Friedrich C. Dalman

A model of glucocorticoid receptor unfolding and stabilization by a heat shock protein complex

It has recently been reported that incubation of avian progesterone receptors, mouse glucocorticoid receptors, or the viral tyrosine kinase pp60src with rabbit reticulocyte lysate reconstitutes their association with the 90 kDa heat shock protein, hsp90. The reassociation is thought to require unfolding of the steroid receptor or pp60src before hsp90 can bind. The unfoldase activity may be provided by hsp70, which is also present in the reconstituted receptor heterocomplex. In this paper we review evidence that hsp70 and hsp90 are associated in cytosolic heterocomplexes that contain a limited number of other proteins. From an analysis of known receptor-hsp interactions and a predicted direct interaction between hsp90 and hsp70 we have developed an admittedly very speculative model of glucocorticoid receptor unfolding and stabilization. One important feature of the model is that the receptor becomes attached to a heat shock protein heterocomplex rather than undergoing independent unfolding and stabilization events. The model requires that hsp70 and hsp90 bind directly to the receptor at independent sites. Importantly, the model accommodates the stoichiometry of 2 hsp90 per 1 molecule of receptor that has been assayed in the untransformed GR heterocomplex in cytosols prepared from hormone-free cells.

A Conserved Proline in the hsp90 Binding Region of the Glucocorticoid Receptor Is Required for hsp90 Heterocomplex Stabilization and Receptor Signaling

Studies of hsp90 in yeast have supported the notion that this chaperone plays a critical role in signaling by steroid receptors. One limitation to these studies is that yeast expressing hsp90 mutants may also be deficient in fundamental cellular functions of the chaperone required for steroid-dependent induction of transcription. In this work, we have prepared mutants of the glucocorticoid receptor (GR) that permit analysis of hsp90 binding and transcriptional activity in cells with normal chaperone function. Our previous data supported a model in which hsp90 binds to the receptor steroid binding domain according to a two-site model. By amino acid mutagenesis of these two sites, we have now generated three receptor mutants and analyzed their function. Upon their translationin vitro, all three mutants interacted with hsp90 similarly to the wild-type receptor. However, one mutant, P643A (GRo), was of particular interest because, although it showed normal steroid binding and transformation to a glucocorticoid response element-specific DNA binding form, it was remarkably deficient in nuclear translocation and transcriptional function at 37 °C. Furthermore, GRo·hsp90 heterocomplexes formed in vivo or assembled under cell-free conditions were much less stable than wild-type GR·hsp90 heterocomplexes. Our results demonstrate that Pro-643 plays a critical role in both stabilizing the receptor·hsp90 complex and in permitting an efficient nuclear translocation and, thus, support the concept that the chaperone is an integral component of the steroid-receptor signaling pathway.

Glucocorticoid receptor phosphorylation in mouse L-cells.

This paper summarizes our observations on the phosphorylation state of untransformed and transformed glucocorticoid receptors isolated from 32P-labeled L-cells. The 300-350-kDa 9S untransformed murine glucocorticoid receptor complex is composed of a 100-kDa steroid-binding phosphoprotein and one or possibly two units of the 90-kDa heat shock protein (hsp90), which is also a phosphoprotein. Transformation of this complex to the 4S DNA-binding state is accompanied by dissociation of hsp90. When receptors in cytosol are transformed by heating at 25 degrees C, there is no gross change in the degree of phosphorylation of the steroid-binding protein. Both receptors that are bound to DNA after transformation under cell-free conditions and receptors that are located in the nucleus of cells incubated at 37 degrees C in the presence of glucocorticoid are labeled with 32P. The results of experiments in which the 32P-labeled receptor was submitted to limited proteolysis suggest that the 16-kDa DNA-binding domain is phosphorylated and that the 28-kDa steroid-binding domain is not.

The Functional Relevance of the Heteromeric Structure of Corticosteroid Receptorsa

Steroid receptors are transacting factors that must move through the cytoplasm to the nucleus to interact with the DNA. They are members of a superfamily with a well-known topology. The carboxy terminal region represents the ligand-binding domain and subsumes the specific recognition site for ligands. In the middle of the protein is the DNA-binding domain, which contains so-called cysteine-rich zinc fingers and is critical for DNA interactions. The amino terminal domain is thought to be important for transcriptional modulation. These domains appear to be functionally separable, and numerous chimeras have been constructed which retain appropriate binding selectivities and DNA recognition properties.’.* In this paper, we shall focus on two of the steroid receptors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), which bind the stress hormones cortisoVcorticosterone and their analogs. They play a key role in the negative feedback mechanisms of the hypothalamic-pituitary-adrenal axis, and in the control of its circadian rhythm. It is now generally accepted that the association of heat shock proteins (hsps) with the steroid receptors is important for the stabilization of the steroid-binding protein in a non-DNA-binding and transcriptional inactive form. This “repressor” function has been typically considered to be the main, if not the only, role of the associated proteins on the receptor fun~ t ion .~ According to this view, GR would appear to be a unique steroid receptor, in that it additionally requires the association of hsps for adopting a high steroid-binding affinity state.” We recently found similar requirements for the MR.8 From a broader perspective, heat shock or stress proteins are considered to be part of the family of molecular chaperones. The members of this family were recently defined by Hendrick & Hart19 as “proteins that bind to and stabilize an otherwise unstable conformer of another protein, and by controlled binding and release of the substrate protein, facilitate its correct folding in vivo: be it folding, oligomeric assembly, transport to a particular subcellular compartment, or controlled switching between activelinactive conformations.” Within this context, the interaction between steroid-binding proteins and chaperones seems to illustrate only a particular example of the more general importance of chaperones in cell physiology. An interesting