scholarly journals Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line

1987 ◽  
Vol 247 (3) ◽  
pp. 731-738 ◽  
Author(s):  
S L Swope ◽  
A Schonbrunn
Keyword(s):  
Islet Cells ◽  
Scatchard Analysis ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Single Class ◽  
Relative Potencies ◽  
Stimulate Insulin Release ◽  
Bombesin Receptors

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

scholarly journals ATP:Mg2+ shapes condensate properties of rRNA-NPM1 in vitro nucleolus model and its partitioning of ribosomes

2021 ◽  
Author(s):  
N. Amy Yewdall ◽  
Alain A. M. André ◽  
Merlijn H. I. van Haren ◽  
Frank H. T. Nelissen ◽  
Aafke Jonker ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Enzymatic Reaction ◽  
Atp Depletion ◽  
Gel Point ◽  
Dependent Manner ◽  
Biophysical Properties ◽  
Temperature Dependent ◽  
Quantitative Fluorescence

Nucleoli have viscoelastic gel-like condensate dynamics that are not well represented in vitro. Nucleoli models, such as those formed by nucleophosmin 1 (NPM1) and ribosomal RNA (rRNA), exhibit condensate dynamics orders of magnitude faster than in vivo nucleoli. Here we show that an interplay between magnesium ions (Mg2+) and ATP governs rRNA dynamics, and this ultimately shapes the physical state of these condensates. Using quantitative fluorescence microscopy, we demonstrate that increased RNA compaction occurs in the condensates at high Mg2+ concentrations, contributing to the slowed RNA dynamics. At Mg2+ concentrations above 7 mM, rRNA is fully arrested and the condensates are gels. Below the critical gel point, NPM1-rRNA droplets age in a temperature-dependent manner, suggesting that condensates are viscoelastic materials, undergoing maturation driven by weak multivalent interactions. ATP addition reverses the dynamic arrest of rRNA, resulting in liquefaction of these gel-like structures. Surprisingly, ATP and Mg2+ both act to increase partitioning of NPM1-proteins as well as rRNA, which influences the partitioning of small client molecules. By contrast, larger ribosomes form a halo around NPM1-rRNA coacervates when Mg2+ concentrations are higher than ATP concentrations. Within cells, ATP levels fluctuate due to biomolecular reactions, and we demonstrate that a dissipative enzymatic reaction can control the biophysical properties of in vitro condensates through depletion of ATP. This enzymatic ATP depletion also reverses the formation of the ribosome halos. Our results illustrate how cells, by changing local ATP concentrations, may regulate the state and client partitioning of RNA-containing condensates such as the nucleolus.

Cytoplasmic and nuclear progesterone receptors in mouse mammary tumours during pregnancy determined by an improved hydroxylapatite assay

1982 ◽  
Vol 94 (1) ◽  
pp. 111-123 ◽  
Author(s):  
Y. Koseki ◽  
M. E. Costlow ◽  
D. Cole ◽  
A. Matsuzawa
Keyword(s):  
Progesterone Receptors ◽  
Nuclear Extract ◽  
Scatchard Analysis ◽  
Normal Mammary Gland ◽  
Single Class ◽  
Heat Stable ◽  
Nuclear Extracts ◽  
Complete Exchange

The binding of [3H] 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020) to progesterone receptors in cytosol and nuclear extracts (0·6 m-KCl) of the pregnancy-dependent, TPDMT-4 mouse mammary tumour was measured at various stages of pregnancy. Compared with conventional dextran-coated charcoal (DCC) assays, a hydroxylapatite assay with DCC pretreatment and precharging of the cytosol with unlabelled R5020 (4 × 10−8 mol/l, for 3–4 h at 4 °C) showed the highest level of binding. The DCC treatment markedly increased the level of R5020 binding in both cytosol and nuclear extracts by allowing the receptor to bind to hydroxylapatite. The DCC pretreatment apparently removed a heat-stable and non-dialysable factor which prevented the receptor from binding to the hydroxylapatite. Using this assay R5020 binding reached a steady state in 24 h at 4 °C, with complete exchange of radioactive for non-radioactive ligand by 20 h. Nuclear extracts did not require precharging and complete exchange was more rapid. Scatchard analysis (without precharging) disclosed a single class of binding sites with a dissociation constant for cytosol of 3·2 ± 0·8 (s.e.m.) × 10−9 mol/l (n = 3) and 4·7 ± 0·6 × 10−9 mol/l (n = 5) for the nuclear extract. Binding was hormone-specific and progesterone translocated binding from the cytoplasm to the nucleus both in vivo and in vitro. Translocation, however, led to a substantial loss of total (nuclear + cytoplasmic receptors. During pregnancy, cytoplasmic progesterone receptor levels were unchanged and low compared to nuclear progesterone receptors which increased by sevenfold from days 1 to 11 and then decreased at day 16. Compared with recent data on cytoplasmic progesterone receptors in normal mammary gland, our results suggested that this tumour may have a reduced sensitivity to the down-regulatory activity of progesterone. This lesion may, in part, account for the failure of the tumour to differentiate during pregnancy.

Myosin VIIA is specifically associated with calmodulin and microtubule-associated protein-2B (MAP-2B)

2001 ◽  
Vol 354 (2) ◽  
pp. 267-274 ◽  
Author(s):  
Penio T. TODOROV ◽  
Rachel E. HARDISTY ◽  
Steve D. M. BROWN
Keyword(s):  
Binding Sites ◽  
Strong Association ◽  
Mouse Kidney ◽  
Scatchard Analysis ◽  
Tail Region ◽  
Immunoblotting Analysis ◽  
Kd Values ◽  
Myosin Viia

Myosin VIIA is a motor molecule with a conserved head domain and tail region unique to myosin VIIA, which probably defines its unique function in vivo. In an attempt to further characterize myosin VIIA function we set out to identify molecule(s) that specifically associate with it. We demonstrate that 17 and 55kDa proteins from mouse kidney and cochlea co-purify with myosin VIIA on affinity columns carrying immobilized anti-myosin VIIA antibody. N-terminal sequencing and immunoblotting analysis identified the 17kDa protein as calmodulin, whereas MS and immunoblotting analysis identified the 55kDa protein as microtubule-associated protein-2B (MAP-2B). Myosin VIIA can also be co-immunoprecipitated from kidney homogenate using anti-calmodulin or anti-MAP2 (recognizing isoforms 2A and 2B) antibodies, confirming the strong association between calmodulin and myosin VIIA and between MAP-2B and myosin VIIA. Myosin VIIA binds to calmodulin with an apparent Kd of 10-9 M. Scatchard analysis of the binding of myosin VIIA to MAP-2B provided evidence for two binding sites, with Kd values of 10-10 and 10-9 M, which have been mapped to medial and C-terminal tail domains of myosin VIIA. The characterization of the interaction of calmodulin and MAP-2B with myosin VIIA provides new insights into the function of myosin VIIA.

scholarly journals PRIC295, a Nuclear Receptor Coactivator, Identified from PPAR-Interacting Cofactor Complex

PPAR Research ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-16 ◽  
Author(s):  
Sean R. Pyper ◽  
Navin Viswakarma ◽  
Yuzhi Jia ◽  
Yi-Jun Zhu ◽  
Joseph D. Fondell ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Target Genes ◽  
Peroxisome Proliferator ◽  
Dependent Manner ◽  
Peroxisome Proliferator Activated Receptor ◽  
Nuclear Receptor Coactivator ◽  
Evolutionarily Conserved

The peroxisome proliferator-activated receptor- (PPAR) plays a key role in lipid metabolism and energy combustion. Chronic activation of PPAR in rodents leads to the development of hepatocellular carcinomas. The ability of PPAR to induce expression of its target genes depends on Mediator, an evolutionarily conserved complex of cofactors and, in particular, the subunit 1 (Med1) of this complex. Here, we report the identification and characterization of PPAR-interacting cofactor (PRIC)-295 (PRIC295), a novel coactivator protein, and show that it interacts with the Med1 and Med24 subunits of the Mediator complex. PRIC295 contains 10 LXXLL signature motifs that facilitate nuclear receptor binding and interacts with PPAR and five other members of the nuclear receptor superfamily in a ligand-dependent manner. PRIC295 enhances the transactivation function of PPAR, PPAR, and ER. These data demonstrate that PRIC295 interacts with nuclear receptors such as PPAR and functions as a transcription coactivator underin vitroconditions and may play an important role in mediating the effectsin vivoas a member of the PRIC complex with Med1 and Med24.

Generation of synthetic RNA-based thermosensors

2008 ◽  
Vol 389 (10) ◽  
Author(s):  
Torsten Waldminghaus ◽  
Jens Kortmann ◽  
Stefan Gesing ◽  
Franz Narberhaus
Keyword(s):  
Conformational Changes ◽  
Translational Control ◽  
Rational Design ◽  
Dependent Manner ◽  
Temperature Dependent ◽  
Structure Probing ◽  
Bacterial Rna ◽  
Computer Based ◽  
Shine Dalgarno Sequence

AbstractStructured RNAs with fundamental sensory and regulatory potential have been discovered in all kingdoms of life. Bacterial RNA thermometers are located in the 5′-untranslated region of certain heat shock and virulence genes. They regulate translation by masking the Shine-Dalgarno sequence in a temperature-dependent manner. To engineer RNA-based thermosensors, we used a combination of computer-based rational design andin vivoscreening. After only two rounds of selection, several RNA thermometers that are at least as efficient as natural thermometers were obtained. Structure probing experiments revealed temperature-dependent conformational changes in these translational control elements. Our study demonstrates that temperature-controlled RNA elements can be designed by a simple combined computational and experimental approach.

Effects of glucocorticosteroids on prolactin and growth hormone production and characterization of the intracellular hormone receptors in rat pituitary tumour cells

1980 ◽  
Vol 95 (3) ◽  
pp. 319-327 ◽  
Author(s):  
Oddvar Naess ◽  
Egil Haug ◽  
Kaare Gautvik
Keyword(s):  
Growth Hormone ◽  
Hormone Receptors ◽  
Pituitary Tumour ◽  
Tumour Cells ◽  
Scatchard Analysis ◽  
Hormone Production ◽  
Single Class ◽  
High Affinity ◽  
Rat Pituitary

Abstract. The effect of corticosterone and dexamethasone on the production of growth hormone and prolactin was studied in rat pituitary tumour cells (GH3-cells) in culture. Corticosterone and dexamethasone caused a dose-dependent stimulation of growth hormone synthesis, and the highest concentration (10−6 mol/l) increased growth hormone levels to 250% of controls. This concentration, however, decreased prolactin synthesis to 25% of the control values. The cytosol fractions from monolayer cultures as well as from tumours of GH3-cells were found to possess receptor molecules for glucocorticoid hormones, having a sedimentation constant close to 8 S in a salt-free buffer and 4 S in the presence of 0.5 mol/l KCL. Isoelectric point of the receptor was 5.8. Scatchard analysis showed one single class of binding sites with high affinity (Kd 2.1 ± 0.4 (sd × 10−9 mol/l). Studies on the steroid specificity revealed that dexamethasone had the highest affinity for the receptor. Corticosterone, cortisol and progesterone had also high affinity, whereas testosterone and oestradiol-17β had no significant affinity for the receptors. After in vivo administration of [3H]dexamethasone to GH3 tumour-bearing rats, radioactivity could be extracted from purified nuclei bound to 4 S macromolecules. The presence of receptors for glucocorticosteroid hormones in the GH3-cells, suggests that these hormones may alter growth hormone and prolactin production at the anterior pituitary level.

scholarly journals Sphingosine 1-phosphate-regulated transcriptomes in heterogenous arterial and lymphatic endothelium of the aorta

2019 ◽  
Author(s):  
Eric Engelbrecht ◽  
Michel V. Levesque ◽  
Liqun He ◽  
Michael Vanlandewijck ◽  
Anja Nitzsche ◽  
...  
Keyword(s):  
Cellular Heterogeneity ◽  
Dependent Manner ◽  
Branch Points ◽  
Transcriptional Responses ◽  
Gpcr Signaling ◽  
Aortic Endothelial Cells ◽  
Sphingosine 1 Phosphate ◽  
G Protein Coupled

AbstractDespite the medical importance of G protein-coupled receptors (GPCRs), in vivo cellular heterogeneity of GPCR signaling and downstream transcriptional responses are not understood. We report the comprehensive characterization of transcriptomes (bulk and single-cell) and chromatin domains regulated by sphingosine 1-phosphate receptor-1 (S1PR1) in adult mouse aortic endothelial cells. First, S1PR1 regulates NFkB and nuclear glucocorticoid receptor pathways to suppress inflammation-related mRNAs. Second, spatially distinct S1PR1 signaling in the aorta is associated with heterogenous endothelial cell (EC) subtypes. For example, a transcriptomically distinct arterial EC population at vascular branch points (aEC1) exhibits ligand- independent S1PR1/ß-arrestin coupling. In contrast, circulatory S1P-dependent S1PR1/ß-arrestin coupling was observed in non-branch point aEC2 cells that exhibit an inflammatory signature. Moreover, an adventitial lymphatic EC (LEC) population shows suppression of lymphangiogenic and inflammation-related transcripts in a S1P/S1PR1-dependent manner. These insights add resolution to existing concepts of GPCR signaling and S1P biology.

scholarly journals Identification of a Bidirectional Splicing Enhancer: Differential Involvement of SR Proteins in 5′ or 3′ Splice Site Activation

1999 ◽  
Vol 19 (11) ◽  
pp. 7347-7356 ◽  
Author(s):  
Cyril F. Bourgeois ◽  
Michel Popielarz ◽  
Georges Hildwein ◽  
James Stevenin
Keyword(s):  
Splice Site ◽  
Sr Proteins ◽  
Dependent Manner ◽  
Wild Type ◽  
Adenovirus E1a ◽  
Differential Involvement ◽  
Splicing Enhancer

ABSTRACT The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5′ splice sites and of one major or one minor 3′ splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5′ splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5′ splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3′ splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5′ splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.

Receptors for angiotensin I1 on platelets from man

1984 ◽  
Vol 66 (6) ◽  
pp. 725-731 ◽  
Author(s):  
Yuan Ding ◽  
Christopher J. Kenyon ◽  
Peter F. Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Capacity ◽  
Specific Binding ◽  
Venous Blood ◽  
Kinetic Studies ◽  
Free Fluid ◽  
Scatchard Analysis ◽  
Ang Ii ◽  
Temperature Dependent ◽  
Single Class

1. Platelets were prepared from peripheral venous blood on iso-osmotic density gradients of Percoll, resulting in a good recovery of cells (50–80%) which were relatively free of contaminating blood cells (erythrocyte <0.1%, leucocyte <0.1%). 2. At 22°C, specific binding of 125labelled angiotensin II (300 pmol/l) was time and temperature dependent, saturable, reversible and linear with cell concentration. 3. Scatchard analysis of saturation curves revealed a single class of binding sites with Kd 1.5 ± 0.4 × 10−10 mol/l and total binding capacity 6.3 ± 1.2 receptorslplatelet. Similar values (Kd 2.4 ± 0.7 × 10−10 mol/l and binding capacity 6.5 ± 1.0 receptors/cell) were obtained by displacement analysis. From kinetic studies the forward and reverse rate constants were 3.1 × 108 mol min−1 1−1 and 3.6 × 10−2/min giving a Kd of 1.2 × 10−10mol/l. 4. The relative binding potencies for angiotensin I1 and analogues were: [Sar1, Thr8]ANC II > ANG II > ANG III > [Sar1, Ala8]ANG II > ANG I. 5. Incubation with an extracellular marker (51Cr-labelled EDTA) demonstrated that binding of angiotensin II to platelets was not due to free fluid endocytosis.

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