PMC

(A) Comparison between human and mouse TRIM indica-ting a strong conservation of the extracellular domain, the transmembrane region, and the first 10 cytoplasmic amino acids (boxed). (B) Similarity between the putative dimerization motifs in the transmembrane domains of ζ, and TRIM. The transmembrane domains are indicated by solid lines, and the putative dimerization motif by a dotted box. The GenBank/EMBL/DDBJ accession no. for mouse TRIM is AJ29769.

Characterization of Jurkat clones stably overexpressing TRIM. (A) and (B) CD3ε and TCR-α/β expression (A) and subcellular localization of ζ (B) in a representative stably transfected Jurkat T clone (pos.; clone 2D1) compared with a transfectant not expressing TRIM (neg.; clone 2C3). (C) Mean channels of CD3ζ expression in six vector transfectants and in six TRIM transfectants as determined by flow cytometry. (D) Enhanced TCR-mediated Ca²⁺ flux in Jurkat T cells stably expressing F-TRIM and the F-15.5 mutant.

Interaction between TRIM and the TCR in peripheral blood T cells. (A) Top: T cells were incubated with CD3ε (OKT3) or anti-HLA I (W6/32) mAbs followed by induction of cap formation using Texas red–labeled donkey anti–mouse Ab. Cells were fixed and the subcellular localization of TRIM determined using polyclonal TRIM Ab and Cy2-labeled donkey anti–rabbit antiserum. Bottom: subcellular localization of SKAP-HOM (left) and LPAP (right) in TCR-capped peripheral blood T cells. (B) T cells were incubated with mAbs directed at CD3, CD4, CD5, and CD28 (all of the IgM isotype) to induce cap formation. Cells were fixed and the distribution of TRIM determined using biotinylated TRIM mAb (TRIM-4) followed by streptavidin Texas red. In parallel, cap formation was monitored by incubating the IgM-treated cells with a FITC-labeled goat anti–mouse IgM antiserum.

Overexpression of TRIM in Jurkat cells results in upregulation of TCR expression. (A) Jurkat T cells were transiently transfected with empty vector or with a cDNA construct encoding FLAG-tagged wild-type TRIM. 16 h later, expression of the TCR-α/β, CD3ε, CD28, or HLA class I on the cell surface was determined by flow cytometry. (B) Jurkat T cells were transfected with empty vector or with a cDNA coding for F-TRIM. After 40 h, cell surface molecules expressed by vector transfectants and TRIM transfectants were biotinylated. Cells were then lysed in digitonin-containing buffer and subjected to CD3ε immunoprecipitation. After washing, the precipitated molecules immunoprecipitates were boiled in SDS-containing buffer and the released material subjected to reprecipitation using either polyclonal Abs directed at CD3γ, CD3δ, or CD3ε, or a mAb directed at ζ. The secondary immunoprecipitates were separated on 14% reducing SDS-PAGE, blotted onto nitrocellulose, which was then probed with peroxidase-labeled streptavidin followed by ECL detection.

Overexpression of TRIM in Jurkat T cells alters the subcellular localization of ζ. (A) Lysates of Jurkat T cells transfected with empty vector or a cDNA coding for F-TRIM were analyzed for the expression of CD3ε (using mouse mAb SP34), TCR-ζ (using mouse mAb 1C10), and F-TRIM (anti-FLAG) by Western blotting. (B) Jurkat T cells were transfected with empty vector alone (left) or with a cDNA construct encoding F-TRIM (right). 40 h later, the subcellular localization of ζ (using mAb 6B10.2) was determined by confocal laser scan microscopy. Nuclei were stained with propidium iodine. (C) Cells were transfected as in B and the subcellular localization of ζ (mAb 6B10.2, green fluor-escence, Cy2) or F-TRIM determined by confocal laserscan analysis. F-TRIM was detected using a biotinylated anti-FLAG Ab. Bottom: specificity control showing that overexpression of the transmembrane adapter protein SIT does not alter the subcellular localization of ζ. Note that additional data indicating that overexpression of SIT also does not influence the expression levels of the TCR on the cell surface are shown in .

Inhibition of spontaneous TCR internalization by TRIM. (A) Vector transfectants or TRIM transfectants were treated for 90 min with BFA at 37°C. Subsequently, the expression of the TCR-α/β heterodimer was determined by indirect immunofluorescence. ctrl., control. (B and C) Vector transfectants or TRIM transfectants were externally labeled on ice with PE-labeled CD3ε mAb UCHT-1. After washing the temperature was shifted to 37°C for the indicated periods of time. After incubation at 37°C, the temperature was again lowered to 4°C and cells were externally labeled with the anti-TCR mAb C305 (IgM) followed by FITC-labeled goat anti–mouse IgM serum. Cells were then analyzed by flow cytometry (B) or by confocal laserscanning analysis (C). sTCR, surface TCR. (D) TCR internalization is only downregulated in Jurkat T cells overexpressing TRIM. Jurkat T cells transfected with a plasmid encoding TRIM were labeled on ice with UCHT1-PE as described above. After washing, cells were either incubated on ice (left) or incubated for 15 min at 37°C (right). Cells were then permeabilized and stained with biotinylated TRIM-4 mAb followed by detection using streptavidin Cy2.

TRIM preferentially associates with the TCR-ζ chain. (A) 3 × 10⁷ Jurkat T cells were lysed in digitonin-containing buffer and subjected to TRIM or CD3ε (OKT-3) immunoprecipitation. Two different monoclonal TRIM Abs (TRIM-4 and TRIM-7) were used for immunoprecipitation. Precipitates were analyzed by sequential anti-CD3ε (a polyclonal rabbit antiserum), anti-ζ (hamster mAb H146), and anti-TRIM (a polyclonal rabbit antiserum) Western blotting. (B) Jurkat cells were transiently transfected with empty vector or with a cDNA construct encoding FLAG-tagged wild-type TRIM. 40 h after transfection, cells were lysed in digitonin-containing buffer and subjected to anti-CD3ε or anti-FLAG immunoprecipitation followed by sequential anti-CD3γ, anti-CD3δ, anti-CD3ε, or TCR-ζ (hamster mAb H146) Western blotting. (C) COS cells were transiently transfected with a cDNA coding for human TCR-ζ together with a construct encoding FLAG-tagged wild-type TRIM (F-TRIM). After 40 h of incubation, double immunofluorescence was applied on the transfected cells using anti-ζ (mAb 6B10.2, green fluorescence, Cy2) and anti-TRIM (the affinity-purified polyclonal antiserum, red fluorescence, Texas red) Abs. (D) A portion of the transfectants was lysed in digitonin-containing buffer and subjected to ζ immunoprecipitation (using mAb 6B10.2) followed by anti-TRIM immunoblotting (using the polyclonal antiserum). (E) Wild-type (WT) Jurkat T cells, as well as the Jurkat variants 18B3 (lacking expression of TCR-α), JBN (lacking expression of TCR-β), and JGN (lacking expression of CD3γ) were assessed for the subcellular localization of TRIM (using mAb TRIM-4) by confocal laserscan microscopy.

The molecular basis for association between TRIM and TCR-ζ. (A) Scheme depicting the various TRIM/LAT chimeras that were analyzed in this figure. The chimeras are composed of parts TRIM (labeled with T) or LAT (labeled with L). The three-letter code stands for the extracellular (EC), transmembrane (TM), and intracellular (IC) domains, respectively. (B) Jurkat T cells were transfected with cDNAs encoding F-TRIM or the F.15.5 mutant in which the potential tyrosine based signaling motifs (Y⁶³GNL, Y⁷⁹EQM, and Y¹¹⁰ASL) were mutated to phenylalanine (F). 40 h after transfection, the cells were briefly treated with pervanadate (2′). Then, anti-FLAG immunoprecipitates were prepared which were first analyzed by anti-PTYR immunoblotting. Blots were then stripped and reprobed with anti-FLAG Ab. Note that mutation of the three tyrosines slightly alters the molecular weight of the F-15.5 mutant. (C) cDNA constructs encoding the various TRIM/LAT chimeras shown in A were transfected into Jurkat T cells by electroporation. 40 h later, the expression of CD3ε on the cell surface was determined by flow cytometry. (D) In parallel, cells were lysed in digitonin-containing buffer, and subjected to ζ (mAb 6B10.2) immunoprecipitation and nonreducing SDS-PAGE followed by anti-FLAG immunoblotting. Note that the differences in molecular weight of the TRIM/LAT chimeras results from the different length of the TRIM and LAT extracellular and cytoplasmic domains, respectively. (E) Subcellular localization of ζ and TRIM/ζ colocalization in Jurkat T cells transfected with cDNA encoding F-TRIM or the F-TTL chimera. Note that the TTL chimera is not properly transported to the cell membrane but colocalizes with ζ inside the cell.