Cloning of ligand targets: systematic isolation of SH3 domain-containing proteins.
Based on the prevalence of modular protein domains, such as Src homology 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 modular domains based on their ligand-binding activity. Using operationally defined SH3 ligands from combinatorial peptide libraries, we screened a series of mouse and human cDNA expression libraries. We found that 69 of the 74 clones isolated encode at least one SH3 domain. These clones encode 18 different SH3-containing proteins, 10 of which have not been described previously. The isolation of entire repertoires of modular domain-containing proteins will prove invaluable in genome analysis and in bringing new targets into drug discovery programs.
Sparks
A B
AB
Department of Biology, University of North Carolina, Chapel Hill 27599, USA.
Hoffman
N G
NG
McConnell
S J
SJ
Fowlkes
D M
DM
Kay
B K
BK
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018909:src Homology Domains
D008024:Ligands; D011506:Proteins