Requirement of hormone for thermal conversion of the glucocorticoid receptor to a DNA-binding state

A central question arising from the model of eukaryotic gene regulation by steroid hormone receptors is whether or not proteins represent pre-existing gene regulatory proteins that are activated on exposure to the extracellular signal. It has been generally believed that the ligand-binding of steroid hormone receptors triggers an allosteric change in receptor structure, manifested by an increased affinity of the receptor for DNA in vitro and nuclear target elements in vivo, as monitored by nuclear translocation. But this model has been challenged by recent reports indicating that glucocorticoid and progesterone receptors bind specifically in vitro to target DNA sequences even in the absence of hormone. On the other hand, it appears that the hormone induces protection in vivo of the glucocorticoid response element of the tyrosine amino transferase gene. Here we show that under conditions permitting minimal in vitro manipulation, the steroid-free glucocorticoid receptor in crude cytosol associates with the hsp90 heat shock protein (relative molecular mass Mr approximately equal to 90,000) to form a large 300K complex, rather than the 94K liganded receptor monomer. More importantly, we have developed an assay to demonstrate the requirement of hormone to dissociate the 300K complex by heat treatment. Specific DNA-binding activity of the receptor becomes apparent in this process, showing that DNA binding occurs but is inhibited in the large heteromeric complex. We propose a model in which receptor function is repressed by association of the receptor with hsp90. Dissociation of this complex is induced by the binding of steroid and is apparently an irreversible process.     

Steroid-free glucocorticoid receptor binds specifically to mouse mammary tumour virus DNA

Steroid hormones are thought to modulate gene expression through their interaction with receptor proteins. The intracellular localization of unoccupied receptor proteins has been a subject of controversy: free glucocorticoid receptor appears to reside in the cytoplasm and moves to the cell nucleus only after binding the steroid. The purified hormone-bound glucocorticoid receptor has been shown to bind selectively to hormone regulatory elements (HRE) in the vicinity of hormonally-inducible promoters and, in particular, in the long terminal repeat (LTR) region of mouse mammary tumour virus (MMTV). We have tackled the question of whether the hormone itself is required for the interaction of the receptor protein with the HRE. Using monoclonal antibodies to the receptor we find that upon heat-activation the steroid-free glucocorticoid receptor present in rat liver cytosol binds specifically in vitro to the HRE of MMTV. No qualitative differences in the DNaseI-footprints were detected when hormone-free receptor was compared to the hormone-receptor complex or even receptor complexed with the hormone antagonist RU486. We conclude that the steroid ligand is not an absolute requirement for generating the conformation of the glucocorticoid receptor that allows its interaction with the HRE in vitro. An alternative function of the hormone in vivo could be to modulate nuclear partitioning of the receptor.     

Binding sites for growth hormone releasing factor on rat anterior pituitary cells

Growth hormone releasing factors (GRFs) have been isolated from human pancreatic tumours (hGRF) and rat hypothalamus (rhGRF). The response to GRF at the pituitary level can be modulated by other factors, including glucocorticoids, thyroid hormones, somatostatin and other neuropeptides and somatomedins. Glucocorticoids enhance GRF-induced growth hormone (GH) secretion in primary cultures of rat anterior pituitary cells, and the synthetic glucocorticoid dexamethasone has recently been shown to increase the amounts of GH released in freely moving rats in response to submaximal doses of intravenous GRF. To investigate whether somatotroph sensitivity to GRF is modulated at its receptor level, we have developed a radioreceptor assay using an iodinated analogue of hGRF as radioligand. We report here that the relative binding affinities of rGRF, hGRF and the two analogues are correlated with their in vitro biological potencies. Further, the number of GRF binding sites is drastically decreased in cells deprived of glucocorticoids either in vivo or in vitro.     

Characterization of DNA sequences through which cadmium and glucocorticoid hormones induce human metallothionein-IIA gene

Deletion experiments have defined two stretches of DNA (genetic elements), lying close to the promoter for a human gene for metallothionein, that separately mediate the induction of the gene by heavy metal ions, particularly cadmium, and by glucocorticoid hormones. The element responsible for induction by cadmium is duplicated, yet a single copy is fully functional; the element responsible for induction by glucocorticoid hormones is coincident with the DNA-binding site for the glucocorticoid hormone receptor.     

Glucocorticoid receptors lacking hormone-binding activity are bound in nuclei of ATP-depleted cells

The glucocorticoid receptor binding capacity of rat thymus cells disappears when the cells are depleted of ATP by anaerobiosis, and rapidly reappears when ATP levels are restored. Loss and recovery of binding capacity occurs even when protein synthesis is suppressed with cycloheximide. In view of this and similar work in other cell systems, we proposed that in cells deprived of ATP the receptor is present in a form--the 'null receptor' form, as we shall call it--that cannot bind hormone. Although many subsequent observations support this idea, no direct evidence has appeared for the existence of the null receptor. We have attempted to detect the null receptor in WEHI-7 mouse thymoma cells with a monoclonal antibody to the glucocorticoid receptor. Here we report that the null receptor is bound in the nuclei of ATP-depleted cells, and is present in amounts comparable to those of receptors in normal cells.     

Domain structure of human glucocorticoid receptor and its relationship to the v-erb-A oncogene product

The interaction of steroids with their nuclear receptors induces a cascade of regulatory events that results from the activation of specific sets of genes by the hormone/receptor complex. Steroids, either acting alone or possibly synergistically with other growth factors, can influence the DNA synthesis and proliferation of specific target cells, initiate developmental pathways and activate expression of the differentiated phenotype. Moreover, steroid hormones have been implicated in abnormal growth regulation both in tumours and tumour-derived cell lines. The identification of complementary DNAs encoding the human glucocorticoid receptor (hGR) predicts two protein forms (alpha and beta; 777 and 742 amino acids long, respectively) which differ at their carboxy termini. We report here that both forms of the receptor are related, with respect to their domain structure, to the v-erb-A oncogene product of avian erythroblastosis virus (AEV), which suggests that steroid receptor genes and the c-erb-A proto-oncogene are derived from a common primordial regulatory gene. Therefore, oncogenicity by AEV may result, in part, from the inappropriate activity of a truncated steroid receptor or a related regulatory molecule encoded by v-erb-A. This suggests a mechanism by which transacting factors may facilitate transformation. We also identify a short region of hGR that is homologous with the Drosophila homoeotic proteins encoded by Antennapedia and fushi tarazu.