We all know how T-cell receptors (TCRs) initiate antigen-induced signal transduction — right? Ligand-induced receptor clustering results in cross-phosphorylation of cytoplasmic immunoreceptor tyrosine-based activation (ITAM) motifs of associated CD3 chains, which recruit signalling molecules. But, what about the possibility of a ligand-induced conformational change in the TCR? G-protein-coupled receptors do it, and now in Cell, Gil et al. report that the TCR can do it too. They show that ligand engagement of TCR–CD3 induces a conformational change in CD3ɛ that recruits the adaptor protein NCK.

Using a yeast two-hybrid system, Gil and colleagues showed that non-ITAM regions of CD3ɛ interact with NCK after TCR–CD3 triggering. Precipitation experiments confirmed that CD3ɛ is the only CD3 subunit that interacts with NCK. NCK co-precipitates with TCR–CD3 from anti-CD3-stimulated, but not unstimulated, Jurkat T cells. This result was confirmed in vivo. It seems that the engagement of TCR–CD3 modifies this complex to recruit NCK to CD3ɛ. A cell-free assay was used to discount contributions from other cellular factors, and a monovalent Fab of anti-CD3 antibody was used to rule out receptor crosslinking, which indicates that a conformational change is responsible.

The authors went on to show that the amino-terminal SRC-homology 3 domain (SH3.1) of NCK binds to the proline-rich sequence (PRS) of CD3ɛ. Using deletion mutants of the CD3ɛ cytoplasmic tail, they showed that deletion of the PRS abolishes NCK binding. The expression of isolated domains of NCK was used to determine that only SH3.1 can bind CD3ɛ and that this binding is inducible.

How is NCK binding associated with TCR tyrosine phosphorylation? Surprisingly, the conformational change that allows NCK binding occurs independently of phosphorylation. Furthermore, after engagement of TCR–CD3, CD3ɛ can associate with NCK before the tyrosine phosphorylation of CD3ζ and other substrates is detected. The common belief that ITAM phosphorylation is the earliest event in TCR signalling needs rethinking. The early association of NCK with TCR–CD3 is physiologically relevant, because inhibition of this interaction decreases the number of T-cell–APC conjugates and inhibits maturation of the immunological synapse.

On the basis of these results, Gil et al. propose that ligand engagement of TCR–CD3 exposes a PRS in CD3ɛ that recruits NCK. This is consistent with the observation that the entire cytoplasmic tails of CD3 subunits have been conserved throughout evolution, and not just the ITAMs. They suggest that full T-cell activation requires a combination of ligand-induced crosslinking, leading to phosphorylation, and ligand-induced conformational change, allowing NCK binding. This is a radical change to our assumptions about TCR signalling.