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At least 19 recordsLinked to original sources

PubMed2004

Study of the mouse sortilin gene: Effects of its transient silencing by RNA interference in TM4 Sertoli cells.

Using databases of the mouse genome in combination with a sequence deduced from a mouse sortilin cDNA originated in our laboratory, we found the sortilin gene to map to a region of chromosome 3. The mouse sortilin gene contains 19 short exons separated by introns of various sizes. The study elucidated the exon-intron boundaries. Some introns extend over more than 24 kb. In the cytoplasmic domain of the translation product, we found a dileucine motif and three other motifs known to constitute the active sorting signal of the mannose 6-phosphate receptor (M6P-R). We also tested the hypothesis that sortilin is involved in the sorting of prosaposin (SGP-1) to the lysosomes. Prosaposin was initially identified in Sertoli cells, found in large amounts in the lysosomal compartment and implicated in the degradation of residual bodies released by the spermatids during spermiation. Interestingly, the targeting of prosaposin to the lysosomes is independent of the M6P-R. This investigation demonstrated that sortilin was required for the trafficking of prosaposin to the lysosomes in TM4 cells. The requirement of sortilin was shown using a siRNA probe to block the translation of sortilin mRNA. Sortilin-deficient cells were not able to route prosaposin to the lysosomal compartment but continue to transport cathepsin B, since this hydrolase uses the M6P-R to be routed to the lysosomes. These results indicate that sortilin appears to be involved in the lysosomal trafficking of prosaposin.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

The lysosomal trafficking of acid sphingomyelinase is mediated by sortilin and mannose 6-phosphate receptor.

Acid sphingomyelinase (ASM), a member of the saposin-like protein (SAPLIP) family, is a lysosomal hydrolase that converts sphingomyelin to ceramide. Deficiency of ASM causes a variant form of Niemann-Pick disease. The mechanism of lysosomal targeting of ASM is poorly known. Previous studies suggest that ASM could use in part the mannose 6-phosphate receptor (M6P-Rc). Sortilin, a type I transmembrane glycoprotein that belongs to a novel family of receptor proteins, presents structural features of receptors involved in lysosomal targeting. In this study we examined the hypothesis that sortilin may be implicated in the trafficking of ASM to the lysosomes. Using a dominant-negative sortilin construct lacking the cytoplasmic tail, which is essential to recruit adaptor proteins and clathrin, we demonstrated that sortilin is also involved in the lysosomal targeting of ASM. Confocal microscopy revealed that truncated sortilin partially inhibited the lysosomal trafficking of ASM in COS-7 cells and abolished the lysosomal targeting of ASM in I-cells. Pulse-chase experiments corroborated that sortilin is involved in normal sorting of newly synthesized ASM. Furthermore, over-expression of truncated sortilin accelerated and enhanced the secretion of ASM from COS-7 cells and I-cells. Co-immunoprecipitation assays confirmed the interaction between sortilin and ASM. In conclusion, ASM uses sortilin as an alternative receptor to be targeted to the lysosomes.

Adaptor Proteins, Vesicular Transport↗

PubMed2001

The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS domain of the GGA2 sorting protein.

Sortilin belongs to a growing family of multiligand type-1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilin's intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane. To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail. We report that sorting signals conforming to YXX and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans-Golgi network, showing little or no recycling. Furthermore, we found that cation-independent mannose 6-phosphate receptor (MPR300)-sortilin chimeras, expressed in mannose 6-phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized beta-hexosaminidase and beta-glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi-endosome sorting. Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p-mediated sorting of yeast carboxypeptidase Y.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

The lysosomal trafficking of sphingolipid activator proteins (SAPs) is mediated by sortilin.

Most soluble lysosomal proteins bind the mannose 6-phosphate receptor (M6P-R) to be sorted to the lysosomes. However, the lysosomes of I-cell disease (ICD) patients, a condition resulting from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases, have near normal levels of several lysosomal proteins, including the sphingolipid activator proteins (SAPs), GM2AP and prosaposin. We tested the hypothesis that SAPs are targeted to the lysosomal compartment via the sortilin receptor. To test this hypothesis, a dominant-negative construct of sortilin and a sortilin small interfering RNA (siRNA) were introduced into COS-7 cells. Our results showed that both the truncated sortilin and the sortilin siRNA block the traffic of GM2AP and prosaposin to the lysosomal compartment. This observation was confirmed by a co-immunoprecipitation, which demonstrated that GM2AP and prosaposin are interactive partners of sortilin. Furthermore, a dominant-negative mutant GGA prevented the trafficking of prosaposin and GM2AP to lysosomes. In conclusion, our results show that the trafficking of SAPs is dependent on sortilin, demonstrating a novel lysosomal trafficking.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin.

Brain-derived neurotrophic factor (BDNF) is best characterized for critical roles in neuronal survival, differentiation, and synaptic modulation mediated by the TrkB receptor tyrosine kinase. Developmentally regulated death signaling by BDNF has also been demonstrated via activation of p75NTR. Because recent studies suggest that proNGF, the precursor form of NGF, is more active than mature NGF in inducing apoptosis after binding to p75NTR and a coreceptor, sortilin, we asked whether the precursor of BDNF (proBDNF) is also a proapoptotic ligand in the nervous system. proBDNF is secreted by cultured neurons, and recombinant proBDNF binds to sortilin. In sympathetic neurons coexpressing sortilin and p75NTR, we found that proBDNF is an apoptotic ligand that induces death at subnanomolar concentrations. In contrast, mature BDNF, but not proBDNF, is effective in inducing TrkB phosphorylation. proBDNF effects are dependent on cellular coexpression of both p75NTR and sortilin, because neurons deficient in p75NTR are resistant to proBDNF-induced apoptosis, and competitive antagonists of sortilin block sympathetic neuron death. Moreover, addition of preformed complexes of soluble sortilin and proBDNF failed to induce apoptosis of cells coexpressing both sortilin and p75NTR, suggesting that interaction of proBDNF with both receptors on the cell surface is required to initiate cell death. Together with our past findings, these data suggest that the neurotrophin family is capable of modulating diverse biological processes via differential processing of the proneurotrophins.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference.

To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.

Journal Article↗

PubMed2005

Sortilin is essential and sufficient for the formation of Glut4 storage vesicles in 3T3-L1 adipocytes.

Impaired translocation of the glucose transporter isoform 4 (Glut4) to the plasma membrane in fat and skeletal muscle cells may represent a primary defect in the development of type 2 diabetes mellitus. Glut4 is localized in specialized storage vesicles (GSVs), the biological nature and biogenesis of which are not known. Here, we report that GSVs are formed in differentiating 3T3-L1 adipocytes upon induction of sortilin on day 2 of differentiation. Forced expression of Glut4 prior to induction of sortilin leads to rapid degradation of the transporter, whereas overexpression of sortilin increases formation of GSVs and stimulates insulin-regulated glucose uptake. Knockdown of sortilin decreases both formation of GSVs and insulin-regulated glucose uptake. Finally, we have reconstituted functional GSVs in undifferentiated cells by double transfection of Glut4 and sortilin. Thus, sortilin is not only essential, but also sufficient for biogenesis of GSVs and acquisition of insulin responsiveness in adipose cells.

3T3-L1 Cells↗

PubMed2002

Sortilin is upregulated during osteoblastic differentiation of mesenchymal stem cells and promotes extracellular matrix mineralization.

Osteoblasts and adipocytes are derived from a common precursor in bone marrow, the mesenchymal stem cell (MSC). Factors driving human MSCs (hMSCs) to differentiate down the two lineages play important roles in determining bone density because it has been shown that bone volume loss associated with osteoporosis and aging is accompanied by reduced osteoblastic bone formation and increased marrow adipose tissue. The genes upregulated in hMSCs during osteogenic differentiation were screened using cDNA microarrays and were semi-quantitated by real-time RT-PCR. One of the genes identified was sortilin, which was upregulated one day after osteogenic induction and remained upregulated for a week. The overexpression of sortilin in hMSCs using an adenovirus vector resulted in the acceleration of mineralization during osteogenic differentiation without affecting alkaline phosphatase activity. Lipoprotein lipase (LPL), produced by adipocytes, is bound by sortilin, which may mediate its endocytosis. By adding LPL to osteogenic induction medium, osteoblastic mineralization was inhibited in a dose-dependent manner. Interestingly, sortilin overexpression abolished the LPL-mediated suppression of osteogenic differentiation. hMSCs exist in marrow where LPL-producing adipose cells are abundant and where osteogenesis is negatively regulated by LPL. Sortilin has a counter effect of promoting osteogenesis by acting as a scavenger of LPL.

Adaptor Proteins, Vesicular Transport↗

PubMed1998

The carboxy-terminal domain of the receptor-associated protein binds to the Vps10p domain of sortilin.

Binding of the receptor-associated protein (RAP) to the newly identified putative sorting receptor, sortilin, was analyzed by surface plasmon resonance analysis of recombinant RAP and sortilin domains and compared with binding to megalin and low density lipoprotein receptor-related protein (LRP). The data show that the RAP-binding site in sortilin is localized in the cysteine-rich lumenal part homologous to yeast vacuolar protein-sorting 10 protein (Vps10p), and the sortilin-binding site in RAP is localized in the carboxy-terminal domain III of the three homologous domains in RAP. Whereas sortilin bound only RAP domain III, megalin and LRP bound all RAP domains with the functional affinity order: domain III >domain I > domain II.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

Sortilin is essential for proNGF-induced neuronal cell death.

Sortilin (approximately 95 kDa) is a member of the recently discovered family of Vps10p-domain receptors, and is expressed in a variety of tissues, notably brain, spinal cord and muscle. It acts as a receptor for neurotensin, but predominates in regions of the nervous system that neither synthesize nor respond to this neuropeptide, suggesting that sortilin has additional roles. Sortilin is expressed during embryogenesis in areas where nerve growth factor (NGF) and its precursor, proNGF, have well-characterized effects. These neurotrophins can be released by neuronal tissues, and they regulate neuronal development through cell survival and cell death signalling. NGF regulates cell survival and cell death via binding to two different receptors, TrkA and p75NTR (ref. 10). In contrast, proNGF selectively induces apoptosis through p75NTR but not TrkA. However, not all p75NTR-expressing cells respond to proNGF, suggesting that additional membrane proteins are required for the induction of cell death. Here we report that proNGF creates a signalling complex by simultaneously binding to p75NTR and sortilin. Thus sortilin acts as a co-receptor and molecular switch governing the p75NTR-mediated pro-apoptotic signal induced by proNGF.

Adaptor Proteins, Vesicular Transport↗

PubMed1998

Sortilin is the major 110-kDa protein in GLUT4 vesicles from adipocytes.

Vesicles containing the glucose transporter GLUT4 from rat adipocytes contain a major protein of 110 kDa. We have isolated this protein, obtained the sequences of peptides, and cloned a large portion of its cDNA. This revealed that the protein is sortilin, a novel membrane protein that was cloned in another context from a human source while this work was in progress. Subcellular fractionation of rat and 3T3-L1 adipocytes, together with GLUT4 vesicle isolation, showed that sortilin was primarily located in the low density microsomes in vesicles containing GLUT4. Insulin caused a 1.7-fold increase in the amount of sortilin at the plasma membranes of 3T3-L1 adipocytes, as assessed by cell surface biotinylation. The expression of sortilin in 3T3-L1 cells occurred only upon differentiation. Previous characterization of sortilin has led to the suggestion that it functions to sort lumenal proteins from the trans Golgi. The significance of its insulin-stimulated increase at the cell surface and of its expression upon differentiation will require definitive delineation of its function.

3T3 Cells↗

PubMed2004

Functional organization of the sortilin Vps10p domain.

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.

Adaptor Proteins, Vesicular Transport↗

PubMed2007

The luminal Vps10p domain of sortilin plays the predominant role in targeting to insulin-responsive Glut4-containing vesicles.

In fat and skeletal muscle cells, insulin-responsive vesicles, or IRVs, deliver glucose transporter Glut4 and several associated proteins to the plasma membrane in response to hormonal stimulation. Although the protein composition of the IRVs is well studied, the mechanism of their formation is unknown. It is believed, however, that the cytoplasmic tails of the IRV component proteins carry targeting information to this compartment. To test this hypothesis, we have studied targeting of sortilin, one of the major IRV constituents. We have found that the reporter protein consisting of the cytoplasmic tail of sortilin and EGFP is co-localized with ectopically expressed Glut4 in the perinuclear compartment of undifferentiated 3T3-L1 cells that do not form insulin-responsive vesicles. Upon cell differentiation, this reporter protein does not enter the IRVs; moreover, it loses its perinuclear localization and becomes randomly distributed throughout the whole intracellular space. In contrast, the tagged luminal Vps10p domain of sortilin demonstrates partial co-localization with Glut4 in both undifferentiated and differentiated cells and is targeted to the IRVs upon cell differentiation. Using chemical cross-linking and the yeast two-hybrid system, we show that sortilin interacts with Glut4 and IRAP in the vesicular lumen. Our results suggest that luminal interactions between component proteins play an important role in the process of IRV biogenesis.

3T3-L1 Cells↗

PubMed2005

Sortilin controls intracellular sorting of brain-derived neurotrophic factor to the regulated secretory pathway.

Brain-derived neurotrophic factor (BDNF), after activity-dependent secretion from neurons, modulates critical nervous system functions. Recently, a variant in the human bdnf gene, resulting in a valine to methionine substitution in the prodomain, has been shown to lead to defective regulated secretion from neurons and memory impairment. Here, we report a novel function for a Vps10p domain protein, sortilin, in controlling BDNF sorting to the regulated secretory pathway. Sortilin interacts specifically with BDNF in a region encompassing the methionine substitution and colocalizes with BDNF in secretory granules in neurons. A truncated form of sortilin causes BDNF missorting to the constitutive secretory pathway without affecting neurotrophin-4 (NT-4) secretion. In addition, sortilin small interfering RNA introduced into primary neurons also led to BDNF missorting from the regulated to the constitutive secretory pathway. Together, these data suggest a mechanism to understand the defect associated with variant BDNF and provide a framework, based on divergent presynaptic regulation of sorting to secretory pathways, to explain how two ligands for tropomyosin-related kinase B, BDNF and NT-4, can mediate diverse biological responses.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

The trafficking of prosaposin (SGP-1) and GM2AP to the lysosomes of TM4 Sertoli cells is mediated by sortilin and monomeric adaptor proteins.

Prosaposin (SGP-1) and GM2 activator protein (GM2AP) are soluble sphingolipid activator proteins (SAPs) that are targeted to the lysosomal compartment of Sertoli cells to aid hydrolases in the breakdown of glycosphingolipids. To reach the lysosome, most soluble proteins must interact with the mannose 6-phosphate receptor (MPR). To be sorted from the Golgi, the MPR must bind to the Golgi associated, gamma-adaptin homologous, ARF binding proteins (GGAs), a group of monomeric adaptor proteins responsible for the recruitment of clathrin. It is well established, however, that the lysosomes of I-cell disease (ICD) patients have near normal levels of several lysosomal proteins, including prosaposin and GM2AP. ICD results from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases. Thus, prosaposin and GM2AP can traffic to lysosomes in a MPR independent manner. Previous work has demonstrated that an interaction with sphingomyelin in the Golgi membrane is necessary for the targeting of prosaposin by an unknown receptor. Using a TM4 Sertoli cell line, we tested the hypothesis that prosaposin and GM2AP are targeted to the lysosomal compartment via the sortilin receptor, which has been recently shown to have a GGA binding motif. Interestingly, dominant-negative GGAs, unable to bind clathrin to shuttle from the Golgi, prevented the trafficking of prosaposin and GM2AP to lysosomes. A dominant negative construct of sortilin lacking the GGA binding domain retained prosaposin and GM2AP in the Golgi. In conclusion, our results showed that the trafficking of prosaposin and GM2AP to the lysosome is dependent on sortilin.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Sortilin, SorCS1b, and SorLA Vps10p sorting receptors, are novel gamma-secretase substrates.

BACKGROUND: The mammalian Vps10p sorting receptor family is a group of 5 type I membrane homologs (Sortilin, SorLA, and SorCS1-3). These receptors bind various cargo proteins via their luminal Vps10p domains and have been shown to mediate a variety of intracellular sorting and trafficking functions. These proteins are highly expressed in the brain. SorLA has been shown to be down regulated in Alzheimer's disease brains, interact with ApoE, and modulate Abeta production. Sortilin has been shown to be part of proNGF mediated death signaling that results from a complex of Sortilin, p75NTR and proNGF. We have investigated and provide evidence for gamma-secretase cleavage of this family of proteins. RESULTS: We provide evidence that these receptors are substrates for presenilin dependent gamma-secretase cleavage. Gamma-secretase cleavage of these sorting receptors is inhibited by gamma-secretase inhibitors and does not occur in PS1/PS2 knockout cells. Like most gamma-secretase substrates, we find that ectodomain shedding precedes gamma-secretase cleavage. The ectodomain cleavage is inhibited by a metalloprotease inhibitor and activated by PMA suggesting that it is mediated by an alpha-secretase like cleavage. CONCLUSION: These data indicate that the alpha- and gamma-secretase cleavages of the mammalian Vps10p sorting receptors occur in a fashion analogous to other known gamma-secretase substrates, and could possibly regulate the biological functions of these proteins.

Journal Article↗

PubMed1999

Expression of the 100-kDa neurotensin receptor sortilin during mouse embryonal development.

Recently, sortilin a non G-protein-coupled receptor has been identified as the 100-kDa neurotensin receptor. In this paper we describe the expression of its gene during mouse embryonal development. We show that the nervous system is the main location of sortilin gene expression and that with ongoing development the forebrain exhibits the highest accumulation of transcripts.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

Multi-tasking by the p75 neurotrophin receptor: sortilin things out?

Signalling by the p75 neurotrophin receptor has been implicated in diverse neuronal responses, including increased differentiation or survival, inhibition of regeneration, and initiation of apoptotic cell death. These numerous roles are matched by, but are not yet correlated with, a multiplicity of extracellular ligands and intracellular interactors. Membrane proteins such as sortilin, a member of the Vps10p family of sorting receptors, and the glycosylphosphatidylinositol-linked Nogo receptor (NgR) and the associated adaptor lingo 1 have recently been added to the list of p75-interacting modulators. Other studies have described intramembranal cleavage of p75 and the potential nuclear targeting of cleavage fragments or of the complete receptor after it has been internalized into a putative signalling endosome. These findings suggest that some of the diversity in p75 activities might be due to differential subcellular localization and transport of p75 receptor complexes. We therefore argue that cell-biology-driven approaches are now required to make sense of p75 signalling.

Adaptor Proteins, Vesicular Transport↗