From the 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
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ABSTRACT |
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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.
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.
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 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.
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.
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).
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
(H1
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.
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.
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
H1
We dramatically decreased apoptosis induced through UNC5H1 by deleting
the NRAGE binding domain; however, we noticed that H1
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.
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.
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, NF 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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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).
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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.
DDmyc) does not affect the interaction with HA-NRAGE in cells,
but deleting the entire intracellular domain of UNC5H1 (H1
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).
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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.
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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.
sac, a truncation that removes much of the intracellular domain,
and found that H1
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 (H1
DD) induces apoptosis.
Our results show that H1
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 (H1
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.
sac still
causes apoptosis above the level in control cells expressing GFP (Fig.
4B). Therefore, we removed the entire intracellular domain
of UNC5H1 (H1
ICD) to determine whether this completely abrogates the
death signal. Fig. 4B shows that H1
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 (H1
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.
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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).
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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
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.
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ACKNOWLEDGEMENTS |
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The initial observation that UNC5Hs induce apoptosis was made in the laboratory of Marc Tessier-Lavigne. We thank Sareina Wu for technical assistance.
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FOOTNOTES |
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* 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.
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ABBREVIATIONS |
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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.
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