Detection of potential ligands for nuclear receptors in cellular extracts.

In response to external stimuli, steroid receptors are directly influenced to transactivate gene expression. Assuming they exist, identification of ligands for orphan steroid receptors is a key to understanding their physiology. In the orphan subgroup of the steroid receptor superfamily, the putative carboxyl terminal ligand-binding domain (LBD) is well conserved among members of the superfamily, which suggests a role in ligand binding. A consequence of ligand binding is the induction of a significant conformational change within the LBD which is necessary for the transactivation function. This characteristic conformational change can be detected by partial proteolytic digestion and has been localized by mutational analysis and epitopic mapping of the LBD using monoclonal antibodies. Based on this finding, a sensitive in vitro assay was developed for the rapid screening and identification of potential ligands for orphan receptors. We examined the patterns of conformational changes in the androgen receptor, glucocorticoid receptor, and progesterone receptor induced by binding of their cognate agonists and antagonists. We demonstrated that the conformational changes induced by ligands can serve as characteristic and reliable markers to distinguish between the ligand-bound and apoprotein states of a receptor. The sensitivity and feasibility of employing this assay to detect new endogenous ligands using fractionated cellular extracts were also tested. The results strongly suggest that unknown compounds can be defined as potential ligands for orphan receptors using this approach.