Abstract
Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP’s essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP’s ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.
Highlights
The prion protein is a cell surface glycoprotein expressed in many cell types, in the nervous system
When expressed on the surface of non-adhesive Drosophila S2 cells, vertebrate PrPs establish homophilic trans-interactions, thereby triggering the formation of weak cell contacts and subsequently accumulating at these sites [34]. We used this experimental paradigm to analyze how PrP protein domains and posttranslational modifications contribute to its role in cell contact formation
Aside from confirming the essential role of the GPI-anchor, these data indicate that the repetitive and globular domains are essential for PrP accumulation at newly formed cell-cell contacts
Summary
The prion protein is a cell surface glycoprotein expressed in many cell types, in the nervous system. The physiological role of PrP and its connection to prion neurotoxicity remain open questions. Further studies suggest that the mechanistic basis of these functions is the ability of PrP to modulate intracellular signaling [7,8,9,10,11,12]. Fish and mammalian PrPs share a common protein domain organization (Fig. 1) [13,14]: A flexible N-terminal half (repetitive domain) and a well-structured C-terminal half (globular domain) connected by a short and highly conserved stretch (hydrophobic region). The immature polypeptide undergoes the cleavage of an N-terminal signal peptide and becomes tethered to the plasma membrane via the addition of a C-terminal glycosylphosphatidylinositol (GPI) anchor. Formation of one disulfide bond and attachment of two N-linked oligosaccharide chains take place
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