Stoichiometry and intracellular fate of TRIM-containing TCR complexes

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.

WASH Knockout T Cells Demonstrate Defective Receptor Trafficking, Proliferation, and Effector Function

“…Importantly, previous work has demonstrated that the expression of a mutated recycling defect ␤2 integrin is directed into the late endosome compartment (67). In addition, recent work has proposed differential endosomal trafficking for members of the TCR/CD3 complex (68,69), and yet the mechanisms behind this trafficking remain unclear. Our data suggest that WASH may be a key regulator of TCR/CD3 complex trafficking in activated T cells and of LFA-1 in either the naive or activated state and that WASH is involved in efficient recycling of these receptors back to the cell surface.…”

Organization of the resting TCR in nanoscale oligomers

“…The current consensus on the composition of the TCR complex is that it is comprised of TCRαβ, CD3γε, CD3δε, and CD3ζ 2 dimers. In addition, some TCRs include a dimer of the TM protein TRIM . Therefore, the minimal stoichiometry possible is αβγεδεζζ.…”

Cholesterol and Sphingomyelin Drive Ligand-independent T-cell Antigen Receptor Nanoclustering

“…It consists of the antigen-recognizing TCR␣␤ (or TCR␥␦) heterodimer and the signal-transducing CD3 dimers: CD3⑀␥, CD3⑀␦, and (1, 2). The basic functional unit of the TCR, defined as the monomeric TCR, has a TCR␣␤CD3␦⑀␥⑀ stoichiometry (3)(4)(5)(6).…”