Soheil Meshinchi

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.

Evidence that removal of an endogenous metal that stabilizes the untransformed glucocorticoid receptor in cytosol allows ligand-independent receptor transformation☆

Cytosol preparations contain an endogenous heat-stable factor which stabilizes the glucocorticoid receptor in its untransformed, non DNA-binding form. Elution of a partially purified preparation of this stabilizing factor through a metal chelating resin (Chelex-100) leads to the loss of its ability to inhibit temperature-mediated transformation of the receptor. Sodium molybdate mimicks the ability of this endogenous metal to stabilize the untransformed receptor, and it too is adsorbed by Chelex resin. When an L-cell cytosol preparation containing the glucocorticoid receptor is passed through a column of Chelex-100 resin and then incubated at 15 degrees C, the receptor is rapidly transformed to the DNA-binding state, regardless of whether it is steroid-bound or not. In contrast, whole cytosol containing endogenous metals is transformed to the DNA-binding state only when the receptor is both steroid-bound and exposed to elevated temperature. these data suggest that a metal (or metals) may be involved in conferring the property of ligand-dependency to the transformation process.

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).