scholarly journals Insulin-Like Growth Factor-I Provokes Functional Antagonism and Internalization of β1-Adrenergic Receptors

Endocrinology ◽  
2007 ◽  
Vol 148 (6) ◽  
pp. 2653-2662 ◽  
Author(s):  
Shai Gavi ◽  
Dezhong Yin ◽  
Elena Shumay ◽  
Hsien-yu Wang ◽  
Craig C. Malbon
Keyword(s):  
Cardiac Myocytes ◽  
Tyrosine Kinases ◽  
Adrenergic Receptors ◽  
Adrenergic Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Intracellular Camp ◽  
Ovary Cells ◽  
Igf I ◽  
Protein Kinase Akt

Hormones that activate receptor tyrosine kinases have been shown to regulate G protein-coupled receptors, and herein we investigate the ability of IGF-I to regulate the β1-adrenergic receptor. Treating Chinese hamster ovary cells in culture with IGF-I is shown to functionally antagonize the ability of expressed β1-adrenergic receptors to accumulate intracellular cAMP in response to stimulation by the β-adrenergic agonist Iso. The attenuation of β1-adrenergic action was accompanied by internalization of β1-adrenergic receptors in response to IGF-I. Inhibiting either phosphatidylinositol 3-kinase or the serine/threonine protein kinase Akt blocks the ability of IGF-I to antagonize and to internalize β1-adrenergic receptors. Mutation of one potential Akt substrate site Ser412Ala, but not another Ser312Ala, of the β1-adrenergic receptor abolishes the ability of IGF-I to functionally antagonize and to sequester the β1-adrenergic receptor. We also tested the ability of IGF-I to regulate β1-adrenergic receptors and their signaling in adult canine cardiac myocytes. IGF-I attenuates the ability of β1-adrenergic receptors to accumulate intracellular cAMP in response to Iso and promotes internalization of β1-adrenergic receptors in these cardiac myocytes.

Expression, purification and characterization of secreted recombinant human insulin-like growth factor-I (IGF-I) and the potent variant des(1–3)IGF-I in Chinese hamster ovary cells

1991 ◽  
Vol 6 (3) ◽  
pp. 231-239 ◽  
Author(s):  
P. McKinnon ◽  
M. Ross ◽  
J. R. E. Wells ◽  
F. J. Ballard ◽  
G. L. Francis
Keyword(s):  
Growth Factor ◽  
Human Insulin ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Recombinant Human Insulin ◽  
Ovary Cells ◽  
Factor I ◽  
Igf I ◽  
Recombinant Peptides

ABSTRACT Recombinant human insulin-like growth factor-I (hIGF-I) and a biologically potent variant lacking the N-terminal tripeptide (des(1–3)IGF-I) were produced from transfected Chinese hamster ovary cells. The constructs encoding the signal peptide, sequence of the mature peptide and a C-terminal extension peptide were expressed under the control of a Rous sarcoma virus promoter. Successfully transfected clones secreting correctly processed recombinant hIGF-I or des(1–3)IGF-I were selected by their secretion of IGF-I-like activity into the culture medium. The recombinant peptides were purified to homogeneity as assessed by high-performance liquid chromatography and N-terminal sequence analysis. The purified recombinant peptides exhibited biological potencies equivalent to authentic IGF-I and des(1–3)IGF-I respectively.

scholarly journals Characterization of Neuropeptide B (NPB), Neuropeptide W (NPW), and Their Receptors in Chickens: Evidence for NPW Being a Novel Inhibitor of Pituitary GH and Prolactin Secretion

Endocrinology ◽  
2016 ◽  
Vol 157 (9) ◽  
pp. 3562-3576 ◽  
Author(s):  
Guixian Bu ◽  
Dongliang Lin ◽  
Lin Cui ◽  
Long Huang ◽  
Can Lv ◽  
...  
Keyword(s):  
Amino Acids ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Prolactin Secretion ◽  
Luciferase Reporter ◽  
Intracellular Camp ◽  
Pituitary Cells ◽  
Reporter System ◽  
Ovary Cells ◽  
The Central Nervous System

The 2 structurally and functionally related peptides, neuropeptide B (NPB) and neuropeptide W (NPW), together with their receptor(s) (NPBWR1/NPBWR2) constitute the NPB/NPW system, which acts mainly on the central nervous system to regulate many physiological processes in mammals. However, little is known about this NPB/NPW system in nonmammalian vertebrates. In this study, the functionality and expression of this NPB/NPW system and its actions on the pituitary were investigated in chickens. The results showed that: 1) chicken NPB/NPW system comprises an NPB peptide of 28 amino acids (cNPB28), an NPW peptide of 23 or 30 amino acids (cNPW23/cNPW30), and their 2 receptors (cNPBWR1 and cNPBWR2), which are highly homologous to their human counterparts. 2) Using a pGL3-CRE-luciferase reporter system, we demonstrated that cNPBWR2 expressed in Chinese hamster ovary cells can be potently activated by cNPW23 (not cNPB28), and its activation inhibits the intracellular cAMP signaling pathway, whereas cNPBWR1 shows no response to peptide treatment, suggesting a crucial role of cNPBWR2 in mediating cNPW/cNPB actions. 3) Quantitative real-time PCR revealed that cNPW and cNPB are widely expressed in chicken tissues, including hypothalamus, whereas cNPBWR1 and cNPBWR2 are mainly expressed in brain or pituitary. 4) In accordance with abundant cNPBWR2 expression in pituitary, cNPW23 could dose dependently inhibit GH and prolactin secretion induced by GHRH and vasoactive intestinal polypeptide, respectively, in cultured chick pituitary cells, as monitored by Western blotting. Collectively, our data reveal a functional NPB/NPW system in birds and offer the first proof that NPW can act directly on pituitary to inhibit GH/prolactin secretion in vertebrates.

scholarly journals Phosphorylation of IGFBP-1 at Discrete Sites Elicits Variable Effects on IGF-I Receptor Autophosphorylation

Endocrinology ◽  
2013 ◽  
Vol 154 (3) ◽  
pp. 1130-1143 ◽  
Author(s):  
Majida Abu Shehab ◽  
Cristiana Iosef ◽  
Robert Wildgruber ◽  
Girish Sardana ◽  
Madhulika B. Gupta
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Inhibitory Effects ◽  
Ovary Cells ◽  
Igf I ◽  
Igf Binding ◽  
Igf Binding Protein

Abstract We previously demonstrated that hypoxia and leucine deprivation cause hyperphosphorylation of IGF-binding protein-1 (IGFBP-1) at discrete sites that markedly enhanced IGF-I affinity and inhibited IGF-I-stimulated cell growth. In this study we investigated the functional role of these phosphorylation sites using mutagenesis. We created three IGFBP-1 mutants in which individual serine (S119/S169/S98) residues were substituted with alanine and S101A was recreated for comparison. The wild-type (WT) and mutant IGFBP-1 were expressed in Chinese hamster ovary cells and IGFBP-1 in cell media was isolated using isoelectric-focusing-free-flow electrophoresis. BIACore analysis indicated that the changes in IGF-I affinity for S98A and S169A were moderate, whereas S119A greatly reduced the affinity of IGFBP-1 for IGF-I (100-fold, P < .0001). Similar results were obtained with S101A. The IGF-I affinity changes of the mutants were reflected in their ability to inhibit IGF-I-induced receptor autophosphorylation. Employing receptor-stimulation assay using IGF-IR-overexpressing P6 cells, we found that WT-IGFBP-1 inhibited IGF-IRβ autophosphorylation (∼2-fold, P < .001), possibly attributable to sequestration of IGF-I. Relative to WT, S98A and S169A mutants did not inhibit receptor autophosphorylation. S119A, on the other hand, greatly stimulated the receptor (2.3-fold, P < .05). The data with S101A matched S119A. In summary, we show that phosphorylation at S98 and S169 resulted in milder changes in IGF-I action; nonetheless most dramatic inhibitory effects on the biological activity of IGF-I were due to IGFBP-1 phosphorylation at S119. Our results provide novel demonstration that IGFBP-1 phosphorylation at S119 can enhance affinity for IGF-I possibly through stabilization of the IGF-IGFBP-1 complex. These data also propose that the synergistic interaction of distinct phosphorylation sites may be important in eliciting more pronounced effects on IGF-I affinity that needs further investigation.

scholarly journals Transmembrane and cytoplasmic domains of syndecan mediate a multi-step endocytic pathway involving detergent-insoluble membrane rafts

2000 ◽  
Vol 351 (3) ◽  
pp. 607-612 ◽  
Author(s):  
Ilia V. FUKI ◽  
Marie E. MEYER ◽  
Kevin Jon WILLIAMS
Keyword(s):  
Tyrosine Kinases ◽  
Core Protein ◽  
Chinese Hamster Ovary Cells ◽  
Heparan Sulphate ◽  
Chinese Hamster ◽  
Endocytic Pathway ◽  
Membrane Rafts ◽  
Cholesterol Depletion ◽  
Cytoplasmic Domains ◽  
Ovary Cells

Syndecan heparan sulphate proteoglycans directly mediate a novel endocytic pathway. Using Chinese hamster ovary cells expressing the human syndecan 1 core protein or a chimaeric receptor, FcR-Synd, consisting of the ectodomain of the IgG Fc receptor Ia linked to the transmembrane and cytoplasmic domains of syndecan 1, we previously reported that efficient internalization is triggered by ligand clustering, requires intact actin microfilaments and tyrosine kinases, proceeds with a t1/2 of approx. 1h and is distinct from coated-pit pathways. We have now examined the involvement of cholesterol-rich, detergent-insoluble membrane rafts. On clustering, 125I-labelled IgG bound to FcR-Synd rapidly became insoluble in cold Triton X-100, well before endocytosis. Insolubility of clustered FcR-Synd ligand did not require the syndecan ectodomain, linkage of the cytoplasmic tail to the cytoskeleton, or energy-dependent cellular metabolism. Pretreatment of cells with cyclodextrin to deplete cholesterol from rafts abolished insolubility of the clustered ligand and inhibited endocytosis in a dose-responsive fashion. Similar results were obtained with 125I-labelled lipoprotein lipase bound to authentic cell-surface syndecan. In contrast, the 39kDa receptor-associated protein (RAP), a coated-pit ligand, was more than 80% soluble in cold Triton even after internalization; cellular cholesterol depletion failed to substantially affect the internalization of 125I-RAP. Overall, our results indicate a multi-step endocytic process consisting of ligand binding, clustering, energy-independent lateral movement into detergent-insoluble membrane rafts and finally recruitment of actin and tyrosine kinases to bring the ligands into the cell.

scholarly journals Involvement of phospholipase D in insulin-like growth factor-I-induced activation of extracellular signal-regulated kinase, but not phosphoinositide 3-kinase or Akt, in Chinese hamster ovary cells

2003 ◽  
Vol 369 (2) ◽  
pp. 363-368 ◽  
Author(s):  
Yoshiko BANNO ◽  
Yoh TAKUWA ◽  
Momoko YAMADA ◽  
Noriko TAKUWA ◽  
Kenji OHGUCHI ◽  
...  
Keyword(s):  
Growth Factor ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Wild Type ◽  
Ovary Cells ◽  
Akt Activation ◽  
Extracellular Signal ◽  
Igf I ◽  
Phosphoinositide 3 Kinase

Available evidence suggests the involvement of phospholipase D (PLD) in cell proliferation and survival. Phosphoinositide 3-kinase (PI 3-kinase)/Akt and extracellular signal-regulated kinases (ERKs) are signalling molecules that have essential roles in cell proliferation and survival. We previously demonstrated that sphingosine 1-phosphate (S1P)-induced PLD activation via the G-protein-coupled receptor endothelial differentiation gene (EDG) 3/S1P3 was involved in S1P-induced stimulation of PI 3-kinase and Akt. In the present study, we examined the involvement of two PLD isozymes, PLD1 and PLD2, in insulin-like growth factor (IGF)-I receptor tyrosine kinase-mediated stimulation of PI 3-kinase/Akt and ERKs. IGF-I and to a lesser degree S1P stimulated PI 3-kinase activity in Chinese hamster ovary cells overexpressing EDG3/S1P3. IGF-I-induced ERK phosphorylation was suppressed by butan-1-ol, but not butan-2-ol, whereas no effect of butanol was observed in IGF-I-induced Akt activation in S1P3-overexpressing Chinese hamster ovary cells. Overexpression of wild-type PLD1 and PLD2 substantially potentiated S1P-, but not IGF-I-, induced activation of PI 3-kinase and Akt, whereas overexpression of the catalytically inactive mutant of PLD1 or PLD2 did not affect the responses to either agonist. On the other hand, overexpression of wild-type PLD1 and PLD2 potentiated IGF-I- and, to much smaller extents, S1P-induced ERK stimulation. ERK activation by IGF-I as well as S1P was dependent on Ras, but Akt activation by IGF-I was not dependent on Ras. These results suggest that PLDs are involved in growth factor regulation of at least two signalling pathways, PI 3-kinase/Akt and ERKs, depending on the class of cell-surface receptors.

scholarly journals Expression of three human beta-adrenergic-receptor subtypes in transfected chinese hamster ovary cells

1991 ◽  
Vol 196 (2) ◽  
pp. 357-361 ◽  
Author(s):  
Keri M. TATE ◽  
Marie-M. BRIEND-SUTREN ◽  
Laurent J. EMORINE ◽  
Colette DELAVIER-KLUTCHKO ◽  
Stefano MARULLO ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Adrenergic Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Receptor Subtypes ◽  
Beta Adrenergic Receptor ◽  
Beta Adrenergic ◽  
Ovary Cells
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