␣ and TCR␤ subunits and the signal-transducing CD3␥, CD3␦, CD3 , and CD3 (CD247) (1, 2). The differentiation of T cells is controlled by the transduction of pre-TCR and TCR signals. Differentiation of immature CD4 ϩ CD8 ϩ double-positive (DP) to mature CD4 ϩ or CD8 ϩ single-positive thymocytes is initiated when the TCR-␣␤ heterodimer engages the peptide͞MHC (pMHC) (2). A unique feature of the TCR is its ability to scan structurally similar pMHC ligands and transmit distinct biochemical signals depending on the strength of the ligand recognized (3, 4). In developing thymocytes, weak TCR ligands induce positive selection, and stronger ligands induce negative selection (5). However, mature T cells are tolerant to weak TCR ligands in the periphery, and they respond to strong ligands by activating a program of cell proliferation and cell differentiation. The differential responsiveness of immature and mature T cells to weak ligands constitutes the cornerstone of self-tolerance that is acquired in the thymus.Many studies have focused on how the TCR␣␤ heterodimer communicates ligand engagement to the CD3 subunits, how the CD3 subunits translate this information into a specific cellular response, and how the TCR complex discriminates between strong and weak ligands. The engagement of the TCR with a weak ligand in mature T cells results in a distinct pattern of intracellular signaling compared with that induced by a full-agonist peptide. For example, stimulation with a weak ligand induces only partial phosphorylation of the CD3 and CD3 chains and the recruitment, but not the phosphorylation, of the -associated protein of 70 kDa, ZAP70 (6, 7). Hence, weak ligands activate only a subset of the downstream signaling pathways that are activated by an agonist peptide (8). Weak ligands bind to the TCR with a lower affinity and a higher dissociation rate than full a...

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“…COS Cell Transfections and Immunoprecipitation-COS cell transfections and immunoprecipitation were performed as previously described (12).…”

Section: Methodsmentioning

confidence: 99%

“…Immunofluorescence and Microscopy-COS cells were fixed and stained for immunofluorescence as previously described (12).…”

Section: Methodsmentioning

confidence: 99%

“…The CD3⑀ cytoplasmic tail, highly conserved (11,12), can be tentatively subdivided into three regions; the N-terminal region contains a basic amino acid cluster, the central region contains a proline-rich sequence, and the C-terminal region contains the ITAM (13). The proline-rich sequence contains the SH3-binding consensus motif XPPXP, and the C-terminal region contains the YXXLXXR endoplasmic reticulum (ER) retention sequence, which partially overlaps the ITAM (14,15).…”

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confidence: 99%

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Intracellular Redistribution of Nucleolin upon Interaction with the CD3ε Chain of the T Cell Receptor Complex

Gil

Gutiérrez

Alarcón

2001

Journal of Biological Chemistry

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T cell activation through the antigen receptor (TCR) involves the cytoplasmic tails of the CD3 subunits CD3␥, CD3␦, CD3⑀, and CD3. 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 CD3⑀ cytoplasmic tail to isolate proteins that specifically interact with this subunit. In this way, we identified the shuttling protein nucleolin as a specific CD3⑀-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 CD3⑀. Transfection in COS cells of wild type CD3⑀, but not of nonbinding mutants of CD3⑀, 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 CD3⑀. We also found that nucleolin associated with the TCR complex. This association was increased upon TCR engagement, suggesting that the CD3⑀/nucleolin interaction may have a role in T cell activation.T cells respond to antigen via a polypeptide complex composed of ligand-binding T cell receptor (TCR) 1 ␣ and ␤ chains (or ␥ and ␦ in ␥␦ T cells) and the CD3 subunits CD3␥, CD3␦, CD3⑀, and CD3 (1, 2). Unlike the TCR chains, the CD3 components have long cytoplasmic tails that associate with cytoplasmic signal transduction molecules. This association is mediated at least in part by a double tyrosine-based motif present in a single copy in the CD3␥, CD3␦, and CD3⑀ chains and in three copies in CD3 (3). This motif, named immune-receptor tyrosine-based activation motif (ITAM), becomes tyrosine phosphorylated during T cell activation by the Src family proteintyrosine kinases . Tyrosine phosphorylated ITAM become docking sites for the Syk family proteintyrosine kinase ZAP70 and other signal-transducing molecules. It is well established that antibody-mediated engagement of protein chimeras containing the cytoplasmic tail of either CD3 or CD3⑀ results in T cell activation (7-10). These data indicate that the cytoplasmic tail of one of these subunits can be sufficient to induce T cell activation. Regarding the role of CD3 subunits in T cell activation, most of the attention has been focused on the ITAM. However, the cytoplasmic tails of the CD3 subunits contain other evolutionarily conserved features that suggest ITAM-independent roles for them.The CD3⑀ cytoplasmic tail, highly conserved (11, 12), can be tentatively subdivided into three regions; the N-terminal region contains a basic amino acid cluster, the central region contains a proline-rich sequence, and the C-terminal region contains the ITAM (13). The proline-rich sequence contains the SH3-binding consensus motif XPPXP, and the C-terminal region contains the YXXLXXR endoplasmic reticulum (ER) retention sequ...

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Characterization of the Region Involved in CD3 Pairwise Interactions within the T Cell Receptor Complex

Cited by 57 publications

References 35 publications

Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

Importance of the CD3γ Ectodomain Terminal β-Strand and Membrane Proximal Stalk in Thymic Development and Receptor Assembly

A conformational change senses the strength of T cell receptor–ligand interaction during thymic selection

Intracellular Redistribution of Nucleolin upon Interaction with the CD3ε Chain of the T Cell Receptor Complex

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