We have provided a historical perspective on a body of steroid receptor research dealing with the structure and physiological significance of the untransformed 9S receptor that has often confused both novice and expert investigators. The frequent controversies and equivocations of earlier studies were due to the fact that the native, hormone-free state of these receptors is a large multiprotein complex that resisted description for many years because of its unstable and dynamic nature. The untransformed 9S state of the steroid and dioxin receptors has provided a unique system for studying the function of the ubiquitous, abundant, and conserved heat shock protein, hsp90. The hormonal control of receptor association with hsp90 provided a method of manipulating the receptor heterocomplex in a manner that was physiologically meaningful. For several steroid receptors, binding to hsp90 was required for the receptor to be in a native hormone-binding state, and for all of the receptors, hormone binding promoted dissociation of the receptor from hsp90 and conversion of the receptor to the DNA-binding state. Although the complexes between tyrosine kinases and hsp90 were discovered earlier, the hormonal regulation or steroid receptor association with hsp90 permitted much more rapid and facile study of hsp90 function. The observations that hsp90 binds to the receptors through their HBDs and that these domains can be fused to structurally different proteins bringing their function under hormonal control provided a powerful linkage between the hormonal regulation of receptor binding to hsp90 and the initial step in steroid hormone action. Because the 9S receptor hsp90 heterocomplexes could be physically stabilized by molybdate, their protein composition could be readily studied, and it became clear that these complexes are multiprotein structures containing a number of unique proteins, such as FKBP51, FKBP52, CyP-40, and p23, that were discovered because of their presence in these structures. Further analysis showed that hsp90 itself exists in a variety of native multiprotein heterocomplexes independent of steroid receptors and other 'substrate' proteins. Cell-free systems can now be used to study the formation of receptor heterocomplexes. As we outlined in the scheme of Fig. 1, the multicomponent receptor-hsp90 heterocomplex assembly system is being reconstituted, and the importance of individual proteins, such as hsp70, p60, and p23, in the assembly process is becoming recognized. It should be noted that our understanding of the mechanism and purpose of steroid receptor heterocomplex assembly is still at an early stage. We can now speculate on the roles of receptor-associated proteins in receptor action, both as individuals and as a group, but their actual functions are still vague or unknown. We can make realistic models about the chaperoning and trafficking of steroid receptors, but we don't yet know how these processes occur, we don't know where chaperoning occurs in the cell (e.g. Is it limited to the cytoplasm? Is it a diffuse process or does chaperoning occur in association with structural elements?), and, with the exception of the requirement for hormone binding, we don't know the extent to which the hsp90-based chaperone system impacts on steroid hormone action. It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell. Because this assembly system is apparently present in all eukaryotic cells, it probably performs an essential function for many proteins. The bacterial homolog of hsp90 is not an essential protein, but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70 (698). (ABSTRACT TRUNCATED)

/pdf/steroid-receptor-interactions-with-heat-shock-protein-and-1iq19twpoc.pdf

Steroid receptor interactions with heat shock protein and immunophilin chaperones.

Molybdate inhibition of glucocorticoid receptor inactivation and transformation.

Separation of the hormone- and DNA-binding sites of the hepatic glucocorticoid receptor by means of proteolysis.

https://www.jbc.org/content/258/1/417.full.pdf

The glucocorticoid receptor in GH1 cells. Evidence from dense amino acid labeling and whole cell studies for an equilibrium model explaining the influence of hormone on the intracellular distribution of receptor.

AT physiological temperatures glucocorticoids1–5, in common with other steroid hormones6, initially interact with normal target cells such as the rat thymocyte by forming hormone–receptor complexes that seem first to be located in the cytoplasm and then rapidly become bound to the nucleus. At low temperatures (0–4 °C) the hormones, when incubated either with target cells or with cytosols from such cells, form ‘non-activated’ complexes that do not bind to nuclei; subsequent warming of the cells1–5, or of the cytosols together with nuclei2,7–11, leads to the formation of nuclear-bound complexes. As shown originally with oestrogens12 and later with glucocorticoids2,7–10,13, if cytosols with non-activated complexes formed at 0–4 °C are warmed to 20–37 °C in the absence of nuclei, the complexes become ‘activated’, and will then bind to nuclei even at low temperatures. Activation, which can also be brought about by increased ionic strength7–9,13, gel filtration, dilution and other treatments14, has been studied extensively and is known to be accompanied by several changes other than enhanced affinity for nuclei. For glucocorticoid–receptor complexes the changes include enhanced affinity for DNA7,10,13–15 and altered mobility on phosphocellulose16, DEAE-Sephadex17 and DEAE-cellulose18 columns. This last property is the one we have used in our experiments. Despite the many published studies on activation, formation of non-activated complexes and subsequent activation have apparently never been demonstrated in cells or cytosols exposed to steroids at normal body temperatures only. Consequently, it is not known whether non-activated complexes and activation have any significant role in physiological conditions, and at least one well known scheme of steroid hormone action19 does not include the activation step. It is therefore possible that when steroid hormones bind to cytoplasmic receptors at physiological temperatures they immediately form activated complexes. The results described here show that for glucocorticoids this alternative can be excluded: when these steroids interact with receptors in rat thymus cells at 37 °C they initially form non-activated complexes, and subsequently give rise to activated complexes.

E Milgrom

Papers

Mechanism and kinetics of the thermal activation of glucocorticoid hormone receptor complex.