scholarly journals Insulin-Like Growth Factor I Increases Rat Peptide YY Promoter Activity through Sp1 Binding Sites

Endocrinology ◽  
2004 ◽  
Vol 145 (2) ◽  
pp. 659-666 ◽  
Author(s):  
Guiyun Wang ◽  
Andrew B. Leiter ◽  
Ella W. Englander ◽  
George H. Greeley
Keyword(s):  
Binding Sites ◽  
Peptide Yy ◽  
Firefly Luciferase ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Pheochromocytoma Cells ◽  
Protein Levels ◽  
Igf I ◽  
Dose Dependent Fashion ◽  
Luc Reporter Gene

Abstract Studies in rodents demonstrate that the mitogen, IGF-I, stimulates intestinal peptide YY (PYY) expression. To investigate whether the stimulatory influence of IGF-I is exerted at the level of gene transcription, rat PYY 5′-upstream sequences (−2800/+37 bp, −770/+37 bp, −127/+37 bp) fused to the firefly luciferase (luc) reporter gene were transfected into rat pheochromocytoma cells (PC12) and luc activity measured after IGF-I treatment. IGF-I increased transcriptional activity of all constructs similarly; the PYY (−127/+37 bp)-luc construct was used in subsequent experiments. IGF-I increased PYY (−127/+37 bp)-luc activity in a time- and dose-dependent fashion. Sequence analysis detected five putative Sp1 binding sites in the −127/+37-bp sequence. EMSA and supershift experiments using two oligonucleotide fragments of the −127/+37 region showed that Sp1 and Sp3 proteins bound to putative Sp1 sites. Overexpression of Sp1 greatly increased PYY (−127/+37 bp)-luc activity and site-directed mutagenesis of putative Sp1 binding sites decreased basal and IGF-I-induced elevations in PYY (−127/+37 bp)-luc activity. IGF-I treatment also increased Sp1 protein levels and binding activity. Blockade of the IGF-I receptor (IGF-IR) with an IGF-IR antibody decreased the stimulatory influence of IGF-I on Sp1 protein levels and PYY (−127/+37 bp)-luc activity. Together, these findings indicate that IGF-I functions as a positive regulator of PYY gene expression and that the stimulatory effect may be mediated by Sp1 proteins that bind to the proximal PYY promoter region.

Overlap of IGF- and heparin-binding sites in rat IGF-binding protein-5

2000 ◽  
Vol 24 (1) ◽  
pp. 43-51 ◽  
Author(s):  
H Song ◽  
J Beattie ◽  
IW Campbell ◽  
GJ Allan
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Heparin Binding ◽  
Igf I ◽  
Heparin Binding Domain ◽  
Igf Binding

Using site-directed mutagenesis, we have undertaken a study of a potential IGF-binding site in the C-terminal domain of rat IGFBP-5, lying close to or within a previously described heparin-binding domain (residues 201-218) in this protein. After analysis of binding activity using three different methods - ligand blotting, solution phase equilibrium binding and biosensor measurement of real-time on- and off-rates - we report that the mutation of two highly conserved residues within this region (glycine 203 and glutamine 209) reduces the affinity of the binding protein for both IGF-I and IGF-II, while having no effect on heparin binding. In addition, we confirm that mutation of basic residues within the heparin-binding domain (R201L, K202E, K206Q and R214A) results in a protein that has attenuated heparin binding but shows only a small reduction in affinity for IGF-I and -II. Previous findings have described the reduction in affinity of IGFBP-5 for IGFs that occurs after complexation of the binding protein with heparin or other components of the extracellular matrix (ECM) and have postulated that such an interaction may result in conformational changes in protein structure, affecting subsequent IGF interaction. Our data suggesting potential overlap of heparin- and IGF-binding domains argue for a more direct effect of ECM modulation of the affinity of IGFBP-5 for ligand by partial occlusion of the IGF-binding site after interaction with ECM.

Expression and binding activity of the glucocorticoid receptor are upregulated in septic muscle

2002 ◽  
Vol 282 (2) ◽  
pp. R509-R518 ◽  
Author(s):  
Xiaoyan Sun ◽  
David R. Fischer ◽  
Timothy A. Pritts ◽  
Curtis J. Wray ◽  
Per-Olof Hasselgren
Keyword(s):  
Skeletal Muscle ◽  
Glucocorticoid Receptor ◽  
Binding Sites ◽  
Cecal Ligation ◽  
Binding Activity ◽  
Scatchard Analysis ◽  
Regulatory Effect ◽  
Hormone Binding ◽  
Experimental Conditions ◽  
Protein Levels

We examined the influence of sepsis, induced by cecal ligation and puncture in rats, on the protein and gene expression and hormone binding activity of the glucocorticoid receptor (GR) in skeletal muscle. Sepsis resulted in increased GR mRNA and protein levels and upregulated hormone binding activity in extensor digitorum longus and soleus muscles. Scatchard analysis suggested that the increased GR hormone binding activity reflected an increased number of hormone binding sites, whereas receptor affinity for glucocorticoids was unchanged. The GR antagonist RU-38486 blocked the sepsis-induced increase in GR expression and hormone binding activity, implicating a positive regulatory effect of glucocorticoids on GR expression and binding activity under the present experimental conditions. The results suggest that glucocorticoid-dependent metabolic changes in skeletal muscle during sepsis may reflect not only high circulating glucocorticoid levels but increased amounts and hormone binding activity of the GR as well.

Biphasic increase in c-jun mRNA is required for induction of AP-1-mediated gene transcription: differential effects of muscarinic and thrombin receptor activation

1992 ◽  
Vol 12 (10) ◽  
pp. 4742-4750
Author(s):  
J Trejo ◽  
J C Chambard ◽  
M Karin ◽  
J H Brown
Keyword(s):  
Dna Binding ◽  
Gene Transcription ◽  
Time Course ◽  
Firefly Luciferase ◽  
Receptor Activation ◽  
Binding Activity ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Firefly Luciferase Gene ◽  
The Difference

Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.

scholarly journals Mutagenesis Studies and Structure-function Relationships for GalNAc/Gal-Specific Lectin from the Sea Mussel Crenomytilus grayanus

Marine Drugs ◽  
2018 ◽  
Vol 16 (12) ◽  
pp. 471 ◽  
Author(s):  
Svetlana Kovalchuk ◽  
Nina Buinovskaya ◽  
Galina Likhatskaya ◽  
Valery Rasskazov ◽  
Oksana Son ◽  
...  
Keyword(s):  
Binding Sites ◽  
In Silico Analysis ◽  
Binding Activity ◽  
Cancer Diagnostics ◽  
Site Directed Mutagenesis ◽  
Carbohydrate Binding ◽  
Synthetic Analog ◽  
Crenomytilus Grayanus ◽  
Mussel Crenomytilus Grayanus ◽  
The Individual

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with β-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties.

Glucocorticoids and appearance of 1,25-dihydroxyvitamin D3 receptor in rat intestine

1983 ◽  
Vol 244 (3) ◽  
pp. E230-E235
Author(s):  
E. R. Massaro ◽  
R. U. Simpson ◽  
H. F. DeLuca
Keyword(s):  
Vitamin D ◽  
Binding Sites ◽  
Specific Binding ◽  
Vitamin D Supplementation ◽  
Binding Activity ◽  
Rat Intestine ◽  
Dihydroxyvitamin D3 ◽  
Protein Levels ◽  
1,25 Dihydroxyvitamin D3 ◽  
Hydrocortisone Administration

The ontogenesis of the 1,25-dihydroxyvitamin D3 specific binding activity in intestine was examined in vitamin D-deficient and replete rats. The absence of binding activity in intestines during the first two postnatal weeks was not influenced by vitamin D supplementation. The concentration of binding sites peaked on day 18 in vitamin D-replete rats and preceded that in the deficient group by approximately 1 wk. The influence of glucocorticoids on 1,25-dihydroxyvitamin D3-binding protein levels was examined by sequential hydrocortisone administration and adrenalectomy. Subcutaneous hydrocortisone administration before day 14 postpartum did not induce binding activity. The concentration of binding sites was significantly increased to 369 +/- 60 fmol/mg of protein by hydrocortisone injections from days 15 to 17 postpartum when compared with an average of 182 +/- 16 fmol/mg of protein in littermate controls. Hydrocortisone administration did not further increase receptor levels in rats injected from days 19 to 21. Bilateral adrenalectomy on day 17 postpartum significantly decreased the concentration of binding sites. It is concluded that adrenal glucocorticoids play an important role in the developmental appearance of 1,25-dihydroxyvitamin D3 specific binding activity in the postnatal rat intestine.

scholarly journals Biphasic increase in c-jun mRNA is required for induction of AP-1-mediated gene transcription: differential effects of muscarinic and thrombin receptor activation.

1992 ◽  
Vol 12 (10) ◽  
pp. 4742-4750 ◽  
Author(s):  
J Trejo ◽  
J C Chambard ◽  
M Karin ◽  
J H Brown
Keyword(s):  
Dna Binding ◽  
Gene Transcription ◽  
Time Course ◽  
Firefly Luciferase ◽  
Receptor Activation ◽  
Binding Activity ◽  
Protein Levels ◽  
Dna Binding Activity ◽  
Firefly Luciferase Gene ◽  
The Difference

Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.

Pheochromocytoma cells PC12 contain binding sites for CART (cocaine- and amphetamine-regulated transcript)

2007 ◽  
Author(s):  
Jana Maixnerová ◽  
Darja Blokešová ◽  
Resha Matyšková ◽  
Renata Haugvicová ◽  
Eva Šloncová ◽  
...  
Keyword(s):  
Binding Sites ◽  
Pheochromocytoma Cells

scholarly journals Putative ligand binding sites of two functionally characterized bark beetle odorant receptors

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jothi K. Yuvaraj ◽  
Rebecca E. Roberts ◽  
Yonathan Sonntag ◽  
Xiao-Qing Hou ◽  
Ewald Grosse-Wilde ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Bark Beetle ◽  
Pest Control ◽  
Binding Sites ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
Odorant Receptors ◽  
Functional Evolution ◽  
General Importance ◽  
Insight Into

Abstract Background Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

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