The Yeast Three - Hybrid System : A Method for Detecting Ligand - Protein Interactions

A primary area of investigation in pharmacology and medicine is the study of ligand-receptor interactions. The pharmacological basis of drug action is often the consequence of non-covalent interactions between small organic molecules and their high affinity receptors. In addition to their therapeutic potential these pharmacologically active ligands have recently been utilized as tools in the dissection of complex signal transduction pathways. Although such an approach holds unparalleled utility there currently exists no efficient methodology for the rapid identification of novel ligand-receptor interactions. We have developed a method which utilizes yeast interaction trap/two-hybrid technology to rapidly identify proteins which interact with small organic ligands. This methodology, termed the Yeast Three-Hybrid System, uses a hybrid ligand as molecular glue to reconstitute transcription by bringing together two transcriptional modules as in the Yeast Two-Hybrid System. The feasibility of the Yeast Three-Hybrid 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 of FKBP12 fused to a transcriptional activation domain activated reporter genes when plated onto medium containing the dexamethasone-FK506 heterodimer. Using this system we were able to screen a number of cDNA libraries and selectively isolate overlapping clones of FKBP12. These results demonstrate that the Yeast Three-Hybrid System is a powerful new technology and can be used to rapidly discover receptors for pharmacologically active small organic ligands. Thesis Supervisor: Jun Liu Title: Assistant Professor of Chemistry and Biology