Role of Sortlin in regulating proteolytic activation of BMP4 during embryogenesis

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DESCRIPTION (provided by applicant): The long term goal of this project is to investigate the role of Sortilin as a regulator of bone morphogenetic proteins (BMPs), and how disruptions in Sortilin-dependent trafficking may contribute to a spectrum of birth defects that arise when BMP signaling is misregulated. Sortilin is one of a family of intracellular receptors that are structurally related to yeast vacuolar protein sorting 10 protein. This family of proteins directs lysosomal sorting but also has been shown to modulate the trafficking of several nonlysosomal proteins in the biosynthetic pathway. BMP4 is a signaling molecule that plays critical roles in lineage selection and differentiation of almost all cell types during embryogenesis. BMP4 is generated as a latent precursor, which is cleaved by furin to generate an active ligand. Indirect evidence suggests that proteolytic activation of BMP4 is temporally restricted during development. The investigators' preliminary data show that Sortilin interacts with BMP4 via sequences within its prodomain, and that co-expression of Sortilin with proBMP4 prevents cleavage of this precursor. Conversely, inhibiting the expression of endogenous Sortilin in Xenopus embryos leads to patterning defects that resemble those that arise when BMP4 signaling is perturbed. Their data support the hypothesis that Sortilin prevents premature activation of the BMP pathway during embryogenesis by binding to and inhibiting cleavage of proBMP4. There are two key questions that the investigators will ask to address this hypothesis and to set the stage for longer term studies: 1) Are Sortilin and BMP4 co-localized in intracellular compartment(s) distinct from those that contain furin? The investigators will compare subcellular localization of BMP4, Sortilin, and furin in transfected Hela cells by immunostaining and subcellular fractionation. They will also ask whether BMP4 can be cleaved in the presence of a chimeric protein consisting of the luminal domain of Sortilin and the cytoplasmic tail of furin, which contains all of the information necessary for subcellular routing of furin. 2) Is BMP signaling disrupted in embryos in which Sortilin expression is misregulated? Xenopus embryos will be injected with Sortilin RNA or antisense morpholino oligonucleotides to up- or down-regulate its expression, respectively. The investigators will then determine whether altering Sortilin levels results in complementary changes in BMP4 processing, phosphorylation of the downstream signaling component, Smad1 and BMP dependent embryonic patterning events.