Polarity of constitutive and regulated von Willebrand factor secretion by transfected MDCK-II cells.
Von Willebrand factor (vWF), synthesized by endothelial cells, is both rapidly secreted by the constitutive pathway and stored in Weibel-Palade bodies. Secretion from these organelles occurs upon activation of the protein kinase C signal transduction pathway and yields highly multimerized vWF. Highly multimerized vWF acts as a more effective adhesive ligand than the lower molecular weight forms that are constitutively secreted. We employed the extensively characterized polar Madin-Darby Canine Kidney II (MDCK-II) epithelial cell line, stably transfected with full-length vWF cDNA or deletion mutants thereof, to gain insight in the polarity of vWF secretion by either one of the two pathways. Immunofluorescence analysis and metabolic labeling experiments revealed that multimeric "wild-type" vWF is stored in MDCK-II cells and released upon stimulation with phorbol esters. Furthermore, we show that 62.0 +/- 3.8% of constitutively secreted and 83.2 +/- 6.6% of the regulated secreted wild-type vWF is encountered at the apical side of the cell. The polarity of the constitutive secretion of deletion mutant vWFdelD'D3 is similar to that of constitutively secreted wild-type vWF, whereas deletion mutant vWFdelD1D2 displays no polar secretion (50.1 +/- 5.7% apical).
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Department of Biochemistry, University of Amsterdam, Academic Medical Center, The Netherlands.
Fontijn
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van Mourik
J A
JA
Pannekoek
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D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D018076:DNA, Complementary; D004285:Dogs; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014841:von Willebrand Factor / Q000096:biosynthesis* / Q000235:genetics
D018076:DNA, Complementary; D014841:von Willebrand Factor; D013755:Tetradecanoylphorbol Acetate