Abstract
BackgroundStudying the stoichiometry and intracellular trafficking of the T cell antigen receptor (TCR) is pivotal in understanding its mechanisms of activation. The αβTCR includes the antigen-binding TCRαβ heterodimer as well as the signal transducing CD3εγ, CD3εδ and ζ2 subunits. Although the TCR-interacting molecule (TRIM) is also part of the αβTCR complex, it has not been included in most reports so far.ResultsWe used the native antibody-based mobility shift (NAMOS) assay in a first dimension (1D) blue native (BN)-PAGE and a 2D BN-/BN-PAGE to demonstrate that the stoichiometry of the digitonin-solublized TRIM-containing αβTCR is TCRαβCD3ε2γδζ2TRIM2. Smaller αβTCR complexes possess a TCRαβ CD3ε2γδζ2 stoichiometry. Complexes of these sizes were detected in T cell lines as well as in primary human and mouse T cells. Stimulating the αβTCR with anti-CD3 antibodies, we demonstrate by confocal laser scanning microscopy that CD3ε colocalizes with ζ and both are degraded upon prolonged stimulation, possibly within the lysosomal compartment. In contrast, a substantial fraction of TRIM does not colocalize with ζ. Furthermore, TRIM neither moves to lysosomes nor is degraded. Immunoprecipitation studies and BN-PAGE indicate that TRIM also associates with the γδTCR.ConclusionsSmall αβTCR complexes have a TCRαβ CD3ε2γδζ2 stoichiometry; whereas those associated with one TRIM dimer are TCRαβ CD3ε2γδζ2TRIM2. TRIM is differentially processed compared to CD3 and ζ subunits after T cell activation and is not degraded. The γδTCR also associates with TRIM.
Highlights
Studying the stoichiometry and intracellular trafficking of the T cell antigen receptor (TCR) is pivotal in understanding its mechanisms of activation
Using the native antibody-based mobility shift (NAMOS) assay, we recently revealed the stoichiometry of the basic abTCR complex extracted from T cell membranes which is TCRab CD3ε2gδζ2 [24,25,26,27]
After separation by blue native (BN)-PAGE, the eluted abTCR complexes were detected with an anti-ζ antiserum
Summary
Studying the stoichiometry and intracellular trafficking of the T cell antigen receptor (TCR) is pivotal in understanding its mechanisms of activation. The abTCR includes the antigen-binding TCRab heterodimer as well as the signal transducing CD3εg, CD3εδ and ζ2 subunits. The TCR-interacting molecule (TRIM) is part of the abTCR complex, it has not been included in most reports so far. The gδTCR is expressed on gδ T cells and is involved in their development and activation. GδTCR ligands are largely undefined, but include protein and non-protein substances [1,2]. Its ligands are non-self peptides presented by major histocompatibility complex molecules (MHC) on the surface of antigen presenting cells (APC). The heterodimeric TCRab contains variable immunoglobulin (Ig) domains, which bind to of the antigen, the CD3 and ζ subunits of the abTCR serve as signal transducing elements. The gδTCR is composed of the same signal transducing elements while carrying the antigen binding TCRgδ heterodimer
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