| Summary: | <p>Abstract</p> <p>Background</p> <p>Studying 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 ζ<sub>2 </sub>subunits. Although the TCR-interacting molecule (TRIM) is also part of the αβTCR complex, it has not been included in most reports so far.</p> <p>Results</p> <p>We 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 TCRαβCD3ε<sub>2</sub>γδζ<sub>2</sub>TRIM<sub>2</sub>. Smaller αβTCR complexes possess a TCRαβ CD3ε<sub>2</sub>γδζ<sub>2 </sub>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.</p> <p>Conclusions</p> <p>Small αβTCR complexes have a TCRαβ CD3ε<sub>2</sub>γδζ<sub>2 </sub>stoichiometry; whereas those associated with one TRIM dimer are TCRαβ CD3ε<sub>2</sub>γδζ<sub>2</sub>TRIM<sub>2</sub>. TRIM is differentially processed compared to CD3 and ζ subunits after T cell activation and is not degraded. The γδTCR also associates with TRIM.</p>
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