scholarly journals Recombinant human endothelin-converting enzyme ECE-1b is located in an intracellular compartment when expressed in polarized Madin–Darby canine kidney cells

1998 ◽  
Vol 333 (2) ◽  
pp. 439-448 ◽  
Author(s):  
Arezou AZARANI ◽  
Guy BOILEAU ◽  
Philippe CRINE
Keyword(s):  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Mdck Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Converting Enzyme ◽  
Madin Darby Canine Kidney ◽  
Endothelin Converting Enzyme ◽  
Darby Canine Kidney ◽  
Canine Kidney

Endothelin-converting enzyme (ECE) is a phosphoramidon-sensitive membrane-bound metalloprotease responsible for the conversion of big-endothelins into endothelins [Yanagisawa, Kurihara, Kimura, Tomobe, Kobayashi, Mitsui, Yazaki, Goto and Masaki (1988) Nature (London) 332, 411–415]. Several distinct isoforms of ECE have been cloned and identified. ECE-1a, b and c have the same ectodomain and differ only by their cytosolic tails [Schweizer, Valdenaire, Nelböck, Deuschle, Edwards, Stumpf and Löffler (1997) Biochem. J. 328, 871–877]. The ectodomain common to ECE-1 a, b and c shares extensive sequence similarities with neprilysin, a major kidney brush border metallopeptidase. To study the sorting of ECE in polarized cells, ECE-1b cDNA was expressed by transfection in polarized Madin–Darby canine kidney (MDCK) cells. Cell-surface biotinylation and immunofluorescence studies showed that ECE-1b is not expressed on the cell-surface but was rather located in intracellular compartments that could also be labelled with anti-Rab-5 and Rab-7 antibodies and was thus tentatively identified as early and late endosomes. Similar results were also obtained when ECE-1b was expressed in non-polarized Chinese hamster ovary cells for comparison purposes. When MDCK or Chinese hamster ovary transfected cells were pre-treated with the ECE inhibitor phosphoramidon, a 3-fold increase in the level of ECE-1b was observed both by Western blotting and by enzymic activity. However, no change in the level of neprilysin or the β-chain of meprin, two apical membrane metallopeptidases, was observed in MDCK cells transfected under similar conditions. Northern blotting showed that the increase in the level of ECE-1b was not owing to changes in the ECE mRNA transcription rate or stability. Rather, pulse-chase experiments followed by immunoprecipitation showed a decrease in the rate of degradation of ECE-1b in phosphoramidon-treated cells. Half-lives were determined to be 2.8 and 7.5 h for non-treated and phosphoramidon-treated cells, respectively. Confocal microscopy showed accumulation of ECE-1b immunoreactive material in the lysosomes of phosphoramidon-treated cells. Taken together, these results suggest that ECE-1b turns over very rapidly between endosomal and lysosomal compartments and that lysosomal degradation of the enzyme is slowed down by phosphoramidon.

scholarly journals The N-terminal segment of endothelin-converting enzyme (ECE)-1b contains a di-leucine motif that can redirect neprilysin to an intracellular compartment in Madin–Darby canine kidney (MDCK) cells

1999 ◽  
Vol 341 (1) ◽  
pp. 119-126 ◽  
Author(s):  
Françoise CAILLER ◽  
Jacques P. ZAPPULLA ◽  
Guy BOILEAU ◽  
Philippe CRINE
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Converting Enzyme ◽  
Madin Darby Canine Kidney ◽  
Intracellular Compartment ◽  
Endothelin Converting Enzyme ◽  
Darby Canine Kidney ◽  
Canine Kidney

Endothelin-converting enzyme (ECE)-1 is a membrane-bound metallopeptidase of the neprilysin (NEP) family. ECE-1 is responsible for the conversion of inactive big-endothelins into active endothelins. Three different isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) have been identified. They differ in their N-terminal cytosolic regions, have distinct tissue distribution and intracellular localization. ECE-1a and ECE-1c are both located at the cell surface whereas ECE-1b is targeted to an intracellular compartment. To better understand the nature of the signal responsible for the targeting of ECE-1b to the intracellular compartment, we have constructed several ECE/NEP chimaeric proteins and expressed them by transfection into Madin-Darby canine kidney (MDCK) cells. This allowed us to identify a nine amino acid segment in the cytosolic tail of ECE-1b that is sufficient to relocate NEP from the cell surface to an intracellular compartment. Site-directed mutagenesis on these chimaeras led to the identification of two leucine residues as part of the intracellular retention signal.

Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin–Darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor

2002 ◽  
Vol 361 (3) ◽  
pp. 653-661
Author(s):  
Abdelkarim ABOUSALHAM ◽  
Tom C. HOBMAN ◽  
Jay DEWALD ◽  
Michael GARBUTT ◽  
David N. BRINDLEY
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mdck Cells ◽  
Chinese Hamster ◽  
Coated Vesicle ◽  
Vesicle Formation ◽  
Madin Darby Canine Kidney ◽  
Adp Ribosylation Factor ◽  
Darby Canine Kidney ◽  
Canine Kidney

Differential effects of acetyl(C2-) ceramide (N-acetylsphingosine) were studied on coated vesicle formation from Golgi-enriched membranes of Chinese hamster ovary (CHO) and Madin—Darby canine kidney (MDCK) cells. C2-ceramide blocked the translocation of ADP-ribosylation factor-1 (ARF-1) and protein kinase C-α (PKC-α) to the membranes from CHO cells, but not those of MDCK cells. Consequently, C2-ceramide blocked the stimulation of phospholipase D1 (PLD1) by the cytosol and guanosine 5′-[γ-thio]triphosphate (GTP[S]) in membranes from CHO cells. Basal specific activity of PLD1 and the concentration of ARF-1 were 3–4 times higher in Golgi-enriched membranes from MDCK cells compared with CHO cells. Moreover, PLD1 activity in MDCK cells was stimulated less by cytosol and GTP[S]. PLD2 was not detectable in the Golgi-enriched membranes. Incubation of intact CHO cells or their Golgi-enriched membranes with C2-ceramide also inhibited COP1 vesicle formation by membranes from CHO, but not MDCK, cells. Specificity was demonstrated, since dihydro-C2-ceramide had no significant effect on ARF-1 translocation, PLD1 activation or vesicle formation in membranes from both cell types. C2-ceramide also decreased the secretion of virus-like particles to a greater extent in CHO compared with MDCK cells, whereas dihydro-C2-ceramide had no significant effect. The results demonstrate a biological effect of C2-ceramide in CHO cells by decreasing ARF-1 and PKC-α binding to Golgi-enriched membranes, thereby preventing COP1 vesicle formation.

scholarly journals Expression of Podocalyxin Inhibits Cell–Cell Adhesion and Modifies Junctional Properties in Madin-Darby Canine Kidney Cells

2000 ◽  
Vol 11 (9) ◽  
pp. 3219-3232 ◽  
Author(s):  
Tetsuro Takeda ◽  
William Y. Go ◽  
Robert A. Orlando ◽  
Marilyn Gist Farquhar
Keyword(s):  
Cell Adhesion ◽  
Cho Cells ◽  
Direct Evidence ◽  
Mdck Cells ◽  
Chinese Hamster ◽  
Cell Aggregation ◽  
Madin Darby Canine Kidney ◽  
Adhesion Properties ◽  
Darby Canine Kidney ◽  
Canine Kidney

Podocalyxin is a major membrane protein of the glomerular epithelium and is thought to be involved in maintenance of the architecture of the foot processes and filtration slits characteristic of this unique epithelium by virtue of its high negative charge. However, until now there has been no direct evidence for podocalyxin's function. Podocalyxin is a type 1 transmembrane sialoprotein with an N-terminal mucin-like domain. To assess its function, we cloned rat podocalyxin and examined the effects of its expression on the cell adhesion properties of stably transfected Chinese hamster ovary (CHO)-K1 and Madin-Darby canine kidney (MDCK) cells and inducible ecdysone receptor–expressing (EcR)-CHO cells. In a cell aggregation assay, CHO-K1 cells expressing high levels of podocalyxin showed complete inhibition of cell aggregation, and MDCK transfectants showed greatly reduced aggregation (∼60–80%) compared with parental cells. In EcR-CHO cells, the expression level of podocalyxin induced by increasing levels of ecdysone analogue correlated closely with the antiadhesion effect. The inhibitory effect of podocalyxin was reversed by treatment of the cells with Arthrobacter ureafacienssialidase, indicating that sialic acid is required for inhibition of cell adhesion. Overexpression of podocalyxin also affected transepithelial resistance and the distribution of junctional proteins in MDCK cells by an unknown mechanism that may involve interaction with the actin cytoskeleton. These results provide direct evidence that podocalyxin functions as an antiadhesin that maintains an open filtration pathway between neighboring foot processes in the glomerular epithelium by charge repulsion.

scholarly journals Microtubule-acting drugs lead to the nonpolarized delivery of the influenza hemagglutinin to the cell surface of polarized Madin-Darby canine kidney cells.

1987 ◽  
Vol 104 (2) ◽  
pp. 231-241 ◽  
Author(s):  
M J Rindler ◽  
I E Ivanov ◽  
D D Sabatini
Keyword(s):  
Golgi Apparatus ◽  
Cell Surface ◽  
G Protein ◽  
Mdck Cells ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Vesicular Stomatitis ◽  
Ha Protein ◽  
Darby Canine Kidney ◽  
Canine Kidney

The synchronized directed transfer of the envelope glycoproteins of the influenza and vesicular stomatitis viruses from the Golgi apparatus to the apical and basolateral surfaces, respectively, of polarized Madin-Darby canine kidney (MDCK) cells can be achieved using temperature-sensitive mutant viruses and appropriate temperature shift protocols (Rindler, M. J., I. E. Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100:136-151). The microtubule-depolymerizing agents colchicine and nocodazole, as well as the microtubule assembly-promoting drug taxol, were found to interfere with the normal polarized delivery and exclusive segregation of hemagglutinin (HA) to the apical surface but not with the delivery and initial accumulation of G on the basolateral surface. Immunofluorescence analysis of permeabilized monolayers of influenza-infected MDCK cells treated with the microtubule-acting drugs demonstrated the presence of substantial amounts of HA protein on both the apical and basolateral surfaces. Moreover, in cells infected with the wild-type influenza virus, particles budded from both surfaces. Viral counts in electron micrographs showed that approximately 40% of the released viral particles accumulated in the intercellular spaces or were trapped between the cell and monolayer and the collagen support as compared to less than 1% on the basolateral surface of untreated infected cells. The effect of the microtubule inhibitors was not a result of a rapid redistribution of glycoprotein molecules initially delivered to the apical surface since a redistribution was not observed when the inhibitors were added to the cells after the HA was permitted to reach the apical surface at the permissive temperature and the synthesis of new HA was inhibited with cycloheximide. The altered segregation of the HA protein that occurs may result from the dispersal of the Golgi apparatus induced by the inhibitors or from the disruption of putative microtubules containing tracks that could direct vesicles from the trans Golgi apparatus to the cell surface. Since the vesicular stomatitis virus G protein is basolaterally segregated even when the Golgi elements are dispersed and hypothetical tracks disrupted, it appears that the two viral envelope glycoproteins are segregated by fundamentally different mechanisms and that the apical surface may be incapable of accepting vesicles carrying the G protein.

scholarly journals Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery.

1989 ◽  
Vol 109 (5) ◽  
pp. 2117-2127 ◽  
Author(s):  
M P Lisanti ◽  
A Le Bivic ◽  
M Sargiacomo ◽  
E Rodriguez-Boulan
Keyword(s):  
Steady State ◽  
Epithelial Cell ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
Steady State Distribution ◽  
State Distribution ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

We used domain-selective biotinylation/125I-streptavidin blotting (Sargiacomo, M., M. P. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. 1989 J. Membr. Biol. 107:277-286), in combination with lectin precipitation, to analyze the apical and basolateral glycoprotein composition of Madin-Darby canine kidney (MDCK) cells and to explore the role of glycosylation in the targeting of membrane glycoproteins. All six lectins used recognized both apical and basolateral glycoproteins, indicating that none of the sugar moieties detected were characteristic of the particular epithelial cell surface. Pulse-chase experiments coupled with domain-selective glycoprotein recovery were designed to detect the initial appearance of newly synthesized glycoproteins at the apical or basolateral cell surface. After a short pulse with a radioactive precursor, glycoproteins reaching each surface were biotinylated, extracted, and recovered via precipitation with immobilized streptavidin. Several basolateral glycoproteins (including two sulfated proteins) and at least two apical glycoproteins (one of them the major sulfated protein of MDCK cells) appeared at the corresponding surface after 20-40 min of chase, but were not detected in the opposite surface, suggesting that they were sorted intracellularly and vectorially delivered to their target membrane. Several "peripheral" apical proteins were detected at maximal levels on the apical surface immediately after the 15-min pulse, suggesting a very fast intracellular transit. Finally, domain-selective labeling of surface carbohydrates with biotin hydrazide (after periodate oxidation) revealed strikingly different integral and peripheral glycoprotein patterns, resembling the Con A pattern, after labeling with sulfo-N-hydroxy-succinimido-biotin. The approaches described here should be useful in characterizing the steady-state distribution and biogenesis of endogenous cell surface components in a variety of epithelial cell lines.

scholarly journals Identification of a putative tyrosine-O-sulphate (TyrS) receptor possibly functioning in the biosynthetic transport of tyrosine-sulphated proteins in Madin-Darby canine kidney cells

1993 ◽  
Vol 294 (2) ◽  
pp. 407-417 ◽  
Author(s):  
J Liu ◽  
J R Han ◽  
C C Liu ◽  
M Suiko ◽  
M C Liu
Keyword(s):  
Cell Surface ◽  
Time Course ◽  
Mdck Cells ◽  
Binding Protein ◽  
Normal Growth ◽  
Madin Darby Canine Kidney ◽  
Intracellular Location ◽  
Cell Lysate ◽  
Darby Canine Kidney ◽  
Canine Kidney

By employing an affinity-gel fractionation technique coupled to Western-blot analysis, we have identified a 175 kDa tyrosine-O-sulphate (TyrS)-binding protein present in Madin-Darby canine kidney (MDCK) cells. The binding of this TyrS-binding protein to TyrS covalently bonded to Sepharose gel was found to be pH-dependent, being strong from pH 8.0 down to pH 6.5 and increasingly weak at pH 6.0 and below. Results obtained from Triton X-114 temperature-induced phase separation and sodium carbonate buffer (pH 11) extraction experiments indicated that the TyrS-binding protein is an integral membrane protein. This 175 kDa TyrS-binding protein was found to be present in association with a major tyrosine-sulphated protein, the apically secreted 80 kDa glycoprotein (gp 80), in cell lysate prepared from MDCK cells maintained under normal growth conditions. When the cell lysate used was prepared from MDCK cells pretreated with 20 mM sodium chlorate, a metabolic sulphation inhibitor, the complex formed between the two proteins could no longer be detected, indicating that the binding of the TyrS-binding protein is through the TyrS residue(s) of gp 80. Both cell-surface biotinylation and cell-surface trypsinization studies demonstrated the predominantly, if not exclusively, intracellular location of the TyrS-binding protein. Furthermore, radioactive pulse-chase experiments revealed that the newly synthesized radiolabelled fibronectin and gp 80 were present in complexes with the TyrS-binding protein in MDCK cells pulse-labelled with [35S]methionine or [35S]sulphate. Exogenous [35S]methionine-labelled gp 80 added to the medium, on the other hand, was not found to be present in association with the TyrS-binding protein in MDCK cells over a 2-h time course. These results strongly suggested the identity of the 175 kDa TyrS-binding protein as a putative ‘TyrS receptor’, possibly functioning in the biosynthetic transport of tyrosine-sulphated proteins in MDCK cells.

scholarly journals Transcellular Transport of Leptin by the Short Leptin Receptor Isoform ObRa in Madin-Darby Canine Kidney Cells*

Endocrinology ◽  
2000 ◽  
Vol 141 (6) ◽  
pp. 1955-1961 ◽  
Author(s):  
Stanley M. Hileman ◽  
Jens Tornøe ◽  
Jeffrey S. Flier ◽  
Christian Bjørbæk
Keyword(s):  
Cell Surface ◽  
Leptin Receptor ◽  
Mdck Cells ◽  
Short Form ◽  
Madin Darby Canine Kidney ◽  
Apical Cell Membrane ◽  
Transcellular Transport ◽  
The Brain ◽  
Darby Canine Kidney ◽  
Canine Kidney

Abstract Leptin is an adipocyte-derived hormone that acts in specific regions of the brain to regulate body weight and neuroendocrine function. The mechanism by which leptin enters the brain is unknown. We previously reported that rat brain microvessels, which constitute the blood-brain barrier, contain large amounts of messenger RNA encoding a short form of the leptin receptor (ObRa), suggesting that this site may be important for receptor-mediated transport of leptin into the brain. The purpose of this study was to determine whether ObRa is capable of transcellular transport of intact leptin. A transwell system in which Madin-Darby Canine Kidney (MDCK) cells stably expressing ObRa are grown in a monolayer was used to determine receptor distribution on apical or basolateral cell surfaces and the capacity for directional transport of 125I-leptin. Binding of 125I-leptin was greater on the apical vs. the basolateral cell surface and transport of 125I-leptin occurred only in the apical to basolateral direction. 11% of transported radioactivity appearing in the basolateral chamber represented intact leptin as assessed by TCA precipitation analysis and by SDS-PAGE. Parental MDCK cells did not express leptin receptors and did not bind or transport 125I-leptin. Epidermal growth factor (EGF) binding and transport via endogenous EGF receptors in MDCK cells also was assessed. In contrast to leptin, specific binding of 125I-EGF occurred primarily on the basolateral cell surface and transport of 125I-EGF occurred predominantly in the basolateral to apical direction. These data show that ObRa is preferentially targeted to the apical cell membrane in MDCK cells and that leptin transport occurs, albeit at a low rate, in a unidirectional manner in the apical to basolateral direction. These findings may be relevant to the putative role of ObRa in receptor-mediated transport of leptin from the circulation into the brain.

scholarly journals Serine phosphorylation site of the 46-kDa mannose 6-phosphate receptor is required for transport to the plasma membrane in Madin-Darby canine kidney and mouse fibroblast cells.

1997 ◽  
Vol 8 (4) ◽  
pp. 567-576 ◽  
Author(s):  
P Breuer ◽  
C Körner ◽  
C Böker ◽  
A Herzog ◽  
R Pohlmann ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Mdck Cells ◽  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Mannose 6 Phosphate ◽  
Darby Canine Kidney ◽  
Canine Kidney

Up to 4% of the human 46-kDa mannose 6-phosphate receptor (MPR46) expressed in Madin-Darby canine kidney (MDCK) cells are localized at the cell surface. At steady state, the expression of MPR46 on the apical surface of filter-grown MDCK cells is about sixfold lower than on the basolateral surface. The cytoplasmic domain of the MPR46 is phosphorylated on serine 56 at low stoichiometry. By expressing mutant MPR46 we have shown that the MPR46 phosphorylation site is required for delivery to the plasma membrane. In addition, mutant MPR46 expressed in MPR-deficient mouse embryonic fibroblasts were not detected at the cell surface and their ability to sort newly synthesized cathepsin D was not altered. Since the loss of MPR46 phosphorylation correlates with the lack of cell surface expression, phosphorylation of serine 56 may either function as a direct plasma membrane targeting signal or inhibit MPR46 recycling from endosomes to Golgi, resulting in trafficking to the cell surface.

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