CLONING OF LIGAND TARGETS - SYSTEMATIC ISOLATION OF SH3 DOMAIN-CONTAINING PROTEINS

Based on the prevalence of modular protein domains, such as Src homolo gy domain 3 and 2 (SH3 and SH2), among important signaling molecules, we have sought to identify new SH3 domain-containing proteins. However , modest sequence similarity among these domains restricts the use of DNA-based methods for this purpose. To circumvent this limitation, we have developed a functional screen that permits the rapid cloning of m odular domains based on their ligand-binding activity. Using operation ally defined SH3 ligands from combinatorial peptide libraries, we scre ened a series of mouse and human cDNA expression libraries. We found t hat 69 of the 74 clones isolated encode at least one SH3 domain. These clones encode 18 different SH3-containing proteins, in of which have not been described previously. The isolation of entire repertoires of modular domain-containing proteins will prove invaluable in genome ana lysis and in bringing new targets into drug discovery programs.