During the past few years, it has become clear that several of the steroid hormone receptors are phosphoproteins containing sulfhydryl moieties that are critical for receptor function. A variety of studies have also shown that cytosols prepared from several tissues contain at least three endogenous factors that interact with steroid receptors to affect either their ligand binding or DNA binding functions. The long-term objective of this work is to study the roles played by both covalent receptor modification and by the endogenous factors in determining the steroid-binding conformation and DNA-binding activity of glucocorticoid receptors. The steroid hormones are used in the therapy of a wide variety of diseases and understanding the mechanisms by which their receptors may be regulated constitutes a fundamental problem in molecular endocrinology. The first endogenous cytosolic factor under study is a heat-stable activity that converts glucocorticoid receptors from a nonbinding to a steroid-binding state. We have recently shown that this factor is thioredoxin and that glucocorticoid receptors are maintained in their reduced, steroid-binding state by a NADPH-dependent, thioredoxin-mediated reducing system. In this proposal we intend: 1) to determine if thioredoxin binds to the receptors; and 2) to determine if thioredoxin acts solely in reduction or if it must be bound to the receptor for the receptor to function. The second endogenous factor is a small, heat-stable component that we have shown acts like molybdate to stabilize unoccupied receptors and to block transformation of the steroid-receptor complex to the DNA-binding state. In this proposal we present a series of experiments designed to: 1) identify the composition of the stabilizing factor; 2) to synthesize the factor from a cytosolic """"""""precursor""""""""; 3) to determine more completely its mechanism of action. The third endogenous factor is a heat-labile macromolecule that binds to transformed steroid-receptor complexes and prevents their binding to DNA. We have extensively purified this factor and in this proposal we present experiments designed to determine: 1) if the inhibitor is a normal component of the untransformed glucocorticoid-receptor complex; and 2) if the inhibitor interacts with other steroid receptors to limit DNA binding.
