Mapping of the Interaction Site between Sortilin and the p75 Neurotrophin Receptor Reveals a Regulatory Role for the Sortilin Intracellular Domain in p75 Neurotrophin Receptor Shedding and Apoptosis*

Background: Sortilin and p75NTR induce neuronal apoptosis by binding pro-neurotrophins during development and following neuronal injury. Results: Sortilin interacts with an extracellular juxtamembrane 23-amino acid sequence of p75NTR. Conclusion: Despite binding being mediated through extracellular interactions, the intracellular domain of sortilin regulates p75NTR shedding and apoptosis. Significance: Mapping may allow design of compounds inhibiting neuronal cell death by blocking the interaction between sortilin and p75NTR. Neurotrophins comprise a group of neuronal growth factors that are essential for the development and maintenance of the nervous system. However, the immature pro-neurotrophins promote apoptosis by engaging in a complex with sortilin and the p75 neurotrophin receptor (p75NTR). To identify the interaction site between sortilin and p75NTR, we analyzed binding between chimeric receptor constructs and truncated p75NTR variants by co-immunoprecipitation experiments, surface plasmon resonance analysis, and FRET. We found that complex formation between sortilin and p75NTR relies on contact points in the extracellular domains of the receptors. We also determined that the interaction critically depends on an extracellular juxtamembrane 23-amino acid sequence of p75NTR. Functional studies further revealed an important regulatory function of the sortilin intracellular domain in p75NTR-regulated intramembrane proteolysis and apoptosis. Thus, although the intracellular domain of sortilin does not contribute to p75NTR binding, it does regulate the rates of p75NTR cleavage, which is required to mediate pro-neurotrophin-stimulated cell death.

Neurotrophins comprise a group of neuronal growth factors that are essential for the development and maintenance of the nervous system. However, the immature pro-neurotrophins promote apoptosis by engaging in a complex with sortilin and the p75 neurotrophin receptor (p75 NTR ). To identify the interaction site between sortilin and p75 NTR , we analyzed binding between chimeric receptor constructs and truncated p75 NTR variants by co-immunoprecipitation experiments, surface plasmon resonance analysis, and FRET. We found that complex formation between sortilin and p75 NTR relies on contact points in the extracellular domains of the receptors. We also determined that the interaction critically depends on an extracellular juxtamembrane 23-amino acid sequence of p75 NTR . Functional studies further revealed an important regulatory function of the sortilin intracellular domain in p75 NTR -regulated intramembrane proteolysis and apoptosis. Thus, although the intracellular domain of sortilin does not contribute to p75 NTR binding, it does regulate the rates of p75 NTR cleavage, which is required to mediate pro-neurotrophin-stimulated cell death.
Neurotrophins including NGF, BDNF, and neurotrophin-3 (NT3) 3 constitute a group of neuronal growth factors that are important for the development, maintenance, and differentiation of the nervous system (1). However, neurotrophins can also be released in an immature form known as pro-neurotrophins (proNTs), which display distinct and often opposing biological activities to those of their mature counterparts (2)(3)(4). Hence, by forming a heterotrimeric complex with sortilin and the p75 neurotrophin receptor (p75 NTR ), proNGF and proBDNF induce apoptosis during development and cell senescence, as well as following neuronal injury (5)(6)(7). In addition, proNT3 has recently been found to promote apoptosis in superior and spiral ganglion neurons by binding sortilin and p75 NTR (8,9). Binding of proNTs is achieved by binding of the pro-domain by sortilin, whereas p75 NTR interacts with the mature part of the neurotrophin (5). However, sortilin and p75 NTR also exist as a preformed receptor complex, interacting in the absence of ligand (5).
Sortilin is a neuronal type I receptor belonging to the Vps10p domain receptor family (10,11). Extracellularly, sortilin contains a ligand-binding 10-bladed ␤-propeller followed by a 10CC module (12). A short cytoplasmic tail contains several internalization and sorting motifs, which facilitate the trafficking and sorting functions of sortilin (13). p75 NTR is a member of the TNF receptor superfamily and is best known for its role in neuronal apoptosis and neurodegeneration (14). Its extracellular domain consists of four tandemly arranged ligand-binding domains followed by a ϳ60-amino acid juxtamembrane stalk region. As for other members of the TNF receptor superfamily, a death domain is found in the cytoplasmic domain linked to the transmembrane sequence via a 60-amino acid p75 NTR -spe-cific "chopper" module (15). The p75 NTR intracellular domain (ICD) has been reported to promote apoptosis, as well as cell survival in a cell type-specific manner if released into the cytosol (16 -19). This is achieved by regulated intramembrane proteolysis (RIP) whereby the stalk region of p75 NTR is first cleaved by the ␣-secretase TACE/ADAM17, generating the C-terminal fragment. The remaining membrane-tethered stub is subsequently cleaved by presenilin-dependent ␥-secretase, releasing the p75 NTR ICD to the cytosol for signaling (20).
We have previously demonstrated that sortilin increases p75 NTR affinity for proNTs. For example, cells expressing p75 NTR alone bind proNGF with an approximate K d value of ϳ15 nM, whereas co-expression of sortilin increases affinity for proNGF ϳ100-fold to K d ϭ ϳ160 pM (5). This dramatically decreases the effective concentration of proNTs required for apoptotic signaling by p75 NTR . Here we identify the extracellular domains in sortilin and p75 NTR that are responsible for receptor heterodimerization. Although the intracellular domain of sortilin is not involved in receptor interactions, we found that it regulates RIP of p75 NTR and proNT-stimulated apoptotic signaling, suggesting additional roles for sortilin in p75 NTR -mediated apoptosis.
To make deletion and truncation expression constructs of p75 NTR (p75 NTR ⌬ C1 (1-29, 66 -425), p75 NTR ⌬ C1,2 (1-29, 109 -425), p75 NTR ⌬ C1,2,3,4 (1-29, 190 -425), p75 NTR ⌬ stalk (1-227, 251-425), p75 NTR stalk,tm,icd, (228 -425), and p75 NTR ICD (274 -425)) and YFP-or CFP-tagged versions of these (34), a modified pCDNA3 (Invitrogen) backbone was used. The rat p75 NTR signal peptide including a Kozak sequence (nucleotides Ϫ29 to ϩ87) was inserted between the KpnI and EcoRV restriction sites, generating the vector pCDNA3-SP. p75 NTR coding sequences were amplified under standard PCR conditions and cloned into pCDNA3-SP using the primer-generated EcoRV and NheI sites. In cases where p75 NTR variants were fused to a fluorophore, YFP and CFP were amplified by PCR from peYFP-N1 and peCFP-N1 (Clontech), using primers incorporating 5Ј EcoRV and NheI restriction sites and a 3Ј stop codon and a HindIII site. Enhanced YFP and CFP were cloned in frame between the EcoRV and HindIII restriction sites of pCDNA3-SP, generating the vectors pCDNA3-YFP/CFP. p75 NTR coding sequences were amplified by PCR with a 5Ј EcoRV and a 3Ј NheI restriction site. p75 NTR coding sequences were then cloned between the EcoRV and NheI restriction sites of pCDNA3-YFP/CFP to generate in-frame fusion proteins. p75 NTR tm,icd (251-425) was constructed as previously described (24) and fused to YFP as described above. To generate sortilin-YFP, full-length human sortilin, including the sequences encoding the signal and pro-peptides, and the Kozak sequence were amplified by PCR, thereby generating a 3Ј-Kpn site and a 5Ј-Nhe site. The fragment was inserted into pCDNA3-YFP. The p75 NTR -Gal4 reporter construct for the p75 NTR cleavage reporter assay was cloned by inserting rat p75 NTR cDNA into the pcDNA3-GVP vector containing Gal4 DNA binding and VP16 transcription activating domains and a neomycin selection cassette. The plasmid was digested with MunI, and a Gal4-responsive 9ϫUAS Luc2P cassette from the pGL4.35 reporter vector (Promega) was inserted by blunt end cloning after filling in the recessed 3Ј ends with Klenow fragment.
Antibodies and Proteins-Anti-neurotensin receptor-3, recognizing the extracellular domain of sortilin, was purchased from BD Transduction Laboratories (612100) and anti-p75 NTR antibody (ab10494) from Abcam. Anti-p75 NTR ICD 9992, for detecting p75 NTR ICD, was a generous gift from Professor Moses V. Chao (Skirball Institute of Biomolecular Medicine, New York University School of Medicine). Anti-IL 2 R␣ (I6152) and anti-HA tag (H6908) antibodies were from Sigma. Anti-GFP (11814460001) was purchased from Roche Applied Science. Recombinant His 6 -tagged soluble human sortilin (solsortilin) was produced and purified as previously described (22). The luminal domain of human p75 NTR (Met 1 -Asn 250 ) and human RET receptor tyrosine kinase (Met 1 -Arg 635 ), all fused to the Fc region of IgG 1 , were purchased from R & D Systems. Recombinant human furin cleavage-resistant proNGF, proNT3, and wild type proBDNF were a gift from Professor Elisabeth Schwarz (Institute for Biotechnology, Martin-Luther-Universität, Halle-Wittenberg) (25). Recombinant mouse proNGF used for RN22 cell death assays was purchased from Chemicon. Mouse NGF was from Austral Biologicals (GF-022) and neurotensin from Sigma (N6383).
Co-immunoprecipitation-Stably or transiently transfected HEK293 cells were incubated with or without ligand in PBS (with 1 mM CaCl 2 and MgCl 2 ) for 90 min at room temperature and then treated with the reducible protein cross-linker dithiobis succinimidylpropionate (Pierce) according to the manufacturer's instructions. After washing, the cells were lysed on ice for 10 min in TNE buffer (20 mM Tris, pH 8, 10 mM EDTA) supplemented with 1% Nonidet P-40 and complete protease inhibitor mixture (Roche Applied Science). Samples were immunoprecipitated overnight at 4°C by use of Gammabind G-Sepharose beads (Amersham Biosciences) coupled with anti-p75 NTR (10494) or anti-GFP antibodies. Nonspecific binding was removed by washing beads five times in TBS supplemented with 0.05% Tween 20, and the proteins were eluted by boiling samples in reducing sample buffer (20 mM dithioerythritol, 2.5% SDS). Protein samples were subjected to reducing SDS-PAGE, Western blotting, and visualization using the Fuji film LAS1000 imaging system. The blots were stripped in 62.5 mM Tris-HCl, pH 6.7, 100 mM 2-mercaptoethanol, and 2% SDS for 30 min at 50°C under rotation. Co-immunoprecipitation performed with p75 NTR variants was visualized with the Odyssey infrared imaging system.
␤-Galactosidase Complementation Assay-HEK293 cells were seeded into white 96-well tissue culture plates with clear bottoms (Sigma) at a density of 30,000 cells/well. The cells were then transfected with sortilin-␤-gal⌬␣ and/or HA-p75 NTR -␤-gal⌬ and after 48 h incubated with or without proNTs for 30 min at 37°C. ␤-galactosidase activity was assessed by adding 100 l of Gal-Screen substrate (50 l of both solution A and solution B) (Applied Biosystems) to each well. The plates were incubated at room temperature for 1 h, and luminescence was measured with a Victor 3 1420 multilabel counter.
Surface Plasmon Resonance Analysis-Analyses were performed on a Biacore 3000 instrument equipped with CM5 sensor chips and activated as previously described (22). solsortilin was immobilized at a density of ϳ72 fmol/mm 2 in 10 mM sodium acetate, pH 4.0, and the remaining sites were blocked with 1 M ethanolamine, pH 8.5. Injection of proteins was done in running buffer at 25°C (10 mM HEPES, pH 7.4, containing 150 mM NaCl, 1.5 mM CaCl 2 , 1 mM EGTA, and 0.005% P20). Binding was expressed in relative response units as the response obtained from the flow cell containing immobilized receptor minus the response obtained when using an activated and blocked but uncoupled flow cell.
Cell Population FRET Assay-The FRET of p75 NTR molecules within a population of HEK293 cells was determined 48 h post-transfection of triplicate wells of transiently transfected cells and untransfected cells (to determine background fluores-cence). The cells were washed once in PBS, pH 7.4, harvested in PBS, and centrifuged for 30 s at 10,000 ϫ g. Cell pellets were resuspended in 200 l of PBS and loaded into 96-well black microtiter plates (Greiner) for immediate analysis. Fluorescence was recorded simultaneously for the donor fluorophore at 430 Ϯ 10 nm/480 Ϯ 10 nm and the acceptor fluorophore at 485 nm/530 Ϯ 10 nm, and FRET was measured at 430 Ϯ 10 nm/530 Ϯ 10 nm on a POLARstar OPTIMA multidetection microplate reader (BMG Labtech). FRET was calculated on cells as a FRET ratio, which is the fractional increase in YFP emission caused by FRET (26) according to the formula described in Ref. 34.
Receptor Processing Experiments-HEK293 cells or stably transfected polyclonal RN22 cells were seeded into poly-Llysine-coated 24-well culture tissue plates at a density of 40,000 cells/well. HEK293 cells were transfected after 24 h and used for shedding experiments after an additional 48 h. Prior to treatment with 200 nM phorbol 12-myristate 13-acetate (PMA) (Calbiochem) cells were incubated for 1 h with 200 nM compound E (Alexis Biochemicals) or 1 M epoxomicin (Sigma) in DMEM supplemented with 10% FCS. The cells were then incubated for 3 h (HEK293 cells) or 6 h (RN22 cells), after which they were lysed in TNE buffer supplemented with 1% Nonidet P-40 and complete protease inhibitor mixture and subjected to Western blotting.
Primary cultures of superior cervical ganglion neurons were established from postnatal day 3-5 sortilin knock-out mice (7) as described in Ref. 27. Dissociated neurons were seeded into poly-L-ornithine (Sigma) and 3 g/ml laminin (Invitrogen) coated wells in neurobasal A medium supplemented with 1% nonessential amino acids, 1% GlutaMAX (all from Gibco), 0.2% B27 (Invitrogen), and 10 ng/ml NGF. 24 h post-seeding, the cells were starved for 4 h and subsequently added 1 M epoxomicin, 10 ng/ml NGF and 10% FCS for 1 h followed by the addition of 200 nM PMA for 3 h. The cells were harvested in TNE buffer supplemented with 1% Nonidet P-40 and complete protease inhibitor mixture and subjected to Western blotting using HRP-conjugated secondary antibodies and Supersignal West Femto Sensitivity Substrate from Pierce.
RN22 Cell Death Assay-Polyclonal stable RN22 cell lines were seeded into poly-L-lysine-coated black 96-well tissue culture plates (PerkinElmer) at a density of 7500 cells/well. After 24 h, the cells were washed three times in DMEM without phenol red, supplemented with 100 units of penicillin and 100 g/ml streptomycin, 1% GlutaMAX (Invitrogen), 1.5 g/ml insulin, 50 g/ml transferrin, 30 nM selenium, and 30 nM triiodothyronine (all from Sigma) and incubated with or without proNGF in a final volume of 100 l of the same medium. Following 72 h of incubation, the number of dead and live cells was scored with the MultiTox-Fluor multiplex cytotoxicity assay (Promega) measuring fluorescence with a Victor 3 1420 multilabel counter. To correct for differences in cell numbers among wells, the death signal was related to the life signal from the same well.
Luciferase Assay-HEK293 cells stably co-expressing p75 NTR -Gal4 and luciferase under the UAS promoter were seeded in 24-well tissue culture plates at a density of 30,000 cells/well. After 24 h, some cells were transiently transfected with variants of sortilin. 72 h post-seeding, the cells were analyzed for luciferase activity using the luciferase assay from Promega. Prior to analysis, the cultures were treated 1 h with epoxomicin (1 M) and in some cases with the ␣-secretase inhibitor TAPI-2 (20 M) followed by the addition of PMA (200 nM) for 3 h. Luminescence was measured with a POLARstar OPTIMA plate reader.
Statistics-The results were analyzed by Student's t test, with p Ͻ 0.05 being considered a significant level of difference. In Figs. 5 and 6, the effect of wild type sortilin was compared with control and with the effect of sortilin mutants.

RESULTS
ProNGF Increases Affinity between Sortilin and p75 NTR -To map binding between sortilin and p75 NTR , we established a coimmunoprecipitation protocol using stably or transiently transfected HEK293 cells. These cells were then treated with the cell-permeable, reducible protein cross-linker dithiobis succinimidylpropionate and lysates subjected to co-immunoprecipitation, reducing SDS-PAGE, and Western blotting. For our studies, we used the well characterized sortilin variant sortilin mut , which contains two disrupted internalization motifs, one being the tyrosine-based YXXØ sequence ( 792 YSVL) and the other being a deletion of the C-terminal dileucine motif ( 829 LL) (13). Whereas wild type sortilin is mainly localized in perinuclear compartments (10,28), sortilin mut is predominantly expressed on the cell surface (13). Results obtained using this construct are therefore directly comparable with those obtained with chimeric receptor constructs lacking intracellular domain sequences, which are also mainly expressed on the cell surface. First, we established that sortilin mut and p75 NTR bind and evaluated the effect of both proNGF and NGF on this interaction. Following cross-linking and immunoprecipitation of p75 NTR , sortilin mut was clearly coimmunoprecipitated, showing that sortilin mut and p75 NTR interact in the absence of ligand ( Fig. 1) consistent with previous reports (5,7). Treatment with 25 nM NGF, prior to crosslinking, had no effect on sortilin mut -p75 NTR complex formation. In contrast, 25 nM proNGF increased the interaction 2.3-fold Ϯ 0.4 (n ϭ 4, p Ͻ 0.04), consistent with the notion that proNGF is a shared ligand between sortilin and p75 NTR , whereby the pro-domain of proNGF binds sortilin and the mature domain binds p75 NTR (5).
p75 NTR Binding Properties of the Extracellular and Transmembrane Domain of Sortilin-We next evaluated whether the extracellular and transmembrane domains of sortilin are required for it to bind to p75 NTR . To do this, co-immunoprecipitations were carried out using a truncated sortilin variant lacking the entire cytoplasmic domain (sortilin tailless ). HEK293 cells co-expressing sortilin tailless and p75 NTR were cross-linked in the absence or presence of 25 nM proNGF, and lysates were immunoprecipitated with anti-p75 NTR antibodies. Sortilin tailless clearly bound p75 NTR , demonstrating that residues within the extracellular domain and/or the transmembrane region account for complex formation in the absence of ligand ( Fig. 2A). As expected, the interaction between sortilin tailless and p75 NTR was again further increased in the presence of proNGF (2.1-fold Ϯ 0.3, n ϭ 4, p Ͻ 0.02).
To confirm these findings in another cellular context, we developed a sensitive protocol for monitoring sortilin and p75 NTR complex formation using the ␤-galactosidase complementation assay (23). This assay is based on two weakly interacting subunits of ␤-galactosidase that upon dimerization reconstitute the active site measured by luminescence (23). We substituted the cytoplasmic domains of sortilin and HA-tagged p75 NTR with each of the two ␤-galactosidase subunits (Fig. 2B). The constructs were then transiently transfected into HEK293 cells and ␤-galactosidase activity measured using a chemiluminescent substrate. Whereas expression of each construct alone only resulted in a weak signal, co-expression increased enzyme activity ϳ13-fold, suggesting that the extracellular and/or the transmembrane domain of sortilin and p75 NTR also interact in this paradigm (Fig. 2, C and D). The addition of increasing amounts of proNGF, proBDNF, or proNT3 from as low as 40 pM proNT increased the interaction of sortilin with p75 NTR (Fig. 2E). These results further show that proBDNF and proNT3 bind with very high affinity to sortilin and p75 NTR , similar to what we previously have reported for proNGF (5).
Sortilin and p75 NTR Interact through Their Extracellular Domains-To investigate whether p75 NTR interacts with the extracellular domain of sortilin, we constructed a sortilin-interleukin-2 receptor (IL 2 R) chimeric receptor designated sortilin ecd -IL 2 R tm,icd that combined the extracellular domain of sortilin and the transmembrane region and intracellular domain of IL 2 R (Fig. 3A). The binding properties of sortilin ecd -IL 2 R tm,icd with p75 NTR were then tested in transfected HEK293 cells by coimmunoprecipitation experiments. We found that sortilin ecd -IL 2 R tm,icd readily interacted with p75 NTR and that proNGF increased this interaction 1.9-fold Ϯ 0.1, n ϭ 5, p Ͻ 0.002 (Fig.  3B). These findings were further substantiated by surface plasmon resonance analysis using the commercially available fusion protein of the extracellular domain of p75 NTR linked to FIGURE 1. Effect of pro-and mature NGF on the sortilin-p75 NTR interaction. Representative (n ϭ 4) Western blot (WB) of lysates of HEK293 cells stably expressing either sortilin mut or sortilin mut and p75 NTR incubated with 25 nM proNGF or NGF as indicated. The cells were cross-linked with dithiobis succinimidylpropionate, and the lysates were immunoprecipitated (IP) with anti-p75 NTR antibodies prior to SDS-PAGE and Western blotting. Sortilin is co-immunoprecipitated by p75 NTR , and the interaction is stabilized by proNGF but not NGF. DECEMBER 21, 2012 • VOLUME 287 • NUMBER 52 the constant region of human IgG (p75 NTR ecd -Fc). When p75 NTR ecd -Fc was injected onto a Biacore sensor chip containing immobilized soluble sortilin (sol-sortilin), binding was clearly evident (Fig. 3C). To ensure that binding did not stem from the Fc region of the fusion protein, binding to a RET-Fc tyrosine-kinase fusion protein was probed under similar conditions. RET-Fc did not bind sol-sortilin (Fig. 3C), confirming that the binding between sortilin and p75 NTR relies on residues within these extracellular domains.

Mapping of the Sortilin-p75 NTR -binding Site
We next addressed whether p75 NTR engages the ligandbinding site in the 10-bladed ␤-propeller domain of sortilin. To do this, we carried out surface plasmon resonance competition studies with excess amounts of the tridecapeptide neurotensin.
Neurotensin binds the tunnel of the ␤-propeller of sortilin and thereby sterically blocks access of all known ligands to sortilin (11,12). Because neurotensin has a low molecular weight, binding to immobilized sortilin elicits only a minor signal, in terms of response units. However, compared with the signal obtained from binding of higher molecular weight proteins, competitive binding of neurotensin results in a greatly reduced signal overall. However, we were only able to block binding between sortilin and p75 NTR ecd -Fc to a minor extent, suggesting that p75 NTR predominantly binds sortilin outside the neurotensin binding area (Fig. 3D).
We next determined whether p75 NTR also interacts with the sortilin intracellular domain. To address this, we made an addi-FIGURE 2. p75 NTR binding epitopes within the extracellular and transmembrane domains of sortilin. A, representative (n ϭ 4) Western blot (WB) of immunoprecipitates (IP) and lysates of p75 NTR and sortilin tailless , lacking its intracellular domain, compared with sortilin mut . Stable cultures of HEK293 cells expressing either sortilin tailless or sortilin tailless and p75 NTR were incubated with or without proNGF and cross-linked with dithiobis succinimidylpropionate. Precipitates and crude lysates were then resolved by SDS-PAGE and Western blotted as indicated. B, schematic representation of expression constructs of sortilin and HA-p75 NTR lacking their cytoplasmic domains and fused to one of two weakly interacting subunits of ␤-galactosidase that were used in the ␤-galactosidase complementation assay. Upon dimerization of receptor chimeras, the ␤-galactosidase active site and thus activity is reconstituted. C, measurement of the luminescence of ␤-galactosidase in HEK293 cells transfected with HA-p75 NTR -and sortilin-␤-galactosidase fusion expression constructs. Co-transfected cells produced significantly higher luminescence than single-and nontransfected cells (p Ͻ 0.03). The data represent the means Ϯ S.E. of three experiments. D, representative Western blot (n ϭ 4) of crude lysates from transfectants used in C. E, luminescence of the ␤-galactosidase substrate added to cells transfected with HA-p75 NTR -and sortilin-␤-galactosidase expression constructs and incubated for 30 min with increasing concentrations of proNGF, proBDNF, or proNT3. In contrast to BSA, at 40 pM all three proNTs increased complex formation (means Ϯ S.E. of three to six experiments). tional sortilin-IL 2 R-based chimeric receptor by substituting the intracellular domain of IL 2 R with that from sortilin mut , thereby generating IL 2 R ecd,tm -sortilin mut,icd , (Fig. 3A). The chimera-encoding plasmid was then transfected into HEK293 cells together with p75 NTR , and binding of the sortilin intracellular domain was assessed by co-immunoprecipitation experiments. Binding of full-length IL 2 R to p75 NTR acted as the control. We were not able to co-immunoprecipitate IL 2 R ecd,tm -sortilin mut icd or IL 2 R, demonstrating that the intracellular domain of sortilin does not interact with p75 NTR (Fig. 3E).
Defining a Sortilin-binding Region of the p75 NTR Extracellular Domain-To map the interaction site(s) of the extracellular domain at which p75 NTR binds sortilin, co-immunoprecipitation experiments using various truncated p75 NTR variants and sortilin were carried out. p75 NTR variants include N-terminally truncated forms lacking one or more ligand binding repeats and a p75 NTR variant lacking 23 amino acids (Thr 228 -Asp 250 ) of the extracellular juxtamembrane stalk region (p75 NTR ⌬ stalk ) (Fig.  4A). Following co-immunoprecipitation, we found that all truncated p75 NTR variants bound equally well to sortilin ecd -IL 2 R tm,icd (Fig. 4B) except for p75 NTR ⌬ stalk , which had greatly reduced binding to sortilin ecd -IL 2 R tm,icd . However, in the presence of 25 nM proNGF, p75 NTR ⌬ stalk was co-immunoprecipitated with sortilin ecd -IL 2 R tm,icd (Fig. 4C). Because p75 NTR ⌬ stalk retained the ability to associate with sortilin via the shared ligand proNGF, reduced binding in the absence of proNGF cannot be ascribed to incorrect folding or improper cell surface expression of p75 NTR ⌬ stalk . Rather, these data suggest that the stalk region of p75 NTR is responsible for its constitutive interaction with sortilin.
To substantiate these results, we subjected p75 NTR ⌬ stalk and sortilin to FRET analysis. To do this, full-length p75 NTR and p75 NTR ⌬ stalk were C-terminally fused with CFP to generate p75 NTR -CFP and p75 NTR ⌬ stalk -CFP, respectively. Constructs were transiently transfected into HEK293 cells together with sortilin fused to YFP (i.e., sortilin-YFP). Because constitutive ligand-independent homodimerization of p75 NTR is not mediated through the intracellular domain (29), FRET originating from cells co-transfected with the ICD of p75 NTR fused to either CFP or YFP (i.e., p75 NTR ICD -CFP and p75 NTR ICD -YFP) served as a negative control. The extent of FRET between p75 NTR -CFP and sortilin-YFP was increased 3-fold relative to background; however, the extent of FRET between p75 NTR ⌬ stalk -CFP and sortilin-YFP was significantly lower, being equivalent to that of p75 NTR ICD -CFP and p75 NTR ICD -YFP (Fig. 4D). We also assessed the binding properties of YFP-tagged p75 NTR receptor constructs by co-immunoprecipitation using sortilin ecd -IL 2 R tm,icd as a binding partner. Similar to FRET analysis, p75 NTR ⌬ stalk -YFP had a reduced binding to sortilin ecd -IL 2 R tm,icd compared with p75 NTR -YFP (Fig. 4E). However, it is noteworthy that a deletion of the stalk region of p75 NTR abrogates binding to sortilin as measured by FRET, indicating that the extracellular domains alone mediate the binding between sortilin and p75 NTR .
Next, we sought to determine whether the 23 amino acids in the stalk region of p75 NTR alone are sufficient for binding to the sortilin extracellular domain. Co-immunoprecipitation experiments were therefore performed using a truncated variant of p75 NTR -YFP designated p75 NTR stalk,tm,icd -YFP containing only the stalk, transmembrane, and intracellular domains of p75 NTR . Because sortilin ecd -IL 2 R tm,icd was used as a binding partner, only the 23 amino acids of the p75 NTR stalk region overlap with sortilin (Fig. 4A). We found that the presence of the stalk region fully rescued binding of the p75 NTR extracellular domain with sortilin ecd -IL 2 R tm,icd (Fig. 4F). This strongly suggests that the residues Thr 228 -Asp 250 within the C-terminal stalk region of p75 NTR constitute a core-binding surface of the p75 NTR extracellular domain for sortilin.
Sortilin Intracellular Domain Regulates RIP of p75 NTR and proNGF-dependent Apoptosis-Given that the interaction between sortilin and p75 NTR maps to the extracellular domain, we sought to determine whether this domain is sufficient to affect p75 NTR function. Depending on the molecular context,  DECEMBER 21, 2012 • VOLUME 287 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 43803 one function of p75 NTR relates to its ability to induce apoptosis, which requires RIP of p75 NTR (16,17). Because sortilin is essential for proNT-dependent apoptosis through p75 NTR (5-9), we measured the accumulation of p75 NTR ICD by Western blotting within HEK293 cells transfected with wild type sortilin, sortilin mut , and sortilin tailless in the presence of proteasome inhibitors. To induce p75 NTR RIP, experiments were carried out in the presence of phorbol ester (PMA), which increases ␣-secretase activity via the protein kinase C pathway (30). Expression of any sortilin construct caused a nonsignificant increase in the amount of p75 NTR C-terminal fragment. By contrast, the sortilin constructs had significant but differential effects on p75 NTR ICD production. p75 NTR control-transfected cells produced a low but obvious amount of p75 NTR ICD. Cotransfection with wild type sortilin significantly increased the amount of p75 NTR ICD after 3 h (Fig. 5, A and B). Sortilin mut increased formation of p75 NTR ICD to an intermediate nonsignificant level, and formation of p75 NTR ICD in the presence of sortilin tailless was even more reduced.

Mapping of the Sortilin-p75 NTR -binding Site
To confirm these findings in another cellular context, we assessed the effect of sortilin on p75 NTR ICD release using the Gal4/UAS system (31). This assay is based on binding of the nuclear protein Gal4 to the UAS element, which controls the nuclear transcription of luciferase. Because Gal4 is fused to the C-terminal end of p75 NTR , only shedding and release of p75 NTR ICD-Gal4 and subsequent translocation to the nucleus induces transcription of luciferase, which can be measured by a chemiluminescent substrate (Fig. 5C). To validate our assay, HEK293 cells were stably co-transfected with p75 NTR -Gal4 and UAS-luciferase cDNA and treated with PMA. PMA significantly increased luciferase activity compared with that observed in untreated cells, consistent with the notion that PMA increases RIP of p75 NTR (Fig. 5D). Furthermore, the effect of PMA was fully inhibited in the presence of an ␣-secretase inhibitor (TAPI-2), showing that RIP of p75 NTR is required for up-regulation of luciferase transcription and activity (Fig. 5D). To test the effect of sortilin variants, p75 NTR -Gal4 and UASluciferase stably transfected HEK293 cells were transiently transfected with wild type sortilin, sortilin mut , or sortilin tailless and treated with PMA. Wild type sortilin significantly increased luciferase activity compared with that in control transfected cells, an effect observable even without PMA stimulation (Fig. 5, E and  F). Again, sortilin mut and sortilin tailless did not significantly induce p75 NTR ICD-Gal4-mediated transcription above that of the control plasmid.
We next assessed the accumulation of endogenous p75 NTR ICD in rat RN22 Schwannoma cells, which naturally express high levels of p75 NTR and have previously been demonstrated to undergo RIP (16). Because RN22 cells only express very low amounts of endogenous sortilin, we generated polyclonal stable RN22 cell lines for wild type sortilin, sortilin mut , and sortilin tailless and measured the accumulation of p75 NTR ICD by Western blotting following addition of PMA and proteasome inhibitors. As for HEK293 cells, we found that wild type sortilin significantly increased p75 NTR ICD formation, whereas sortilin mut induced a less pronounced nonsignificant accumulation of p75 NTR ICD (Fig. 6, A and B). Sortilin tailless did not affect p75 NTR ICD accumulation, which was again comparable with that observed in untransfected RN22 cells.
Because sortilin apparently regulates RIP of p75 NTR and given that RIP has been associated with neuronal apoptosis (16), we next determined the ability of wild type sortilin, sortilin mut , and sortilin tailless to regulate proNT-dependent apoptosis. We established a proNGF-induced cell death assay using polyclonal stable RN22 sortilin-expressing cell lines. Cells were then incubated with increasing amounts of proNGF, and death was assessed after 72 h using the MultiTox-Fluor multiplex cytotoxicity assay, which measures activity of proteases released from necrotic cells. Untransfected RN22 cells did not die upon proNGF stimulation. However, proNGF significantly stimulated cell death in cells transfected with wild type sortilin in a dose-dependent manner (Fig. 6C). Cells expressing sortilin mut exhibited an intermediate rate of death, whereas cells transfected with sortilin tailless were largely unaffected. This strongly suggests that the intracellular domain of sortilin regulates proNGF-dependent cell death through p75 NTR in RN22 cells.
To test the effect of sortilin on p75 NTR RIP under physiological conditions, we measured accumulation of p75 NTR ICD within primary cultures of SCG neurons isolated from sortilin knock-out mice. SCG neurons endogenously express high amounts of sortilin and p75 NTR , which has previously been shown to undergo RIP upon exposure to PMA (16). In the presence of PMA, neurons devoid of sortilin showed reduced rates of p75 NTR RIP compared with SCG neurons isolated from wild type littermates (Fig. 6D). These findings, together with the results obtained from HEK293 and RN22 cells, collectively suggest that sortilin promotes RIP of p75 NTR , resulting in increased release of p75 NTR ICD and ultimately in apoptosis.

DISCUSSION
Here we have mapped the binding site between sortilin and p75 NTR and found that the interaction relies on residues within their extracellular domains. Although we found no evidence FIGURE 4. The stalk region of p75 NTR binds sortilin. A, schematic representation of truncated and deletion p75 NTR receptor variants used to map the domain of p75 NTR required to interact with sortilin. B, representative (n ϭ 4) Western blots (WB) of immunoprecipitates (IP) and lysates of HEK293 cells that were transiently co-transfected with sortilin ecd -Il 2 R tm,icd and p75 NTR receptor variants. The most C-terminal 23 amino acids of the p75 NTR stalk region interact with sortilin. C, representative (n ϭ 3) Western blots of immunoprecipitates and lysates from HEK293 transiently expressing sortilin ecd -Il 2 R tm,icd and p75 NTR receptor variants as indicated. p75 NTR ⌬ stalk binds exogenous proNGF (25 nM) and thereby interacts with sortilin ecd -Il 2 R tm,icd . D, FRET analysis of sortilin and p75 NTR interactions. HEK293 cells were transiently co-transfected with the indicated constructs and analyzed 48 h post-transfection. Full-length p75 NTR and sortilin induced FRET above background, whereas p75 NTR ⌬ stalk did not, indicating that the stalk region of p75 NTR is required for the interaction with sortilin. The data represent the means Ϯ S.E. of three experiments. E, representative (n ϭ 3) Western blot of lysates and immunoprecipitates of sortilin ecd -IL 2 R tm,icd and YFP-tagged p75 NTR variants demonstrating that p75 NTR ⌬ stalk does not significantly interact with sortilin ecd -IL 2 R tm,icd . Constructs were transiently transfected into HEK293 cells, and p75 NTR -YFP receptor constructs were immunoprecipitated with anti-GFP antibodies. F, representative (n ϭ 3) Western blots of lysates and immunoprecipitates of sortilin ecd -Il 2 R tm,icd and YFP-tagged p75 NTR variants demonstrating that the 23 amino acids of the p75 NTR stalk region are sufficient to rescue extracellular binding of two otherwise nonoverlapping sortilin and p75 NTR constructs. that the sortilin intracellular domain interacts with p75 NTR , we found that it does regulate RIP of p75 NTR and p75 NTR -dependent cell death signaling.
The present results confirm previous findings that co-expression of sortilin and p75 NTR generates a high affinity receptor complex for proNTs, which increases p75 NTR affinity for proNTs by more than 2 orders of magnitude (5). Furthermore, the current study is the first demonstration that picomolar concentrations of proBDNF and proNT3 (ϳ40 pM) increase complex formation between sortilin and p75 NTR , similar to what has been reported for proNGF (5). Mapping of the interaction site between sortilin and p75 NTR revealed that the extracellular domain of sortilin alone is sufficient to mediate an interaction with full-length p75 NTR . Unfortunately, because of the structure of the sortilin extracellular domain (12), it is not possible to truncate the receptor further and still maintain the structural integrity of either the 10-bladed ␤-propeller or the 10CC domain. Thus, we were unable to further map the region of the extracellular domain of sortilin required for its constitutive interaction with p75 NTR . However, based on the finding that addition of a non-neurotrophin sortilin ligand, neurotensin, had little effect on the direct interaction between the extracellular domain of sortilin and p75 NTR , we conclude that the ligand-binding tunnel of the 10-bladed ␤-propeller domain of sortilin is unlikely to contain the p75 NTR interaction site and that the 10CC domain is more likely to mediate this interaction.
Using a variety of truncated p75 NTR receptor constructs, we determined that the extracellular domain of p75 NTR is alone sufficient to mediate the co-association with sortilin in the absence of ligand. Deletion of the cysteine-rich ligand-binding domains of p75 NTR had no effect on sortilin binding; however, deletion of 23 amino acids within the membrane-proximal region of the p75 NTR stalk domain significantly reduced the ability of p75 NTR to interact with sortilin. Remarkably, a construct containing only the most membrane-proximal 23 amino acids of the extracellular domain was sufficient to restore binding between p75 NTR and sortilin, indicating that this is the major sortilin-binding site of p75 NTR . These findings further support our notion that the 10CC domain of sortilin is likely to contain the p75 NTR -binding site because the 10CC domain is FIGURE 6. Sortilin intracellular domain promotes RIP of p75 NTR and proNGF-dependent cell death in RN22 cells. A, representative (n ϭ 6) Western blots (WB) of lysates from RN22 cells that were stably transfected with sortilin constructs as shown and treated for 6 h with 200 nM PMA, 200 nM compound E, or 1 M epoxomicin as indicated. B, densitometric quantification of the amount of p75 NTR ICD in each condition in A relative to the amount of full-length p75 NTR . The data represent the means Ϯ S.E. of six experiments. C, quantification of proNGF-induced cell death relative to no proNT addition in polyclonal RN22 cell lines stably expressing various sortilin constructs. The cells were incubated with proNGF as indicated for 72 h, whereupon cell death was quantified using the MultiTox-Fluor multiplex cytotoxicity assay. *, p Ͻ 0.05; **, p Ͻ 0.01. D, Western blot (n ϭ 3) of lysates from SCG neurons isolated from wild type and sortilin knock-out mice cultured in the presence of 1 M epoxomicin for 1 h followed by the addition of 200 nM PMA for 3 h. CTF, C-terminal fragment; Untransf., untransfected. DECEMBER 21, 2012 • VOLUME 287 • NUMBER 52 expected to be located relatively close to the plasma membrane surface (12).

Mapping of the Sortilin-p75 NTR -binding Site
By use of Western blotting and an assay that measures p75 NTR ICD-dependent transcription of luciferase, we analyzed the effect of sortilin on p75 NTR RIP. We found that sortilin increases RIP of p75 NTR , leading to significantly increased levels of p75 NTR ICD. Moreover, the increased RIP of p75 NTR was accompanied by an increase in cell death signaling mediated by p75 NTR . Our finding that sortilin expression facilitates the generation of the p75 NTR ICD is consistent with our data and published reports that proNTs facilitate both an interaction between p75 NTR and sortilin and the generation of a p75 NTR ICD-dependent cell death signal (5,7,8,16). Importantly, the YXXØ and dileucine trafficking motifs of sortilin were required for sortilin to increase the rate of p75 NTR RIP and to promote cell death in the presence of proNGF in RN22 cells. Interestingly, the dileucine motif together with the YXXØ motif are both required for sortilin endocytosis (13), and the sortilin mutant used herein has both of these motifs specifically mutated. The tailless sortilin mutant also lacks these sequences. Our findings that sortilin mut and sortilin tailless engage in a complex with p75 NTR but fail to stimulate RIP of p75 NTR and RN22 cell death indicate that sortilin-dependent apoptosis is the result of two events, which require high affinity binding of proNTs, as well as endocytosis of sortilin. Because generation of p75 NTR ICD occurs predominately in endosomes where ␥-secretase activity is concentrated (32,33), it is tempting to speculate that sortilin facilitates internalization of p75 NTR or fragments thereof destined for endosomes. However, additional studies would be required to elucidate the mechanism of sortilin trafficking in relation to RIP of p75 NTR and neuronal cell death.
In summary, we have mapped the binding site between sortilin and p75 NTR to the extracellular juxtamembrane stalk region of p75 NTR . The interaction between sortilin and p75 NTR is further strengthened by the pro-apoptotic proNT ligands, which promote generation of the p75 NTR ICD (16). Furthermore, sortilin-dependent release of the p75 NTR ICD correlates with proNT-dependent apoptosis facilitated by sortilin. We therefore conclude that sortilin mediates cell death not only by interacting with p75 NTR as a co-receptor for proNTs but also through generation of p75 NTR cleavage fragments and release of p75 NTR ICD.