23]Intracellular receptor characterization and ligand screening by transactivation and hormone-binding assays

Abstract

The assessment of receptor hormone-binding activity remains today an essential tool for the characterization of both the receptors with known ligands and the putative orphan receptors. The chapter discusses the utilization of transactivation and hormone-binding assays for the characterization of known receptors and presents a brief overview of their application in the drug screening of these known receptors. Specifically, the six retinoid receptors, including the three known retinoic acid receptors (RARs) and the three retinoid X receptors (RXRs), and their known retinoid isomer ligands, all- trans -retinoic acid (tRA) and 9- cis -retinoic acid (9cRA), are presented as an example system in the chapter. These receptors have been characterized by both hormone-binding assays and transactivation assays, the latter performed in both mammalian cells and yeast. Both hormone-binding and transactivation assays are indispensable tools for the characterization of receptors, for screening potential drug candidate ligands for a known receptor, and for screening compounds that are potential orphan receptor ligands. Because the receptors exhibit modular functional domain structure, the hormone-binding domain (HBD) of a known receptor can be replaced with the HBD of an orphan receptor (within the context of the intact known receptor) to generate a chimeric receptor. The chimeric receptor transcribes a reporter enzyme plasmid containing the hormone response element (HRE) of the known receptor in response to a ligand that binds to the HBD of the orphan receptor.