Topographic recognition of cyclic hydrocarbons and related compounds by receptors for androgens, estrogens, and glucocorticoids

The structural requirements for the interaction of about 80 cyclic hydrocarbons and related compounds with the androgen receptor of rat ventral prostate, the estrogen receptor of human breast tumor MCF-7 cells, and the glucocorticoid receptor of rat liver were examined by comparing their abilities to compete with radioactive hormones for binding to the respective receptors. The results indicate that the receptor-binding affinity of a compound is dependent on its electronic configuration and geometrical similarity to a portion of a natural steroid hormone which can be recognized by local ligand-binding sites in the receptor. For the estrogen receptor, beta-phenols are more active than the corresponding alpha-phenols, whereas nonphenolic compounds are totally inactive. For androgen and glucocorticoid receptors, alpha-phenols are more active than beta-phenols. The androgen receptor can interact stereospecifically with nonoxygenated and nonalkylated cyclic hydrocarbons, such as 10,11-dihydro-5H-dibenzo[a,d] cycloheptene or 9,10-dihydrophenanthrene, which can, in vivo, inhibit the androgen-dependent growth of the male accessory reproductive organs. The affinities of naphthalene, anthracene, phenanthrene, biphenyl, and adamantane toward glucocorticoid and androgen receptors can be enhanced by acetylation or ethanolization of these ligands. Our results also indicate that, while the hormonal action of a steroid may be dependent on the interaction of a functional group on the hormone with a specific group on the receptor, the presence of such a group may not be required for the antagonistic activity of a compound that can physically block hormone binding to the receptor. Thus, many small molecules that were hitherto considered to be biologically inert may interact with steroid receptors specifically and affect hormonal activities in vivo.     

Differential In Vivo Regulation of Steroid Hormone Receptor Activation by Cdc37p

The CDC37 gene is essential for the activity of p60^v-src when expressed in yeast cells. Since the activation pathway for p60^v-src and steroid hormone receptors is similar, the present study analyzed the hormone-dependent transactivation by androgen receptors and glucocorticoid receptors in yeast cells expressing a mutant version of the CDC37 gene. In this mutant, hormone-dependent transactivation by androgen receptors was defective at both permissive and restrictive temperatures, although transactivation by glucocorticoid receptors was mildly defective only at the restrictive temperature. Cdc37p appears to function via the androgen receptor ligand-binding domain, although it does not influence receptor hormone-binding affinity. Models for Cdc37p regulation of steroid hormone receptors are discussed.     

Hormone-induced changes in the in vitro DNA-binding activity of the chicken progesterone receptor

Previous analyses have indicated that steroid hormone receptors undergo an allosteric change in structure upon binding by the steroid ligand. This structural change was envisioned as an intramolecular unmasking of the protein's DNA-binding domain, thus allowing the receptor to function in gene regulation. We report an analysis of the effect of hormone on the DNA-binding activity of the chicken progesterone receptor. Using an isocratic elution of DNA affinity columns we show that unliganded receptor (aporeceptor) can bind a 23-basepair progesterone response element with high affinity and a high degree of sequence preference. Hormone causes a 1.5-fold increase in affinity for the PRE sequence and a 2-fold decrease in affinity for non-specific DNA. Kinetic analysis of the off-rate of receptor-DNA complexes is consistent with this minor effect of hormone. In addition, gel retardation analysis of receptor-progesterone response element complexes further substantiates that hormone is not required for sequence-specific DNA binding. These results indicate that hormone is not necessary for the progesterone receptor to fold into a conformation that recognizes specific gene regulatory sequences.     

雄激素受体的作用机制

主要概述了雄激素受体的作用机制,特别对影响雄激素受体特异性的因素进行探讨.雄激素受体（AR）属于甾体激素受体超家族,能通过配体依赖方式与特异的DNA序列结合,调控基因转录.     

Identification and characterization of a ligand-regulated nuclear export signal in androgen receptor

Androgen receptor (AR) belongs to the steroid receptor superfamily that regulates gene expression in a ligand-dependent fashion. AR is localized to the cytoplasm in the absence of androgen and translocates into the nuclei to activate gene expression in the presence of ligand. Regulation of AR nuclear import and export represents an essential step in androgen action. A nuclear localization signal (NLS) has been identified in the DNA-binding domain and hinge region of AR and other steroid receptors. Studies on nuclear export of AR, however, are limited, and what might be the underlying mechanism regulating the intracellular localization of steroid receptors is unclear. Our studies have identified a leptomycin B-insensitive nuclear export signal (NESAR) in the ligand-binding domain of AR, which is active in the absence of androgen and repressed upon ligand binding. Consistent with its androgen-sensitivity, NESAR contains amino acid residues in the immediate vicinity of the bound ligand. NESAR is necessary for AR nuclear export and is dominant over the NLS in the DNA-binding domain and hinge region in the absence of hormone. Our findings suggest that androgen can regulate NESAR and, subsequently, the NLS of the AR, providing a mechanism by which androgen regulates AR nuclear/cytoplasmic shuttling. Estrogen receptor alpha and mineralocorticoid receptor also contain functional NES, suggesting that this ligand-regulated NES is conserved among steroid receptors.     

The Mr 90,000 heat shock protein-free androgen receptor has a high affinity for steroid, in contrast to the glucocorticoid receptor

Transformed and bacterially expressed glucocorticoid receptors free from Mr 90,000 heat shock protein (hsp90) have a 100-fold lower steroid-binding affinity than the hsp90-bound nontransformed receptor, suggesting that hsp90 is needed for high-affinity steroid binding [Nemoto, T., Ohara-Nemoto, Y., Denis, M., & Gustafsson, J.-A. (1990) Biochemistry 29, 1880-1886]. To investigate whether or not this phenomenon is common to all steroid receptors, we investigated the steroid-binding affinities of bacterially expressed and transformed androgen receptors. The C-terminal portion of the rat androgen receptor containing the putative steroid-binding domain was expressed as a fusion protein of protein A in Escherichia coli. The recombinant protein bound a synthetic androgen, [3H]R1881, with high affinity (Kd = 0.8 +/- 0.3 nM). Glycerol gradient analysis revealed that the recombinant protein sedimented at around the 3S region irrespective of the presence of molybdate, indicating that the receptor is present in monomeric form. The steroid-free transformed androgen receptor was obtained by exposure of rat submandibular gland cytosol to 0.4 M NaCl in the absence of steroid. High-performance ion-exchange liquid chromatography analysis showed that the transformed androgen receptor bound to [3H]R1881 with high affinity. Thus these observations indicate that, in contrast to the glucocorticoid receptor, hsp90 is not required for the high-affinity steroid binding of the androgen receptor. In addition, the hsp90-free androgen receptor prebound with radioinert R1881 was efficiently relabeled with [3H]R1881, while the triamcinolone acetonide-bound, transformed glucocorticoid receptor failed in ligand exchange. The inability to achieve ligand exchange probably reflects the low steroid-binding affinity of this entity.     

Creation of "super" glucocorticoid receptors by point mutations in the steroid binding domain

Almost all modifications of the steroid binding domain of glucocorticoid receptors are known to cause a reduction or loss of steroid binding activity. Nonetheless, we now report that mutations of cysteine 656 of the rat receptor, which was previously suspected to be a crucial amino acid for the binding process, have produced "super" receptors. These receptors displayed an increased affinity for glucocorticoid steroids and a decreased relative affinity for cross-reacting steroids such as progesterone and aldosterone. The increased in vitro affinity of the super receptors was maintained in a whole cell bioassay. These results indicate that additional modifications of the glucocorticoid receptor, and probably the other steroid receptors, may further increase the binding affinity and/or specificity.     

Multihormone regulation of MMTV-LTR in transfected T-47-D human breast cancer cells

Multihormonal regulation on the long terminal repeat (LTR) region of mouse mammary tumour virus (MMTV) has been studied using T-47-D human breast cancer cells stably transfected with the steroid sensitive LTR-C3 chimaeric gene. The specificity of steroid action on transfected LTR sequences has been compared with regulation of endogenous cellular markers. We conclude that the hormone response element of the LTR can be induced by physiological concentrations of androgen, progestin and glucocorticoid. 17 beta-Oestradiol did not regulate the LTR at physiological levels but an effect was found at 10(-6) M. This effect was not inhibited by antioestrogen nor was it reproduced by the synthetic oestrogen diethylstilboestrol suggesting such effects do not occur via the oestrogen receptor. The antioestrogens tamoxifen and transhydroxytamoxifen do not induce the LTR. No significant steroid competition was found in LTR regulation: whilst oestradiol did not act at physiological concentration it did not interfere with induction by androgen, progestin or glucocorticoid. Such gene regulation did not simply follow receptor status of the cells nor was it reflected in patterns of growth regulation by steroids. The implications of these findings on the mechanism of steroid hormone action are discussed.     

Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor

Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.