Abstract
Decision making in cellular ensembles requires the dynamic release of signaling molecules from the producing cells into the extracellular compartment. One important example of molecules that require regulated release in order to signal over several cell diameters is the Hedgehog (Hh) family, because all Hhs are synthesized as dual-lipidated proteins that firmly tether to the outer membrane leaflet of the cell that produces them. Factors for the release of the vertebrate Hh family member Sonic Hedgehog (Shh) include cell-surface sheddases that remove the lipidated terminal peptides, as well as the soluble glycoprotein Scube2 that cell-nonautonomously enhances this process. This raises the question of how soluble Scube2 is recruited to cell-bound Shh substrates to regulate their turnover. We hypothesized that heparan sulfate (HS) proteoglycans (HSPGs) on the producing cell surface may play this role. In this work, we confirm that HSPGs enrich Scube2 at the surface of Shh-producing cells and that Scube2-regulated proteolytic Shh processing and release depends on specific HS. This finding indicates that HSPGs act as cell-surface assembly and storage platforms for Shh substrates and for protein factors required for their release, making HSPGs critical decision makers for Scube2-dependent Shh signaling from the surface of producing cells.
Highlights
Gpcs can directly control spatiotemporal Hh release from producing cells in an HS-dependent manner in vitro and in vivo[26], supporting the idea that HSPGs act as assembly and storage sites for Hh ligands, but can recruit factors required for their regulated release[27,28]
We wondered whether the soluble glycoprotein Scube2 [signal peptide, CUB domain, epidermal growth factor (EGF)-like protein 2] is one release factor that is attracted to the Sonic hedgehog (Shh) source cluster in such a way
By combining biochemistry, confocal heteroprotein imaging and genetics, we demonstrate that Scube[2] recruitment and activity require specific HSPG expression at the surface of Shh source cells and that clustered basic amino acids located in the Scube[2] spacer domain associate the molecule with heparin and HS in vitro
Summary
By combining biochemistry, confocal heteroprotein imaging and genetics, we demonstrate that Scube[2] recruitment and activity require specific HSPG expression at the surface of Shh source cells and that clustered basic amino acids located in the Scube[2] spacer domain associate the molecule with heparin and HS in vitro Consistent with this finding, heparin competition or HS degradation strongly impairs Scube2-dependent Shh release from the cell surface; the release of acidic lipidated proteins not associated with HS is Scube[2] independent. Hierarchical Shh/Scube2/sheddase association at the surface of morphogen-producing cells represents the first example of a signal-releasing extracellular signalosome, as defined by a multiprotein complex of signaling elements whose association and proteolytic activities are regulated by an HSPG scaffold This raises the exciting possibility that HSPG-dependent decision making ensures the specificity and speed of ectodomain release for the numerous other sheddase substrates expressed on one given cell
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