Emery H. Bresnick

Relationship between glucocorticoid receptor steroid-binding capacity and association of the Mr 90,000 heat shock protein with the unliganded receptor.

Treatment of rat liver cytosol with hydrogen peroxide (H2O2) or sodium molybdate (MoO4(2-)) inhibits thermal inactivation of glucocorticoid receptor steroid-binding capacity at 25 degrees C. Dithiothreitol (DTT) prevents the stabilization of receptors by H2O2. Heating (25 degrees C) of immune pellets formed by immunoadsorption of L-cell murine glucocorticoid receptor complexes to protein-A-Sepharose with an anti-receptor monoclonal antibody (BuGR2) results in dissociation of the M 90,000 heat shock protein (hsp90) from the steroid binding protein. Such thermal-induced dissociation of hsp90 is inhibited by H2O2. Pretreatment of immunoadsorbed receptor complexes with the thiol derivatizing agent, methyl methanethiosulfonate (MMTS) prevents the ability of H2O2 to stabilize the hsp90-receptor interaction. These data suggest a role for hsp90 in maintaining an active steroid-binding conformation of the glucocorticoid receptor.

Speculations on the Role of the 90 kDa Heat Shock Protein in Glucocorticoid Receptor Transport and Function

In their untransformed state, several of the steroid receptors have been shown to be associated with the 90 kDa heat shock protein (hsp90). The receptor-hsp90 association has been demonstrated both in cytosol preparations and in intact cells. Exposure of cytosolic glucocorticoid receptors (GR) to hormone is accompanied by temperature-dependent dissociation of the receptor from hsp90 and by the simultaneous generation of the DNA binding state. Recent observations from our laboratory suggest that association of the GR with hsp90 is required for the receptor to have a competent steroid binding site. As the dissociated components do not reassociate in cytosol, it is clear that the GR is not in free equilibrium with hsp90, and we speculate that hsp90 becomes bound to the GR during receptor translation. In intact cells, hsp90 is associated with microtubules. We propose that these hsp90-containing microtubular structures are responsible for transporting the GR from the cytoplasm to the nucleus. The 9S, untransformed receptor complex may be derived from such a receptor transport system. Receptors, like the estrogen or progesterone receptor, which are located in the nucleus in the hormone-free state, may have arrived at some type of “docking” position at the terminus of the transport pathway from which they cannot progress until they have undergone hormone-mediated dissociation from hsp90.

Inhibition of Glucocorticoid Receptor Conversion to the DNA-Binding State and Inhibition of Subunit Dissociation

When steroid-bound glucocorticoid receptors present in cell-free lysates are exposed either to elevated temperatures or to increased ionic strength, they are transformed from a state that does not bind to DNA to one that does bind to DNA (1). This transformation event is accompanied by a shift in the size of the receptor from an oligomer of approximately 320,000 Da to a monomer of approximately 100,000 Da (2–4). Numerous proposals have been made to explain the oligomeric nature of the untransformed glucocorticoid receptor (see Ref. 1 for review). Among these, is the idea that the receptor oligomer is a tetramer of identical steroid-binding subunits (1). We now know, however, that the untransformed glucocorticoid receptor in mouse L-cell cytosol is a heteromer which contains a 100-kDa steroid-binding phosphoprotein and a 90-kDa non-steroid-binding phosphoprotein (5). In addition, we have used immunological techniques to show that the non-steroid-binding subunit of the L-cell receptor complex is the murine 90-kDa heat shock protein (6). These results are in agreement with those recently reported for the progesterone receptor complex in chick oviduct cytosol. The affinity purified untransformed progesterone receptor complex contains a 90-kDa phosphoprotein which does not bind progestin (7,8).