Abstract
A Vps10p domain makes up the entire luminal part of Sortilin, and this type of domain is the hallmark of a new family of neuronal receptors that target a variety of ligands, including neurotrophins and neuropeptides. We have shown that two structural features of the Vps10p domain, the N-terminal propeptide and the C-terminal segment of ten conserved cysteines (10CC), are key elements in the function of Sortilin. The propeptide has two functions. (i) It binds the mature part of Sortilin and prevents ligands in the biosynthetic pathway from binding to the uncleaved proreceptor, and (ii) it facilitates receptor transport in early Golgi compartments by a mechanism that does not depend on its ability to prevent ligand binding. In contrast, other Vps10p domain receptors, such as SorLA and SorCS3, do not need their propeptide for normal and swift processing. The 10CC segment constitutes an exchangeable module containing five conserved disulfide bridges, and using module-shuffling and truncations, we have shown that the 10CC segment is a major ligand-binding region in Sortilin.
Highlights
A vacuolar protein-sorting 10 protein (Vps10p) domain makes up the entire luminal part of Sortilin, and this type of domain is the hallmark of a new family of neuronal receptors that target a variety of ligands, including neurotrophins and neuropeptides
The segment Trp17-Pro37 showed a high affinity for mature Sortilin, and the heptapeptide Gly22-Arg28 was found to inhibit binding of the wt propeptide by about 70% (Fig. 1B)
In mammals, corresponding domains are only found in the five members of the Vps10p-D receptor family that are widely expressed in neuronal tissues [25,26,27]
Summary
Vps10p-D, Vps10p domain; Vps10pSorLA, the Vps10p-D of SorLA; 10CC, ten conserved cysteines; BFA, brefeldin A; BSS, balanced salt solution; CHO, Chinese hamster ovary; Endo-H, endoglycosidase H; GDNF, glia-derived neurotrophic factor; GST, glutathione S-transferase; NGF, nerve growth factor; NGFpro, prodomain of NGF; RAP, receptor-associated protein; s-, indicates soluble minireceptors consisting of the luminal part of given receptors (e.g. s-Sortilin); wp, without propeptide; wt, wild-type; HPLC, high pressure liquid chromatography; ER, endoplasmic reticulum; SPR, surface plasmon resonance. Propeptide cleavage conditions both Sortilin and SorLA for ligand binding, and their propeptides bind the mature receptors with high affinity and inhibit binding of all currently known Vps10p-D ligands These findings imply that the propeptides might be types of “intrinsic” chaperones that assist in protein folding or serve to prevent the newly synthesized Vps10p-D receptors from premature interaction with ligands in the biosynthetic pathway. All ligands compete for binding, and the propeptides of Sortilin and SorLA both inhibit binding of any known ligand to either of the two receptors [7, 20] This implies that the binding sites within the Vps10p-D(s) are closely situated and located in a region with a relatively high degree of structural conservation. By analysis of soluble minireceptors in transfected cells, we demonstrated that Sortilin, in contrast to SorLA and SorCS3, depends on its propeptide to expedite transport in the biosynthetic pathway
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