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The transmembrane adaptor protein TRIM regulates T cell receptor (TCR) expression and TCR-mediated signaling via an association with the TCR zeta chain.

Kirchgessner H, Dietrich J, Scherer J, Isomäki P, Korinek V, Hilgert I, Bruyns E, Leo A, Cope AP, Schraven B - J. Exp. Med. (2001)

Bottom Line: T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells.Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-zeta chains and to a lesser extent via the CD3-straightepsilon/gamma heterodimer.Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Institute for Immunology, Ruprecht-Karls University Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-zeta chains and to a lesser extent via the CD3-straightepsilon/gamma heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca(2+) mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

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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.
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Figure 5: 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.

Mentions: We next wished to determine how TRIM interacts with the TCR/CD3/ζ complex. To assess this question, we analyzed TRIM and CD3ε immunoprecipitates prepared from digitonin lysates of Jurkat T cells for the relative amounts of CD3ε and TCR-ζ, respectively, by Western blotting. Fig. 5 A depicts that identical amounts of CD3ε and TCR-ζ are detectable in CD3ε immunoprecipitates. In contrast, under the same experimental conditions, ∼10 times more ζ than CD3ε coprecipitate with TRIM (as judged from densitometric analysis of the shown blot). Similar results as shown here for Jurkat T cells are obtained when the immunoprecipitates are prepared from resting peripheral blood T cells (not shown). This suggests that TRIM interacts with the TCR preferentially via the TCR-ζ chain.


The transmembrane adaptor protein TRIM regulates T cell receptor (TCR) expression and TCR-mediated signaling via an association with the TCR zeta chain.

Kirchgessner H, Dietrich J, Scherer J, Isomäki P, Korinek V, Hilgert I, Bruyns E, Leo A, Cope AP, Schraven B - J. Exp. Med. (2001)

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.
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Related In: Results  -  Collection

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Figure 5: 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.
Mentions: We next wished to determine how TRIM interacts with the TCR/CD3/ζ complex. To assess this question, we analyzed TRIM and CD3ε immunoprecipitates prepared from digitonin lysates of Jurkat T cells for the relative amounts of CD3ε and TCR-ζ, respectively, by Western blotting. Fig. 5 A depicts that identical amounts of CD3ε and TCR-ζ are detectable in CD3ε immunoprecipitates. In contrast, under the same experimental conditions, ∼10 times more ζ than CD3ε coprecipitate with TRIM (as judged from densitometric analysis of the shown blot). Similar results as shown here for Jurkat T cells are obtained when the immunoprecipitates are prepared from resting peripheral blood T cells (not shown). This suggests that TRIM interacts with the TCR preferentially via the TCR-ζ chain.

Bottom Line: T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells.Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-zeta chains and to a lesser extent via the CD3-straightepsilon/gamma heterodimer.Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

View Article: PubMed Central - PubMed

Affiliation: Institute for Immunology, Ruprecht-Karls University Heidelberg, D-69120 Heidelberg, Germany.

ABSTRACT
T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-zeta chains and to a lesser extent via the CD3-straightepsilon/gamma heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca(2+) mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

Show MeSH
Related in: MedlinePlus