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.
Licitra
E J
EJ
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139, USA.
Liu
J O
JO
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