scholarly journals The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain

1994 ◽  
Vol 124 (5) ◽  
pp. 705-715 ◽  
Author(s):  
RJ Garippa ◽  
TW Judge ◽  
DE James ◽  
TE McGraw
Keyword(s):  
Cell Surface ◽  
Transferrin Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Amino Terminus ◽  
Ovary Cells ◽  
Major Function ◽  
Amino Terminal ◽  
Human Transferrin Receptor

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.

scholarly journals Truncations of the C-terminal cytoplasmic domain of MG160, a medial Golgi sialoglycoprotein, result in its partial transport to the plasma membrane and filopodia

1998 ◽  
Vol 111 (2) ◽  
pp. 249-260 ◽  
Author(s):  
J.O. Gonatas ◽  
Y.J. Chen ◽  
A. Stieber ◽  
Z. Mourelatos ◽  
N.K. Gonatas
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Type I ◽  
Ovary Cells

MG160, a type I cysteine-rich membrane sialoglycoprotein residing in the medial cisternae of the rat Golgi apparatus, is highly homologous to CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand located at the cell surface of mouse myeloid cells and recently detected in the Golgi apparatus as well. The mechanism for the transport of MG160 from the Golgi apparatus to the cell surface is unknown. In this study we found that differential processing of the carboxy-terminal cytoplasmic domain (CD), consisting of amino acids Arg1159 Ile Thr Lys Arg Val Thr Arg Glu Leu Lys Asp Arg1171, resulted in the partial transport of the protein to the plasma membrane and filopodia. In Chinese hamster ovary cells (CHO), stably transfected with the entire cDNA encoding MG160, the protein was localized in the Golgi apparatus. However, when the terminal Arg1171 or up to nine distal amino acids were deleted, the protein was distributed to the plasma membrane and filopodia as well as the Golgi apparatus. This report shows that the CD of an endogenous type I Golgi protein is important for its efficient retention and identifies a unique residue preference in this process. Cleavage within the CD of MG160 may constitute a regulatory mechanism for the partial export of the protein from the Golgi apparatus to the plasma membrane and filopodia.

scholarly journals Functional expression of the human transferrin receptor cDNA in Chinese hamster ovary cells deficient in endogenous transferrin receptor.

1987 ◽  
Vol 105 (1) ◽  
pp. 207-214 ◽  
Author(s):  
T E McGraw ◽  
L Greenfield ◽  
F R Maxfield
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Transferrin Receptor ◽  
Diphtheria Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Functional Expression ◽  
Genetically Engineered ◽  
Ovary Cell ◽  
Ovary Cells

Transferrin (Tf) receptor-variant Chinese hamster ovary cells have been isolated by selection for resistance to two Tf-toxin conjugates. The hybrid toxins contain Tf covalently linked to ricin A chain or a genetically engineered diphtheria toxin fragment. The Tf-receptor-variant (TRV) cells do not have detectable cell-surface Tf receptor; they do not bind fluorescein-Tf or 125I-Tf. TRV cells are at least 100-fold more resistant to the Tf-diphtheria toxin conjugate than are the parent cells. The TRV cells have retained sensitivity to native diphtheria toxin, indicating that the increased resistance to the conjugate is correlated with the loss of Tf binding. The endocytosis of fluorescein-labeled alpha 2-macroglobulin is normal in TRV cells, demonstrating that the defect does not pleiotropically affect endocytosis. Since these cells lack endogenous Tf receptor activity, they are ideally suited for studies of the functional expression of normal or altered Tf receptors introduced into the cells by cDNA transfection. One advantage of this system is that Tf binding and uptake can be used to monitor the behavior of the transfected receptor. A cDNA clone of the human Tf receptor has been transfected into TRV cells. In the stably expressing transfectants, the behavior of the human receptor is very similar to that of the endogenous Chinese hamster ovary cell Tf receptor. Tf binds to cell surface receptors, and is internalized into the para-Golgi region of the cell. Iron is released from Tf, and the apo-Tf and its receptor are recycled back to the cell surface. Thus, the TRV cells can be used to study the behavior of genetically altered Tf receptors in the absence of interfering effects from endogenous receptors.

Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX

2001 ◽  
Vol 358 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Philippe ULSEMER ◽  
Catherine STRASSEL ◽  
Marie-Jeanne BAAS ◽  
Jean SALAMERO ◽  
Sylvette CHASSEROT-GOLAZ ◽  
...  
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Surface Expression ◽  
Platelet Glycoprotein ◽  
Ovary Cells ◽  
Confocal Immunofluorescence Microscopy ◽  
Vessel Injury ◽  
Von Willebrand

The multisubunit leucine-rich glycoprotein (GP) Ib–IX–V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard–Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse–chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbα undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbα and surface expression of the complex. Biosynthetic studies were then performed on a Bernard–Soulier variant based on previous description of abnormal GPIbα size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbα. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments (‘ERGIC’) and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.

The cytoplasmic domain of syndecan-1 is not required for association with Triton X-100-insoluble material

1994 ◽  
Vol 107 (6) ◽  
pp. 1571-1581 ◽  
Author(s):  
H.M. Miettinen ◽  
M. Jalkanen
Keyword(s):  
Extracellular Matrix ◽  
Heparan Sulfate ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Wild Type Mouse ◽  
Cytoplasmic Domain ◽  
Insoluble Residue ◽  
Wild Type ◽  
Ovary Cells ◽  
Triton X

Cell surface heparan sulfate proteoglycans such as syndecan-1 bind various extracellular matrix proteins and have been suggested to interact with the cytoskeleton. Such interactions are thought to be important for stabilizing cell morphology. Syndecan-1 resists extraction with Triton X-100. This insolubility was reported not to be affected by removal of the glycosaminoglycan chains, suggesting that the insolubility is not due to binding to the extracellular matrix, but rather to an association with the actin cytoskeleton (Rapraeger, A., Jalkanen, M. and Bernfield, M. (1986) J. Cell Biol. 103, 2683–2696). To examine further the interaction of syndecan-1 with the Triton X-100-insoluble residue, we expressed wild-type mouse syndecan-1 and a cytoplasmic deletion mutant (tail-less) in Chinese hamster ovary cells. We observed that both the wild-type and the tail-less syndecan-1 were partly insoluble in Triton X-100. The insolubility was not affected by increasing temperature (37 degrees C or 50 degrees C) or by cytochalasin D. Removal of the glycosaminoglycan chains from the ectodomain, however, resulted in complete Triton X-100 solubility, unlike previous reports. Syndecan-1 could also be released into the Triton X-100-soluble fraction by addition of heparin or heparan sulfate to the extraction medium. We conclude that the cytoplasmic domain of syndecan-1 is not responsible for Triton X-100 insolubility. Instead, our results indicate that Triton X-100 insolubility is caused by an interaction of syndecan-1 molecules with other cellular and/or extracellular molecules mediated by the heparan sulfate chains.

Surface polypeptides of the Chinese hamster ovary cell; an immunochemical study

1976 ◽  
Vol 54 (2) ◽  
pp. 185-191 ◽  
Author(s):  
M. Behar-Bannelier ◽  
R. L. Juliano
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Immunochemical Study ◽  
Ovary Cells ◽  
Molecular Weights ◽  
Label Technique

Antibodies elicited by the injection of live Chinese hamster ovary cells (CHO) into rabbits precipitated four major components from detergent extracts of CHO membranes. The four components, of molecular weights 200 000, 125 000, 95 000 and 41 000 daltons, corresponded to cell surface components identified by the lactoperoxidase surface label technique.

scholarly journals CYCLIC CHANGES IN THE CELL SURFACE

1974 ◽  
Vol 60 (2) ◽  
pp. 434-441 ◽  
Author(s):  
Leighton P. Everhart ◽  
Robert W. Rubin
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary Cells ◽  
Surface Membrane ◽  
Chinese Hamster ◽  
Thymidine Uptake ◽  
Ovary Cells ◽  
Dependent Inhibition ◽  
Thymidine Transport ◽  
Cyclic Changes ◽  
Inhibition Of Dna Synthesis

Cytochalasin B (CB) shows a marked concentration-dependent inhibition of the incorporation of [3H]thymidine into Chinese hamster ovary cells. This inhibition was shown to result from an inhibition of thymidine uptake, not from an inhibition of DNA synthesis. Cells normally acquire the capacity to transport thymidine as they move from the G1 stage of the cell cycle into the S phase. If CB is added to cells while they are in G1, they do not acquire the ability to transport thymidine as they enter S. However, the addition of CB to cells that are already in S has no effect on their ability to transport thymidine. These results are discussed in terms of a model in which elements involved in thymidine transport enter the cell surface membrane as the cells move from G1 to S. It is proposed that CB prevents this structural transition by binding to the cell surface.

scholarly journals Rapid cell surface appearance of endocytic membrane proteins in Chinese hamster ovary cells.

1981 ◽  
Vol 1 (3) ◽  
pp. 261-268 ◽  
Author(s):  
B Storrie ◽  
T D Dreesen ◽  
K M Maurey
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Gel Electrophoresis ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Appearance ◽  
Ovary Cells ◽  
One Dimensional

Lactoperoxidase was used to selectively radiolabel endocytic membrane. CHO cells were incubated with enzyme at 37 degrees C for 10 min to permit lactoperoxidase internalization. Radioiodination was done at 4 degrees C. About 90% of the radioiodinated products pelleted at 100,000 X g. From 12 to 15 different electrophoretic species were detected by one-dimensional gel electrophoresis. When cells labeled by internalized lactoperoxidase were warmed to 37 degrees C, the incorporated radioactivity was lost in a biphasic manner with an overall t1/2 of approximately 20 h. Upon warming cells to 37 degrees C, the labeled species became sensitive to pronase or trypsin digestion. The increase in protease sensitivity was progressive over a 10- to 20-min period. Maximally 45% of the initially intracellular radiolabel could be released. A digest of exterior-radioiodinated cells released 50% of the incorporated radioiodine. These observations strongly suggest a rapid shuttling of approximately 90% of the radioiodinated membrane species initially present within the cell to the cell surface.

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