A Motif within the T Cell Receptor α Chain Constant Region Connecting Peptide Domain Controls Antigen Responsiveness

Assembly of the T cell receptor (TCR) with its dimeric signaling modules, CD3␦⑀, CD3␥⑀, and , is organized by transmembrane (TM) interactions. Each of the three assembly steps requires formation of a three-helix interface involving one particular basic TCR TM residue and two acidic TM residues of the respective signaling dimer. The extracellular domains of CD3␦⑀ and CD3␥⑀ contribute to assembly, but TCR interaction sites on CD3 dimers have not been defined. The structures of the extracellular domains of CD3␦⑀ and CD3␥⑀ demonstrated parallel ␤-strands ending at the first cysteine in the CXXCXEXXX motif present in the stalk segment of each CD3 chain. Mutation of the membrane-proximal cysteines impaired assembly of either CD3 dimer with TCR, and little complex was isolated when all four membrane-proximal cysteines were mutated to alanine. These mutations had, however, no discernable effect on CD3␦⑀ or CD3␥⑀ dimerization. CD3␦⑀ assembled with a TCR␣ mutant that lacked both immunoglobulin domains, but shortening of the TCR␣ connecting peptide reduced assembly, consistent with membrane-proximal TCR␣-CD3␦⑀ interactions. Chelation of divalent cations did not affect assembly, indicating that coordination of a cation by the tetracysteine motif was not required. The membrane-proximal cysteines were within close proximity but only formed covalent CD3 dimers when one cysteine was mutated. The four cysteines may thus form two intrachain disulfide bonds integral to the secondary structure of CD3 stalk regions. The three-chain interaction theme first established for the TM domains thus extends into the membraneproximal domains of TCR␣-CD3␦⑀ and TCR␤-CD3␥⑀. The T cell receptor (TCR)3 controls T cell development and function and is assembled from six distinct component polypeptides into an eight-chain complex. The TCR heterodimer is responsible for ligand recognition but lacks cytoplasmic signaling domains. Instead, it associates through noncovalent interactions with three dimeric signaling modules, CD3␦⑀, CD3␥⑀, and (1-11). The chain only has a short nine-amino acid extracellular domain and forms covalent dimers through a cysteine at position 2 of the predicted transmembrane (TM) domain (3, 12, 13). The CD3␥, -␦, and -⑀ chains have more substantial extracellular components, and their Ig domains form noncovalent heterodimers through parallel ␤-strands, which create a ␤-sheet along the dimer interface. In each CD3 chain, a nine-amino acid segment with a highly conserved CXXCXEXXX motif connects this ␤-strand to the TM domain (1, 14 -18).Assembly of the TCR heterodimer with the three dimeric signaling modules requires proper placement of a total of nine ionizable TM residues, the three basic TM residues of the TCR heterodimer (7, 19) and a pair of acidic TM residues in each signaling module (4). Each basic TCR TM residue serves a specific role in the assembly process; lysine residues located close to the center of the TM domains of TCR␣ and TCR␤ serve as interaction sites for the CD3␦⑀ and CD3␥⑀ dimers, respectively, whereas the ar...

Section: Discussionsupporting

confidence: 88%

A Membrane-proximal Tetracysteine Motif Contributes to Assembly of CD3δϵ and CD3γϵ Dimers with the T Cell Receptor

Call

Wucherpfennig

2006

“…Cell Lines and Transfections, Luciferase Assays, Confocal Microscopy-The mouse T-cell hybridoma 58hCD4 expressing the 3BBM74 TCR (35), kindly provided by Ed Palmer, and the human T-lymphoma line Jurkat were grown in RPMI supplemented with 7.5% defined bovine serum (HyClone Laboratories, Logan, UT). The neuronal line PC12 (ATCC) was grown in 5% bovine serum and 5% equine serum (HyClone Laboratories).…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of a Novel Nuclear Factor of Activated T-cells-1 Isoform Expressed in Mouse Brain

Plyte

Boncristiano

Fattori

et al. 2001

The nuclear factor of activated T-cells (NFAT) family transcription factors play a key role in the control of cytokine gene expression in T-cells. Although initially identified in T-cells, recent data have unveiled unanticipated roles for NFATs in the development, proliferation, and differentiation of other tissues. Here we report the identification, cDNA cloning, and functional characterization of a new isoform of NFAT1 highly expressed in mouse brain. This isoform, which we named NFAT1-D, is identical to NFAT1 throughout the N-terminal regulatory domain and the portion of the Rel domain which includes the minimal region required for specific binding to DNA and interaction with AP-1. The homology stops sharply upstream of the 3-boundary of the Rel homology domain and is followed by a short unique C-terminal region. NFAT1-D was expressed at high levels in all brain districts and was found as a constitutively active transcription complex. Transfection of a NFAT/luciferase reporter in the neuronal cell line PC12, which also expresses NFAT1-D, showed that these cells expressed a constitutive NFAT activity that was enhanced after nerve growth factor-induced differentiation but was resistant to the immunosuppressant cyclosporin A. NFAT1-D was, however, inducibly activated in a cyclosporin A-sensitive manner when expressed in Tcells, suggesting that the activity of NFAT proteins might be controlled by their specific cellular context. Initially described as a transcriptional complex that bound a T-cell antigen receptor (TCR)1 response element on the interleukin (IL)-2 gene enhancer, nuclear factor of activated T-cells (NFAT) is a family of transcription factors crucially involved in the regulation of cytokine gene expression in T-cells (1). NFAT activity is strongly unregulated after TCR triggering; however, receptor engagement can be bypassed by a combination of phorbol esters and calcium ionophores, which activate protein kinase C and induce a rise in intracellular calcium ions, respectively. This dual requirement reflects the subunit composition of NFAT factors, which includes a cytoplasmic and a nuclear component. In resting cells, NFAT is found as a cytosolic protein phosphorylated on serine residues. After an elevation in intracellular calcium ions, the calmodulin-dependent phosphatase calcineurin is activated and dephosphorylates NFAT, exposing a nuclear localization sequence near the N terminus of NFAT and resulting in its translocation to the nucleus (2-5). This process is exquisitely sensitive to the immunosuppressants cylosporin A (CsA) and FK506, which interact with specific cytosolic receptors and form complexes that bind with high affinity to calcineurin and lock it in an inactive conformation (6 -8). In the nucleus NFAT assembles in cooperative DNAbinding complexes with dimers of the AP-1 family of transcription factors (4, 9). Complementation studies using constitutively active mutants of signaling proteins have shown that the calcium/calcineurin and the Ras/protein kinase C/mitogen-activated protein ki...

“…Surface biotinylation was performed as described previously with minor alterations (27). Briefly, 2 ϫ 10 7 cells were resuspended in 10 ml of carbonate buffer (25 mM NaHCO 3 , pH 8.3, 50 mM NaCl, 1 mM MgCl 2 ) containing 100 g/ml Sulfo-NHS-biotin (Pierce).…”

Section: Methodsmentioning

confidence: 99%

The Phosphorylation State of CD3γ Influences T Cell Responsiveness and Controls T Cell Receptor Cycling

Dietrich¹,

Bäckström²,

Lauritsen³

et al. 1998

Self Cite

The T cell receptor (TCR) is internalized following activation of protein kinase C (PKC) via a leucine (Leu)-based motif in CD3␥. Some studies have indicated that the TCR is recycled back to the cell surface following PKC-mediated internalization. The functional state of recycled TCR and the mechanisms involved in the sorting events following PKC-induced internalization are not known. In this study, we demonstrated that following PKC-induced internalization, the TCR is recycled back to the cell surface in a functional state. TCR recycling was dependent on dephosphorylation of CD3␥, probably mediated by the serine/threonine protein phosphatase-2A, but independent on microtubules or actin polymerization. Furthermore, in contrast to ligand-mediated TCR sorting, recycling of the TCR was independent of the tyrosine phosphatase CD45 and the Src tyrosine kinases p56Lck and p59 Fyn . Studies of mutated TCR and chimeric CD4-CD3␥ molecules demonstrated that CD3␥ did not contain a recycling signal in itself. In contrast, the only sorting information in CD3␥ was the Leu-based motif that mediated lysosomal sorting of chimeric CD4-CD3␥ molecules. Finally, we found a correlation between the phosphorylation state of CD3␥ and T cell responsiveness. Based on these observations a physiological role of CD3␥ and TCR cycling is proposed.