Structural insights into proteolytic activation of the human Dispatched1 transporter for Hedgehog morphogen release

Abstract

The membrane protein Dispatched (Disp), which belongs to the RND family of small molecule transporters, is essential for Hedgehog (Hh) signaling, by catalyzing the extracellular release of palmitate- and cholesterol-modified Hh ligands from producing cells. Disp function requires Furin-mediated proteolytic cleavage of its extracellular domain, but how this activates Disp remains obscure. Here, we employ cryo-electron microscopy to determine atomic structures of human Disp1 (hDisp1), before and after cleavage, and in complex with lipid-modified Sonic hedgehog (Shh) ligand. These structures, together with biochemical data, reveal that proteolytic cleavage opens the extracellular domain of hDisp1, removing steric hindrance to Shh binding. Structure-guided functional experiments demonstrate the role of hDisp1–Shh interactions in ligand release. Our results clarify the mechanisms of hDisp1 activation and Shh morphogen release, and highlight how a unique proteolytic cleavage event enabled acquisition of a protein substrate by a member of a family of small molecule transporters.

Two-way Dispatched function in Sonic hedgehog shedding and transfer to high-density lipoproteins

“…Our updated Hh shedding model thus unifies previously disparate models of Disp-, sheddase-, and LPP-mediated Shh solubilization into a comprehensive system. This comprehensive system is fully consistent with published reports on in vivo Disp functions 5, 6, 7, 8, 28 , in vivo Scube2 functions 29, 30, 31 , the in vitro role of the Scube2 CUB domain 23, 25 , the Disp structure 32, 33 , and the required N-terminal – but not C-terminal – shedding during in vivo Hh solubilization 34, 35, 36 . Our model is also consistent with the established importance of Hh C-cholesterol for Hh association into “large punctate” structures visible by light microscopy that may represent Hh-LPP complexes 37, 38, 39 , C-cholesterol-dependent Hh spreading 10, 40 , and previously established in vivo roles of LPPs in Hh biofunction 13, 14, 15 .…”

The Inseparable Relationship Between Cholesterol and Hedgehog Signaling

“…The transport activity of DISP is dependent on the ubiquitous TM sodium (Na + ) gradient across the PM, suggesting that it functions as a Na + -driven antiporter for cholesteroylated SHH release from cells (49). Finally, several cryo-EM structures of DISP confirm the structural similarities between DISP, PTCH, NPC1, and the broader RND family, including residues putatively involved in interactions with monovalent cations (50)(51)(52)(53). The structures provide insight into how DISP accommodates a protein substrate for transport (Figure 3).…”

A Residual N-Terminal Peptide Enhances Signaling of Depalmitoylated Hedgehog to the Patched Receptor

“…This result is consistent with the published finding that furin-mediated removal of eleven amino acids from the N-terminus of a mutated Shh, including amino acids that we determined to be critical for receptor activation, severely reduces Ptch binding and signaling [ 46 ]. The result also suggests that furin resistance of the wild-type N-terminal CW sequence, although rich in basic amino acids, is critical for protecting Shh during its secretion to the plasma membrane and during furin-mediated Disp activation at the cell surface [ 59 , 60 ] to maintain its biofunction.…”

“…It is well known that all vertebrate and invertebrate Disp family members contain a sterol sensing domain (SSD) that is conserved in proteins that bind, transport, or respond to cellular sterols, such as SREBP cleavage activating protein (SCAP) and NPC1 [ 62 , 63 , 64 ]. The SSD extends into a hydrophobic surface channel that has been proposed to function as an open “slide” for lipophiles [ 65 ] for subsequent transfer to an acceptor [ 59 ]. This acceptor is likely to be a serum lipoprotein, as supported by the published concept that the fly lipoprotein called lipophorin carries cholesteroylated Hh in vivo [ 58 , 61 , 66 ] and a previous report showing that C-cholesterol is necessary and sufficient for Disp-mediated protein export [ 30 ].…”