scholarly journals Specific role for p85/p110β in GTP-binding-protein-mediated activation of Akt

2005 ◽  
Vol 392 (3) ◽  
pp. 607-614 ◽  
Author(s):  
Hiroshi Kubo ◽  
Kaoru Hazeki ◽  
Shunsuke Takasuga ◽  
Osamu Hazeki
Keyword(s):  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Point Mutations ◽  
Chinese Hamster ◽  
Middle Part ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Intact Cells ◽  
Ovary Cells ◽  
Gtp Binding

We prepared CHO (Chinese hamster ovary) cells expressing both IR (insulin receptor) and A1R (A1 adenosine receptor). Treatment of the cells with insulin or PIA [N6-(2-phenylisopropyl)adenosine], a specific A1R agonist increased Akt activity in the cells in a PI3K- (phosphoinositide 3-kinase) dependent manner. Transfection of p110β into the cells augmented the action of PIA with little effect on insulin. Introduction of a pH1 vector producing shRNA (short hairpin RNA) that targets p110β abolished PIA-induced Akt activation. By contrast, an shRNA probe targeting p110α did not impair the effects of PIA. The effect of PIA in p110α-deficient cells was attenuated effectively by both Δp85 and βARK-CT (β-adrenergic receptor kinase-C-terminal peptide). A Δp85-derived protein possessing point mutations in its two SH2 domains did not impair PIA action. These results suggest that tyrosine-phosphorylated proteins and Gβγ (βγ subunits of GTP-binding protein) are necessary for the specific function of p110β in intact cells. The p110β-middle (middle part of p110β) may play an important role in signal reception from GPCRs (GTP-binding-protein-coupled receptor), because transfection of the middle part impaired PIA sensitivity.

scholarly journals The Small GTP-binding Protein R-Ras Can Influence Integrin Activation by Antagonizing a Ras/Raf-initiated Integrin Suppression Pathway

1999 ◽  
Vol 10 (6) ◽  
pp. 1799-1809 ◽  
Author(s):  
Tariq Sethi ◽  
Mark H. Ginsberg ◽  
Julian Downward ◽  
Paul E. Hughes
Keyword(s):  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Integrin Activation ◽  
Gtp Binding Protein ◽  
Ovary Cells ◽  
Downstream Effector ◽  
Gtp Binding ◽  
Downstream Effectors ◽  
Rapid Modulation

The rapid modulation of ligand-binding affinity (“activation”) is a central property of the integrin family of cell adhesion receptors. The small GTP-binding protein Ras and its downstream effector kinase Raf-1 suppress integrin activation. In this study we explored the relationship between Ras and the closely related small GTP-binding protein R-Ras in modulating the integrin affinity state. We found that R-Ras does not seem to be a direct activator of integrins in Chinese hamster ovary cells. However, we observed that GTP-bound R-Ras strongly antagonizes the Ras/Raf-initiated integrin suppression pathway. Furthermore, this reversal of the Ras/Raf suppressor pathway does not seem to be via a competition between Ras and R-Ras for common downstream effectors or via an inhibition of Ras/Raf-induced MAP kinase activation. Thus, R-Ras and Ras may act in concert to regulate integrin affinity via the activation of distinct downstream effectors.

scholarly journals ADP-ribosylation of the GTP-binding protein Rho by Clostridium limosum exoenzyme affects basal, but not N-formyl-peptide-stimulated, actin polymerization in human myeloid leukaemic (HL60) cells

1994 ◽  
Vol 299 (3) ◽  
pp. 775-779 ◽  
Author(s):  
G Koch ◽  
J Norgauer ◽  
K Aktories
Keyword(s):  
Actin Polymerization ◽  
Binding Protein ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Intact Cells ◽  
Hl60 Cells ◽  
Adp Ribosylation ◽  
Gtp Binding ◽  
Long Term Treatment ◽  
Formyl Peptide

Treatment of human myeloid leukaemic (HL60) cells with Clostridium limosum exoenzyme, which inactivates the small GTP-binding protein Rho by ADP-ribosylation, decreased the basal F-actin content. Inhibition of F-actin occurred after long-term treatment (24 h) of intact HL60 cells or after introduction of the toxin by electropermeabilization in a toxin-concentration-dependent manner. Concomitantly with the decrease in the basal F-actin content, the GTP-binding protein Rho was ADP-ribosylated in intact cells. However, Clostridium limosum toxin had no inhibitory effect on N-formyl-peptide-induced actin polymerization. Moreover, the relative N-formyl-peptide-stimulated polymerization was substantially enhanced in cells treated with Clostridium limosum transferase. In contrast with Clostridium limosum exoenzyme, component C21 of the Clostridium botulinum C2 toxin, which ADP-ribosylates G-actin, depolymerized basal F-actin and inhibited N-formyl-peptide-induced actin polymerization in electropermeabilized HL60 cells. These findings indicate that Rho proteins are involved in the basal, but not the ligand-evoked, actin polymerization in HL60 cells.

scholarly journals Recombinant human GM-CSF induces leukocytosis and activates peripheral blood polymorphonuclear neutrophils in nonhuman primates

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 206-213 ◽  
Author(s):  
P Mayer ◽  
C Lam ◽  
H Obenaus ◽  
E Liehl ◽  
J Besemer
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
White Blood Cells ◽  
Chinese Hamster ◽  
Coli Strain ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Ovary Cells ◽  
Gm Csf ◽  
Cell Counts

The in vivo efficacy of glycosylated and nonglycosylated recombinant human granulocyte macrophage colony-stimulating factor (rh GM-CSF) expressed in Chinese hamster ovary cells and Escherichia coli respectively was studied in rhesus monkeys following a daily subcutaneous (SC; three times) or intravenous (IV; over six hours) dose for seven consecutive days. The monkeys responded to the rh GM-CSF with a prompt (within 24 hours) rise in circulating white blood cells (WBCs). Thereafter the total cell counts increased steadily in a dose- dependent manner with repeated dosing to numbers six times over the pretreatment levels. Overall, granulocyte counts increased fivefold, lymphocytes twofold to fourfold, and monocytes threefold to fourfold. Platelets and erythrocytes were unaffected. Within 1 week after the end of treatment the leukocytosis had disappeared. Of the two routes of treatment, SC (three times daily)-administered rh GM-CSF was more effective than the same dose given by a six-hour IV infusion. In addition to inducing leukocytosis, parenterally administered rh GM-CSF primed mature circulating granulocytes for enhanced oxidative metabolism and killing of an E coli strain. These results show that exogenously administered glycosylated or nonglycosylated rh GM-CSF is both an effective stimulator of leukocytosis and a potent activator of the phagocytic function of mature granulocytes in monkeys.

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin

1993 ◽  
Vol 13 (4) ◽  
pp. 2061-2068
Author(s):  
H Shirataki ◽  
K Kaibuchi ◽  
T Sakoda ◽  
S Kishida ◽  
T Yamaguchi ◽  
...  
Keyword(s):  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Bovine Brain ◽  
Target Protein ◽  
Dependent Manner ◽  
Gtp Binding Protein ◽  
Reading Frame ◽  
Gtp Binding ◽  
Putative Target

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

Stereoselective Interaction of Ketamine with Recombinant [micro sign], [small kappa, Greek], and [small delta, Greek] Opioid Receptors Expressed in Chinese Hamster Ovary Cells 

1999 ◽  
Vol 90 (1) ◽  
pp. 174-182 ◽  
Author(s):  
Kazuyoshi Hirota ◽  
Hirobumi Okawa ◽  
Balraj L. Appadu ◽  
David K. Grandy ◽  
Lakshmi A. Devi ◽  
...  
Keyword(s):  
Opioid Receptors ◽  
Room Temperature ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Dependent Manner ◽  
Ovary Cells ◽  
Racemic Ketamine ◽  
Dose Dependent

Background The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). Methods CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. Results Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. Conclusions Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors.

scholarly journals EVIDENCE FOR PLEIOTROPIC CHANGES IN LINES OF CHINESE HAMSTER OVARY CELLS RESISTANT TO CONCANAVALIN A AND PHYTOHEMAGGLUTININ-P

1973 ◽  
Vol 56 (3) ◽  
pp. 666-675 ◽  
Author(s):  
J. A. Wright
Keyword(s):  
Concanavalin A ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Step ◽  
Cross Resistance ◽  
Wild Type ◽  
Con A ◽  
Intact Cells ◽  
Ovary Cells ◽  
Resistant Lines

Lines of Chinese hamster ovary cells resistant to the lectins concanavalin A (Con A) and phytohemagglutinin-P (PHA-P) have been isolated and characterized. Lines were isolated by a stepwise, a single-step, or a cycling single-step procedure, from both mutagen-treated and untreated cultures. The resistant lines showed a higher efficiency of colony formation in the presence of the appropriate lectin than did the wild-type parental line. The cell lines resistant to Con A did not exhibit any detectable cross resistance to PHA-P, nor did the PHA-resistant cells exhibit cross resistance to Con A. The toxicity of Con A from the wild-type and Con A-resistant lines was reduced in the presence of methyl α-D-glucopyranoside; this effect was not seen with the PHA-resistant line. Using 125I-labeled Con A, it was found that Con A was bound preferentially to the surface of intact cells, and that the amount of labeled Con A bound to intact cells was similar for the wild-type and lectin-resistant lines. The Con A-resistant lines were found to be more susceptible to the toxic effects of a number of different compounds, including cyclic AMP and its dibutyryl derivative, sodium butyrate, high concentrations of glucose, phenethyl alcohol, phenol, ouabain, and testosterone. It appears that, in these lines, acquisition of resistance to Con A gave rise to pleiotropic effects which were detected by changes in the sensitivity of the cells to a variety of agents.

scholarly journals A Real-Time, Fluorescence-Based Assay for Measuring µ-Opioid Receptor Modulation of Adenylyl Cyclase Activity in Chinese Hamster Ovary Cells

2013 ◽  
Vol 19 (2) ◽  
pp. 223-231 ◽  
Author(s):  
Alisa Knapman ◽  
Fe Abogadie ◽  
Peter McIntrye ◽  
Mark Connor
Keyword(s):  
Adenylyl Cyclase ◽  
Real Time ◽  
Opioid Receptor ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dependent Manner ◽  
Simultaneous Application ◽  
Ovary Cells ◽  
Receptor Modulation

Inhibition of adenylyl cyclase (AC) activity is frequently used to measure µ-opioid receptor (MOR) activation. We sought to develop a simple, rapid assay of AC activity in whole cells that could be used to study MOR signaling. Chinese hamster ovary cells expressing human MOR (CHO-MOR cells) were grown in 96-well plates and loaded with membrane potential–sensitive fluorescent dye. CHO-MOR cells were treated with the AC activator forskolin (FSK), with or without simultaneous application of MOR agonists, and the resulting change in fluorescence was measured. CHO-MOR cells hyperpolarized in response to application of FSK ( pEC50, 7.3) or calcitonin ( pEC50, 9.4). A submaximally effective concentration of FSK (300 nM) caused a 52% ± 2% decrease in fluorescence. Simultaneous application of the opioids DAMGO ( pEC50, 7.4; Emax, 56%), morphine ( pEC50, 7.0; Emax, 61%); and buprenorphine ( pEC50, 8.6; Emax, 24%) inhibited the FSK response in a dose-dependent manner while having no effect by themselves. The effects of DAMGO were blocked by pertussis toxin. This assay represents a simple, robust method for real-time observation of AC inhibition by MOR in CHO cells. It represents an appealing alternative to end-point assays that rely on cAMP accumulation and can avoid potential confounds associated with rapid desensitization of MOR signaling.

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