UNC5H1 Induces Apoptosis via Its Juxtamembrane Region through an Interaction with NRAGE*

Megan E. WilliamsDagger , Phyllis StricklandDagger , Ken Watanabe§, and Lindsay HinckDagger

From the Dagger  Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, California 95064 and § Department of Geriatric Research, National Institute for Longevity Sciences, Aichi 474-8522, Japan

Received for publication, January 14, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The UNC5Hs are axon guidance receptors that mediate netrin-1-dependent chemorepulsion, and dependence receptors that mediate netrin-1-independent apoptosis. Here, we report an interaction between UNC5H1 and NRAGE. Our experiments show that this interaction is responsible for apoptosis induced by UNC5H1, and this level of apoptosis is greater than the amount induced by either UNC5H2 or UNC5H3. We mapped the NRAGE binding domain of UNC5H1 to its ZU-5 domain and show that this region, in addition to an adjacent PEST sequence, is required for UNC5H1-mediated apoptosis. Chimeric UNC5H2 and UNC5H3 receptors, containing the NRAGE binding domain and PEST sequence of UNC5H1, bind NRAGE and cause increased levels of apoptosis. UNC5H1 expression does not induce apoptosis in differentiated PC12 cells, which down-regulate NRAGE, but induces apoptosis in native PC12 cells that endogenously express high levels of NRAGE and in differentiated PC12 cells when NRAGE is overexpressed. Together, these results demonstrate a mechanism for UNC5H1-mediated apoptosis that requires an interaction with the MAGE protein NRAGE.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Apoptosis plays a critical role in determining the size and shape of the vertebrate nervous system (1). The execution phase of the apoptotic program in neurons is well characterized and, as with most cell types, depends on the activation of intracellular proteases, primarily caspases (2). In contrast, it is not well understood how cues from the environment regulate this process during development of the nervous system.

UNC-5 was originally characterized in Caenorhabditis elegans as a gene required for axonal repulsion in netrin/UNC-6 responsive neurons (3, 4). The vertebrate members of this family (UNC5H1, 2, and 3) (5, 6), together with C. elegans UNC-5 (4) and Drosophila Unc5 (7), comprise a subgroup of the Ig superfamily of receptors. The UNC5s contain two Ig and two thrombospondin type-I repeats in the extracellular domain. In addition, their cytoplasmic domains contain regions of homology with other proteins: 1) a ZU-5 domain homologous to Zona Occludens-1, a protein implicated in tight-junction formation (8); and 2) a C-terminal death domain, a domain first identified as the pro-apoptotic region of tumor necrosis factor receptor-1 (9, 10). In a netrin-1 gradient, a complex of UNC5H1 and DCC mediates repulsion (11), although there is evidence suggesting that short range repulsion by netrin-1 may be mediated by UNC5 alone (3, 7).

NRAGE (Dlxin-1, MAGE-D1) is a recently identified molecule belonging to the MAGE (melanoma antigen) protein family. There are currently over 25 MAGE proteins in humans, characterized by the presence of a MAGE homology domain. The expression of many MAGE proteins is restricted to cancer cells (12); however, recent studies have revealed a role for two MAGE proteins in the nervous system. One MAGE family member, necdin, is thought to maintain the differentiated state of post-mitotic neurons by preventing entry into the cell cycle (13, 14). Another MAGE family member, NRAGE, is expressed in the nervous system during early development in proliferative neural populations (15). Recent studies have reported two major functions for NRAGE, as a transcriptional regulator for the dlx/msx family of transcription factors (16, 17) and as a regulator of apoptosis. The first study to implicate NRAGE in apoptosis found that NRAGE binds the nerve growth factor receptor p75NTR, blocks cell cycle progression, and promotes p75NTR-mediated apoptosis (18). Subsequently, it was found to utilize two mechanisms to induce apoptosis in cells. One involves NRAGE-dependent degradation of the survival protein XIAP (X-linked inhibitor of apoptosis) (19) and the other involves NRAGE-dependent activation of the c-Jun N-terminal kinase signaling pathway and caspases (20).

Recently a role has begun to emerge for netrin-1 receptors in apoptosis. In this paper, we report that UNC5H1 mediates more than twice the amount of cell death as UNC5H2 and UNC5H3, and we identify NRAGE as a specific binding partner for UNC5H1. UNC5H1 binds NRAGE in vitro and can be co-immunoprecipitated from cells that endogenously express both proteins. Both the apoptotic region and NRAGE binding domain of UNC5H1 map to its juxtamembrane region that contains a PEST sequence and ZU-5 domain. We find that chimeric UNC5H2 and UNC5H3 proteins containing the PEST and ZU-5 sequence of UNC5H1 bind NRAGE and induce increased levels of apoptosis. UNC5H1 and NRAGE co-localize at the cell membrane in heterologous cells and are co-expressed in several regions of the developing nervous system. Using PC12 cells, we show that UNC5H1 expression induces apoptosis in native, mitotically active cells that endogenously express high levels of NRAGE but not in differentiated cells that sharply down-regulate NRAGE. We also show that UNC5H1 induces apoptosis in these differentiated PC12 cells when NRAGE is over-expressed. Taken together our data identify a novel signaling mechanism for UNC5H1-mediated apoptosis that requires an interaction with NRAGE.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Constructs and Reagents-- Full-length rat unc5h1 and unc5h2 were cloned into pSecTagB (Invitrogen), which contains a C-terminal myc tag as described previously (6, 11). All other unc5h mutant constructs were generated in pSecTagB by PCR cloning using the flanking restriction sites HindIII to XbaI and placed in-frame with the myc tag included within the vector. H2/H1apo and H3/H1apo were constructed using PCR to delete the juxtamembrane region of UNC5H2 (amino acids Asp407-Cys625) and UNC5H3 (amino acids Glu410-Cys612), and this region was replaced with a NotI site. Then, the apoptotic region of UNC5H1 (amino acids Leu391-Cys579) was generated by PCR with flanking NotI sites and inserted into the new NotI site in unc5h2 and unc5h3. unc5h3 was also cloned in-frame with the myc tag of pSecTagB using the EcoRI and XbaI sites. Full-length mouse nrage was cloned into pcDNA3 (Invitrogen) with a 5' HA1 tag as described previously (16). unc5h1myc and HA-nrage, including a consensus Kozak and start site, were cloned into the sindbis virus vector pSinRep5 (Invitrogen). All PCR products were verified by sequencing.

Cell Culture and Transfections-- COS cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and transfected using FuGENE (Roche Molecular Biochemicals). PC12 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 5% fetal bovine serum, and 30% PC12 conditioned medium and grown on collagen-coated dishes. Cells were differentiated for 14 days by incubating in serum-free Dulbecco's modified Eagle's medium with 50 ng/ml purified NGF (Sigma). sindbis virus expressing UNC5H1 was packaged in baby hamster kidney cells as described by the manufacturer's protocol (Invitrogen), and undiluted virus was used to infect native and differentiated PC12 cells.

Immunostaining and Antibodies-- Cells were fixed for immunostaining in 4% PFA + 0.1% Triton X-100 for 20 min, rinsed in PBS, and blocked in PBS + 3% heat-inactivated goat serum + 0.1% Triton X-100 for 30 min. Primary antibody in blocking buffer was added for 60 min at room temperature followed by three washes for 5 min each. Fluorescent secondary antibody was diluted and incubated on cells for 40 min and washed three times before coverslipping. To determine the percent of pyknotic nuclei using DAPI stain, cells were incubated in DAPI diluted in PBS to 1 µg/ml for 5 min before coverslipping. Cells were visualized under fluorescence, and a total of at least 300 transfected cells were scored for pyknotic nuclei and apoptotic morphology. Each transfection was repeated and scored at least three times in a blind manner by multiple researchers.

The UNC5H1 antibody 6E9 was raised against the extracellular domain of UNC5H1.2 The NRAGE rabbit polyclonal was used in Western blotting at 1:200 (16). Anti-myc 9E10 and anti-HA 12CA5 were used for both immunostaining and Western blotting at 1 µg/ml.

Yeast Two-hybrid-- The E18 mouse brain library was made according to manufacturer's instructions using the Stratagene cDNA synthesis kit and ligating into the EcoRI/XhoI sites in pACT2 (Clontech), which contains a Gal4 activating domain. H1ICD was cloned by PCR into the EcoRI/SalI sites of pBTM116 in-frame with the LexA DNA binding domain (11). DNA was transformed into the L40 yeast strain stably transformed with a LexA-driven HIS3 gene and LexA-driven LacZ gene (21). Transformants were selected for the ability to grow on minus histidine plates supplemented with 30 mM 3-amino1,2,4-triazole and subsequently screened for the ability to produce LacZ.

In Vitro Translation and GST Pull-downs-- Rip60NRAGE in pcDNA3 was translated and labeled with [35S]methionine using the in vitro translation system (Promega). In vitro translated rip60NRAGE was incubated in buffer (0.5% Nonidet P-40, 10 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 5 mM dithiothreitol, protease inhibitors) with 5 µg of GST-H1ICD or GST control bound to glutathione-agarose beads. The samples were rocked for 2 h at 4 °C, washed three times with binding buffer, and fractionated by SDS-PAGE for autoradiography.

Immunoprecipitation and Western Blotting-- COS or PC12 cells were immunoprecipitated as described (11). Briefly, cells were incubated in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% TritonX-100, 10% glycerol) containing aprotinin, leupeptin, and phenylmethylsulfonyl fluoride at 1 µg/ml each. The sample was rocked at 4 °C for 40 min and then pelleted in a microfuge at 14,000 rpm (full speed) for 20 min. The supernatant was then incubated with antibody prebound to protein A/G (Santa Cruz Biotechnology, Inc.) for 6 h at 4 °C. Western blots were visualized using ECL detection (Amersham Biosciences).

In Situ Hybridizations-- A portion of the rat unc5h1 intracellular domain (1480-2080 bp) was subcloned from cDNA into pBlueScript II. Similarly, the N terminus (1-873 bp) and C terminus (2025-2325 bp) of nrage cDNA were cloned into pBlueScript II. These constructs were used to make digoxigenin-labeled riboprobes.

Brain tissue was prepared by immersion in 4% PFA for 2 h at room temperature, infiltrated with 30% sucrose, embedded in tissue freezing medium (Triangle Biomedical Sciences), and frozen at -80 °C. 20-µm sections were brought to room temperature and post-fixed in 4% PFA. Next, sections were digested with 10 µg/ml proteinase K for 2 min, stopped in 0.2% glycine, and fixed in 4% PFA for 5 min. Tissue was acetylated in acetic anhydride for 10 min and permeabilized with PBS with 0.1% Triton X-100 for 30 min. Prehybridization solution (50% deionized formamide, 4× SSC, 1× Denhardt's, 1 mg/ml tRNA, 0.5 mg/ml herring sperm DNA) was added for 2 h at room temperature. Hybridization solution including either digoxigenin-labeled UNC5H1 (900 ng/ml) or NRAGE (500 ng/ml) was added to serial sections and incubated overnight at 65 °C. Washes in 2× SSC/50% formamide for 1 h and 0.2× SSC for 1 h were followed by standard anti-digoxigenin detection protocols (Roche Molecular Biochemicals) with detection in BM Purple.

Statistics and Image Analysis-- Statistical analyses including ANOVA with Tukey's post-test for multiple comparisons were performed using R version 1.6.1 and Microsoft Excel. NIH image 1.63 software was used to determine relative protein levels following Western analysis and enhanced chemiluminescence detection.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

UNC5H1 Expression Induces Apoptosis at Significantly Higher Levels Than Its Vertebrate Homologues, UNC5H2 and UNC5H3-- Based on earlier observations that the UNC5Hs may be involved in apoptotic processes (11, 22), we directly assessed the ability of UNC5H1, UNC5H2, and UNC5H3 to induce apoptosis in heterologous cells. Apoptosis was assayed by immunohistochemistry to identify transfected cells and DAPI labeling to identify pyknotic nuclei, a standard indicator of apoptosis. The identification of pyknotic nuclei is not affected by differences in the efficiency of transient transfections, because cells are observed at single cell resolution and equal numbers of transfected cells are counted. GFP was transfected as a control to determine the basal rate of death due to culturing, transfecting, and overexpressing protein in COS cells. Fig. 1A shows a representative image of UNC5H1 expressing cells that display condensed, fragmented nuclei and loss of adhesion, indicative of dying cells. This cell morphology is distinctly different compared with cells expressing UNC5H2 and UNC5H3 that maintain extensive cell processes and intact nuclei, indicative of healthy cells (Fig. 1A). Quantification of apoptotic nuclei reveals that UNC5H1 expression causes a dramatic increase in apoptotic cells (69%) compared with the GFP control (12%) (p = 0.001; Fig. 1B). UNC5H2 (24%) and UNC5H3 (16%) induce apoptosis over control cells (p = 0.01 for each; Fig. 1B) but at significantly lower levels than UNC5H1. Because UNC5H1 expression induces the most robust apoptosis in our assays, we focused on understanding the mechanism underlying the ability of UNC5H1 to induce apoptosis.


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Fig. 1.   UNC5H1 expression induces apoptosis. A, COS cells expressing UNC5H1myc (a and b), UNC5H2myc (c and d), or UNC5H3myc (e and f) were immunostained with anti-Myc antibody to visualize UNC5Hs (a, c, and e, red) and simultaneously with DAPI to visualize nuclei (b, d, and f, blue). Representative examples of pyknotic (b) and normal (d and f) nuclei are shown in the upper left-hand boxes. Scale bar, 10 µm. B, the percent of apoptosis in cells expressing each UNC5H are shown. All transfected cells within multiple non-overlapping fields of view (at least 300 cells) were counted and scored for apoptosis based on pyknotic nuclear morphology by DAPI staining. Error bars indicate S.D. (n >=  3).

The Apoptotic Domain of UNC5H1 Interacts with the Pro-apoptotic MAGE Protein NRAGE-- To understand the signaling mechanism used by UNC5H1, a yeast two-hybrid screen was performed on a cDNA library generated from E18 murine brain tissue using the intracellular domain of UNC5H1 as bait. From 14 positive clones, we identified one, rip60NRAGE, as the C-terminal half of the pro-apoptotic protein NRAGE (Dlxin-1, MAGE-D1) (16, 18). The NRAGE amino acid sequence contains a repeat region consisting of 25 repeats of a WQXPXX consensus sequence followed by a MAGE homology domain. The rip60NRAGE clone begins at amino acid 411 and continues through the poly(A) tail of NRAGE and contains five WQXPXX repeats, the entire MAGE domain, and the C-terminal tail (Fig. 2A).


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Fig. 2.   UNC5H1 interacts with NRAGE. A, schematic diagram showing the interaction in yeast of NRAGE deletion constructs with H1ICD, and UNC5H1 deletion constructs with rip60NRAGE. "++" indicates an excellent ability to grow on histidine selection, "+" indicates some growth, and "-" indicates no growth. B, GST or GST-H1ICD were incubated with in vitro transcribed and translated [35S]rip60NRAGE. Glutathione-agarose beads were used to pull down the GST constructs, the samples were separated by SDS-PAGE, and autoradiography was used to detect co-precipitating rip60NRAGE (upper panel). The Coomassie-stained gel shows that equal amounts of both GST proteins were used (lower panel). C, COS cells were transfected with HA-nrage and the indicated unc5h myc-tagged constructs. After 24 h, cells were immunoprecipitated with anti-myc antibodies. The upper panels show the co-immunoprecipitated NRAGE. The membranes were stripped and reprobed with anti-myc (middle panels). The bottom panels show equal amounts of cell lysates blotted with anti-HA as a control. "*" indicates the UNC5H intracellular cleavage product D, COS cells were transfected and immunostained for HA-NRAGE (a, green) and UNC5H1myc (b, red). Merged images indicate overlapping expression (c, yellow). Scale bar, 10 µm.

The interacting domains between UNC5H1 and NRAGE were mapped in yeast. The C-terminal tail of NRAGE, the last 100 amino acids, is required for binding the intracellular domain of UNC5H1 (H1ICD), but is not sufficient to interact on its own (Fig. 2A). Neither the WQXPXX repeat nor the MAGE domain alone were sufficient to interact with H1ICD; however, adding the C-terminal tail back onto the MAGE domain restores binding (Fig. 2A). Therefore, the C-terminal tail of NRAGE may directly bind UNC5H1, or the tail is needed for the MAGE domain to fold properly, allowing the MAGE domain to directly bind H1ICD.

Similarly, we mapped the NRAGE binding site of UNC5H1. Deletions of H1ICD from the C terminus, including the death domain, interact with rip60NRAGE (Fig. 2A). Increasingly, C-terminal truncations of UNC5H1 interacted less well, possibly because they were subject to degradation. Therefore, we generated several N-terminal truncations of H1ICD and tested them for the ability to interact with rip60NRAGE. We found that removing the ZU-5 domain abolished binding with rip60NRAGE (Fig. 2A) and conclude that this region is necessary for the interaction between UNC5H1 and NRAGE.

UNC5H1 possesses a unique ability to induce apoptosis at significantly higher levels than either UNC5H2 or UNC5H3 (Fig. 1B). Therefore, we used the yeast two-hybrid system to test whether the NRAGE interaction is specific to UNC5H1 or whether it interacts with other UNC5Hs. Fig. 2A shows that, in yeast, rip60NRAGE does not interact with either UNC5H2 or UNC5H3 and thus, our results suggest that UNC5H1 interacts specifically with NRAGE through its ZU-5 domain.

To confirm that the UNC5H1-NRAGE interaction in yeast is the result of a direct protein-protein interaction, we performed an in vitro GST pull down assay. A purified GST-H1ICD fusion protein revealed a strong interaction with in vitro translated rip60NRAGE, whereas GST alone shows little nonspecific binding, indicating that the interaction between UNC5H1 and NRAGE is direct (Fig. 2B).

Next, we tested whether UNC5H1 interacts with NRAGE in cells by co-immunoprecipitation. COS cells were transiently transfected with full-length unc5h1myc and full-length HA-nrage. Fig. 2C shows that immunoprecipitating with anti-Myc pulls down HA-NRAGE only when UNC5H1myc is present and not when cells are transfected with empty vector. Deleting the death domain of UNC5H1 (H1Delta DDmyc) does not affect the interaction with HA-NRAGE in cells, but deleting the entire intracellular domain of UNC5H1 (H1Delta ICDmyc) abolishes the interaction (Fig. 2C). These results confirm our observations in yeast and identify the ZU-5 domain as the NRAGE binding domain. To explore the specificity of the interaction between NRAGE and UNC5H1 in cells, we also tested whether UNC5H2myc or UNC5H3myc co-immunoprecipitate with HA-NRAGE. Our results show that UNC5H2myc binds HA-NRAGE but at consistently lower levels than UNC5H1myc (Fig. 2C). Quantification of NRAGE from Western blots shows that UNC5H2myc co-immunoprecipitates ~70% (±13) less NRAGE than UNC5H1myc, and no interaction between UNC5H3myc and HA-NRAGE is detected (Fig. 2C).

Last, we examined the cellular localization of UNC5H1 and NRAGE in COS cells. Previous studies have shown that NRAGE is localized to multiple cellular compartments, including the cytosol, nucleus, and at the cell membrane (16, 18). Because UNC5H1 is a transmembrane protein, we examined whether NRAGE is localized to the membrane in the presence of UNC5H1. Co-expression of UNC5H1myc and HA-NRAGE in COS cells shows strong co-localization between the two proteins at the cell membrane (Fig. 2D).

UNC5H1 and NRAGE Display Overlapping Expression Patterns in Vivo-- For the interaction between UNC5H1 and NRAGE to be physiologically relevant, the two proteins must be present in the same place at the same time. It is already known that both unc5h1 and nrage are highly expressed in the nervous system (6, 15, 16, 18, 23, 24). To determine specific sites of overlapping expression, we performed in situ hybridization analysis on serial sections from embryonic brain tissue. unc5h1 and nrage mRNA are found together in the E16 striatum and both are largely excluded from the neighboring subventricular zone (Fig. 3, A and C). The outer layers of the E16 cortex, including the cortical plate and the marginal zone, stain strongly for both unc5h1 and nrage transcripts (Fig. 3, E and F). We have noted several other sites of overlapping expression during development, including the E18 hippocampus (Fig. 3, G and H) and olfactory bulb and E11 motor neurons (data not shown). These data demonstrate that unc5h1 and nrage are co-localized in the nervous system, suggesting they may function together in vivo.


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Fig. 3.   UNC5H1 and NRAGE have overlapping expression patterns in vivo. In situ hybridization reveals UNC5H1 and NRAGE expression in the E16 striatum (A and C), E16 cortex (E and F), and E18 hippocampus (G and H). Sense controls for UNC5H1 (B) and NRAGE (D) are shown. Scale bars, 200 µm. SVZ, subventricular zone; st, striatum; CP, cortical plate; VZ, ventricular zone; D, dorsal; V, ventral; M, medial; L, lateral.

UNC5H1 Mediates Apoptosis via Its Juxtamembrane Region That Includes a PEST Sequence and the NRAGE Binding Domain-- The initial observation that UNC5H1 induces apoptosis was made in COS cells. Therefore, if NRAGE is required for UNC5H1-mediated apoptosis, COS cells must express NRAGE endogenously. To test this hypothesis, we performed a Western blot on COS cell lysate using a polyclonal antibody against NRAGE. Fig. 4A shows that NRAGE appears to be abundant in COS cell lysates, and thus we decided to perform a structure/function analysis on the ability of UNC5H1 to induce apoptosis in these cells.


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Fig. 4.   The juxtamembrane region of UNC5H1 is required for full apoptotic signaling and enhances apoptosis by UNC5H2 and UNC5H3. A, 25 µg of lysate from untransfected COS cells was probed using an anti-NRAGE antibody. Lysate from COS cells transfected with HA-nrage was used as a positive control. B and C, the percent of apoptosis in cells expressing each UNC5H1 mutant are graphed, and a schematic representation of the constructs is shown above each bar graph. All transfected cells within multiple non-overlapping fields of view (at least 300 cells) are counted and scored for apoptosis based on pyknotic nuclear morphology by DAPI staining. Error bars indicate S.D. (n >=  3). "*" indicates p < 0.05, "**" indicates p < 0.005 compared with wild type UNC5Hs. One-way ANOVA with Tukey's post-test to compare multiple groups computes an overall significant p value of <1.0-10. D, COS cells were transfected with HA-nrage and the indicated unc5h myc-tagged constructs. After 24 h, cells were immunoprecipitated with anti-Myc antibodies. The upper panel shows co-immunoprecipitated NRAGE using an anti-HA antibody. The membrane was then stripped and reprobed with anti-Myc. The bottom panel shows equal amounts of cell lysates blotted with anti-HA as a control.

To examine whether the interaction between UNC5H1 and NRAGE is required for cell death, we constructed several mutants of UNC5H1 and assayed these constructs for the ability to induce apoptosis. First, we assayed H1Delta sac, a truncation that removes much of the intracellular domain, and found that H1Delta sac induces significantly less apoptosis than full-length UNC5H1 (p = 0.002; Fig. 4B). Although this mutant removes the NRAGE binding domain, it also removes the death domain, a region of UNC5H1 shown by others to be required for UNC5H1-mediated apoptosis (22). To address this, we asked whether a construct deleted in only the death domain (H1Delta DD) induces apoptosis. Our results show that H1Delta DD does not significantly impede the ability of UNC5H1 to induce death in COS cells (Fig. 4B). Next, we made an UNC5H1 construct deleted in the DCC binding domain (H1Delta DB), because the UNC5H1 intracellular domain interacts with the netrin-1 receptor DCC (11), which may also be pro-apoptotic (25). One possibility is that cell death induced by UNC5H1 is the result of a signal through endogenous DCC. Our results show that deleting the DCC binding domain has no effect on the ability of UNC5H1 to induce apoptosis (Fig. 4B). Together, these results indicate that the ZU-5/NRAGE binding domain is required for UNC5H1-mediated apoptosis and rule out a role for both the death and DCC binding domains.

We dramatically decreased apoptosis induced through UNC5H1 by deleting the NRAGE binding domain; however, we noticed that H1Delta sac still causes apoptosis above the level in control cells expressing GFP (Fig. 4B). Therefore, we removed the entire intracellular domain of UNC5H1 (H1Delta ICD) to determine whether this completely abrogates the death signal. Fig. 4B shows that H1Delta ICD eliminates UNC5H1 apoptosis to control levels. Sequence analysis of this 116-amino acid region between the end of the transmembrane domain and the start of the ZU-5 domain revealed the presence of a PEST sequence that is not conserved in either UNC5H2 or UNC5H3 (26, 27). When we delete the PEST domain (H1Delta PEST) the UNC5H1 apoptotic signal is eliminated. These results suggest that most of the UNC5H1 death signal requires the presence of the NRAGE binding domain (ZU-5); however, some signaling requires only the unique PEST sequence of UNC5H1. Taken together, we identified the juxtamembrane region, consisting of the adjacent PEST and ZU-5 domains, as the primary signaling region in UNC5H1-mediated apoptosis and NRAGE binding.

UNC5H1 expression induces the highest percent of apoptosis (69%) in cells whereas UNC5H2 expression induces less than half (24%), and UNC5H3 expression induces even less (16%) (Fig. 1B). The same pattern is true for the UNC5Hs ability to interact with NRAGE. UNC5H1 directly binds NRAGE whereas UNC5H2 binds NRAGE relatively weakly, and UNC5H3 shows no binding (Fig. 2C). These results suggest that the ability of UNC5Hs to induce apoptosis depends on their interaction with NRAGE. Thus, we asked whether the apoptotic domain of UNC5H1, when present on the homologous receptors, enhances either the apoptotic signal or NRAGE binding. To accomplish this, we constructed two chimeric receptors (H2/H1apo and H3/H1apo) in which the juxtamembrane region including the ZU-5 domain of UNC5H2 and UNC5H3 was replaced with the juxtamembrane region from UNC5H1 (Fig. 4C). Our data show that the H2/H1apo chimera significantly increases the percent of apoptotic cells compared with wild type UNC5H2 to levels close to full-length UNC5H1 (p < 0.0001; Fig. 4C). The H3/H1apo chimera produced a small but significant increase in the percent of apoptotic cells compared with wild type UNC5H3 (Fig. 4C, p < 0.01). These results suggest that the apoptotic region defined here, consisting of the PEST sequence and ZU-5 domain, is a pro-apoptotic signaling determinant specific to UNC5H1.

Next, we determined whether myc-tagged chimeric receptors, H2/H1apo and H3/H1apo, co-immunoprecipitate with HA-NRAGE. Because the apoptotic domain of UNC5H1 includes the interaction domain with NRAGE, we reasoned that the chimeric receptors, which show an increased ability to induce cell death, would show an enhanced affinity for NRAGE. In support of this hypothesis, both H2/H1apo and H3/H1apo show significantly increased NRAGE binding compared with their wild type homologues (Fig. 4D). We find that H2/H1apo binds an average of 5.4 (±3.0) times more NRAGE than UNC5H2. We also find that H3/H1apo interacts strongly with NRAGE whereas UNC5H3 does not (Fig. 4D). Although both chimeric molecules show a strong interaction with NRAGE and induce a significant amount of apoptosis, the increase in death is much less for H3/H1apo than for H2/H1apo. This suggests that UNC5H3 is different from UNC5H1 and UNC5H2, and factors other than the inability to bind NRAGE may prevent UNC5H3 from inducing robust apoptosis (Fig. 1B). The experiments on chimeric receptors, which cause both an increase in apoptosis and an increase in the ability to bind NRAGE over wild type receptors, strongly support the idea that NRAGE binding facilitates UNC5H1-mediated apoptosis.

UNC5H1 Interacts with NRAGE and Induces Apoptosis in Native but Not Differentiated PC12 Cells-- To determine whether UNC5H1 interacts with NRAGE in untransfected cells, we used PC12 cells, because they endogenously express both proteins (18, 23)3 and can be differentiated using NGF. First, to examine endogenous UNC5H1 protein, we characterized the specificity of the monoclonal antibody 6E9. Using Myc-tagged constructs, we show that 6E9 specifically immunoprecipitates UNC5H1myc but not UNC5H2myc or UNC5H3myc (Fig. 5A). Next, we immunoprecipitated equal amounts of native and 14-day differentiated PC12 cells with anti-UNC5H1 (6E9) and Western blotted with a rabbit polyclonal against NRAGE. Fig. 5B shows that NRAGE specifically co-immunoprecipitates with anti-UNC5H1 in native PC12 cells but not in differentiated PC12 cells. Thus, UNC5H1 and NRAGE interact when expressed at physiological levels in mitotically active but not in differentiated PC12 cells.


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Fig. 5.   Endogenous UNC5H1 and NRAGE interact in PC12 cells. A, COS cells were transfected with myc-tagged unc5h1, 2, or 3. Cells were then immunoprecipitated and Western blotted using anti-Myc antibodies as a positive control or immunoprecipitated with UNC5H1 monoclonal antibody 6E9 and blotted using anti-Myc antibodies. Only cells expressing UNC5H1 and not cells expressing UNC5H2 or UNC5H3 were immunoprecipitated using 6E9. B, co-immunoprecipitation of endogenous UNC5H1 and NRAGE. Native (nPC12) and 14-day NGF-differentiated (dPC12) PC12 cells were immunoprecipitated with anti-UNC5H1 (6E9) antibody and Western blotted with an anti-NRAGE antibody (top panel). Endogenous NRAGE expression in native versus differentiated PC12 cells is shown by Western blotting cell lysates with an anti-NRAGE antibody (middle panel). The total protein concentration for each lysate was determined by Bradford assays, and equal protein for each lysate was loaded as indicated by the Coomassie-stained gel (lower panel).

A Western blot using an anti-NRAGE antibody on native and differentiated PC12 lysates reveals that NRAGE is down-regulated after differentiation (Fig. 5B). After 14 days of NGF treatment, differentiated PC12 cells express 70% (±10) less NRAGE than native PC12 cells, and this may explain why NRAGE is not co-immunoprecipitated with UNC5H1 in differentiated cells. Because NRAGE is down-regulated in differentiated PC12 cells, we hypothesized that UNC5H1 overexpression would induce apoptosis in native PC12 cells, similar to COS cells, but not in differentiated PC12 cells. To test this, we infected PC12 cells with UNC5H1myc using sindbis virus and examined apoptosis using Tdt-mediated dUTP nick-end labeling (TUNEL). The experiment revealed a striking difference in the percent of apoptosis between UNC5H1 expressing native and differentiated PC12 cells. 82% of the native PC12 cells expressing UNC5H1 were TUNEL-positive compared with just 3% of native cells infected with GFP as a control (Fig. 6, A and B). In contrast, only 1% of differentiated PC12 cells expressing UNC5H1 are apoptotic (Fig. 6, A and B). Because differentiated PC12 cells down-regulate NRAGE, we used sindbis virus to overexpress HA-NRAGE in these cells. We found that expression of NRAGE alone resulted in a low level of TUNEL-positive nuclei (10%). Interestingly, although overexpression of NRAGE in differentiated cells did not cause much cell death, the cells appear to have retracted their processes that were induced upon differentiation. In contrast, co-expression of NRAGE and UNC5H1 resulted in a significant increase in the percent of TUNEL-positive cells (48%) (Fig. 6, A and B). These experiments strongly suggest that NRAGE expression is required for UNC5H1 to induce an apoptotic signal.


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Fig. 6.   UNC5H1 induces apoptosis in native but not differentiated PC12 cells. A, native (nPC12) and 14-day-differentiated (dPC12) PC12 cells infected with UNC5H1myc or HA-NRAGE sindbis virus were immunostained for UNC5H1 (green) and NRAGE (blue) expression and apoptotic nuclei using TUNEL (red). Merged images are shown. Scale bar, 50 µm. B, the percent of TUNEL-positive nuclei for native and differentiated PC12 cells expressing UNC5H1, NRAGE, and GFP was determined. All infected cells within multiple non-overlapping fields of view are counted and scored for apoptosis using the TUNEL assay. Error bars indicate standard deviation (n = 3; "*" indicates p < 0.0001 compared with GFP control).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This paper provides the first evidence of an interaction between UNC5H1 and NRAGE. Currently, very little is known about the immediate downstream signals induced by any of the UNC5Hs in either apoptosis or axon guidance. The identification of NRAGE as a binding partner for UNC5H1 provides further clues toward understanding the mechanism of UNC5H1-mediated apoptosis, because studies have recently begun to shed light on NRAGE signaling.

Our analysis of the role UNC5Hs play in apoptosis first revealed a striking difference between the ability of UNC5H1 to induce death compared with UNC5H2 or UNC5H3. UNC5H1 induces apoptosis at more than twice the level of UNC5H2, four times greater than UNC5H3, and six times greater than controls. We also found that UNC5H1 interacts with NRAGE significantly stronger than UNC5H2 or UNC5H3. Through the use of chimeric receptors, we show that both of these properties, apoptosis induction and NRAGE binding, specifically require the juxtamembrane region of UNC5H1. The juxtamembrane region of UNC5H1 consists of a short PEST sequence immediately followed by a ZU-5 domain. In contrast, UNC5H2, UNC5H3, and C. elegans UNC-5 do not contain a PEST sequence and have an insertion of ~20 amino acids in length preceding the ZU-5 domain. These sequence differences may explain the discrepancies between the UNC5Hs ability to bind NRAGE and induce apoptosis.

We find that the UNC5H1 PEST sequence, amino acids His568-Arg480, is capable of inducing significant apoptosis even when the remainder of the intracellular domain is deleted. PEST sequences, stretches of amino acids rich in proline, glutamic acid, serine, and threonine, have been shown to function in a variety of cellular processes, most notably in promoting protein degradation by the proteosome (26, 27). It is intriguing that the NGF receptor p75NTR also contains a PEST sequence, and, like UNC5H1, the PEST is located near the transmembrane region (28). This region on p75NTR, termed Chopper to distinguish it from the death domain, is capable of inducing apoptosis in cells (28). UNC5H1 and p75NTR both require their juxtamembrane region for the interaction with NRAGE (18). Thus, UNC5H1 and p75NTR display several structural similarities, and it is possible that NRAGE induces apoptosis through similar mechanisms for both receptors.

The identification of the juxtamembrane region of UNC5H1 as a required component for full apoptotic signaling and NRAGE binding was initially surprising, because UNC5H1 contains a C-terminal death domain that has been shown to be required for apoptosis (22). Death domains received their name, because they are responsible for apoptosis induced by the canonical death receptors, tumor necrosis factor receptor-1 and Fas (9, 29, 30). Since then, additional death domain containing proteins have been identified. Some of these proteins function in diverse cellular processes unrelated to apoptosis (e.g. ankyrin, NFkappa B, human N5), and others can induce apoptosis independent of their death domain (e.g. p75NTR, UNC5H1) (10, 28, 31-33). Therefore, it may be more appropriate to think of the death domain of UNC5H1 in more general terms as a protein-protein interaction domain.

Several studies show that NRAGE functions as a pro-apoptotic molecule. It interacts with p75NTR to induce NGF-dependent apoptosis (18), and subsequent studies identified two different mechanisms underlying NRAGE-mediated apoptosis. One is by promoting the degradation of the survival protein XIAP (X-linked inhibitor of apoptosis) (19), and the second is by activating the c-Jun N-terminal kinase and caspase signaling pathway, both of which are known signaling cascades for programmed cell death (20). It is not known whether these two mechanisms operate independently, perhaps in different cell types, or whether NRAGE can induce multiple signals to ensure the demise of the cell. We find that native PC12 cells, which express endogenous UNC5H1 and NRAGE, also express relatively high levels of XIAP (data not shown). The presence of this pro-survival protein may be one reason why native PC12 cells do not undergo apoptosis despite expressing endogenous levels of UNC5H1 and NRAGE. In such a model, the overexpression of UNC5H1 may initiate a strong apoptotic signaling cascade via NRAGE that degrades or silences the XIAP survival signal, leading to cell death in native PC12 cells.

NRAGE also regulates transcription through the dlx/msx homeodomain family of transcription factors (16, 17). NRAGE protein can be found in the cytosol and nucleus, possibly facilitating an interaction with transcriptional regulators. When NRAGE is co-expressed with UNC5H1, however, it is primarily found at the cell membrane, seemingly sequestered away from transcription factors in the nucleus. Interestingly, we find that UNC5H1 undergoes a specific cleavage by calpain to release the entire intracellular domain into the cytosol (Fig. 2C) (data not shown). Thus, cleavage of UNC5H1 may provide a way for an UNC5H1·NRAGE complex to communicate with transcriptional regulators.

NRAGE is primarily expressed in proliferative neural subpopulations and not in differentiated neurons (15). Similarly, we show that NRAGE expression is relatively high in native PC12 cells but down-regulated in differentiated PC12 cells. This decrease in NRAGE expression likely explains why UNC5H1 overexpression alone does not induce apoptosis in differentiated neurons. We show the requirement for NRAGE in UNC5H1-mediated apoptosis by overexpressing NRAGE with UNC5H1 in these cells and demonstrating that, together, they induce apoptosis. Prior to differentiation, many neural precursors are rapidly proliferating and competing for survival factors. These periods in development are often accompanied by apoptosis to ensure that appropriate cell numbers are maintained. Apoptosis in response to external survival cues may require a receptor, such as UNC5H1, and the presence of NRAGE to transmit the death signal. Once a cell has begun to differentiate, studies have shown that some neurons use UNC5Hs to mediate axon guidance or cell migration (3, 7, 11, 34, 35). Down-regulation of NRAGE would ensure that a differentiated neuron responding to guidance cues is no longer able to send an apoptotic signal through UNC5H1.

    ACKNOWLEDGEMENTS

The initial observation that UNC5Hs induce apoptosis was made in the laboratory of Marc Tessier-Lavigne. We thank Sareina Wu for technical assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants from NS39572-01 (to L. H.) and MH12813-02 (to M. W.), a University of California Cancer Research Coordinating Committee grant, and the March of Dimes Birth Defects Foundation Grant 5-FY99-765.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 831-459-5253; Fax: 831-459-3139; E-mail: hinck@biology.ucsc.edu.

Published, JBC Papers in Press, February 21, 2003, DOI 10.1074/jbc.M300415200

2 S. Faynboym, L. Hinck, E. D. Leonardo, and M. Tessier-Lavigne, unpublished information.

3 M. E. Williams and L. Hinck, unpublished information.

    ABBREVIATIONS

The abbreviations used are: HA, hemagglutinin; NGF, nerve growth factor; PBS, phosphate-buffered saline; GST, glutathione S-transferase; ANOVA, analysis of variance; GFP, green fluorescent protein; TUNEL, Tdt-mediated dUTP nick-end labeling; DCC, deleted in colorectal cancer; PFA, paraformaldehyde.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Nijhawan, D., Honarpour, N., and Wang, X. (2000) Annu. Rev. Neurosci. 23, 73-87[CrossRef][Medline] [Order article via Infotrieve]
2. Yuan, J., and Yankner, B. A. (2000) Nature 407, 802-809[CrossRef][Medline] [Order article via Infotrieve]
3. Hedgecock, E. M., Culotti, J. G., and Hall, D. H. (1990) Neuron 4, 61-85[Medline] [Order article via Infotrieve]
4. Leung-Hagesteijn, C., Spence, A. M., Stern, B. D., Zhou, Y., Su, M. W., Hedgecock, E. M., and Culotti, J. G. (1992) Cell 71, 289-299[Medline] [Order article via Infotrieve]
5. Ackerman, S. L., Kozak, L. P., Przyborski, S. A., Rund, L. A., Boyer, B. B., and Knowles, B. B. (1997) Nature 386, 838-842[CrossRef][Medline] [Order article via Infotrieve]
6. Leonardo, E. D., Hinck, L., Masu, M., Keino-Masu, K., Ackerman, S. L., and Tessier-Lavigne, M. (1997) Nature 386, 833-838[CrossRef][Medline] [Order article via Infotrieve]
7. Keleman, K., and Dickson, B. J. (2001) Neuron 32, 605-617[Medline] [Order article via Infotrieve]
8. Schultz, J., Milpetz, F., Bork, P., and Ponting, C. P. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5857-5864[Abstract/Free Full Text]
9. Tartaglia, L. A., Ayres, T. M., Wong, G. H., and Goeddel, D. V. (1993) Cell 74, 845-853[Medline] [Order article via Infotrieve]
10. Hofmann, K., and Tschopp, J. (1995) FEBS Lett. 371, 321-323[CrossRef][Medline] [Order article via Infotrieve]
11. Hong, K., Hinck, L., Nishiyama, M., Poo, M. M., Tessier-Lavigne, M., and Stein, E. (1999) Cell 97, 927-941[Medline] [Order article via Infotrieve]
12. Barker, P. A., and Salehi, A. (2002) J Neurosci. Res. 67, 705-712[CrossRef][Medline] [Order article via Infotrieve]
13. Taniura, H., Taniguchi, N., Hara, M., and Yoshikawa, K. (1998) J. Biol. Chem. 273, 720-728[Abstract/Free Full Text]
14. Hayashi, Y., Matsuyama, K., Takagi, K., Sugiura, H., and Yoshikawa, K. (1995) Biochem. Biophys. Res. Commun. 213, 317-324[CrossRef][Medline] [Order article via Infotrieve]
15. Kendall, S. E., Goldhawk, D. E., Kubu, C., Barker, P. A., and Verdi, J. M. (2002) Mech. Dev. 117, 187-200[CrossRef][Medline] [Order article via Infotrieve]
16. Masuda, Y., Sasaki, A., Shibuya, H., Ueno, N., Ikeda, K., and Watanabe, K. (2001) J. Biol. Chem. 276, 5331-5338[Abstract/Free Full Text]
17. Sasaki, A., Masuda, Y., Iwai, K., Ikeda, K., and Watanabe, K. (2002) J. Biol. Chem. 277, 22541-22546[Abstract/Free Full Text]
18. Salehi, A. H., Roux, P. P., Kubu, C. J., Zeindler, C., Bhakar, A., Tannis, L. L., Verdi, J. M., and Barker, P. A. (2000) Neuron 27, 279-288[Medline] [Order article via Infotrieve]
19. Jordan, B. W., Dinev, D., LeMellay, V., Troppmair, J., Gotz, R., Wixler, L., Sendtner, M., Ludwig, S., and Rapp, U. R. (2001) J. Biol. Chem. 276, 39985-39989[Abstract/Free Full Text]
20. Salehi, A. H., Xanthoudakis, S., and Barker, P. A. (2002) J. Biol. Chem. 277, 48043-48050[Abstract/Free Full Text]
21. Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Cell 74, 205-214[Medline] [Order article via Infotrieve]
22. Llambi, F., Causeret, F., Bloch-Gallego, E., and Mehlen, P. (2001) EMBO J. 20, 2715-2722[Abstract/Free Full Text]
23. Shiras, A., Sengupta, A., and Shepal, V. (2001) Mol. Cell. Biol. Res. Commun. 4, 313-319[CrossRef][Medline] [Order article via Infotrieve]
24. Barrett, C., and Guthrie, S. (2001) Mech. Dev. 106, 163-166[CrossRef][Medline] [Order article via Infotrieve]
25. Mehlen, P., Rabizadeh, S., Snipas, S. J., Assa-Munt, N., Salvesen, G. S., and Bredesen, D. E. (1998) Nature 395, 801-804[CrossRef][Medline] [Order article via Infotrieve]
26. Rechsteiner, M., and Rogers, S. W. (1996) Trends Biochem. Sci. 21, 267-271[CrossRef][Medline] [Order article via Infotrieve]
27. Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368[Medline] [Order article via Infotrieve]
28. Coulson, E. J., Reid, K., Baca, M., Shipham, K. A., Hulett, S. M., Kilpatrick, T. J., and Bartlett, P. F. (2000) J. Biol. Chem. 275, 30537-30545[Abstract/Free Full Text]
29. Ashkenazi, A., and Dixit, V. (1998) Science 281, 1305-1308[Abstract/Free Full Text]
30. Green, D. (1998) Cell 94, 695-698[Medline] [Order article via Infotrieve]
31. Coulson, E. J., Reid, K., Barrett, G. L., and Bartlett, P. F. (1999) J. Biol. Chem. 274, 16387-16391[Abstract/Free Full Text]
32. Feinstein, E., Kimchi, A., Wallach, D., Boldin, M., and Varfolomeev, E. (1995) Trends Biochem. Sci. 20, 342-344[CrossRef][Medline] [Order article via Infotrieve]
33. Rabizadeh, S., Oh, J., Zhong, L. T., Yang, J., Bitler, C. M., Butcher, L. L., and Bredesen, D. E. (1993) Science 261, 345-348[Medline] [Order article via Infotrieve]
34. Finger, J. H., Bronson, R. T., Harris, B., Johnson, K., Przyborski, S. A., and Ackerman, S. L. (2002) J. Neurosci. 22, 10346-10356[Abstract/Free Full Text]
35. Przyborski, S. A., Knowles, B. B., and Ackerman, S. L. (1998) Development 125, 41-50[Abstract/Free Full Text]


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