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 Nicotinic acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway

2008 ◽  
Vol 181 (7) ◽  
pp. 1179-1193 ◽  
Author(s):  
Sudha Kumari ◽  
Virginia Borroni ◽  
Ashutosh Chaudhry ◽  
Baron Chanda ◽  
Ramiro Massol ◽  
...  
Keyword(s):  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Actin Polymerization ◽  
Neuromuscular Junctions ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Endocytic Pathway ◽  
Nicotinic Acetylcholine ◽  
Ovary Cells ◽  
Major Mechanism

Endocytosis of the nicotinic acetylcholine receptor (AChR) is a proposed major mechanism of neuromodulation at neuromuscular junctions and in the pathology of synapses in the central nervous system. We show that binding of the competitive antagonist α-bungarotoxin (αBTX) or antibody-mediated cross-linking induces the internalization of cell surface AChR to late endosomes when expressed heterologously in Chinese hamster ovary cells or endogenously in C2C12 myocytes. Internalization occurs via sequestration of AChR–αBTX complexes in narrow, tubular, surface-connected compartments, which are indicated by differential surface accessibility of fluorescently tagged αBTX–AChR complexes to small and large molecules and real-time total internal reflection fluorescence imaging. Internalization occurs in the absence of clathrin, caveolin, or dynamin but requires actin polymerization. αBTX binding triggers c-Src phosphorylation and subsequently activates the Rho guanosine triphosphatase Rac1. Consequently, inhibition of c-Src kinase activity, Rac1 activity, or actin polymerization inhibits internalization via this unusual endocytic mechanism. This pathway may regulate AChR levels at ligand-gated synapses and in pathological conditions such as the autoimmune disease myasthenia gravis.

scholarly journals Steric and not structure-specific factors dictate the endocytic mechanism of glycosylphosphatidylinositol-anchored proteins

2009 ◽  
Vol 186 (4) ◽  
pp. 615-628 ◽  
Author(s):  
Pinkesh Bhagatji ◽  
Rania Leventis ◽  
Jonathan Comeau ◽  
Mohammad Refaei ◽  
John R. Silvius
Keyword(s):  
Mammalian Cells ◽  
Protein Targeting ◽  
Folate Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fundamental Property ◽  
Membrane Lipid ◽  
Endocytic Pathway ◽  
Ovary Cells ◽  
Lipid Markers

Diverse glycosylphosphatidylinositol (GPI)-anchored proteins enter mammalian cells via the clathrin- and dynamin-independent, Arf1-regulated GPI-enriched early endosomal compartment/clathrin-independent carrier endocytic pathway. To characterize the determinants of GPI protein targeting to this pathway, we have used fluorescence microscopic analyses to compare the internalization of artificial lipid-anchored proteins, endogenous membrane proteins, and membrane lipid markers in Chinese hamster ovary cells. Soluble proteins, anchored to cell-inserted saturated or unsaturated phosphatidylethanolamine (PE)-polyethyleneglycols (PEGs), closely resemble the GPI-anchored folate receptor but differ markedly from the transferrin receptor, membrane lipid markers, and even protein-free PE-PEGs, both in their distribution in peripheral endocytic vesicles and in the manner in which their endocytic uptake responds to manipulations of cellular Arf1 or dynamin activity. These findings suggest that the distinctive endocytic targeting of GPI proteins requires neither biospecific recognition of their GPI anchors nor affinity for ordered-lipid microdomains but is determined by a more fundamental property, the steric bulk of the lipid-anchored protein.

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.

scholarly journals The Cytoplasmic Domain of the Integrin α9 Subunit Requires the Adaptor Protein Paxillin to Inhibit Cell Spreading but Promotes Cell Migration in a Paxillin-independent Manner

2001 ◽  
Vol 12 (10) ◽  
pp. 3214-3225 ◽  
Author(s):  
Bradford A. Young ◽  
Yasuyuki Taooka ◽  
Shouchun Liu ◽  
Karen J. Askins ◽  
Yasuyuki Yokosaki ◽  
...  
Keyword(s):  
Cell Migration ◽  
Intracellular Signaling ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Adaptor Protein ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Cytoplasmic Domains ◽  
Independent Manner ◽  
Ovary Cells

The integrin α9 subunit forms a single heterodimer, α9β1. The α9 subunit is most closely related to the α4 subunit, and like α4 integrins, α9β1 plays an important role in leukocyte migration. The α4 cytoplasmic domain preferentially enhances cell migration and inhibits cell spreading, effects that depend on interaction with the adaptor protein, paxillin. To determine whether the α9 cytoplasmic domain has similar effects, a series of chimeric and deleted α9 constructs were expressed in Chinese hamster ovary cells and tested for their effects on migration and spreading on an α9β1-specific ligand. Like α4, the α9 cytoplasmic domain enhanced cell migration and inhibited cell spreading. Paxillin also specifically bound the α9 cytoplasmic domain and to a similar level as α4. In paxillin −/− cells, α9 failed to inhibit cell spreading as expected but surprisingly still enhanced cell migration. Further, mutations that abolished the α9-paxillin interaction prevented α9 from inhibiting cell spreading but had no effect on α9-dependent cell migration. These findings suggest that the mechanisms by which the cytoplasmic domains of integrin α subunits enhance migration and inhibit cell spreading are distinct and that the α9 and α4 cytoplasmic domains, despite sequence and functional similarities, enhance cell migration by different intracellular signaling pathways.

scholarly journals Hepatitis C virus core protein induces apoptosis and impairs cell-cycle regulation in stably transformed chinese hamster ovary cells

Hepatology ◽  
2000 ◽  
Vol 31 (6) ◽  
pp. 1351-1359 ◽  
Author(s):  
Masao Honda ◽  
Shuichi Kaneko ◽  
Takeo Shimazaki ◽  
Eiki Matsushita ◽  
Kenichi Kobayashi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Cycle Regulation ◽  
Core Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Virus Core ◽  
Cycle Regulation

Enhancement of the Cytotoxicity of Liposomal Ricin by the Carboxylic Ionophore Monensin and the Lysosomotropic Amine NH4Cl in Chinese Hamster Ovary Cells

2006 ◽  
Vol 25 (5) ◽  
pp. 349-359 ◽  
Author(s):  
Seemha Bharadwaj ◽  
Shailendra Singh Rathore ◽  
Prahlad C. Ghosh
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Endocytic Pathway ◽  
Ovary Cells ◽  
Selective Elimination ◽  
Monensin A ◽  
A Chain ◽  
Ionophore Monensin

Ricin was encapsulated in negatively charged liposomes and its effect on the cytotoxicity was compared with native ricin in Chinese hamster ovarian (CHO) cells. The cytotoxicity of ricin, as measured by a marker protein synthesis (incorporation of 3H-leucine), was reduced markedly (300-fold) following encapsulation in liposomes. Lactose, a potent inhibitor of ricin cytotoxicity, had no effect on the binding, internalization, and cytotoxicity of liposomal ricin, indicating that liposomal ricin enter into mammalian cells by an alternative route, bypassing galactose-mediated endocytic pathway. Both monensin (a carboxylic ionophore) and NH4Cl (a lysosomotropic amine) markedly enhances the cytotoxicity of liposomal ricin, indicating endocytotic uptake of liposomal ricin. The degree of potentiation of the cytotoxicity of liposomal ricin by both monensin and NH4Cl was significantly higher (441- and 51-fold) as compared to native ricin (62.5- and 12.5-fold). The extent of exocytosis of free ricin was found to be much higher as compared to liposomal ricin; on the other hand, the extent of degradation of free and liposomal ricin was identical. Consequently, the intracellular level of liposomal ricin was increased to 3.5-fold. This higher level of intracellular liposomal ricin may allow more efficient ricin A-chain release into the cytosol under the influence of NH4Cl and monensin. Monensin-induced potentiation of liposomal ricin was prevented by brefeldin A, indicating that in the presence of monensin, the liposomal ricin was efficiently routed through the Golgi apparatus en route to the cytosol. Thus, liposomal ricin in combination with monensin may have potential application for selective elimination of malignant cells.

scholarly journals Glycosaminoglycan synthesis is depressed during mitosis and elevated during early G1.

1985 ◽  
Vol 101 (3) ◽  
pp. 1086-1093 ◽  
Author(s):  
S F Preston ◽  
C S Regula ◽  
P R Sager ◽  
C B Pearson ◽  
L S Daniels ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Biochemical Analysis ◽  
Core Protein ◽  
Chinese Hamster Ovary Cells ◽  
Membrane Vesicle ◽  
Chinese Hamster ◽  
Glycosaminoglycan Synthesis ◽  
Ovary Cells ◽  
Interphase Cells ◽  
G1 Cells

[35S]Sulfate incorporation was measured in populations of Chinese hamster ovary cells enriched for mitotics, early G1 cells, and interphase monolayers or suspensions. Incorporation was determined by biochemical analysis of extracts and quantitative autoradiography of thick sections. 90% of [35S]sulfate was incorporated into glycosaminoglycan (GAG). Incorporation was depressed fourfold in mitotics and stimulated by from two- to three-fold in early G1 cells relative to mixed interphase cells. GAG synthesis was maintained into late G2. Thus, the rate of GAG biosynthesis was correlated temporally with the detachment and reattachment of cells to substrate. Inhibitors of protein synthesis brought about the rapid arrest of GAG biosynthesis. However, xylosides, which bypass the requirement for core protein, did not bring oligosaccharide sulfation in mitotics to interphase levels. These observations indicate an inhibition of Golgi processing and are consistent with a generalized defect of membrane vesicle-mediated transport during mitosis.

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

Malignancy-related characteristics of wild type and drug-resistant chinese hamster ovary cells

Pathology ◽  
1993 ◽  
Vol 25 (3) ◽  
pp. 268-276 ◽  
Author(s):  
Wanda B. Mackinnon ◽  
Marlen Dyne ◽  
Rebecca Hancock ◽  
Carolyn E. Mountford ◽  
Adrienne J. Grant ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Drug Resistant ◽  
Wild Type ◽  
Ovary Cells
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