Receptor-Dependence of the Transcription Read-Out in a Small-Molecule Three-Hybrid System

Small‐molecule three‐hybrid systems show promise as an in vivo alternative to affinity chromatography for detecting small‐molecule–protein interactions. While several three‐hybrid systems have been reported, little has been done to characterize these systems and, in particular, to test the assumption that the protein–small‐molecule interaction can be varied without disrupting the transcription read‐out. Recently we reported a dexamethasone–methotrexate chemical inducer of dimerization (CID) for use in the yeast three‐hybrid system, based on the well‐studied ligand–receptor pairs dexamethasone (Dex)–glucocorticoid receptor (GR) and methotrexate (Mtx)–dihydrofolate reductase (DHFR). Here we describe our first efforts to characterize this system, by focusing on a comparison of the activity of a bacterial and a mammalian DHFR as a test case of the influence of the ligand–receptor pair on the transcription read‐out. By using a lacZ reporter gene, the activity of several GR and DHFR protein chimeras with different orientations and linker sequences and Dex–Mtx CIDs with different chemical linkers have been compared. In addition, Western analyses and in vivo biochemical assays have been carried out to confirm the integrity of the GR and DHFR protein chimeras. The transcription read‐out is found to be much more sensitive to the structure of the protein chimeras than the CID. The most surprising result is that the levels of transcription activation are consistently higher with the bacterial than the mammalian DHFR, despite the fact that both proteins bind Mtx with an inhibition constant (KI) in the low pM range. These results set the stage for understanding three‐hybrid systems at the biochemical level so that they can be used to detect ligand–receptor pairs with a range of structures and dissociation constants. Three‐hybrid systems show promise as an in vivo alternative to affinity chromatography for screening based on small‐molecule–protein interactions. Despite this potential, there has been little biochemical characterization of these systems. Here we look at the influence of the ligand–receptor interaction on the transcription read‐out in a dexamethasone–methotrexate yeast three‐hybrid system (see schematic representation).