DETECTION OF POTENTIAL LIGANDS FOR NUCLEAR RECEPTORS IN CELLULAR-EXTRACTS

In response to external stimuli, steroid receptors are directly influe nced to transactivate gene expression. Assuming they exist, identifica tion of ligands for orphan steroid receptors is a key to understanding their physiology. In the orphan subgroup of the steroid receptor supe r-family, the putative carboxyl terminal ligand-binding domain (LBD) i s well conserved among members of the superfamily, which suggests a ro le in ligand binding. A consequence of ligand binding is the induction of a significant conformational change within the LBD which is necess ary for the transactivation function. This characteristic conformation al change can be detected by partial proteolytic digestion and has bee n localized by mutational analysis and epitopic mapping of the LBD usi ng monoclonal antibodies. Based on this finding, a sensitive in vitro assay was developed for the rapid screening and identification of pote ntial ligands for orphan receptors. We examined the patterns of confor mational changes in the androgen receptor, glucocorticoid receptor, an d progesterone receptor induced by binding of their cognate agonists a nd antagonists. We demonstrated that the conformational changes induce d by ligands can serve as characteristic and reliable markers to disti nguish between the ligand-bound and apoprotein states of a receptor. T he sensitivity and feasibility of employing this assay to detect new e ndogenous ligands using fractionated cellular extracts were also teste d. The results strongly suggest that unknown compounds can be defined as potential ligands for orphan receptors using this approach.