Reconstitution of an active surface T3/T-cell antigen receptor by DNA transfer

Ohashi, Pamela S.; Mak, Tak W.; Elsen, Peter van den; Yanagi, Yusuke; Yoshikai, Yasunobu; Calman, Andrew F.; Terhorst, Cox; Stobo, John D.; Weiss, Arthur

doi:10.1038/316606a0

Cited by 289 publications

(163 citation statements)

References 36 publications

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“…PCR products were cut with XbaI and NcoI, cloned into the 4.1 kb XbaI-NcoI fragment of pBluescript-,BWT and confirmed by complete DNA sequencing. The 1.8 kb XbaI-BamHI pBluescript fragment containing the PCR product was subsequently cloned into the 5.9 kb XbaI-BamI{ fragment of the expression vector pT(3Fneo (Ohashi et al, 1985). Subsequently the cells were washed three times in ice-cold PFB containing 10 mM EDTA, 5 mM EGTA and 10 mM NaF and lysed in 1 ml lysis buffer (1% Triton X-100, 10 mM Tris-HCI, pH 7.5, 150 mM NaCl, 10 mM EDTA, 5 mM EGTA, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride) for 30 min on ice.…”

Section: Methodsmentioning

confidence: 99%

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

et al. 1994

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Several cell surface receptors including the T cell receptor (TCR) are phosphorylated and down-regulated following activation of protein kinase C (PKC). Among other substrates the activated PKC in T cells phosphorylates the CD3-y subunit of the TCR. To investigate the role of CD3'y phosphorylation in PKC-mediated TCR downregulation, point mutated CD3,y cDNA was transfected into the CD3'y-negative T cell line JGN and CD3'y transfectants were analysed. Phosphorylation at S126 but not S123 in the cytoplasmic tail of CD3-y was required for PKC-mediated down-regulation of the TCR. Furthermore, analysis of a series of CD3'y truncation mutants indicated that in addition to S126 phosphorylation a motif C-terminal of S126 was required for TCR down-regulation. Point mutation analyses confirmed this observation and demonstrated that a membrane-proximal di-leucine motif (L131 and L132) in the cytoplasmic tail of CD3'y was required for PKC-mediated TCR downregulation in addition to phosphorylation at S126. Incubation of T cells in hypertonic medium known to disrupt normal clathrin lattices severely inhibited PKCmediated TCR down-regulation in non-mutated T cells, indicating that the TCR was down-regulated by endocytosis via clathrin coated pits. Based on the present results and previously published observations on intracellular receptor sorting, a general model for intracellular sorting of receptors containing di-leucineor tyrosine-based motifs is proposed.

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Section: Methodsmentioning

confidence: 99%

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

et al. 1994

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“…For generating PU.1-positive cell lines we constructed a PU.1 expression vector pAWneoPU.1 by inserting the 859 bp PvuII-NaeI fragment of murine PU.1 cDNA from pSGPU.1, described above, into the expression vector pAWneo2 (kindly provided by Dr Arthur Weiss) (Ohashi et al, 1985), which carries neomycin resistance gene. In stable transfections, con¯uent NIH3T3 cell cultures were transfected, as described above, either with PU.1 expression vector, or with the empty expression vector (20 mg each) and incubated overnight in fresh medium.…”

Section: Cell Transfectionsmentioning

confidence: 99%

Differential regulation of interstitial collagenase (MMP-1) gene expression by ETS transcription factors

1997

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Expression of interstitial collagenase (MMP-1) has been detected in stromal ®broblasts of various malignant tumors. Here, we have studied the e ect of three structurally di erent ETS transcription factors (ETS-1, ERGB/Fli-1, and PU.1) on MMP-1 promoter activity in NIH3T3 ®broblasts. ETS-1 increased the activity of 3.8 kb MMP-1 promoter construct up to tenfold, while ERGB/Fli-1 or PU.1 alone had no marked e ect on basal promoter activity. ETS-1 also markedly potentiated enhancement of MMP-1 promoter by both c-Jun and JunB, whereas ERGB/Fli-1 augmented only the e ect of c-Jun. Interestingly, PU.1 abolished induction of MMP-1 promoter by both c-Jun and JunB. Stimulation of MMP-1 promoter by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid was di erentially augmented by ETS-1 and ERGB/Fli-1, and abrogated by PU.1. Cotransfection studies with MMP-1 promoter 5'-deletion constructs revealed that AP-1 site was necessary for PU.1-elicited suppression. As compared to control cell lines, PU.1-positive stable cells exhibited clearly weaker binding of c-Jun and JunD containing AP-1 complexes to MMP-1 promoter AP-1 element, as well as marked reduction in basal level and induction of c-jun mRNA by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid, suggesting a novel mechanism for PU.1-mediated inhibition of AP-1 dependent gene expression. These results show that three structurally distinct ETS transcription factors di erently modulate AP-1 dependent upregulation of MMP-1 gene expression.

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“…Whereas the a, p, y and 8 chains of the TCR are clonotypic and confer ligand specificity, the CD3 subunits are invariant and function as signal transducing molecules through interactions of their cytoplasmic domains with the protein tyrosine kinases ZAP-70 or pse' 0 * (6)(7)(8) Efficient surface expression of a functional TCR-CD3 complex on mature T cells has been shown to require all six chains (9). In the absence of either TCR a or TCR p (10,11), mature T cells bearing functional TCR ap-CD3 complexes do not develop.…”

Section: Introductionmentioning

confidence: 99%

Expression of genes encoding the pre-TCR and CD3 complex during thymus development

MacDonald

Malissen

1995

International Immunology

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The mature TCR is composed of a clonotypic heterodimer (ap" or 76) associated with the Invariant CD3 components (y, 8, e and Q. There is now considerable evidence that more immature forms of the TCR-CD3 complex (consisting of either CD3 alone or CD3 associated with a heterodimer of TCR p and pre-Ta) can be expressed at the cell surface on early thymocytes. These pre-TCR complexes are believed to be necessary for the ordered progression of early T cell development. We have analyzed in detail the expression of both the pre-TCR and CD3 complex at various stages of adult thymus development. Our data indicate that all CD3 components are already expressed at the mRNA level by the earliest identifiable (CD4 to ) thymic precursor. In contrast, genes encoding the pre-TCR complex (pre-Ta and fully rearranged TCR fJ) are first expressed at the CD44'°CD25 + CD4~CD8~ stage. Detectable surface expression of both CD3 and TCR f) are delayed relative to expression of the corresponding genes, suggesting the existence of other (as yet unidentified) components of the pre-TCR complex.

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Reconstitution of an active surface T3/T-cell antigen receptor by DNA transfer

Cited by 289 publications

References 36 publications

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.

Differential regulation of interstitial collagenase (MMP-1) gene expression by ETS transcription factors

Expression of genes encoding the pre-TCR and CD3 complex during thymus development

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