Assembly of multimeric von Willebrand factor directs sorting of P-selectin.
We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)-like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis and storage organelles, was transfected with full-length vWF cDNA or a deletion mutant thereof. Stable transfectants of T24 with full-length vWF cDNA revealed the generation of WPB-like organelles as demonstrated by colocalization of vWF and P-selectin with double-labeling immunofluorescence. In contrast, T24 cells transfected with vWF delD'D3 cDNA, encoding a mutant that is unable to form vWF multimers, displayed only perinuclear vWF staining, whereas no indication was found for the presence of WPB-like organelles. The contents of the organelles in full-length vWF cDNA-transfected T24 cells were released on activation of the protein kinase C pathway, similar to the situation with genuine endothelial cells. The expression of vWF did not affect the biosynthesis of P-selectin, as deduced from the observation that untransfected and vWF cDNA-transfected T24 cells contained the same amount of P-selectin mRNA. We propose that the biosynthesis of multimeric vWF directs the generation of WPB-like organelles, as evidenced by the sequestering and anchoring of P-selectin into these storage granules.
Hop
C
C
Department of Biochemistry, Academic Medical Center, University of Amsterdam, Netherlands.
Guilliatt
A
A
Daly
M
M
de Leeuw
H P
HP
Brinkman
H J
HJ
Peake
I R
IR
van Mourik
J A
JA
Pannekoek
H
H
D001692:Biological Transport / Q000502:physiology; D018076:DNA, Complementary; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D019007:P-Selectin / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms; D020302:Weibel-Palade Bodies / Q000378:metabolism*; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D019007:P-Selectin; D012333:RNA, Messenger; D014841:von Willebrand Factor; D011493:Protein Kinase C