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.     

Antiglucocorticosteroid effects suggest why steroid hormone is required for receptors to bind DNA in vivo but not in vitro

Sequence-specific interaction between steroid hormone receptors (R) and DNA hormone-responsive elements (HRE) takes place in vitro irrespective of the presence of hormone and even when R is liganded with an antagonist. In vivo, in contrast, the presence of hormone is mandatory for glucocorticosteroid (G) receptor-HRE interaction to occur and no HRE occupancy is detected in the presence of an antagonist. One possible explanation is that in vivo R is originally complexed with a protein that prevents its binding to target HREs. The hormone would then induce the dissociation of the oligomer, thus unmasking the functional DNA binding domain of the receptor. The unliganded, non DNA-binding 8S-form of the chick GR is a hetero-oligomer including the relative molecular mass (Mr) 94,000 steroid-binding unit (4S-GR), and the non-steroid-binding, non-DNA-binding 90,000 protein common to all classes of 8S-R and identified as heat-shock protein (hsp 90). We report here that triamcinolone acetonide (TA) promotes the transformation of 8S-GR to 4S-GR complexes both in explants and in cell-free conditions and that the high-affinity antiglucocorticosteroid RU 486 stabilizes the 8S-GR, as assessed by gradient sedimentation and HPLC. However, in vitro TA- and RU 486- 4S-GR showed comparable DNA-binding activity. These results suggest that the lack of affinity for DNA of the 8S form of GR may be attributable in vivo to the interaction of the 4S-GR protein with hsp 90, and that hormone binding might trigger a conformational change which results in the release of active 4S-GR.     

Glucocorticoid and progesterone receptors bind to the same sites in two hormonally regulated promoters

The glucocorticoid receptor of rat liver recognizes nucleotide sequences near the promoter of mouse mammary tumour virus (MMTV) required for hormonal induction in gene transfer experiments. Similar nucleotide sequences have been found in the human metallothionein gene IIA and in the chicken lysozyme gene, the later induced also by oestrogen, progesterone and androgens. In microinjection experiments, deletion of only 44 base pairs (bp) of the lysozyme promoter (from -208 to -164) results in coordinated loss of progesterone and glucocorticoid-dependent gene expression. We show here that purified glucocorticoid receptor from rat liver and progesterone receptor from rabbit uterus yield similar or overlapping exonuclease III footprints in the promoter regions of MMTV and chicken lysozyme. Thus, the regulatory elements for different steroid hormones may be similar or at least share structural features.     

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.     

Identification of a new class of steroid hormone receptors

The gonads and adrenal glands produce steroids classified into five major groups which include the oestrogens, progestins, androgens, glucocorticoids and mineralocorticoids. Gonadal steroids control the differentiation and growth of the reproductive system, induce and maintain sexual characteristics and modulate reproductive behaviour. Adrenal steroids also influence differentiation as well as being metabolic regulators. The effects of each steroid depend primarily on its specific receptors, the nature of which could therefore provide a basis for classification of steroid hormone action. The successful cloning, sequencing and expression of the human glucocorticoid (hGR) (ref. 1), oestrogen (hER), progesterone (hPR), and mineralocorticoid (hMR) receptors, complementary DNA, plus homologues from various species, provides the first opportunity to study receptor structure and its influence on gene expression. Sequence comparison and mutational analysis show structural features common to all groups of steroid hormone receptors. The receptors share a highly conserved cysteine-rich region which functions as the DNA-binding domain. This common segment allows the genome to be scanned for related gene products: hMR cDNA for example, was isolated using an hGR hybridization probe. In this study, using the DNA-binding domain of the human oestrogen receptor cDNA as a hybridization probe, we have isolated two cDNA clones encoding polypeptides with structural features suggestive of cryptic steroid hormone receptors which could participate in a new hormone response system.     

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.     

Impaired type II glucocorticoid-receptor function in mice bearing antisense RNA transgene.

Glucocorticoids, in conjunction with their cognate receptors, exert negative-feedback effects on the hypothalamus-pituitary-adrenal axis, suppressing adrenal steroid secretions. Two types of corticosteroid receptor, distinguishable by their ability to bind corticosterone, have been identified as classical mineralocorticoid (type I) and glucocorticoid (type II) receptors by cloning their complementary DNAs. The type I receptor controls the basal circadian rhythm of corticosteroid secretion. Both receptor types are involved in negative feedback, but the type II receptor may be more important for terminating the stress response as it is the only one to be increased in animals rendered more sensitive to corticosteroid negative-feedback effects. Here we create a transgenic mouse with impaired corticosteroid-receptor function by partially knocking out gene expression with type II glucocorticoid receptor antisense RNA. We use this animal to study the glucocorticoid feedback effect on the hypothalamus-pituitary-adrenal axis.