Direct targeting of neutral endopeptidase (EC 3.4.24.11) to the apical cell surface of transfected LLC-PK1 cells and unpolarized secretion of its soluble form.
LLC-PK1 cells were transfected with a cDNA encoding rabbit neutral endopeptidase (NEP; EC 3.4.24.11), an abundant enzyme of the kidney proximal brush border. Clones of cells expressing high levels of the protein were isolated. Selective biotinylation and radioimmunolabelling were used to determine that 85-95% of NEP was localized in the apical domain of filter-grown LLC-PK1 cells. Pulse-chase and selective biotinylation studies revealed that the majority (85%) of newly made NEP was directly targeted to the apical membrane. However, a soluble form of NEP was found to be secreted in approximately equal amounts from both sides of the monolayer when expressed in LLC-PK1 cells. Transfected pro-opiomelanocortin, a pituitary hormone precursor, was secreted almost exclusively into the basolateral medium, suggesting that the bulk flow is to the basolateral membrane. This behaviour contrasts with that observed in MDCK cells, where both the transmembrane and secreted forms of NEP are directly targeted to the apical membrane and where the secretion of pro-opiomelanocortin is unpolarized.
Lanctôt
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Département de Biochimie, Faculté de Médecine, Université de Montréal, Québec, Canada.
Fournier
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Howell
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Boileau
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Crine
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P
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D006168:Guinea Pigs; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D018374:LLC-PK1 Cells / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology*; D015260:Neprilysin / Q000235:genetics* / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D012995:Solubility; D013552:Swine; D014162:Transfection
D018076:DNA, Complementary; D007527:Isoenzymes; D011333:Pro-Opiomelanocortin; D018819:Dipeptidyl Peptidase 4; D015260:Neprilysin