characterized of these modular domains is the Src Ho-Methodology has been developed which gives a spemology 2 (SH2) 2 domain (2). SH2 domains comprise cific measure of the interaction of an SH2 domain with approximately 100 amino acids and are found in both a phosphopeptide ligand using scintillation proximity tyrosine kinases and phosphatases as well as adaptor assay (SPA) technology. Recombinant SH2 domains proteins with no catalytic function. These indepenwere expressed from a T7 RNA polymerase-based vecdently folded domains share the common property of tor in Escherichia coli as fusions to the C-terminus of recognizing phosphorylated tyrosine residues in spethe FK506-binding protein (FKBP) and purified from cific peptide contexts. As many as 100 SH2 domains freeze-thaw lysates in high yield by affinity chromahave been discovered, and in many cases the ligand tography using immobilized phosphopeptides. For specificity and/or functional role have been defined. binding assays the phosphopeptide ligands were syn-An example of an important signal transduction prothesized with a biotin tag and the FKBP fusion cess involving SH2 domains is T-cell activation. Enproteins were noncovalently radiolabeled with comgagement of the extracellular domains of the T-cell remercially available [ 3 H]dihydroFK506. Complexes of ceptor (TCR) triggers rapid induction of intracellular tritiated SH2 fusion protein and biotinyl-phosphopeptyrosine phosphorylation (3, 4). While the cytoplasmic tide were then captured on streptavidin-coated SPA domains of the TCR do not have intrinsic kinase activbeads and counted. The modular protocol is an equilibity, they do have specific sequences critical to signalling rium technique that does not employ washing steps or referred to as immunoreceptor tyrosine-based activaspecialized radiochemical syntheses required in other tion motifs (ITAM). Phosphorylation of both tyrosines binding assays. The utility of the assay has been demin the minimal ITAM consensus sequence YxxL(x) 7-8 onstrated in an examination of the ligand specificity YxxL enables recruitment of the tyrosine kinase ZAP70 of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentially generalizable via a specific interaction with the tandem SH2 domains to any receptor-ligand interaction in which one com-present in that protein. A number of biochemical and ponent can be expressed as a fusion partner with genetic studies implicate an essential role for ZAP70 FKBP and the other component can be captured on a in the propagation of signals which lead to T-cell activa-SPA bead.