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.     

采用染料亲和层析的方法纯化卵泡刺激素

采用染料亲和层析、QAE离子交换层析等纯化步骤，从绝经期妇女尿促性腺激素(HMG)粗品中分离纯化获得高纯度卵泡刺激素(FSH)产品，其中FSH的生物活性达到268.1IU／mg，卵泡刺激素与黄体生长素(LH)的活性比值达到259，产品超过欧洲药典对高纯度卵泡刺激素的要求。此工艺FSH的活性回收率高达65％，而且分离纯化步骤少，简单易行，适合于大规模生产。     

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.     

胸腺肽对不同月龄雌性小鼠血清激素水平的影响

 通过分别给1月龄和3月龄雌性小鼠连续30d皮下注射不同剂量的胸腺肽,研究胸腺肽对血清中促卵泡激素（FSH）、雌二醇（E2）、促甲状腺激素（TSH）和胰岛素（INS）水平的影响。结果表明,各剂量胸腺肽使1月龄小鼠血清FSH、E2水平显著或极显著降低（P<0.05,P<0.01）;TSH水平显著或极显著升高（P<0.05,P<0.01）;INS水平降低,其中高剂量组INS水平显著降低（P<0.05）。各剂量胸腺肽使3月龄小鼠血清FSH水平降低,其中高剂量组FSH水平显著降低（P<0.05）;TSH水平升高,其中高剂量组TSH水平极显著升高（P<0.01）;E2、INS水平降低,但差异不显著。可见,胸腺肽能影响雌性小鼠血清激素水平,从而调节雌性小鼠的内分泌系统。     

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.     

Gonadotropin-releasing hormone analogue binds to luteal cells and inhibits progesterone production.

Although gonadotropin-releasing hormone (GnRH) is believed to mediate the hypothalamic control of pituitary gonadotropin secretion, continuous or repeated administration of GnRH or its agonist analogues has been shown to cause paradoxical antifertility effects in several species, including primates. GnRH-induced interruption of reproductive cycles and pregnancy is associated with diminished progesterone production, implying defective function of the corpus luteum. These luteolytic effects have been attributed to the well recognized desensitising actions of elevated luteinising hormone (LH) levels on ovarian LH receptors and steroidogenesis, subsequent to GnRH-induced gonadotropin release from the anterior pituitary. However, treatment with high doses of exogenous LH did not cause suppression of serum progesterone levels during early pregnancy in rats, whereas a highly active GnRH analogue was effective in this regard. These observations suggested that GnRH and its agonist analogues, given in high or sustained doses, can exert a direct action on the ovary that is independent of the pituitary. This hypothesis was further supported by the ability of GnRH and its agonists to inhibit human chorionic gonadotropin (HCG)-induced ovarian and uterine weight gain in hypophysectomised rats and to delay the onset of puberty in intact female rats. Also, GnRH and its agonist analogues have recently been shown to inhibit steroidogenesis induced by follicle-stimulating hormone (FSH) in cultured granulosa cells, confirming the direct action of such peptides on the ovarian follicle. The marked inhibitory effects of GnRH and its agonists on corpus luteum function suggest that these compounds could exert direct actions by binding to specific receptors on luteal cells. The present experiments, which examine the effects of GnRH agonists on luteal steroidogenesis, demonstrate the existence of such actions and their mediation by specific high-affinity receptor sites present in luteal cell membranes.     

珍珠鸟和家鸡甲状旁腺激素3型受体(PTH3R)的结构与表达图谱分析

本研究采用RT-PCR方法,首先克隆珍珠鸟和家鸡的PTH3R基因全长cDNA序列.结果显示,家鸡PTH3R(cPTH3R)cDNA全长1632bp,编码543个氨基酸,珍珠鸟PTH3R(zPTH3R-w)cDNA序列全长1563bp,编码520个氨基酸,其蛋白均含有信号肽序列、七次跨膜区等特征性结构.此外,在珍珠鸟中还发现一个新剪接变体zPTH3R-v1,其cDNA序列全长1468bp,编码488个氨基酸,其缺失第3外显子进而导致第1跨膜结构域缺失.利用生物信息学方法,我们还对珍珠鸟和家鸡PTH3R蛋白序列进行三维建模.采用RT-PCR方法,本研究也对珍珠鸟PTH3R基因进行组织表达分析.结果显示,zPTH3R及其剪切变体zPTH3R-v1在珍珠鸟脑及外周组织中广泛表达.     

Mechanism of glutamate receptor desensitization

Ligand-gated ion channels transduce chemical signals into electrical impulses by opening a transmembrane pore in response to binding one or more neurotransmitter molecules. After activation, many ligand-gated ion channels enter a desensitized state in which the neurotransmitter remains bound but the ion channel is closed. Although receptor desensitization is crucial to the functioning of many ligand-gated ion channels in vivo, the molecular basis of this important process has until now defied analysis. Using the GluR2 AMPA-sensitive glutamate receptor, we show here that the ligand-binding cores form dimers and that stabilization of the intradimer interface by either mutations or allosteric modulators reduces desensitization. Perturbations that destabilize the interface enhance desensitization. Receptor activation involves conformational changes within each subunit that result in an increase in the separation of portions of the receptor that are linked to the ion channel. Our analysis defines the dimer interface in the resting and activated state, indicates how ligand binding is coupled to gating, and suggests modes of dimer dimer interaction in the assembled tetramer. Desensitization occurs through rearrangement of the dimer interface, which disengages the agonist-induced conformational change in the ligand-binding core from the ion channel gate.     

动物抑制素的作用机制与技术开发（综述）

抑制素的生理功能不仅是抑制促卵泡素分泌,而且与下丘脑或更高级的中枢神经活动有关。生理剂量的抑制素只影响促卵泡素的分泌,对促黄体素分泌基本无作用。抑制素免疫可显著提高动物体内促卵泡素的浓度,对繁殖季节和非繁殖季节的母畜都有作用。本文综述了抑制素免疫在提早母畜性成熟和提高公畜繁殖力,提高母畜排卵率,与其它生殖激素免疫的比较,牛抑制素亚单位重组产品,抑制素在禽类繁殖上的作用,抑制素水平检测及应用等方面研究进展,并阐明了抑制素技术开发的前景。     

The mast cell binding site on human immunoglobulin E

Antibodies of the immunoglobulin E isotype sensitize mast cells and basophils for antigen-induced mediator release by binding through the Fc portion to a high-affinity receptor (Fc epsilon R1, Ka = 10(9)M-1) on the cell surface causing the clinical manifestations of type I hypersensitivity. As the amino acid sequence of the human epsilon chain is now known, attempts have been made to map the Fc epsilon R1 binding site on IgE to a fragment smaller than Fc epsilon using proteolytic cleavage products, none of which proved to be active. Cleavage between the C epsilon 2 and C epsilon 3 domains released two inactive fragments, suggesting that the junction between these segments could be important in receptor binding. This region is protected against protease digestion in the rat IgE complex with the receptor of rat basophilic leukaemia cells. Here we report the mapping of the mast cell receptor binding site on human IgE to a sequence of 76 amino acids at the C epsilon 2/C epsilon 3 junction. Recombinant peptides containing this sequence inhibit passive sensitization of skin mast cells in vivo and sensitize mast cells to degranulation by anti-IgE in vitro almost as efficiently as a myeloma IgE. Fragments containing the separate domains are inactive. Additional sequences are required for rapid assembly of fragments into disulphide-linked dimers, suggesting that a single chain can form the active site. In a three-dimensional model of the human Fc epsilon, the two identical segments are far apart. Each folds to generate a cleft between the C epsilon 2 and C epsilon 3 domains on the surface of the Fc epsilon. The docking of IgE on to mast cells could take place within this cleft.     

Inhibin is absent from azoospermic semen of infertile men

There is ample evidence that the secretion of follicle stimulating hormone (FSH) by the pituitary gland is regulated by a nonsteroidal hormone of gonadal origin, termed inhibin. In several species, testicular extracts, gonadal lymph and seminal plasma have been shown to contain proteins which inhibit FSH release; lack of suitable assays for inhibin has, however, prevented clear definition of its physiological significance. Men whose tests show the histological changes of germinal cell failure usually have raised FSH levels in the blood, but may have normal levels of luteinizing hormone (LH) and testosterone. Such patients would thus be predicted to show reduced inhibin production. To test this, we have measured the FSH inhibitory activity of seminal plasma from azoospermic subjects with raised plasma FSH levels. We report here our confirmation of diminished inhibin levels in seminal plasma of these patients, thus providing convincing support for its physiological role as a modulator of FSH production in man.     

Molecular heterogeneity of benzodiazepine receptors

Benzodiazepines exhibit reversible, stereospecific high affinity binding to mammalian brain membranes, and the respective binding sites for 3H-flunitrazepam represent pharmacologically and clinically relevant receptors for benzodiazepines. Recently it has been demonstrated that reversibly bound 3H-flunitrazepam becomes irreversibly attached to a specific membrane protein with apparent molecular weight of 50,000 when incubations are performed in the presence of UV light. Irreversible binding of 3H-flunitrazepam to this protein had pharmacological properties similar to reversible benzodiazepine receptor binding, indicating that 3H-flunitrazepam is a photoaffinity label for the benzodiazepine receptor. Using irreversible binding of 3H-flunitrazepam and subsequent electrophoretic separation of the labelled proteins in SDS-gels followed by fluorography, we found that in hippocampus and several other brain regions at least two different types of benzodiazepine receptors exist. Each seems to be associated with a gamma-aminobutyric acid (GABA) receptor.