Abstract

T-cell receptors (TCRs) upon binding to peptide–MHC ligands transduce signals in T lymphocytes. Tyrosine phosphorylations in the cytoplasmic domains of the CD3 () and subunits of the TCR complex by Src family kinases initiate the signaling cascades via docking and activation of ZAP-70 kinase and other signaling components. We examined the role of the low-density detergent-insoluble membranes (DIMs) in TCR signaling. Using mouse thymocytes as a model, we characterized the structural organization of DIMs in detail. We then demonstrated that TCR engagement triggered an immediate increase in the amount of TCR/CD3 present in DIMs, which directly involves the engaged receptor complexes. TCR/CD3 recruitment is accompanied by the accumulation of a series of prominent tyrosine-phosphorylated substrates and by an increase of the Lck activity in DIMs. Upon TCR stimulation, the DIM-associated receptor complexes are highly enriched in the hyperphosphorylated p23 chains, contain most of the TCR/CD3-associated, phosphorylation-activated ZAP-70 kinases and seem to integrate into higher order, multiple tyrosine-phosphorylated substrate-containing protein complexes. The TCR/CD3 recruitment was found to depend on the activity of Src family kinases. We thus provide the first demonstration of recuitment of TCR/CD3 to DIMs upon receptor stimulation and propose it as a mechanism whereby TCR engagement is coupled to downstream signaling cascades.