A three-hybrid system for detecting small ligand-protein receptor interactions.
Small ligand-receptor interactions underlie many fundamental processes in biology and form the basis for pharmacological intervention of human diseases in medicine. We report herein a genetic system, named the yeast three-hybrid system, for detecting ligand-receptor interactions in vivo. This system is adapted from the yeast two-hybrid system with which a third synthetic hybrid ligand is combined. The feasibility of this system was demonstrated using as the hybrid ligand a heterodimer of covalently linked dexamethasone and FK506. Yeast expressing fusion proteins of the hormone binding domain of the rat glucocorticoid receptor fused to the LexA DNA-binding domain and FKBP12 fused to a transcriptional activation domain activated reporter genes when plated on medium containing the dexamethasone-FK506 heterodimer. The reporter gene activation is completely abrogated in a competitive manner by the presence of excess FK506. Using this system, we screened a Jurkat cDNA library fused to the transcriptional activation domain in yeast expressing the hormone binding domain of rat glucocorticoid receptor-LexA DNA binding domain fusion protein in the presence of dexamethasone-FK506 heterodimer. We isolated overlapping clones of human FKBP12. These results demonstrate that the three-hybrid system can be used to discover receptors for small ligands and to screen for new ligands to known receptors.
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139, USA.
E J
EJ
Licitra
J O
JO
Liu
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D003907:Dexamethasone / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D017930:Genes, Reporter; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000096:biosynthesis / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D022021:Tacrolimus Binding Proteins; D015533:Transcriptional Activation; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D003907:Dexamethasone; D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D016559:Tacrolimus; D022021:Tacrolimus Binding Proteins; D000099160:LexA Repressor Protein; C102348:dexamethasone-FK506 heterodimer