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INTRODUCTION |
The balance between cell division and programmed cell death, or
apoptosis, within mammals is crucial for normal development (1-5). An
increase in cell death can lead to neurodegenerative diseases, whereas
decreased apoptosis or unfettered cellular proliferation can lead to
cancer (6). Apoptosis is controlled by multiple pathways that integrate
both intra- and extracellular signals, which eventually converge upon
cellular proteases, or caspases (7-9). The tumor necrosis factor
(TNF)1 superfamily (TNFR1,
Fas, etc.) of trans-membrane receptors respond to soluble or
membrane-bound ligands (TNF-
, Fas ligand, TRAIL) to instigate
formation of death-inducing signaling complexes (DISCs), which
ultimately culminates in caspase activation and death (10, 11).
The TRAIL cytokine controls one such apoptotic pathway by binding a
group of extra-cellular receptors belonging to this TNF receptor
superfamily (11). Two members of this group, DR4 (TRAIL-R1) and
KILLER/DR5 (TRAIL-R2, TRICK2), actively promote apoptosis upon
overexpression or TRAIL binding (12-20). TRAIL binding causes the
receptors to trimerize, bringing together a 70-amino acid intracellular
protein/protein interaction motif, termed the death domain (DD) (10,
21, 22). This domain serves as a platform to recruit an adapter protein
which then binds to and activates the initiator caspases (11). In the
case of TRAIL ligand, it has recently been demonstrated that FADD
(Fas-associated death domain) and caspase 8 are required for cell death
signaling (23-26). Both FADD-null and caspase 8-null cells were shown
to be deficient in TRAIL-induced apoptosis (23, 24, 26). Two
anti-apoptotic members or decoy receptors, TRID (DcR1, TRAIL-R3) and
TRUNDD (DcR2, TRAIL-R4), are capable of binding TRAIL but do not
transmit an apoptotic signal due to the lack of a functional DD
(27-30). The balance between these receptors on the cell and the
expression of caspase 8, as well as the presence of intracellular
inhibitors, termed cFLIPs, determine the fate of the cell in response
to TRAIL treatment (31-33).
The importance of the DD in causing an apoptotic response from DR4 or
KILLER/DR5 is underscored by two observations. Experiments in which the
DD is deleted renders them nonfunctional (12-20), and the TRAIL
receptors, TRID and TRUNDD, are deemed decoys due to their absence of a
functional DD (27-30). Studies of the C terminus of TNFR1 including
deletion and alanine mutagenesis of DD revealed that this domain is
critical for apoptotic signaling (34). Homology studies recognized the
presence of a DD within Fas as well (34). Crystal structure analysis of
the Fas DD revealed six anti-parallel
-helices (35). Mutagenesis of
the Fas DD, as well as similar studies with the pro-apoptotic adapter
molecule FADD, revealed the importance of helices 2 and 3 for
self-association and protein/protein interactions (35, 36). The
interactions appear to act in an antiparallel fashion with FADD helix
2/Fas helix 3 and Fas helix 2/FADD helix 3 comprising the bulk of the
electrostatic interactions (36).
Naturally occurring mutants of DD-containing receptors have been
described. The lprcg mouse, which develops an
autoimmune lymphoproliferative disorder, contains a I238N alteration
changing an isoleucine to an asparagine within the DD of the mouse Fas
receptor (37). Mutations of DD-containing receptors have also been
found in human cancers. Rare loss-of-function alterations were
initially described in the TRAIL receptor KILLER/DR5 in head and neck
cancer specimens. A single-base insertional mutation led to a
frameshift in the DD of KILLER/DR5, leading to premature termination of
translation and a resulting DD-truncated functionally deficient
receptor (38.). More recently, a number of mutations have been reported
in the DD of KILLER/DR5 in non-small cell lung cancers in 11 of 104 (10.6%) specimens tested (39). These alterations included eight
missense, one nonsense, one splice site, and one silent change within
exons 9 and 10 of the human KILLER/DR5 gene (39). None of the
substitution changes were functionally characterized, although three of
the eight missense alterations involved a C-to-T transition at base
pair 1087 leading to an L334F (leucine to phenylalanine change).
Leu-334 is the homologous position to the lpr mutation in
the mouse Fas receptor gene. Alteration in cancer of other receptors
has been reported and includes homozygous deletion of DR4 in
nasopharyngeal cancer (40), overexpression of DcR3 (a Fas decoy
receptor) in colon and lung cancers (41), and overexpression of DcR1
(TRID decoy) in gastrointestinal tumors (42).
Because of their involvement in cancers and the potential therapeutic
applications of TRAIL and TRAIL receptor signaling, we undertook a
site-directed mutagenesis strategy to identify important aspects of DD
transduction of a cell death signal. No detailed mutational analysis or
structure-function study has been reported for either of the two
pro-apoptotic TRAIL receptors DR4 and KILLER/DR5. Moreover, no detailed
comparisons with other TNF receptor family members have been performed
in terms of conservation of structural features versus
functional outcome in death signaling. Thus, the present studies
involved the generation of a series of point mutants within human
KILLER/DR5 leading to 18 different amino acid substitutions by alanine
at positions chosen based on alignments with family members, and
information derived from structural evidence with other family members.
Finally, we investigated the functional significance of six known DD
substitutions isolated from non-small cell lung cancer specimens. Our
results provide interesting comparisons into the relative importance of
charged versus hydrophobic DD amino acid residues and death
signaling. Moreover, we show that one tumor-derived KILLER/DR5 mutant
displayed a more severe loss-of-function phenotype, whereas a different tumor-derived mutant displayed a much more modest defect in death signaling as compared with their respective alanine substitutions. Finally, a number of tumor-derived mutants were shown to retain apoptotic capability when overexpressed. The present studies provide novel insights into specific structural determinants within the DD that
signal downstream caspase activation by the KILLER/DR5 death receptor protein.
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MATERIALS AND METHODS |
Alanine Scanning Mutagenesis of KILLER/DR5--
The KILLER/DR5
cDNA was cloned in frame into pcDNA3.1-Myc-HisA
(Invitrogen)
as an EcoRI/HindIII fragment. This C-terminally tagged Myc-His plasmid was subsequently mutagenized using the QuikChange site-directed mutagenesis kit (Stratagene). Due to the size
of the template (6.7 kilobases), certain changes were made to the
manufacturer's protocol to yield polymerase chain reaction product:
inclusion of 10% glycerol, 5% Me2SO, and 100 ng of
template in polymerase chain reaction, decrease of annealing temperature to 50 °C, and 18 cycles of amplification. Mutations were
verified by sequencing, and in each case the entire cDNA was
checked for the absence of second site mutations.
Transfections, in Vitro Translation, and Western
Blotting--
The colon cancer cell line SW480 was maintained and
transfected as described previously (13). Protein extracts were
harvested in 1× Laemmli sample buffer 16 h after transfection.
PARP (Roche Molecular Biochemicals; 1:2000) and Myc (Santa Cruz; 1:500)
immunoblots were performed following SDS-polyacrylamide gel
electrophoresis. Horseradish peroxidase-conjugated secondary antibody
(Pierce; 1:5000) treatments were followed by enhanced chemiluminescence (Amersham Pharmacia Biotech). In vitro translation reactions
were carried out using 1 µg of wild-type or mutant KILLER/DR5 plasmid DNA and the TNT T7-coupled reticulocyte lysate system (Promega).
GFP/PI Fluorescence-activated Cell Sorting Analysis for
Sub-G1 Peak--
SW480 cells were transfected with a 1:10
ratio of EGFP-spectrin (43) and KILLER/DR5 expression plasmid.
Transfections were harvested at 18 h after transfection,
processed, and analyzed for the presence of a sub-G1 peak
as described previously (40). 10,000 GFP-positive cells were analyzed
per experiment, and three independent experiments were performed for
each mutant. Percentage of apoptosis was calculated by subtracting
transfection-induced sub-G1 peak (vector transfection) from
each wild-type or mutant KILLER/DR5 sub-G1 peak. Figures
for Tables II and III were generated by setting wild-type death to
100% (~40% sub-G1 peak at 18 h) in order to
compare point mutants to the wild-type protein.
Blue Cell Method--
The blue cell method was performed as
previously described (44). Calculations for Table I were carried out by
setting the number of vector-transfected blue cells to 100% (at least
300 cells for each independent experiment), and the wild-type and point
mutants were determined as a percentage of vector transfected.
TRAIL DISC Immunoprecipitation--
293 HEK cells were plated to
achieve 80% confluence at the time of transfection in a T75. 30 µg
of the indicated KILLER/DR5 plasmid was transfected by calcium
phosphate for 16 h to minimize loss of transfected cells. The
cells were then trypsinized, spun down, and resuspended in 2 ml of
complete medium supplemented with 50 ng/ml His tagged-TRAIL and 1 µg/ml anti-6-histidine antibody (R&D Systems) for 15 min at 37 °C.
For untreated samples, only the TRAIL was excluded. The cells were
washed twice with ice-cold phosphate-buffered saline and lysed for 30 min on ice in TRAIL DISC IP lysis buffer (30 mM Tris, pH
7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100). The
lysates were cleared twice by centrifugation at 4 °C. The
supernatants were immunoprecipitated overnight with 30 µl of Protein
A/G Plus-agarose (Santa Cruz) at 4 °C to isolate the TRAIL DISC. The
complexes were subsequently washed four times with TRAIL DISC IP lysis
buffer and eluted with Immunopure Gentle Ag/Ab elution buffer (Pierce)
with 0.1 M dithiothreitol at room temperature for 2 h.
The protein complexes were methanol/chloroform-precipitated and
resolved on 15% SDS-polyacrylamide gels. Caspase 8 (Cell Signaling; 1:1000) and FADD (Upstate Biotechnology; 1:2000) Western blots were
performed to measure recruitment of these endogenous proteins to the
TRAIL DISC along with the exogenously expressed KILLER/DR5 (Myc; Santa
Cruz) protein.
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RESULTS |
Design of KILLER/DR5 DD Alanine Scanning Mutants Based on Homology
to DDs of the TNFR Superfamily--
Recent mutational and crystal
structure studies with Fas and FADD (35, 36) have revealed the
importance of helices 2 and 3 of both proteins. Electrostatic
interactions between surface residues are thought to mediate DD/DD
interactions. DD mutants were designed, based on a number of selection
criteria including residue charge, hydrophobicity, conservation, and
demonstrated functional significance in other receptor systems. Due to
the noted importance of helices 2 and 3 within Fas and FADD, we focused our mutagenesis on charged residues in order to identify residues crucial for receptor/adapter interactions. Fig.
1 depicts an amino acid alignment of the
death domains of selected members of the TNF superfamily. Eighteen
residues within KILLER/DR5, which were targeted for alanine replacement
mutagenesis, are noted with an asterisk along with the amino
acid position of the short form of the KILLER/DR5 protein.

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Fig. 1.
Alignment of DD from TNF receptor superfamily
members. Numbers denote residues in KILLER/DR5 that
were targeted in alanine scanning mutagenesis. Bold
line below Fas DD denotes -helices determined from Fas
crystal structure studies (35).
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Fig. 2A shows protein
expression of each construct following transfection into SW480 colon
cancer cells for 16 h. Most of the alanine mutants expressed
protein at levels equivalent to wild type KILLER/DR5. Selected mutants
showed an increase in protein expression levels in addition to the
appearance of a lower mobility form of the protein. Interestingly,
these mutants with higher levels of expression and the larger form
appeared to be either partially or completely defective in inducing
apoptosis (see data below). This would explain the higher levels of the
transfected protein as the cell was able to withstand the presence of
these non-toxic proteins. The lower mobility form could also be
explained due to the higher levels of transfected protein being made
and withstood by the cell. Fig. 2B compares transfected cell
lysates to in vitro translated proteins, which do not
undergo processing of the signal peptide. The TNT reactions yield a
lower mobility form of KILLER/DR5, which comigrates with the upper band
observed in the transfected cell lysates of mutants, W325A and R330A.
In addition, cotransfection of the anti-apoptotic gene,
FLIPS, along with wild type KILLER/DR5 protects the cells
from death and both the processed and unprocessed forms of the
wild-type protein can be detected by Western blot analysis (data not
shown).

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Fig. 2.
Protein expression of wild-type
(W.T.) and alanine mutant KILLER/DR5 constructs.
A, 0.5 × 106 SW480 cells were transiently
transfected for 16 h, harvested, and equivalent amounts of protein
were run on a 15% SDS-polyacrylamide gel. The gel was then transferred
and probed with an anti-Myc antibody, followed by a horseradish
peroxidase-conjugated secondary and ECL. B, comigration of
in vivo upper KILLER/DR5 band with unprocessed in
vitro translation (IVT) product. The unlabeled in
vitro translation reaction was carried out on 1 µg of plasmid as
per manufacturer's instructions (Promega). The in vitro
translation reactions were run out along with transfected lysates and
processed as in A.
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Conserved Hydrophobic Residues Are More Critical for Apoptotic
Signaling as Compared with Conserved Charged Positions in the
KILLER/DR5 DD--
In order to functionally characterize the mutant
receptors, we employed three methods to assess cell death following
transfection. The first method involved cotransfection of the mutant
receptor along with pCMV-
gal. Blue cells were counted after 40 h in order to determine relative cell survival as compared with
vector-transfected cells (number of blue cells for vector was set as
equivalent to 100% survival; Table I).
To specifically measure apoptosis, we used a cotransfection based flow
cytometric assay to functionally assess apoptotic signaling of each
substitution mutant. Each mutant was cotransfected with an
EGFP-spectrin construct at a 10:1 ratio (mutant plasmid:EGFP-spectrin
plasmid) into SW480 cells for 18 h and the presence of a
sub-G1 peak was used to quantitate apoptotic signaling. The
use of the spectrin-bound GFP allowed for the identification of
specifically transfected cells, and the spectrin fusion permits cell
membrane retention of GFP even if cells lose membrane permeability due
to cell death (43). Fig. 3 shows an
example of a typical experiment with GFP-positive cells in the
left column and GFP-negative cells in the
right column. Vector-transfected (GFP(+)) cells
displayed minimal toxicity (as compared with the GFP(
) counterparts)
associated with the transfection of DNA into the cells. In contrast,
the transfection of wild-type KILLER/DR5 dramatically increased the sub-G1 peak in addition to decreasing the G2
peak as compared with the untransfected cell population (Fig. 3). One
example of a signaling-competent and -incompetent mutant is also shown
for comparison. Table II lists the
results of three independent experiments for each mutant compared with
the wild-type protein after subtracting out transfection-induced death.
Finally, as a further demonstration of each mutant's ability to induce
apoptosis, PARP cleavage was measured following 16 h of
transfection. Fig. 4 illustrates these results with the arrow indicating the cleaved form of the
PARP protein, indicative of apoptosis. Vector-transfected cells showed no sign of PARP cleavage at 16 h, whereas wild-type KILLER/DR5 efficiently caused PARP cleavage (Fig. 4, compare lanes
1 and 2).

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Fig. 3.
Example of flow cytometry/GFP-spectrin based
assay to assess sub-G1 peak following transient
transfection of wild-type (W.T.) and alanine mutant
KILLER/DR5 constructs. SW480 cells were transfected with a 1:10
ratio of GFP-spectrin:mutant plasmid and harvested at 18 h for
flow cytometric analysis. Specifically transfected GFP-positive cells
are shown in the left column, and untransfected
cells are shown in the right column.
Sub-G1 peak, indicative of apoptosis, is denoted by the
percentage shown to the left of the vertical
line in each graph. One example is given for vector,
wild-type KILLER/DR5, signaling competent and incompetent mutant
transfected to demonstrate the assay.
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Fig. 4.
PARP cleavage following transient
transfection of wild-type (W.T.) and alanine mutant
KILLER/DR5 constructs. Lysates from Fig. 2A were
reprobed with an anti-PARP antibody to measure amount of PARP cleavage
induced by each mutant. The arrow denotes the cleaved PARP
product.
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The data from the three methods correlated well with one another
(Tables I and II and Fig. 4) and yielded three groups into which the
alanine mutants could be divided based on phenotype. The first group
contained five mutants, which have a dramatically reduced ability to
induce apoptosis: W325A, R330A, L334A (lpr-like), I339A, and
W360A. The cells expressing these mutants retained ~75% blue cell
staining (as compared with 8% for wild type; Table I), demonstrated at
least a 75% reduction in sub-G1 peak at 18 h in four
out of the five mutants (Table II), and displayed little to no
PARP-cleaving activity at 16 h (Fig. 4). This also correlated with
the observation on the protein level that these five mutants had high
levels of processed and unprocessed receptor that the cells were able
to withstand due to its lack of toxicity (Fig. 2A,
lanes 4, 6, 8,
11, and 17). Interestingly, four of these
(including the lpr-like mutation) represent hydrophobic
residues scattered throughout helices 2, 3, and 4, which when mutated
probably affect overall protein structure due to disruption of the
hydrophobic core. Disruption of the lpr position in either
Fas or FADD results in an inability of the proteins to interact and in
TNFR1 a loss of cytotoxicity. Of these five mutants, only R330A might
play a role in potentially mediating an electrostatic death domain interaction. This charged residue, which is completely conserved in
every proapoptotic TNFR family member, also proved to be critical in
TNFR1, Fas, and FADD (34-36), thereby highlighting its importance in
transducing an apoptotic signal.
The second class of mutants that was identified by this study
demonstrated only a partial loss in cell death signaling: K331A, D336A,
E338A, K340A, K343A, D351A, and L377A. These mutants, when overexpressed, displayed only a 10-25% reduction in cell death as
measured by sub-G1 (Table II) and slightly reduced amount
of PARP cleavage as compared with wild-type (Fig. 4). With the
exception of L377A, these partially defective mutants represent charged residues within helices 2 and 3 and the boundary of helix 4. Interestingly in vitro binding studies with Fas (35) and
FADD (36) revealed a total loss of interaction if a mutation occurred
at the positions corresponding to Lys-331, Asp-336, and Lys-340 and a
partial loss at Lys-343. Therefore, these partial loss-of-function
mutations may indicate residues important in mediating interactions
with an adapter molecule.
The final class of mutants are those that are unaffected by alanine
substitution: Ser-324, Glu-326, Asn-362, Lys-386, Gln-387, and Lys-388.
They function as the wild-type protein does in all three of the
aforementioned assays. In the case of position 326, the homologous
residue in FADD when mutated and tested shows an inability to interact
with Fas in vitro. Examination of charge distribution
reveals that, in the case of the TRAIL receptors, DR4 and KILLER/DR5,
this residue is negatively charged whereas Fas, FADD, and TNFR1 all
retain a positive charge. This residue represents a difference between
the TRAIL receptors and the rest of TNFR family DDs not only in charge
but also in function. The other residues, which are not affected by
mutation, although illustrating examples of subtle differences between
family members, are not conserved in identity or charge and to this
point have not been demonstrated to be important for function in any of
the receptor/adapter systems studied thus far. Meanwhile, mutation of
highly conserved hydrophobic residues throughout the DD renders
KILLER/DR5 nonfunctional and elimination of charged residues presumed
to be the sites of putative protein interactions partially suppresses
the apoptotic signal in an overexpression environment.
Some but Not All Tumor-derived KILLER/DR5 Mutants Display Loss of
Apoptotic Function When Overexpressed--
Due to the incidence of
chromosome 8p21-22 loss in human cancers and the lack of an identified
tumor suppressor gene in the area, groups have turned their attention
toward identifying mutations in DR4 and KILLER/DR5, which have both
been mapped to 8p21. The first report of a tumor associated mutation of
KILLER/DR5 was in a head neck cancer, which resulted in a truncation of
the cytoplasmic domain of the protein (38). In another study, also
looking to assign significance to the KILLER/DR5 gene in chromosome
8p21-22 loss of cancers, Lee et al. reported alterations in
the cytoplasmic domain of the protein in non-small cell lung cancers.
Out of 104 samples, 11 mutations were reported including eight missense
mutations. This provided us with the opportunity to compare our
functional data using alanine mutagenesis to naturally occurring
tumor-derived mutants. Three of the tumor mutations occurred at amino
acid position 334 (L334F), which corresponds to the same residue
altered in the Fas lpr case. Coincidentally, four of the
remaining five mutations were targeted in our original alanine
mutagenesis of the protein: S324F, E326K, E338K, K386N. The remaining
point mutation occurred just four amino acids from the end of the
protein, D407Y.
The tumor-derived mutants were generated in the same manner as the
alanine mutants in order to compare alanine versus
tumor-specific mutant with the wild-type KILLER/DR5 protein. Mutations
were verified by DNA sequencing, and protein expression along with PARP
cleavage was evaluated (Fig. 5 and data
not shown). The most common naturally occurring mutation (L334F; 3 out
of 11), which corresponds to the lpr position, actually
demonstrated a more severe phenotype as an alanine substitution (Table
III). Interestingly, the naturally occurring mutation (L334F) retains the hydrophobicity at the position while decreasing its apoptotic potential by 50%. Conversely, a mutation at a putative protein-protein interaction site (E338K) displayed a complete loss-of-function whereas the alanine mutation displayed only a partial loss-of-function phenotype (Table III). The
tumor-specific mutation at position 338 changes the charge from
negative to positive, explaining the dramatic loss of apoptotic capacity; however, both Fas and FADD normally have a positively charged
lysine at this position. Charge differences such as this between family
members may provide a clue to receptor/adapter specificity.
Nevertheless, the tumor-specific mutant data provides convincing
in vivo evidence that positions Leu-334 and Glu-338 are
important for proper downstream signaling of cell death.

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Fig. 5.
Protein expression and PARP cleavage
induction by tumor-derived KILLER/DR5 mutants. The
upper panel displays protein expression of the
mutants as assessed by anti-Myc Western blot, and the lower
panel measures PARP cleavage induced by each mutant.
W.T., wild type.
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Unexpectedly, the remaining four missense mutations (S324F, E326K,
K386N, D407Y) displayed no loss of apoptotic signaling as assessed by
sub-G1 (Table III) or by PARP cleavage (Fig. 5) in the
overexpression studies. Mutation of these residues in the original
alanine-mutagenesis yielded no phenotype as well, strengthening the
idea of a lesser role played by these sites in the overall structure
and signaling of the molecule. This does not rule out the possibility
that in these tumors, many of which may have 8p LOH and hence reduced
gene dosage, that this mutation may have subtle effects not detectable
in these types of overexpression studies.
Expression of Partial or Complete Loss-of-function Mutants
Diminishes Endogenous FADD and Caspase 8 Recruitment to TRAIL
DISCs--
In addition to the receptor/ligand trimers, the TRAIL DISC
has recently been demonstrated to contain the adapter molecule FADD and
caspase 8 (23-25) in order to propagate the apoptotic signal. Due to
an inability to demonstrate a direct interaction between FADD and
KILLER/DR5 in vitro, we attempted to recapitulate this
interaction in vivo in the context of the DISC and to
determine the effect of exogenous partial and loss-of-function
KILLER/DR5 mutants on FADD and caspase 8 recruitment. 293 HEK cells
were chosen due to their ability to be transfected and relative
resistance to TRAIL-induced apoptosis. Following transfection of the
wild-type or mutant KILLER/DR5 plasmid, the cells were harvested
16 h later and treated with His-tagged TRAIL and a cross-linking
anti-6-histidine antibody for 15 min at 37 °C. The DISC was then
immunoprecipitated and analyzed for the presence of the exogenous
KILLER/DR5 receptor along with FADD and caspase 8. As demonstrated in
Fig. 6A, TRAIL treatment led
to the recruitment of the exogenous KILLER/DR5 receptor into DISCs as
visualized with an anti-myc antibody. Total caspase 8 was provided as a
loading control to ensure equivalent amounts of proteins were treated
and processed for DISC analysis. FADD and caspase 8 (both pro- and the
cleaved p46 form) were detected in the DISC of vector- and wild-type
receptor-transfected 293 cells; however, introduction of either
complete loss-of-function receptor, R330A or L334A, led to a dramatic
decrease in both FADD and caspase 8 recruitment. This observation helps
to explain the lack of apoptosis induction by these mutants in earlier
cell death assays (Tables I and II and Fig. 4).

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Fig. 6.
A, TRAIL DISC IP of transfected 293 cells. A T75 (80% confluent) of 293 HEK cells was transfected for
16 h with the indicated wild-type (WT) or mutant
KILLER/DR5 plasmid followed by a 15-min TRAIL treatment (50 ng/ml) at
37 °C. The DISC immunoprecipitation was carried out overnight at
4 °C followed by SDS-polyacrylamide gel electrophoresis and Western
blot analysis for DISC-associated caspase 8, FADD, and exogenous
KILLER/DR5. Total caspase 8 is provided as a loading control to ensure
equal amounts of protein were used for TRAIL treatment and DISC
analysis. B, TRAIL DISC IP of panel of total and partial
loss-of-function alanine mutants. The DISC IPs were carried out as
specified in A. The ( ) TRAIL lane includes the
anti-His6 antibody and protein A/G-agarose beads as a
control for the specificity of the DISC IP. C, TRAIL DISC IP
of the alanine versus tumor loss-of-function mutants. IPs
were carried out as detailed above. P indicates partial
loss-of-function, and C indicates complete loss-of-function
mutants.
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These observations were extended to the panel of complete and partial
loss-of-function alanine mutations (Fig. 6B) as well as the
tumor mutants (Fig. 6C). Absence of TRAIL treatment did not
result in DISC formation, but TRAIL treatment of vector or wild-type
transfected cells showed a robust recruitment of both FADD and
caspase 8.
Examination of the amount of cleaved caspase 8 in Fig. 6B
clearly correlated with the predicted apoptotic potential of each receptor class. The partial loss-of-function mutants showed a decrease
in both cleaved caspase 8 and FADD as compared with wild-type. The
complete loss-of-function alanine mutants have an even more severe
impairment in FADD and caspase 8 recruitment. Finally, in Fig.
6C, a similar correlation between DISC components and receptor function was observed. The complete loss-of-function mutation,
L334A, showed a decrease in FADD and caspase 8 (pro- and cleaved) as
compared with wild-type; however, the corresponding tumor mutation,
L334F, exhibited increased binding of both FADD and caspase 8 in
accordance with its classification as a partial loss-of-function
mutant. Likewise, the partial loss-of-function mutant, E338A, recruited
more FADD and caspase 8 than its complete loss-of-function tumor
counterpart, E338K. The correlation between FADD/caspase 8 recruitment
to TRAIL DISCs and the cell death data for the complete and partial
loss-of-function mutants supports the importance of these residues
uncovered in the earlier overexpression studies.
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DISCUSSION |
Mutagenesis of the death domain of KILLER/DR5 revealed hydrophobic
residues possibly important for overall protein structure as well as
charged residues which may potentially interact with downstream
effector molecules. The hydrophobic residues tested (Trp-325, Leu-334,
Ile-339, Trp-360, and Leu-377) cause a partial (Leu-377) or complete
loss-of-function when mutated to alanine. The complementary residues of
Leu-334 and Trp-360 within Fas were shown via crystal structure
analysis to comprise part of the hydrophobic core of the death domain
(35). Additional studies suggested that disruption of the Leu-334 Fas
complementary residue (lpr) caused a disruption of the
protein structure (35). Future crystal structure studies may determine
whether these five hydrophobic residues are truly buried within the
protein. Charged residues implicated in mediating electrostatic
interactions include Arg-330 (complete loss), Lys-331, Asp-336,
Glu-338, Lys-340, Lys-343, and Asp-351. These residues (excluding
Glu-338 and Asp-351) were also demonstrated to be important for Fas or
FADD self-aggregation as well as protein partner binding (35, 36),
signifying their importance in death domain signaling in general. It is
possible that mutagenesis of charged residues led only to partial
loss-of-function because multiple residual charges were still able to
stabilize protein-protein interactions.
Careful attention to charge distribution within helices 2 and 3 of the
receptors may begin to explain differences in receptor/adapter specificity. Residue Glu-326 showed a complete loss in Fas/FADD interaction (36), yet no apparent defect was observed when mutated to
alanine in KILLER/DR5. It is interesting to note the difference in this
case because the charge of this particular position varies within the
superfamily of receptors. Only the TRAIL receptors, DR4 and KILLER/DR5,
have a negatively charged amino acid at this position which may begin
to illustrate differences between TRAIL and other TNFR family
receptors. Position 336, also shown to be important in this study,
retains a negative charge in all family members except DR4. Future
mutational analysis of DR4 will determine whether this residue plays a
role in adapter binding by DR4. Lastly, position 338, which has a
partial defect in KILLER/DR5-mediated apoptosis, remains untested in
the Fas or TNFR1 system; however, inspection of the charge at this
position among family members reveals that Fas is the only receptor
with a positive charge. Fas is also the only member of the family
demonstrated to be able to directly bind to the proapoptotic
adapter molecule FADD. Although FADD is present in the DISC of other
receptors such as TNFR1, DR3, and KILLER/DR5 (10, 23-25), a direct
interaction between these molecules and FADD has not been demonstrated
and, in the case of TNFR1, the adapter molecule TRADD is required.
In order to more specifically examine adapter binding to
loss-of-function KILLER/DR5 mutants, we utilized a TRAIL DISC
immunoprecipitation strategy in which various mutants were introduced
and the relative levels of FADD and caspase 8 were assessed. The DD
mutants were actively recruited into TRAIL DISCs after TRAIL treatment,
signifying that cytoplasmic DD loss-of-function mutations had no effect
on ligand binding. The defects, however, could be explained by a decrease in both FADD and caspase 8 recruitment into TRAIL DISCs. As
expected, the amount of caspase 8 and FADD recruitment directly correlated with the amount of apoptosis induction, as assessed by the
cell death assays. These experiments illustrate the concept that one
mutant TRAIL receptor (i.e. DR4 or KILLER/DR5) can
potentially disrupt cell death signaling through the formation of
defective TRAIL DISCs.
The identification of DD tumor point mutants (39) allowed us to
functionally characterize their apoptotic capability and FADD/caspase 8 recruitment in addition to comparing their phenotype to the
corresponding alanine substitution. The most prevalent mutation
occurred in 3 out of 11 samples (L334F) corresponding to the naturally
occurring Fas lpr mutation, demonstrating the conserved
importance of this residue in two receptor systems. The alanine
substitution yielded a mutant receptor with a greater reduction in
apoptotic potential as well as FADD and caspase 8 recruitment as
compared with the tumor-derived mutant (L334F). This divergence in
phenotype may be explained by the maintenance of a hydrophobic residue
in the tumor-specific mutation. Nevertheless, its prevalence in
non-small cell lung cancer points to its importance in maintaining
proper protein folding and death signaling. In contrast, the drastic
charge change of the tumor mutant E338K resulted in a complete
loss-of-death induction by the receptor, whereas the alanine mutant
only partially disrupted cell death signaling. The severity of the
tumor-specific defect E338K points to an overall disruption of the
electrostatic interactions maintained by the residues in helices 2 and 3.
The loss-of-function of these two tumor-specific alleles (L334F, E338K)
is not surprising based on the alanine studies; however, the other
tumor-associated mutations (S324F, E326K, K386N, D407Y) would not be
predicted to affect receptor function. When these mutants were
generated and tested, no phenotype was observed, supporting the
observations made from the alanine mutagenesis. Physiological levels of
these mutant receptors may affect their stability or in the context of
ligand binding may demonstrate defective signaling, but these
preliminary overexpression studies coupled with the lack of
conservation of these residues among family members argue against their
importance in KILLER/DR5 structure/function.
In conclusion, our mutational study of the death domain of KILLER/DR5
identified conserved hydrophobic residues and charged amino acids,
which are crucial for normal cell death signaling downstream of the
receptor. The clustering of specific charged residues critical for
apoptosis that are proposed to be in helices 2 and 3 correlates with
data from other death domain-containing proteins (Fas, FADD) that these
helices are critical for protein/protein interactions. These mutants
may be useful in order to better understand the molecular mechanisms
behind receptor/adapter specificity.