[7] Yeast three-hybrid system for detecting ligand-receptor interactions

In an attempt to speed up the receptor discovery process, the yeast three-hybrid system has been developed that now provides a general and rapid method for detecting ligand–protein interactions in yeast cells. The yeast three-hybrid system is an extension of the two-hybrid system. In addition to the two hybrid fusion proteins needed in the two-hybrid system, the three-hybrid system requires a third hybrid ligand that acts as a chemical inducer of dimerization. The yeast three-hybrid system consists of two pairs of ligand–receptor interactions. As such, it can be conceptually broken into two parts: a conserved ligand–receptor pair common to all three-hybrid screens and a second ligand–receptor pair that represents the interaction of interest. The first conserved pair represents a known and well-established high-affinity interaction. For the second ligand–receptor pair, one component, either the ligand or the receptor, can be unknown. Application of the three-hybrid system to screen a library of either small molecules or receptors would allow the identification of the missing component. Thus, the yeast three-hybrid system can be used to identify new ligands that bind to a known receptor or to identify new receptors for an orphan ligand, the latter of which is of more general interest to those who are trying to identify targets for natural products or synthetic ligands. This chapter provides a practical guide to the yeast three-hybrid system, using as an example the interaction between the immunosuppressive drug FK506 and its binding protein FKBP12.