scholarly journals Effects of Progesterone on Nuclear and Cytosol Steroid Receptor Levels in the Oestrogen-stimulated Uterus: Comparison of the Sheep and Mouse

1982 ◽  
Vol 35 (4) ◽  
pp. 403 ◽  
Author(s):  
GM Stone ◽  
C McCaffery ◽  
BG Miller
Keyword(s):  
Binding Site ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Direct Effect ◽  
Dissociation Constants ◽  
Single Injection ◽  
Steroid Receptor ◽  
Scatchard Analysis ◽  
Mouse Uterus ◽  
Receptor Level

Methods for the measurement of nuclear receptors for oestradiol (Ez) and progesterone (P) in mouse uterus and sheep endometrium have been established. Scatchard analysis of nuclear receptors gave the following dissociation constants (nM) and binding site concentrations (pmol steroid/mg DNA): Ez receptor, mouse 3� 76 and 2� 68, sheep 2� 03 and 5� 37; P receptor, mouse 5� 77 and 2� 52, sheep 5 34 and 3� 58. The effects of a single injection of P on nuclear and cytosol levels of Ez and P receptors have been contrasted in these tissues from Ez-treated mice and sheep. In both species, P treatment resulted, in 30-120 min, in depletion of its own cytosol receptor and accumulation of its nuclear receptor. A significant reduction of cytosol E2 receptor was seen only in the mouse at 24 h. P decreased the nuclear Ez-receptor level in the mouse at 2-8 h, but had no such effect in the sheep. The results indicate that the anti-uterotrophic action of P in mouse uterus is caused by an early direct effect of P on nuclear Ez-receptor retention, and appear also to explain why P is not anti-uterotrophic in sheep uterus.

Sex difference in the cytosolic and nuclear distribution of androgen receptor in mouse submandibular gland

1986 ◽  
Vol 108 (2) ◽  
pp. 267-273 ◽  
Author(s):  
S. Kyakumoto ◽  
R. Kurokawa ◽  
Y. Ohara-Nemoto ◽  
M. Ota
Keyword(s):  
Androgen Receptor ◽  
Nuclear Receptors ◽  
Binding Sites ◽  
Androgen Receptors ◽  
Dissociation Constants ◽  
Scatchard Analysis ◽  
Male And Female ◽  
Short Period ◽  
Almost All ◽  
Androgen Binding

ABSTRACT Cytosol and nuclear androgen receptors in submandibular glands of male and female mice were measured by an exchange assay at 0 °C. The binding of [3H]methyltrienolone to cytosol receptors in females was mostly saturated within a short period of incubation (3 h), whereas the saturation was much slower in males; suggesting that almost all of the cytosol receptors were unoccupied in females and the receptors were partially occupied in males. Nuclear receptors were extracted with pyridoxal 5′-phosphate (5 mmol/l) from nuclear fractions with 93–95% efficiency. The exchange of the bound steroids occurred by 24–48 h at 0 °C, suggesting that most of the nuclear androgen receptor was occupied. The binding was low at higher temperatures, probably due to inactivation of the receptor. Scatchard analysis showed that the apparent dissociation constants of cytosol and nuclear receptors were similar (0·8 and 0·9 nmol/l respectively) in both sexes. On the other hand, the number of androgen-binding sites in the nucleus was much higher in males than in females (1052 fmol/mg DNA and 32 fmol/mg DNA respectively), while the number in the cytosol was higher in females than in males (512 fmol/mg DNA and 368 fmol/mg DNA respectively). These observations show that androgen receptors exist mainly (74%) in the nuclei of males, while they exist mostly (94%) in the cytosol of females. J. Endocr. (1986) 108, 267–273

Prolonged influence of a neonatal cyproterone acetate treatment on renal androgen binding in mice

1988 ◽  
Vol 119 (2) ◽  
pp. 263-268
Author(s):  
G. Veyssiére ◽  
Ch. Gallon ◽  
M. Berger ◽  
Ch. Jean-Faucher ◽  
M. de Turckheim ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Cyproterone Acetate ◽  
Single Injection ◽  
Adult Males ◽  
Male Mice ◽  
Testosterone Levels ◽  
Low Levels ◽  
Androgen Binding

Abstract. To determine whether neonatal endogenous androgens influence adult renal androgen binding, newborn male mice were injected from 1 to 10 days of age with cyproterone acetate and newborn females with testosterone from 1 to 10 days and from 20 to 40 days of age. In controls, at adulthood, the total cellular androgen receptor content was significantly higher in males (1700 200 receptors per cell) than in females (1060 ± 50) and, as expected, the nuclear receptor content was 12-fold higher in males. While the total number of receptors (1650 ± 220 per cell) was unchanged in adult males neonatally treated with cyproterone acetate, their distribution between cytosol and nucleus was similar to that in control females despite normal circulating and renal testosterone levels. The nuclear receptors represented 50, 7 and 11% of the total receptors in control males, control females and cyproterone acetate-treated males, respectively. The very low levels of nuclear receptors present in the kidney of cyproterone acetate-treated males probably explain the decreased sensitivity of this organ to testosterone. The nuclear receptor accumulation measured in adult animals after a single injection of testosterone did not seem to be affected by neonatal hormonal manipulations.

Thionamides and arsenite inhibit specific T3 binding to the hepatic nuclear receptor

1990 ◽  
Vol 68 (3) ◽  
pp. 616-621 ◽  
Author(s):  
Shinko Takagi ◽  
Brian C. W. Hummel ◽  
Paul G. Walfish
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Specific Activity ◽  
Binding Capacity ◽  
High Specific Activity ◽  
Relative Mass ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Therapeutic Effects ◽  
Inhibitory Effects

Methimazole (MMI) and propylthiouracil (PTU) are widely used for the treatment of Graves' disease. However, no studies have been reported on the action of these drugs on binding of L-triiodothyronine (T3) to the nuclear receptor. T3 receptors of rat liver nuclei, prepared by differential centrifugation, were extracted with 0.4 M KCl and 5 mM dithiothreitol (DTT). In the assessment of T3 binding to the DTT-reduced receptor, the hepatic nuclear extract was chromatographed on Superose 6 to remove DTT and isolate proteins of relative mass ≈ 50 000 (chromatographed nuclear receptors (CNRs)), prior to the addition of [125I]T3 of high specific activity (3300 μCi/μg; 1 Ci = 37 GBq). MMI or PTU at 2 mM reduced specific T3 binding to CNR by 84% and 85%, respectively. The inhibitory effects of these reagents and 2 mM sodium arsenite (which complexes dithiols) were additive. Scatchard analyses indicated that neither MMI nor PTU (at 2 mM) significantly altered the affinity constant (Ka) (from 2.41 × 109 to 1.74 × 109 M−1 for PTU and 1.79 × 109 M−1 for MMI), while they both decreased (p < 0.02) maximal binding capacity (from 0.36 ± 0.02 to 0.19 ± 0.02 pmol/mg protein for MMI and 0.17 ± 0.02 pmol/mg protein for PTU). Dose-response curves showed that 50% inhibition was attained at 0.6 mM PTU or 1.0 mM MMI with ≈25% inhibition by both at 0.1 mM. Artefactual binding effects by MMI and PTU on [125I]T3 were excluded by chromatography experiments. Similar results were obtained using nuclear receptors prepared from livers of hyperthyroid rats. Pretreatment of CNR for 1 h with 5 mM methyl methanethiosulfonate (an oxidant of thiol groups) abolished the inhibitory effects of PTU, MMI, or arsenite, but was not inhibitory in itself. From these studies it is concluded that (i) MMI and PTU could exert at least part of their therapeutic effects by inhibiting specific binding of L-T3 to its hepatic nuclear receptor; (ii) these inhibitory effects of MMI and PTU are likely due to an interaction with cysteine residues (some of which are not in a dithiol configuration) that are essential for T3 binding to its receptor; and (iii) binding of T3 is not inhibited by oxidation of receptor thiols to methyl dithiol groups.Key words: nuclear triiodothyronine (T3) receptor, methimazole, propylthiouracil, Scatchard analysis.

Mechanisms of androgen receptor signalling via steroid receptor coactivator-1 in prostate.

2004 ◽  
Vol 11 (1) ◽  
pp. 117-130 ◽  
Author(s):  
S M Powell ◽  
V Christiaens ◽  
D Voulgaraki ◽  
J Waxman ◽  
F Claessens ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Point Mutations ◽  
Steroid Receptor ◽  
Activation Function ◽  
Steroid Receptor Coactivator ◽  
Basal Transcriptional Machinery ◽  
Independent States

The androgen receptor (AR) is a member of the nuclear receptor superfamily. These ligand-activated transcription factors usually contain two activation functions, a ligand-independent activation function 1(AF1) in the divergent N-terminal domain and a ligand-dependent AF2 in the more conserved C-terminal ligand-binding domain. To promote transcription from target promoters, DNA-bound nuclear receptors recruit coactivator proteins that promote transcription by modifying histones within nucleosomes, resulting in altered topology of chromatin to allow access of the basal transcriptional machinery, or stabilising the pre-initiation complex. It is well known that most coactivators interact with AF2 of many nuclear receptors via conserved, helical LxxLL motifs (where L is leucine and x is any amino acid). The AF2 of the AR is very weak, but we were able to demonstrate that its intrinsic ligand-dependent activity is potentiated by steroid receptor coactivator-1 (SRC1) and that this region interacts with coactivators via LxxLL motifs. However, a mutant SRC1 coactivator with no functional LxxLL motifs was still able to potentiate AR activity. We found that SRC1 can also be recruited to (and increase activity of) AF1 of the AR via a conserved, glutamine-rich region. Point mutations within this region abolish SRC1 interaction with AF1 and also abolish or severely impair its ability to potentiate AR activity on all promoters tested. Thus the AR interacts with SRC1 via two different regions and the AF1 interaction is functionally the more important, although the contribution of the two interactions varies in a promoter-dependent fashion. SRC1 then potentiates receptor activity via recruitment of CBP/p300, a histone acetyltranferase. This is important in the context of prostate cancer as SRC1 and other coactivators including CBP are coexpressed with AR in the luminal epithelial cells of the prostate, where over 90% of prostate tumours arise. There is a need for effective second-line prostate cancer therapy aimed at blocking the AR pathway when anti-androgen therapy has failed. Since there is growing evidence that nuclear receptor cofactors may be implicated in the progression of hormone-dependent tumours to hormone-independent states, novel targets could include the interaction of AR with coactivator proteins. We suggest that the N-terminal interaction would be a more specific and effective target in the case of prostate cancer than the LxxLL/AF2 interaction.

Thioredoxin and glutaredoxin enhance the binding of L-triiodothyronine to its hepatic nuclear receptors

1989 ◽  
Vol 67 (8) ◽  
pp. 477-480 ◽  
Author(s):  
Shinko Takagi ◽  
Ganesh B. Bhat ◽  
Brian C. W. Hummel ◽  
Paul G. Walfish
Keyword(s):  
Glutathione Reductase ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Binding Capacity ◽  
Specific Binding ◽  
Thioredoxin Reductase ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Physiological Effects ◽  
Thioredoxin System

The influence of thioredoxin and glutaredoxin on binding of L-triiodothyronine (T3) to the rat hepatic nuclear T3 receptor was compared with that of the exogenous activator dithiothreitol. Specific [125I]T3 binding, the affinity constant, Ka, and the maximal binding capacity, MBC, were measured using whole nuclei, solubilized preparations of receptor, and chromatographed nuclear receptor. Both the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH) and the glutaredoxin system (glutaredoxin, glutathione reductase, glutathione, and NADPH) increased specific binding of T3 to nuclei, solubilized receptor, and chromatographed receptor significantly. Compared with the values obtained in the absence of added thiol (Ka = 1.6 ± 0.1 × 109 M−1 MBC = 1.7 ± 0.06 pM), the thioredoxin and glutaredoxin systems increased Ka by 147 and 112%, respectively, while decreasing MBC by 51 and 45%, respectively, when chromatographed receptor was used. The same tendency was observed with solubilized receptor. However, dithiothreitol increased Ka without affecting MBC when solubilized receptor was used. These results, the first demonstration of endogenous disulphide reductant systems enhancing binding of T3 to its receptor, suggest that the thioredoxin and (or) glutaredoxin systems may modulate the physiological effects of thyroid hormone.Key words: nuclear triiodothyronine (T3) receptor, thioredoxin, glutaredoxin, Scatchard analysis.

scholarly journals Molecular Dynamics of Cobalt Protoporphyrin Antagonism of the Cancer Suppressor REV-ERBβ

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3251
Author(s):  
Taufik Muhammad Fakih ◽  
Fransiska Kurniawan ◽  
Muhammad Yusuf ◽  
Mudasir Mudasir ◽  
Daryono Hadi Tjahjono
Keyword(s):  
Binding Site ◽  
Nuclear Receptor ◽  
Target Genes ◽  
Protoporphyrin Ix ◽  
Binding Energies ◽  
Metal Center ◽  
Complex Nature ◽  
Dynamic Simulations ◽  
Subtle Change ◽  
Cobalt Protoporphyrin

Nuclear receptor REV-ERBβ is an overexpressed oncoprotein that has been used as a target for cancer treatment. The metal-complex nature of its ligand, iron protoporphyrin IX (Heme), enables the REV-ERBβ to be used for multiple therapeutic modalities as a photonuclease, a photosensitizer, or a fluorescence imaging agent. The replacement of iron with cobalt as the metal center of protoporphyrin IX changes the ligand from an agonist to an antagonist of REV-ERBβ. The mechanism behind that phenomenon is still unclear, despite the availability of crystal structures of REV-ERBβ in complex with Heme and cobalt protoporphyrin IX (CoPP). This study used molecular dynamic simulations to compare the effects of REV-ERBβ binding to Heme and CoPP, respectively. The initial poses of Heme and CoPP in complex with agonist and antagonist forms of REV-ERBβ were predicted using molecular docking. The binding energies of each ligand were calculated using the MM/PBSA method. The computed binding affinity of Heme to REV-ERBβ was stronger than that of CoPP, in agreement with experimental results. CoPP altered the conformation of the ligand-binding site of REV-ERBβ, disrupting the binding site for nuclear receptor corepressor, which is required for REV-ERBβ to regulate the transcription of downstream target genes. Those results suggest that a subtle change in the metal center of porphyrin can change the behavior of porphyrin in cancer cell signaling. Therefore, modification of porphyrin-based agents for cancer therapy should be conducted carefully to avoid triggering unfavorable effects.

scholarly journals Electron-paramagnetic-resonance studies on nitrogenase of Klebsiella pneumoniae. Evidence for acetylene- and ethylene-nitrogenase transient complexes

1978 ◽  
Vol 173 (1) ◽  
pp. 277-290 ◽  
Author(s):  
D J Lowe ◽  
R R Eady ◽  
R N F Thorneley
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Klebsiella Pneumoniae ◽  
Binding Site ◽  
Dissociation Constants ◽  
Paramagnetic Resonance ◽  
Fe Protein ◽  
Low Concentrations ◽  
Direct Binding ◽  
Electron Paramagnetic ◽  
Single Binding Site

Klebsiella pneumoniae nitrogenase exhibited four new electron-paramagnetic-resonance signals during turnover at 10 degrees C, pH7.4, which were assigned to intermediates present in low concentrations in the steady state. 57Fe-substituted Mo–Fe protein showed that they arose from Fe–S clusters in the Mo–Fe protein of nitrogenase. The new signals are designated: Ic, g values at 4.67, 3.37 and approx. 2.0; VI, g values at 2.125, 2.000 and 2.000; VII, g values at 5.7 and 5.4; VIII, g values at 2.092, 1.974 and 1.933. The sharp axial signal VI arises from a Fe4S4 cluster at the −1 oxidation level. This signal was only detected in the presence of ethylene and provides the first evidence of an enzyme–product complex for nitrogenase. [13C]Acetylene and [13C]ethylene provided no evidence for direct binding of this substrate and product to the Fe–S clusters giving rise to these signals. The dependence of signal intensities on acetylene concentration indicated two types of binding site, with apparent dissociation constants K less than 16 micron and K approximately 13mM. A single binding site for ethylene (K=1.5mM) was detected. A scheme is proposed for the mechanism of reduction of acetylene to ethylene and inhibition of this reaction by CO.

scholarly journals Characterization of Receptor Interaction and Transcriptional Repression by the Corepressor SMRT

1997 ◽  
Vol 11 (13) ◽  
pp. 2025-2037 ◽  
Author(s):  
Hui Li ◽  
Christopher Leo ◽  
Daniel J. Schroen ◽  
J. Don Chen
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Transcriptional Repression ◽  
Thyroid Hormone Receptor ◽  
Receptor Interaction ◽  
Basal Transcription ◽  
Protein Protein Interaction ◽  
Stable Association ◽  
Interacting Surfaces ◽  
Transcriptional Corepressors

Abstract SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are two related transcriptional corepressors that contain separable domains capable of interacting with unliganded nuclear receptors and repressing basal transcription. To decipher the mechanisms of receptor interaction and transcriptional repression by SMRT/N-CoR, we have characterized protein-protein interacting surfaces between SMRT and nuclear receptors and defined transcriptional repression domains of both SMRT and N-CoR. Deletional analysis reveals two individual nuclear receptor domains necessary for stable association with SMRT and a C-terminal helix essential for corepressor dissociation. Coordinately, two SMRT domains are found to interact independently with the receptors. Functional analysis reveals that SMRT contains two distinct repression domains, and the corresponding regions in N-CoR also repress basal transcription. Both repression domains in SMRT and N-CoR interact weakly with mSin3A, which in turn associates with a histone deacetylase HDAC1 in a mammalian two-hybrid assay. Far-Western analysis demonstrates a direct protein-protein interaction between two N-CoR repression domains with mSin3A. Finally we demonstrate that overexpression of full-length SMRT further represses basal transcription from natural promoters. Together, these results support a role of SMRT/N-CoR in corepression through the utilization of multiple mechanisms for receptor interactions and transcriptional repression.

scholarly journals Inactivation of the Nuclear Receptor Coactivator RAP250 in Mice Results in Placental Vascular Dysfunction

2003 ◽  
Vol 23 (4) ◽  
pp. 1260-1268 ◽  
Author(s):  
Per Antonson ◽  
Gertrud U. Schuster ◽  
Ling Wang ◽  
Björn Rozell ◽  
Elin Holter ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Blood Circulation ◽  
Transcriptional Activity ◽  
Vascular Dysfunction ◽  
Diverse Group ◽  
Placental Development ◽  
Nuclear Receptor Coactivator ◽  
The Past ◽  
Placental Blood

ABSTRACT Coactivators constitute a diverse group of proteins that are essential for optimal transcriptional activity of nuclear receptors. In the past few years many coactivators have been identified but it is still unclear whether these proteins interact indiscriminately with all nuclear receptors and whether there is some redundancy in their functions. We have previously cloned and characterized RAP250 (ASC-2/PRIP/TRBP/NRC), an LXXLL-containing coactivator for nuclear receptors. In order to study its biological role, Rap250 null mice were generated by gene targeting. Here we show that genetic disruption of Rap250 results in embryonic lethality at embryonic day (E) 13.5. Histological examination of placentas revealed a dramatically reduced spongiotrophoblast layer, a collapse of blood vessels in the region bordering the spongiotrophoblast, and labyrinthine layers in placentas from Rap250−/− embryos. These findings suggest that the lethality of Rap250−/− embryos is the result of obstructed placental blood circulation. Moreover, the transcriptional activity of PPARγ is reduced in fibroblasts derived from Rap250−/− embryos, suggesting that RAP250 is an essential coactivator for this nuclear receptor in the placenta. Our results demonstrate that RAP250 is necessary for placental development and thus essential for embryonic development.

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