Selective and Signal-dependent Recruitment of Membrane Proteins to Secretory Granules Formed by Heterologously Expressed von Willebrand Factor

Abstract

von Willebrand factor (vWF) is a large, multimeric protein secreted by endothelial cells and involved in hemostasis. When expressed in AtT-20 cells, vWF leads to the de novo formation of cigar-shaped organelles similar in appearance to the Weibel-Palade bodies of endothelial cells in which vWF is normally stored before regulated secretion. The membranes of this vWF-induced organelle, termed the pseudogranule, are uncharacterized. We have examined the ability of these pseudogranules, which we show are secretagogue responsive, to recruit membrane proteins. Coexpression experiments show that the Weibel-Palade body proteins P-selectin and CD63, as well as the secretory organelle membrane proteins vesicle-associated membrane protein-2 and synaptotagmin I are diverted away from the endogenous adrenocorticotropic hormone-containing secretory granules to the vWF-containing pseudogranules. However, transferrin receptor, lysosomal-associated membrane protein 1, and sialyl transferase are not recruited. The recruitment of P-selectin is dependent on a tyrosine-based motif within its cytoplasmic domain. Our data show that vWF pseudogranules specifically recruit a subset of membrane proteins, and that in a process explicitly driven by the pseudogranule content (i.e., vWF), the active recruitment of at least one component of the pseudogranule membrane (i.e., P-selectin) is dependent on residues of P-selectin that are cytosolic and therefore unable to directly interact with vWF.

Proteomic analysis of secreted membrane vesicles of archaeal Sulfolobus species reveals the presence of endosome sorting complex components

“…vWA domains were originally found in the blood coagulation protein von Willebrand factor where it functions in the formation of protein aggregates (Whittaker and Hynes 2002). In eukaryotes von Willebrand factor is stored in secretory vesicular compartments, so-called Weibel-Palade bodies, where it also recruits other proteins to be secreted (Bierings et al 2007;Blagoveshchenskaya et al 2002).…”

High-pressure freezing provides insights into Weibel-Palade body biogenesis

“…For immunofluorescence studies, antibody staining and confocal microscopy were carried out as previously described (Blagoveshchenskaya et al, 2002). Sheep polyclonal anti-human VWF (Serotec) was used in conjunction with secondary antibodies coupled to fluorophores (Jackson).…”

Differential Kinetics of Cell Surface Loss of von Willebrand Factor and Its Propolypeptide after Secretion from Weibel-Palade Bodies in Living Human Endothelial Cells

“…Nucleofected HUVECs grown on gelatin-coated glass coverslips were processed for immunofluorescence as described previously (21).…”