Apical cell surface expression of rat dipeptidyl peptidase IV in transfected Madin-Darby canine kidney cells

The enterocytic differentiation of Caco-2 cells, a human colon adenocarcinoma cell line, is accompanied by the transcriptionally regulated expression of a subset of proteins and their correct sorting towards the cell surface. In the present work we have explored the possibility that post-translational events may interfere with this process by investigating the short term effects of a potent adenylyl cyclase activator, forskolin, on cell surface expression of dipeptidyl peptidase IV. Previous works have shown that this protein is targeted towards the apical domain through either a direct or an indirect route. Domain specific biochemical experiments demonstrate that cell surface expression of neosynthesized dipeptidyl peptidase IV rapidly decreases after a 1 hour forskolin treatment. Both initial basolateral and apical dipeptidyl peptidase IV membrane delivery were altered by forskolin treatment. Decrease of dipeptidyl peptidase IV cell surface expression was not restricted to this protein, since membrane expression of ‘525’ antigen, a basolateral protein and of sucrase-isomaltase, an apically targeted hydrolase, which unlike dipeptidyl peptidase IV mainly follows a direct route to the brush border membrane, also decreases. In addition endocytosis of proteins from the apical and from the basolateral domain was essentially unchanged, suggesting that forskolin's target may be located on the exocytic pathway. Confocal laser scanning microscopy and immuno-electron microscopy studies demonstrate that, within 5 minutes of forskolin treatment, the cell surface proteins studied accumulate in intracellular vesicles which were co-labeled with a polyclonal antibody raised against Lamp-1, a lysosomal membrane marker. Electron microscopy studies show that these vesicles display an autophagic-like morphology. Finally, biochemical experiments indicate that dibutyryl cAMP does not mimick the forskolin effect, thus suggesting that it is a cAMP-independent phenomenon.

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Iterative sorting of apical and basolateral cargo in Madin–Darby canine kidney cells

Molecular Biology of the Cell ◽

10.1091/mbc.e16-02-0096 ◽

2016 ◽

Vol 27 (14) ◽

pp. 2259-2271 ◽

Cited By ~ 2

Author(s):

Aleksandr Treyer ◽

Mario Pujato ◽

Ximo Pechuan ◽

Anne Müsch

Keyword(s):

Cell Surface ◽

Kidney Cells ◽

Madin Darby Canine Kidney ◽

Golgi Region ◽

Respective Surface ◽

Surface Domains ◽

Sorting Station ◽

Golgi Network ◽

Darby Canine Kidney ◽

Canine Kidney

For several decades, the trans-Golgi network (TGN) was considered the most distal stop and hence the ultimate protein-sorting station for distinct apical and basolateral transport carriers that reach their respective surface domains in the direct trafficking pathway. However, recent reports of apical and basolateral cargoes traversing post-Golgi compartments accessible to endocytic ligands before their arrival at the cell surface and the post-TGN breakup of large pleomorphic membrane fragments that exit the Golgi region toward the surface raised the possibility that compartments distal to the TGN mediate or contribute to biosynthetic sorting. Here we describe the development of a novel assay that quantitatively distinguishes different cargo pairs by their degree of colocalization at the TGN and by the evolution of colocalization during their TGN-to-surface transport. Keys to the high resolution of our approach are 1) conversion of perinuclear organelle clustering into a two-dimensional microsomal spread and 2) identification of TGN and post-TGN cargo without the need for a TGN marker that universally cosegregates with all cargo. Using our assay, we provide the first evidence that apical NTRp75 and basolateral VSVG in Madin–Darby canine kidney cells still undergo progressive sorting after they exit the TGN toward the cell surface.

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Molecular characterization of gp40, a mucin-type glycoprotein from the apical plasma membrane of Madin–Darby canine kidney cells (type I)

Biochemical Journal ◽

10.1042/bj3260099 ◽

1997 ◽

Vol 326 (1) ◽

pp. 99-108 ◽

Cited By ~ 32

Author(s):

Gert ZIMMER ◽

Friedrich LOTTSPEICH ◽

Andrea MAISNER ◽

Hans-Dieter KLENK ◽

Georg HERRLER

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Apical Plasma Membrane ◽

Type I ◽

Kidney Cells ◽

Madin Darby Canine Kidney ◽

A Cell ◽

Polarized Transport ◽

Darby Canine Kidney ◽

Canine Kidney

gp40 has been recently identified as a major apical cell-surface sialoglycoprotein of type-I Madin–Darby canine kidney cells, a cell line widely used for the study of polarized transport. The determination of two internal amino acid sequences of the purified glycoprotein by Edman degradation enabled us to isolate the cDNA encoding the 18.6 kDa protein backbone of gp40. Sequence analysis revealed that gp40 is a type-I membrane protein which has several characteristics in common with glycophorin A and other mucin-type glycoproteins. At least 14 serine/threonine residues were found to be used for O-glycosylation. No potential sites for N-glycosylation were detected. gp40 turned out to represent the canine homologue of a cell-surface antigen expressed by various epithelial and non-epithelial cells in rat and mouse. Potential O-glycosylation sites, transmembrane and cytoplasmic domains were found to be highly conserved in the three species. gp40 was detected in canine lung, intestine, kidney, brain and heart but not in liver and spleen. The subline II of Madin–Darby canine kidney cells was found not to express gp40. Stable expression of gp40 in transfected type-II cells revealed that gp40 is predominantly delivered to the apical plasma membrane. N-Glycans and a glycosylphosphatidylinositol anchor, both proposed apical targeting signals, are absent from gp40, indicating that other determinants are responsible for its polarized transport.

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