TumorNecrosisFactor-relatedApoptosis-inducingLigand (TRAIL ...

Supplemental Material can be found at:
http://www.jbc.org/content/suppl/2012/04/10/M111.306480.DC1.html

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 25, pp. 21265–21278, June 15, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Tumor Necrosis Factor-related Apoptosis-inducing Ligand Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012
(TRAIL) Induces Death Receptor 5 Networks That Are Highly
Organized*□S

Received for publication, September 29, 2011, and in revised form, April 10, 2012 Published, JBC Papers in Press, April 10, 2012, DOI 10.1074/jbc.M111.306480

Christopher C. Valley‡, Andrew K. Lewis‡, Deepti J. Mudaliar‡, Jason D. Perlmutter‡, Anthony R. Braun‡,
Christine B. Karim§, David D. Thomas§, Jonathan R. Brody¶, and Jonathan N. Sachs‡1
From the Departments of ‡Biomedical Engineering and §Biochemistry, Molecular Biology, and Biophysics, University of Minnesota,
Minneapolis, Minnesota 55455 and the ¶Department of Surgery-Division of Surgical Research, Jefferson Medical College,
Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Background: Whether ligand-induced clusters of DR5 have a specific structural organization is unknown.
Results: Ligand binding results in the formation of death receptor dimers that exist within high molecular weight networks.
Conclusion: Ligand-induced DR5 clusters are highly organized networks formed through dimerization of receptor trimers.
Significance: The biophysical character of DR5 networks may have implications for future rational design of DR5-targeted
therapeutics.

Recent evidence suggests that TNF-related apoptosis-induc- body agonists (8 –13) targeting this pathway have potent anti-
ing ligand (TRAIL), a death-inducing cytokine with anti-tumor tumor activity without exhibiting systemic cytotoxicity (7, 11).
potential, initiates apoptosis by re-organizing TRAIL receptors Therefore, given the potential as a promising target in cancer, it
into large clusters, although the structure of these clusters and is critical to characterize the precise mechanism by which death
the mechanism by which they assemble are unknown. Here, we receptors initiate apoptosis to optimize existing TRAIL re-
demonstrate that TRAIL receptor 2 (DR5) forms receptor ceptor-targeted therapeutic strategies (9, 14 –18). However,
dimers in a ligand-dependent manner at endogenous receptor despite the well established sequence of signaling events down-
levels, and these receptor dimers exist within high molecular stream of receptor activation (19, 20), how the initiating events
weight networks. Using mutational analysis, FRET, fluorescence in TRAIL-induced cell death (specifically events in the mem-
microscopy, synthetic biochemistry, and molecular modeling, brane) propagate a death signal remains unknown.
we find that receptor dimerization relies upon covalent and
noncovalent interactions between membrane-proximal resi- The crystal structure of the extracellular domain (ECD) of
dues. Additionally, by using FRET, we show that the oligomeric the TRAIL-DR5 complex (21–23) suggests that three receptor
structure of two functional isoforms of DR5 is indistinguishable. monomers tightly associate with the trimerized TRAIL (24) to
The resulting model of DR5 activation should revise the form a symmetric ligand-receptor complex (Fig. 1A, Protein
accepted architecture of the functioning units of DR5 and the Data Bank code 1d0g, shown is a top view of the complex).
structurally homologous TNF receptor superfamily members. Additionally, based on the last resolved amino acid of the ECD,
the trimeric receptor arrangement places the transmembrane
Tumor necrosis factor (TNF)-related apoptosis-inducing (TM) domains of DR5 ϳ50 Å apart (Fig. 1A, red dashed line),
ligand (TRAIL)2 is a member of the TNF superfamily of ligands and therefore interactions between the TM domains are pre-
(1) that triggers the extrinsic apoptotic pathway via death sumed to have no role in TRAIL-DR5 signaling. The TRAIL-
receptors 4 and 5 (DR4 or TRAIL-R1 and DR5 or TRAIL-R2, DR5 trimeric structure is consistent with the crystal structure
respectively) (2– 4). These type I membrane receptors serve as of LT␣-TNFR1 (25), a related TNF ligand-receptor pair, which
viable targets for cancer therapeutics as pre-clinical models is also a complex of three noninteracting receptor monomers
have demonstrated that recombinant TRAIL (5–7) and anti- and a tightly bound homo-trimeric ligand. Thus, based upon
these crystal structures, it has reasonably been presumed that
* This research was supported in part by grants to J. N. S. (NIH R21 CA152668 the relevant signaling event in the TNF receptor superfamily is
ligand-induced extracellular trimerization and subsequent
and ACS IRG-58-001-49 IRG 49-28) and to D. D. T. (NIH GM27906). trimerization of these receptors’ intracellular regulatory
□S This article contains Methods, “Results,” supplemental Figs. S1–S4, and domains (death domains in the case of DR5). However, crystal
structures and functional studies of downstream domains and
additional references. proteins (including the death domain, FADD, and caspase-8)
1 To whom correspondence should be addressed: Dept. of Biomedical Engi- suggest that the process may instead be dictated by receptor
dimerization, not trimerization (26 –29). An additionally
neering, University of Minnesota, Rm. 7-105 NHH, 312 Church St. SE, Min- intriguing piece of evidence is a dimeric crystal structure of the
neapolis, MN 55455. Tel.: 612-624-7158; Fax: 612-626-6583; E-mail: ECD of TNFR1 in the absence of ligand (30), which is stabilized
[email protected]. by the well studied pre-ligand assembly domain (PLAD) (31–
2 The abbreviations used are: TRAIL, TNF-related apoptosis-inducing ligand; 33) as well as by membrane-proximal residues, for which no
CFP, cyan fluorescent protein; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hy-
droxymethyl)propane-1,3-diol; TM, transmembrane; ECD, extracellular
domain; PLAD, pre-ligand assembly domain; REMD, replica exchange
molecular dynamics; Fmoc, N-(9-fluorenyl)methoxycarbonyl.

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TRAIL-induced DR5 Networks Are Highly Organized

FIGURE 1. A current model for organized ligand receptor networks. A, TRAIL-DR5 crystal structure (center, Protein Data Bank code 1d0g, top view), a Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012
schematic representation (left) and an overlay (right). Dashed red line indicates the previously predicted transmembrane domain separation. B, in the absence
of ligand, DR5 forms pre-ligand trimers via interactions within the pre-ligand assembly domain, although pre-ligand dimers have also been proposed. C,
addition of TRAIL causes a structural reorganization from a preligand state to the ligand-induced trimer state, as inferred by the trimeric ligand-bound crystal
structure, as shown in A. D, ligand-induced structural changes allow for interactions of multiple ligand-receptor trimers resulting in early stage receptor
clustering via dimerization or trimerization of ligand-receptor trimers, shown above and below, respectively. E, combination of trimeric ligand-receptor
interactions (i.e. crystal structure trimer) and receptor-receptor interactions (either dimeric or trimeric) drives receptor clustering and the formation of large
organized receptor networks.

known role exists. In both the TNF receptors and DR5, the Although no experimental data exist to support a molecular
PLAD has been identified as crucial for ligand binding, but model of SPOTS, several hypothetical models of TNF ligand-
there has been no definitive study to clarify the role of the receptor network structure have been presented (41, 42), two of
dimeric TNFR1 complex in signaling. To date, no ligand-inde- which are schematized in Fig. 1. In the absence of ligand, pre-
pendent oligomeric structure of DR5 exists nor is it known formed receptor oligomers, shown as trimeric complexes
whether there is a relevant dimeric form of DR5. (described below as our preferred model for DR5) but also sug-
gested to be dimeric (e.g. in the case of TNFR1), assemble in the
Recently, our understanding of TNF receptors has been fur- plasma membrane via the membrane-distal residues of the
ther complicated by a number of studies that show activation is PLAD (Fig. 1B). Based on available crystal structures (21–23),
associated with supramolecular receptor clustering within the ligand binding first results in the formation of a symmetric
membrane (34 –38), with the most recent example being DR5 trimer complex, which consists of three noninteracting recep-
(39, 40). Visualized in fluorescence microscopy studies, these tor monomers held together by the trimeric TRAIL (Fig. 1C).
ligand-induced aggregates of receptors, previously referred to Further clustering of these trimer complexes then proceeds by
as signaling protein oligomeric transduction structures, or previously unproven interactions. In the case of TNFR1, clus-
SPOTS, are on the order of 300 –500 nm in diameter. Despite tering of trimer complexes has been hypothesized to proceed
considerable speculation about the physical character of via dimeric receptor interactions, one receptor monomer from
SPOTS (41, 42), the mechanism through which clusters form each of the two adjacent interacting trimeric complexes (Fig.
and whether they have a specific structural organization are 1D, top) (42). Additional trimer complexes are similarly added
unknown, leaving open the following significant and funda- thus leading to large network assemblies (Fig. 1E, top). This
mental question. Are SPOTS simply co-localized aggregates model potentially explains the occurrence of both trimeric
formed via random nonspecific interactions between recep- ligand-receptor complexes (i.e. the crystal structure trimer
tors? Or, more interestingly, are they highly structured net- complex) and the unliganded dimeric receptor species (30).
works that possess stabilizing and targetable motifs? Because of The model also suggests that dimeric receptor interactions
the difficulty of studying endogenous membrane receptors in hold together a hexagonal mosaic of trimeric complexes and
their native states, no specific structural information has been focuses on the receptor dimer (i.e. the dimeric receptor-recep-
available to begin to address this question.

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TRAIL-induced DR5 Networks Are Highly Organized

tor interaction embedded within the network of trimeric tion System). Filters for excitation and emission of CFP (430/24 Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012
ligand-receptor complexes) as the intracellular activating unit. and 470/24 nm, respectively) and YFP (500/20 and 535/30 nm,
An alternative model suggests that the network is held together respectively) were controlled using an automated filter wheel
by trimeric, not dimeric, receptor interactions (Fig. 1, D and E, (Ludl MAC6000). Steady-state images were taken in 5–20-s
bottom) (41). intervals throughout the course of photobleaching, which took
an average of 5 min. Images were acquired using MetaMorph
Here, we have evaluated whether TRAIL-induced rearrange- and analyzed using ImageJ software. Instrument-independent
ment of DR5 involves the formation of a stable receptor dimer FRET efficiency was calculated using Equation 1, where FDA is
species and whether that dimeric species exists within large the donor fluorescence in the presence and FD is the donor
structured networks. In the process, we offer the first evidence fluorescence after acceptor photobleaching, as described previ-
that TM ␣-helices within TNF receptors form tightly associ- ously (48). Results for each experiment are based on ϳ50 cells
ated bundles and the first structural and functional interroga- taken from a single transfection over ϳ5 regions of interest,
tion of two distinct, alternatively spliced DR5 isoforms. with each cell treated as an independent sample, and cells hav-
ing fluorescence primarily within the plasma membrane were
EXPERIMENTAL PROCEDURES studied. Reported averages and standard errors were calculated
over the full sample of cells. All FRET experiments were
Cell Culture and Reagents—Jurkat cells were cultured in repeated at least three times to confirm results.
RPMI 1640 media (HyClone). HEK293 cells were cultured in
DMEM (HyClone). BJAB-derived cells (43, 44) were cultured E ϭ 1 Ϫ FDA (Eq. 1)
in RPMI 1640 with HEPES, sodium pyruvate, and L-glutamine FD
(ATCC). All media were supplemented with 10% FBS, L-glu-
tamine, penicillin, and streptomycin. All cultures were main- In a separate experiment, multiple populations of cells were
tained at subconfluence at 37 °C and 5% CO2 in a water- transfected at a CFP/YFP ratio ranging from 1:0.5 to 1:4. FRET
jacketed incubator. FLAG-sTRAIL (residues 114 –281) was was measured using photobleaching as described, and FRET
expressed in Escherichia coli and purified as described previ- efficiency was plotted as a function of YFP intensity. FRET effi-
ously (45) using the pT7-FLAG-1 inducible expression vector ciency versus YFP intensity data were fit using a two-parameter
and anti-FLAG resin (Sigma). DR5 antibody agonist (mAb631), saturable binding model, where the measured FRET efficiency
DR5 surface-staining antibody (mAb6311), and fluorescent (FRET%) is a function of the local YFP intensity, described by
secondary antibody (NL637) were purchased from R&D Sys- Equation 2 and described previously (46, 48 –50).
tems. Antibodies for Western blots, DR5 (3696) and ␤-actin
(5125), were purchased from Cell Signaling Technologies. FRET% ϭ FRET%max ⅐ YFP (Eq. 2)
BJAB DR5-deficient and BJAB DR5-deficient ϩ DR5-S cells YFP ϩ Kd2
were a kind gift from Andrew Thorburn (43, 44).
From this model fit of the data were extracted two parameters
Cloning and DNA Constructs—Complementary DNA as follows: the maximum FRET efficiency (FRET%max) and the
(cDNA) for full-length DR5-S (residues 1– 411) and DR5-L relative dissociation constant (Kd2), which yield information
(residues 1– 440) was cloned into pcDNA3.1(ϩ) for transient about the fluorophore separation (i.e. structure) and binding
expression in HEK293 cells. For transient expression in BJAB affinity, respectively, for comparison of DR5 isoforms and
DR5-deficient cells, DR5-S(1– 411) and DR5-L(1– 440) were mutants. Results shown are from three separate transfections at
inserted into pIRES2-EGFP vector. For FRET analysis, extracel- varying CFP/YFP ratios, and the results were pooled, with each
lular and TM residues for DR5-S(1–211) and DR5-L(1–240) point representing data from a single cell. These experiments
were inserted in-frame into pECFP-N1 and pEYFP-N1 vectors (i.e. multiple transfections at different CFP/YFP ratios) were
using EcoRI and BamHI sites. Both pECFP-N1 and pEYFP-N1 repeated to verify reproducibility, although data from transfec-
vectors contain the monomeric mutation A206K to the CFP or tions done on separate days were not pooled.
YFP preventing constitutive fluorphore clustering (46), and
CFP/YFP shows no affinity for each other (Fig. 3C). Mutation of Confocal Microscopy—Imaging of ligand-receptor clusters in
DR5 constructs was carried out by two-step PCR mutagenesis. Jurkat cells was done as described previously (37, 39). Briefly,
All constructs were verified by sequencing, and expression was cells were washed with PBS and treated with a DR5-specific
verified by Western blot and cell surface staining. CFP and YFP mouse antibody (R&D Systems, MAB631) agonist at 5 ␮g/ml
constructs were additionally verified individually by fluores- for 1 h. After washing, cells were treated with an anti-mouse
cence microscopy using both CFP and YFP filters. pECFP-N1 secondary antibody labeled with either NorthernLights557 or
and pEYFP-N1 constructs were a gift from David D. Thomas NorthernLights637 (R&D Systems) used at 5 ␮g/ml for 30 – 60
and the CFP-YFP tandem constructs was a gift from Stanley G. min and washed. All incubations and washes were done in
Nathenson (47). PBS ϩ 1% FBS and at 4 °C to prevent receptor internalization.
Labeled cells were transferred to pre-chilled polylysine-coated
FRET—Briefly, subconfluent HEK293 cells were transfected 35-mm glass-bottom culture dishes (MatTek Corp.) for ϳ5–10
by the calcium phosphate method of transfection with CFP- min at RT before imaging to allow cells to settle and clusters to
and/or YFP-tagged constructs shown in Fig. 3A with a 2-fold form.
excess of acceptor. Twenty four to 48 h post-transfection, cells
were imaged using a Nikon Eclipse TE200 inverted microscope NorthernLights637-labeled clusters were imaged in an
and a ϫ40 objective lens. Fluorescent proteins were illuminated Olympus IX81 inverted microscope equipped with a FluoView
using a mercury lamp (XCite 120-watt Fluorescence Illumina-

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TRAIL-induced DR5 Networks Are Highly Organized

FV1000 laser scanning confocal head (633 nm laser excitation, Version 2.0, and protein sequences were obtained via the NCBI Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012
645–745 nm emission). Either ϫ60 (1.42NA) or ϫ100 (1.3NA) gene data base.
oil immersion objective lenses were used.
Replica Exchange Molecular Dynamics (REMD) Simulations—
Z-stacks of NorthernLights557-labeled clusters were ac- REMD simulations were run using the CHARMM software
quired using a Zeiss Cell Observer Z1 microscope equipped package (51) and force-field version parameter set 22 (52) with
with a Yokogawa CSU-X1 confocal head using a ϫ100 CMAP correction (53) and the MMTSB toolset (54). All simu-
(1.40NA) oil immersion objective (561 nm laser excitation, lations were run using an implicit bilayer model, generalized
617/73 nm emission). The spinning disc confocal allowed born with switching function (55–56). For the monomer simu-
image acquisition at multiple depths overcoming the sample lations, the bilayer hydrophobic thickness was set to 25, 30, or
photobleaching problem in the laser scanning confocal. 35 Å, with a switching length of 0.3 Å in each case. The starting
Orthogonal sectioning and maximum intensity projections of configuration was a single ideal helix (sequence: GTKHS-
Z-stacks were performed using the Zeiss Axiovision software. GEVPAVEETVTSSPGTPASPCSLSGIIIGVTVAAVVLIVAVF-
VCKSLLWKK), placed in the center of the bilayer, and aligned
Cross-linking and Western Blot Analysis—Jurkat or BJAB with the bilayer normal axis. For the dimer simulations, TM
cells (DR5-deficient and re-expressing either DR5-S or DR5-L) helices (sequence: GTKHSGEVPAVEETVTSSPGTPASPC-
were washed and treated with TRAIL or DR5-specific antibody SLSGIIIGVTVAAVVLIVAVFVCKSLLWKK) were embedded
agonist for 1 h at 4 °C in PBS (pH 8.0) with rotation. Cells were in a bilayer of hydrophobic thickness of 32 Å). REMD simula-
subsequently cross-linked with 0.5–1 mM BS3 (Pierce; a homo- tions were run using 16 replicas, over a temperature range of
bifunctional, amine-reactive, noncleavable, and membrane- 300 – 600 K. A switch was attempted every 2 ps, with an
impermeable cross-linker with an 11.4 Å spacer arm), for observed switching frequency of ϳ25%. All analysis was per-
30 min at room temperature. Cross-linked samples were formed on the lowest temperature replica (300 K). Simulations
quenched with 20 mM Tris-HCl (pH 7.5) for 15 min at room were run using a 2-fs time step for a duration of 10 ns for each
temperature. Cells were pelleted by centrifugation and lysed in state (i.e. a total of 160 ns when considering the number of
RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% replicates).
sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40) supple-
mented with protease inhibitors and 10 mM iodoacetamide for Synthesis, Purification, and Analysis of DR5-TM—Solid-
1 h at 4 °C. Total protein concentration of lysates was deter- phase peptide synthesis of DR5-TM (TPASPCSLSGIIIGVT-
mined by BCA assay (Pierce), and equal amounts of total pro- VAAVVLIVAVFVSKSLLYKK) was carried out with a PE Bio-
tein (typically ϳ100 ␮g) were mixed with 2ϫ Laemmli sample systems PioneerTM using standard double-coupling cycles of
buffer with DTT and ␤-mercaptoethanol, boiled at 100 °C for Fmoc/tBu-chemistry with HATU/DIEA as coupling reagents,
10 min, and loaded on 4 –12% BisTris SDS-polyacrylamide gel in the presence of NMP. The sequence was assembled on
(Invitrogen). For nonreducing conditions, lysates were mixed Fmoc-Lys-PEG-PS resin (initial load of 0.18 mmol/g). The syn-
with 2ϫ Laemmli sample buffer in the absence of DTT and thesis was optimized using pseudoproline dipeptides: Fmoc-
␤-mercaptoethanol, but samples were treated identically oth- Val-Ser (␺Me,Me-Pro)-OH, Fmoc-Val-Thr (␺Me,Me-Pro)-OH,
erwise. Proteins were transferred to nitrocellulose membrane and Fmoc-Leu-Ser (␺Me,Me-Pro)-OH (Novabiochem, San
and probed using antibodies as described. To account for Diego). Final cleavage from the resin and side chain deprotec-
potential nonspecific cross-linking in the presence of ligand, tion was carried out by treatment with reagent K: TFA, phenol,
the same lysates were separated via SDS-PAGE and Coomassie- thioanisole, 1,2-ethanedithiol, water (82.5, 5, 5, 2.5, and 5%), for
stained (see supplemental material). 4 h at 25 °C. The cleavage mixture was filtered, and the resin was
washed with a small amount of reagent K. Combined filtrates
Caspase Activity and Surface Staining—BJAB DR5-deficient were concentrated under nitrogen and precipitated in 30 ml of
cells, five million cells in 400 ␮l of serum-free media, were diethyl ether at 0 °C. Precipitated peptide was collected by cen-
transfected by electroporation (200 V, 975 microfarads) with trifugation and washed with ice-cold diethyl ether. The crude
the indicated plasmid and moved back into 10 ml of complete peptide was dissolved in 50% TFA and purified by HPLC on a
media. Several hours after electroporation, live cells were iso- C-4 column (Vydac, 214TP1010) using a Gold Beckman
lated by Ficoll gradient. Twenty four to 48 h after transfection, Coulter system. Protein elution was achieved with a linear gra-
cells were treated with a DR5-specific antibody agonist dient from 0 to 63% B (95% isopropyl alcohol, 4.9% H2O, 0.1%
(mAb631) at 1 ␮g/ml for 4 h at 37 °C. Caspase activity was TFA) in 40 min at a flow rate of 2.0 ml/min with detection at 220
measured using CaspGLOW Red Active Caspase 8 staining kit nm. The HPLC fractions were collected and analyzed by
(BioVision), gating on live GFP-positive cells by flow cytometry MALDI-TOF MS. The pooled fractions found to be essentially
(FACSCalibur). Additionally, DR5 surface expression was pure were lyophilized to yield 22% based on starting resin.
measured on unstimulated cells by antibody staining using a
DR5 antibody and fluorescent secondary antibody. DR5 surface Analysis of DR5-TM by MALDI-TOF—Mass spectral data
expression was determined by flow cytometry using an identi- were acquired with a Bruker Biflex III Matrix-assisted Laser
cal gating scheme for live GFP-positive cells. Data were ana- Desorption/Ionization Time-of-Flight (MALDI-TOF) system.
lyzed using FlowJo software (Tree Star, Inc.). The sample was co-crystallized with the matrix 3,5-dimethoxy-
4-hydroxycinnamic acid (sinapinic acid), and the data were col-
Prediction of Helical Transmembrane Domains—The se- lected in the linear mode, positive polarity, with an accelerating
quence-based prediction of helical transmembrane domains potential of 19 kV. Each spectrum is the accumulation of 100 –
was based on a hidden Markov model via the TMHMM Server 400 laser shots. Mass spectroscopy for DR5-TM yielded an m/z

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TRAIL-induced DR5 Networks Are Highly Organized

FIGURE 2. DR5 is expressed as two functional isoforms. A, transcription of two alternatively spliced isoforms results in the expression of DR5-S and DR5-L Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012

differing by 29 amino acids (bold) within the extracellular domain and predicted transmembrane domain (underlined). B, DR5-S or DR5-L (or empty vector
control) were transiently transfected into BJAB DR5-deficient (DR5-def) cells and treated with a DR5-specific agonist antibody (␣-DR5) as indicated. Flow
cytometry analysis of caspase-8 activity shows a regain of function and ligand sensitivity with expression of DR5-S or -L. Results represent the level of caspase-8
activity (mean Ϯ S.E.) for three independent experiments. C, surface expression in transiently expressing cells was quantified by surface staining and flow
cytometry to show equal surface expression of DR5-S (blue) and DR5-L (green) and increased levels over vector transfected (red). D, whole-cell lysates from

Jurkat cells, BJAB DR5-def cells, and DR5-def cells re-expressing DR5-S or DR5-L confirm the predicted size of DR5-S (40 kDa) and DR5-L (43 kDa).

value of 3647 [M ϩ H]ϩ, which is in agreement with the which have been shown to express both isoforms at the mRNA
predicted value of 3646 Da. HATU is N-[(dimethylamino)-1H- level (57), also express both isoforms at the protein level (Fig.
1,2,3-triazolo[4, 5-b]pyridino-1-ylmethylene]-N-methylmeth- 2D). Importantly, although we show here the first evidence that
anaminium hexafluorophosphate N-oxide; DIEA is N,N-diiso- both isoforms of DR5 are ligand-sensitive, and DR5-S appears
propylethylamine; and NMP is N-methyl-2-pyrrolidone. to activate caspase-8 to a greater extent than DR5-L (Fig. 2B,
compare DR5-S and DR5-L), we do not suggest that DR5-S is
RESULTS necessarily more active as there are noticeable differences in the
expression level. Whether DR5-S and DR5-L differ in their olig-
DR5 Is Expressed as Two Functional Isoforms That Differ in omeric structure, a primary focus of this study, has not been
Their Transmembrane Domains—DR5 is expressed as two iso- investigated to date. However, given the location of Cys-209 of
forms that differ by the insertion of 87 bp resulting from alter- DR5-L (which, as we will describe below, is located within the
native splicing and the inclusion of an intron (57). Translation contiguous TM ␣-helix), we wondered whether this cysteine
of DR5 with and without this insertion results in the expression residue could be used to identify potential interactions, cova-
of long and short isoforms of DR5 at the protein level, respec- lent and noncovalent, within the DR5 TM domain.
tively, referred to here as DR5-L and DR5-S (Fig. 2A). DR5-L
contains an additional 29 amino acids at the junction between Ligand-induced Dimerization Mediates DR5 Network
the extracellular and predicted transmembrane (TM) domains, Formation—It has been shown previously that TNF ligands
including a cysteine residue at position 209. The organization of (including TRAIL and a DR5 antibody agonist) induce receptor
DR5 networks via ligand-induced dimerization may allow for clustering within the plasma membrane (37, 39, 40). Therefore,
interaction of DR5 TM ␣-helices as receptor TM domains are we first used fluorescence microscopy to identify whether DR5
no longer confined to the 50 Å separation observed in the agonistic antibody binds and induces ligand-receptor clusters.
TRAIL-DR5 complex crystal structure. Therefore, we won- Fluorescent-labeled agonist ligand binds DR5 on the surface of
dered if physical interactions between the TM domains of DR5 Jurkat cells, which endogenously express both DR5-S and
receptor monomers play a role in the stabilization of receptor DR5-L, and forms large clusters representing ligand-receptor
network. complexes within the plasma membrane (Fig. 3A). Images were
taken from a single confocal xy plane at approximately the mid-
We first sought to confirm the function of DR5-L and DR5-S. plane of the cell. Shown are the fluorescent receptor-bound
Both isoforms are functionally active, as expression of either agonist ligand (Fig. 3A, panel i), transmitted light image (Fig.
DR5-S or DR5-L in DR5-deficient cells (43) results in activation 3A, panel ii), and the overlay (Fig. 3A, panel iii) demonstrating
of caspase-8, both in a ligand-independent manner, consistent that cluster formation occurs within the plasma membrane.
with previous results (58), and in a ligand-induced manner (Fig. Estimates of the cluster sizes based on these fluorescent images
2B). Surface staining and flow cytometry confirmed approxi- are on the order of 200 –500 nm in diameter, consistent with
mately equal expression of DR5-S and DR5-L on the cell surface the estimated size of FasL-induced Fas clusters (37). To deter-
relative to the DR5-deficient control cell line (Fig. 2C). Further- mine whether the ligand-receptor clusters are distributed
more, protein lysates from cells expressing exclusively DR5-S throughout the plasma membrane of the entire cell, multiple
or DR5-L illustrate the size difference by SDS-PAGE, running confocal images (in the xy-plane) were acquired at varying
at approximately their predicted molecular masses of 40 kDa
(DR5-S) and 43 kDa (DR5-L), respectively (Fig. 2D). Jurkat cells,

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TRAIL-induced DR5 Networks Are Highly Organized

FIGURE 3. Ligand-induced DR5 dimers within high molecular weight networks. A, Jurkat cells were treated with an agonistic antibody specific to DR5 and Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012

subsequently stained with fluorescent secondary antibody. Confocal microscopy shows the formation of ligand-receptor clusters on the cell surface. Shown is
the fluorescent-labeled agonist (panel i), transmitted light (panel ii), and an overlay (panel iii). Scale bar represents 2 ␮m. B, confocal microscopy of agonist
treated Jurkat cells in x-, y-, and z-dimensions shows ligand-receptor clustering in the xy-, yz-, or xz-focal plane at approximately the mid-line of the cell.

Maximum intensity projection (MIP) in the xy-plane illustrates the size and distribution of these clusters. C, Jurkat cells were treated with TRAIL or DR5-specific
agonist (␣-DR5) and cross-linker (x-link) as indicated, and whole-cell lysates were run under nonreducing SDS-PAGE and probed for DR5. Highlighted are
monomeric (M), dimeric (D), trimeric (T), and oligomeric (O) forms of DR5. D and E, a similar experiment was run using BJAB DR5-deficient (DR5-def) cells ϩDR5-S
or ϩDR5-L using ␣-DR5. Agonist causes the formation of a disulfide dimer in DR5-L cells but not in DR5-S cells, and again this dimer species exists within a high
molecular weight complex. F, Jurkat, BJAB DR5-deficient, DR5-deficient cells ϩDR5-S, and DR5-deficient cells ϩDR5-L were treated with a DR5 agonist, and
lysates were run under nonreducing conditions. Shown is the dimeric form of DR5, present upon treatment with ligand and only when DR5-L is expressed.

depths (in z). Axial reconstruction at the cell mid-plane in each monomers, including interactions between TM domains. To
image clearly shows the distribution of fluorescent ligand-re- date, it is unknown whether ligand-induced structural re-ar-
ceptor clusters throughout the plasma membrane (Fig. 3B, xy-, rangement of DR5 results in stabilization of TM interactions,
yz-, and xy-planes). Maximum intensity projection in the xy- including disulfide bond formation via Cys-209 unique to
plane illustrates the approximate size and distribution through- DR5-L. First, Jurkat cells were treated with TRAIL or DR5 anti-
out the entire spherical cell (Fig. 3B, MIP). Collectively, confo- body agonist as indicated, and cell lysates were analyzed via
cal fluorescence microscopy results demonstrate that agonist nonreducing SDS-PAGE (Fig. 3C) (59, 60). The addition of the
binding to DR5 at the surface of the cell induces the formation membrane-impermeant cross-linker results in the formation of
of large clusters within the membrane, consistent with previous a ligand-independent receptor trimer, at the approximate
studies of DR5 and Fas, a related TNF receptor. molecular weight of three receptor monomer units (ϳ120 kDa,
Fig. 3C, compare 1st and 4th lanes). This result provides the
To begin to characterize the molecular architecture of first structural insight into pre-ligand receptor assembly and
ligand-induced DR5 clusters, and thus to illuminate new details stoichiometry of DR5, which differs from the presumed pre-
regarding the various models of how DR5 oligomers stabilize ligand dimeric state of TNFR1 (55) and motivates our choice in
networks (e.g. those in Fig. 1E), we used cross-linking of surface Fig. 1B. RNAi studies were used to confirm that this band is
proteins with a membrane-insoluble amine-reactive cross- indeed DR5 (see supplemental material).
linker. This approach allowed us to capture the native oligo-
meric states of the receptor as they are in the cell membrane The addition of either TRAIL or antibody agonist causes the
before they are lost on dissociating gels. Based on hypothetical formation of high molecular weight DR5 clusters (200ϩ kDa,
network models (Fig. 1E) and the sequence of the DR5 TM Fig. 3C, 5th and 6th lanes), reflecting the ligand-induced recep-
domain, we hypothesized that TRAIL- or agonist-induced net- tor clusters shown in Fig. 3, A and B. In the absence of cross-
works may be stabilized in part via close contacts between DR5 linker, the addition of TRAIL or agonist produces a distinct

21270 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 25 • JUNE 15, 2012

TRAIL-induced DR5 Networks Are Highly Organized

band at ϳ85 kDa, the expected molecular mass of two DR5 formation. We note that in both Jurkat and BJAB cell lines, Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012
monomer units (Fig. 3C, compare 1st lane with 2nd and 3rd there is an overall increase in DR5 intensity at all nonmono-
lanes), indicating the presence of a disulfide-linked receptor meric molecular weights upon addition of the cross-linker.
dimer species. That this is in fact a DR5 monomer-monomer Experiments with a reversible cross-linker suggest that cross-
interaction is further corroborated with mutational analysis linking does not up-regulate the receptor (data not shown);
and FRET experiments described below. That cross-linking of therefore, this difference in apparent total DR5 levels may more
either TRAIL- or agonist-treated samples causes the disappear- likely be due to epitope masking of certain populations of recep-
ance of this dimer band suggests that ligand-induced DR5 tor and/or poor detection of monomeric DR5 under nonreduc-
dimers are embedded within the high molecular weight clusters ing SDS-PAGE.
and provides the first molecular details of the ligand-induced
rearrangement of DR5 and the higher order structure of the DR5 Dimers Are Stabilized via Covalent and Noncovalent
TRAIL-DR5 network (Fig. 1E) and agonist-DR5 network. Anal- Interactions—Cells expressing DR5-S show no disulfide-linked
ysis under reducing conditions abolishes the DR5 dimer in both dimer; however, the similar presence of high molecular weight
the TRAIL and antibody agonist-treated samples (see supple- clusters indicates that DR5-S may also cluster via ligand-depen-
mental material), indicating that ligand-dependent dimeriza- dent dimerization, albeit exclusively through noncovalent
tion is driven through a cysteine disulfide bond, suggesting this interactions not detected by nonreducing SDS-PAGE. More-
may be DR5-L (further discussed below). over, in both DR5-S- and DR5-L-expressing cells, cross-linking
of surface proteins results in the formation of a ligand-indepen-
We note that the addition of ligand does not result in a dent receptor trimer (Fig. 3, C–E). Therefore, we tested
molecular mass shift of the 120 kDa pre-assembled receptor whether DR5-S and DR5-L are able to form stable receptor
trimer (Fig. 3C, 4th to 6th lanes). Thus, we see no specific band oligomers, including both receptor dimers and trimers,
at the predicted molecular weight of the ligand-receptor trimer through either covalent (i.e. disulfide) or noncovalent interac-
complex (the crystal structure, which should be at ϳ180 kDa). tions in transiently transfected HEK293 cells. To assess disul-
Like the dimer, we interpret this to most likely mean that the fide-linked dimerization, HEK293 cells were transiently trans-
ligand-bound trimeric structure (Fig. 1C) exists only transiently fected with either DR5-S or DR5-L (Fig. 4A), as well as cysteine
on its own before becoming embedded within the DR5 clusters. mutants (Fig. 4B), and lysates were isolated and run under
However, this accounting does not explain the persistence of reducing or nonreducing conditions as indicated. Transient
the 120-kDa trimer band upon addition of TRAIL. It is possible expression of DR5-L results in disulfide-linked dimerization,
that a steady-state level of pre-ligand assembled trimer is main- whereas DR5-S is unable to form disulfide-linked dimers, and
tained in the membrane. Alternatively, this lack of shift in reducing either DR5-S or DR5-L expressing cells produces
molecular weight may correspond to an inability to cross-link entirely monomeric receptors (Fig. 4A). Mutational analysis of
the TRAIL to the trimeric receptor. The distinction between free cysteine residues within the TM domains of DR5-S (Cys-
these various possibilities remains unclear. Numerous attempts 203) and DR5-L (Cys-209 and Cys-232) demonstrates that
to identify TRAIL on these blots has proven unsuccessful, pos- disulfide dimers of DR5-L involve Cys-209, the cysteine residue
sibly due to a lack of sensitivity in detecting ligand bound to unique to DR5-L. Mutation of the TM Cys-203/-232 (a con-
endogenous receptor or due to epitope masking (e.g. as a result served TM cysteine residue in DR5-S and DR5-L, respectively,
of residing in a complex or due to cross-linker masking of the see Fig. 2A) has no effect on dimerization (Fig. 4B).
antibody epitope).
To evaluate noncovalent association of receptors, HEK293
Because Jurkat cells express both isoforms of DR5, we tested cells were transiently transfected with DR5-S or DR5-L, and
whether the observed disulfide-bonded dimer is indeed DR5-L, surface receptors were cross-linked as described and cell lysates
likely given its additional cysteine residue. We generated two analyzed via SDS-PAGE. Interestingly, although noncross-
cell lines derived from a BJAB cell line previously selected for linked receptors are entirely monomeric, cross-linking of sur-
DR5 deficiency (43, 44) expressing either the DR5-S or DR5-L face receptors shows that oligomeric receptor structures are
isoform. The addition of a DR5-specific agonist induces clus- stable within the membrane at high levels of receptor expres-
tering in both DR5-S and DR5-L lines (Fig. 3, D and E), although sion in the absence of ligand (Fig. 4C). Noncovalent dimers of
somewhat more so in the short isoform. Importantly, however, DR5-S are present, although the short isoform has a greater
the ligand-dependent formation of disulfide-linked receptor tendency to form high molecular weight aggregates in the
dimer occurs exclusively in cells expressing DR5-L (Fig. 3, D absence of ligand. That DR5-S exists as a dimeric species when
and E, compare 2nd lane in each Western blot). This result is concentrated at the cell surface suggests that, like DR5-L, it may
recast in a separate noncross-linked Western blot, focusing on exist as a dimer within ligand-receptor networks (despite lack-
the dimer molecular weight from four cell lines (Jurkat, BJAB ing the cysteine) (Fig. 1E). Under these overexpressed condi-
DR5-deficient, BJAB-DR5-S, and BJAB-DR5-L) (Fig. 3F). That tions, DR5-L primarily forms receptor dimers and trimers (Fig.
disulfide-linked dimers of DR5-L do not occur in the absence of 4C), with a notable reduction in higher order molecular weight
ligand despite ligand-independent trimerization suggests the clusters as compared with DR5-S, consistent with the differ-
following. 1) The free cysteine residue in unliganded DR5-L is ence in the BJAB cells (Fig. 3, D and E). The presence of oligo-
inaccessible, either by the receptor conformation or possibly by meric receptor species in transiently expressing cells indicates
being buried in the membrane, and is thus unable to form disul- that overexpression due to the unregulated CMV promoter,
fide linkages. 2) Ligand binding causes conformational changes and thus receptor crowding in the membrane, enables DR5 to
that expose the free cysteine and thus promote disulfide bond adopt an active oligomerized conformation, consistent with the

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TRAIL-induced DR5 Networks Are Highly Organized

FIGURE 4. DR5 dimerization and network formation via covalent and non-covalent interactions. A, HEK293 cells were transiently transfected with DR5-S Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012
or DR5-L, and lysates were run on SDS-PAGE in the absence or presence of reducing agents (red). Transient overexpression of DR5-L, but not DR5-S, in HEK293
cells causes spontaneous disulfide dimer formation. B, cysteine mutagenesis within the transmembrane domain, including Cys-203 in DR5-S and Cys-209 and
Cys-232 in DR5-L, demonstrates that disulfide dimerization of DR5-L occurs via Cys-209, a cysteine residue unique to the long isoform. Highlighted are the
monomeric (M) and dimeric (D) forms of DR5. C, HEK293 cells transiently expressing DR5-S or -L (or vector control) were surface cross-linked (x-link) and run on
SDS-PAGE gel. Surface cross-linking shows the similarities and differences in the organizations of DR5-S and DR5-L. Consistent with stabilization of an active
conformation, dimer formation occurs in both DR5-S and DR5-L under transient overexpression. DR5-S forms high molecular weight clusters, whereas DR5-L
is primarily dimeric and trimeric. Highlighted are monomeric (M), dimeric (D), trimeric (T), and oligomeric (O) forms of DR5. D, full-length YFP-tagged DR5-S
(panel i) shows a high degree of receptor clustering within the membrane, forming large receptor aggregates, consistent with cross-linking experiments.
Full-length YFP-tagged DR5-L (panel ii) shows some degree of clustering in the membrane, but it has a more diffuse pattern than DR5-S. Consistent with
previous studies, removal of the cytosolic domain results in homogeneous localization throughout the plasma membrane, as observed with DR5-S-YFP lacking
a cytosolic domain (panel iii).

observation that overexpression results in ligand-independent cells expressing DR5-S or DR5-L (Fig. 3, B and C) and 293 cells
DR5 activity (58). We note the lower level of background non- (Fig. 4C).
specific bands in transiently expressing HEK293 cells (com-
pared with Jurkat and BJAB results in Fig. 3), which is a result of DR5 Transmembrane ␣-Helices Form Dimeric Bundles in
receptor overexpression relative to other proteins. Both Isoforms—Accordingly, to further measure receptor
structure in the membrane of a living cell, we performed fluo-
To verify these Western blot results that, at overexpressed rescence resonance energy transfer (FRET), using CFP- and
receptor levels, DR5 clusters occur in the absence of ligand, YFP-tagged DR5 and tumor necrosis factor receptor 1 (TNFR1)
DR5-S and DR5-L were tagged with a C-terminal YFP fluoro- as a control (Fig. 5A). Placement of the fluorophore immedi-
phore, and the resulting plasmid transfected into HEK293 cells ately downstream of the TM domain allows for analysis of TM
for fluorescence imaging. The function of both DR5-S-YFP and separation. Similar constructs have been used to study receptor
DR5-L-YFP was confirmed by their ability to activate caspase-8, assembly in DR5, Fas, and TNF systems using FRET (61).
with DR5 agonist augmenting caspase-8 activity, similar to the Although truncation of the cytosolic domain precludes the for-
untagged receptor when expressed in DR5-deficient BJAB cells mation of ligand-independent receptor clusters (Fig. 4D), this
(see supplemental material). Using confocal microscopy, we placement of the fluorophore immediately downstream of the
observe the formation of large clusters of DR5-S-YFP in the TM domain still yields measurable energy transfers. This dem-
plasma membrane in the absence of ligand (Fig. 4D, panel i). onstrates that truncated receptors retain the ability to oli-
Expression of DR5-L-YFP, although somewhat clustered, gomerize via extracellular and transmembrane residues (the
exhibits a more diffuse pattern of fluorescence in the plasma focus of this study), and it further allows for detailed analysis of
membrane (Fig. 4D, panel ii), suggesting a lesser degree of clus- TM domain oligomerization and the relative role of TM resi-
tering consistent with cross-linking results. As a control, dues, including Cys-209. The function of fluorescent protein-
removal of the cytosolic domain results in a diffuse localization tagged DR5 constructs, both full-length and truncated, was
of both DR5-S (Fig. 4D, panel iii) and DR5-L (data not shown) evaluated in BJAB DR5-deficient cells (see supplemental
in the plasma membrane, confirming a potential role of the material).
death domain in cluster formation as described previously (36).
In addition to demonstrating that overexpression of DR5 FRET was measured by acceptor selective photobleaching
results in ligand-independent oligomerization, confocal mi- (62), where an increase in donor fluorescence after selective
croscopy results demonstrate that surface cross-linking of DR5 photobleaching of the acceptor provides a quantitative measure
is nonrandom, as DR5-S and DR5-L show different patterns of of energy transfer efficiency between fluorophores (Fig. 5B).
fluorescence, consistent with results in agonist-treated BJAB Transfection of DR5-CFP and -YFP constructs yields signifi-
cant energy transfer over donor-only controls, with DR5-L,

21272 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 25 • JUNE 15, 2012

TRAIL-induced DR5 Networks Are Highly Organized

FIGURE 5. DR5-S and DR5-L differ in their self-associating affinity but not in their TM domain separation. A, FRET constructs, DR5-S, DR5-L, and TNFR1, Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012

were truncated and labeled shortly after the predicted transmembrane domain. B, acceptor photobleaching FRET in TNFR1, used as a control, shows an

increase in donor (CFP) fluorescence after selective photobleaching of the acceptor (YFP). C, co-transfection of donor and acceptor plasmids in HEK293 cells

shows significant differences in energy transfer between DR5-S and DR5-L. Additionally, hetero-oligomer formation is less favorable than homo-oligomeriza-

tion of either DR5-S or DR5-L. Neither DR5-S nor DR5-L is able to form hetero-oligomeric complexes with TNFR1. Note: * indicates statistically significant with
p Ͻ 0.05; ** indicates statistically significant with p Ͻ 0.001. D, mutation of DR5-L C209A causes a significant reduction in DR5-L FRET to a level indistinguishable
from DR5-S. Note: * indicates statistically significant with p Ͻ 0.05. E–G, transfection of donor and acceptor constructs at varying acceptor (YFP) levels shows
increasing FRET at higher YFP concentration. Each data point represents calculated FRET efficiency at a measured YFP intensity for an individual cell. Curve fits

(solid lines) are based on a two-parameter saturable binding curve. H, parameters from the curve fit, maximum FRET efficiency, and Kd2 suggest that the
fluorophore separation of the DR5-S, DR5-L, and DR5-L C209A in the complexed state are indistinguishable (light bars), but the effect of the cysteine residue is

to reduce the dissociation constant and thus favor oligomerization (dark bars).

0.402, having a significantly higher energy transfer than DR5-S, C209A is identical to that of DR5-S, this mutation did not
0.327 (p Ͻ 0.05) (Fig. 5C). Although co-transfection of DR5-S increase the propensity for heterodimer formation with DR5-S
and DR5-L leads to measurable levels of hetero-oligomeric (data not shown), suggesting that the relatively low hetero-
FRET, 0.164, the amount of energy transfer is significantly oligomerization affinity is not due to the high affinity of DR5-L
lower (p Ͻ 0.001) than in either homo-oligomeric case. The self-association via the disulfide bond. Rather, differences in
hetero-oligomeric interaction of DR5-S or DR5-L with TNFR1 hetero-oligomerization may be a result of isoform-dependent
yields negligible energy transfer, 0.022 and 0.040, respectively differences in the vertical position of the PLAD, protein local-
(Fig. 5C), indicating that the DR5-S/DR5-L hetero-oligomeric ization within the membrane due to TM domain differences, or
interaction, although less favorable than the homo-oligomeric perhaps a difference in affinity due to TM orientation.
interaction, is still present and may represent a physiologically
relevant structure in the plasma membrane. The observation of measurable steady-state FRET efficien-
cies clearly indicates that oligomeric receptor complexes exist
Mutation of DR5-L Cys-209 to alanine results in a significant in the cell membrane. FRET efficiency increases with either the
decrease in the FRET efficiency of DR5-L to a level statistically fraction of receptors in oligomeric complexes or the proximity
indistinguishable from that observed for DR5-S (Fig. 5D). This of donors and acceptors within the complex. Thus, it was
result is consistent with the idea that receptor oligomerization unknown whether the observed differences in FRET efficiency
of DR5-L, directed in part through Cys-209 disulfide dimeriza- between DR5-S, DR5-L and DR5-L C209A are due to a change
tion, results in a structure and/or association kinetics that differ in the equilibrium of association or a change in the structure of
with those of DR5-S. Although the FRET efficiency of DR5-L the dimer (i.e. the separation of the fluorophores). To elucidate

JUNE 15, 2012 • VOLUME 287 • NUMBER 25 JOURNAL OF BIOLOGICAL CHEMISTRY 21273

TRAIL-induced DR5 Networks Are Highly Organized

FIGURE 6. Transmembrane domain clustering of DR5. A, synthetic DR5-L TM peptide was reconstituted in DPC micelles in the absence and presence of Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012

reducing agents and glutaraldehyde cross-linker (x-link) as indicated, run on SDS-PAGE gel, and analyzed by silver stain. Results show that DR5-L TM domain

may form higher order clusters, including TM trimers and tetramers under nonreducing, cross-linked conditions. Samples prepared in the presence of reducing

agents do not recruit additional TM helices, suggesting a possible TM domain role in higher order receptor clustering. Highlighted are monomeric (M), dimeric

(D), and oligomeric (O) forms of the TM peptide. B, replica exchange molecular dynamics simulation of the monomeric DR5-L TM domain embedded in a
membrane shows contiguous ␣-helix formation of four amino acids unique to DR5-L, including Cys-209. Helical residues shown in red are located within
bilayer. C, replica exchange molecular dynamics simulation of the membrane embedded, disulfide-linked DR5-L dimer predicts one possible ␣-helical TM
structure that includes a GG4 (GXXXG) interaction at the dimer interface.

these two independent factors, FRET measurements were that if this is the dimer structure that is embedded within a
made in HEK293 cells at varying YFP concentrations, and FRET supramolecular network, then the TM helices may in fact form
efficiency was plotted as a function of YFP intensity (Fig. 5, a stable dimeric bundle within the membrane. Therefore, we
E–G). FRET efficiency (FRET) increases with increasing YFP next tested whether synthetic DR5 TM peptides, in the absence
intensity ([YFP]), and the data were fit to a hyperbolic saturable of regulation by soluble domains, have the propensity for self-
binding curve of the form FRET ϭ (FRETmax⅐[YFP])/(Kd2 ϩ association. Synthetic DR5 TM peptides were reconstituted
[YFP]) as described previously (46, 50). From this fit two param- into dodecylphosphocholine micelles in the absence and pres-
eters were extracted as follows: the maximum FRET efficiency ence of reducing agents and were then cross-linked, or not, with
(FRETmax, the theoretical limit of FRET efficiency at large glutaraldehyde. We observe the covalent dimerization of TM
[YFP]) and the relative dissociation constant, Kd2 (the YFP con- peptides via Cys-209 disulfide bonding with samples prepared
centration at which half-maximum FRET is observed), yielding in the absence of reducing agents (Fig. 6A). The addition of
information about fluorophore separation and relative binding reducing agents during sample preparation precludes dimer
affinity, respectively. DR5-S and DR5-L C209A show a similar formation. Moreover, cross-linking of reduced samples shows
increase in FRET efficiency with increasing acceptor concen- the DR5 TM domain has only a low propensity to dimerize via
tration, whereas DR5-L shows a sharp increase in energy trans- noncovalent interactions, suggesting that noncovalent interac-
fer at low levels of YFP, thus favoring the complexed state. tions within the TM domain alone may not be sufficient for
Despite differences in the equilibrium of oligomerization, the receptor dimerization. Nonreduced, cross-linked samples
maximum FRET in each case is approximately equal, indicating show that the disulfide-linked dimer species may be able to
that fluorophore separation in the receptor-complexed state is recruit an additional monomer or dimer, indicating some level
the same for DR5-S, DR5-L, and DR5-L C209A (Fig. 5H). These of higher order TM domain clustering through both covalent
results suggest that receptor assembly involves TM dimeriza- and noncovalent interactions, consistent with results in
tion, forming similar oligomeric structures even in the absence HEK293 cells expressing the full-length protein (Fig. 4C).
of disulfide bond formation. Time-resolved FRET analysis using labeled peptides also con-
firms that DR5 TM peptides exist as oligomers in detergent,
In general, interactions between TM domain ␣-helices can both in the presence and absence of reducing agent (data not
provide a driving force for intermolecular assembly and are shown).
involved in the physiologic function and pathogenic dysfunc-
tion in a number of TM signaling molecules (63– 66). However, We predicted the structure of the monomeric and disul-
in the crystal structures of the ECD of the TRAIL-DR5 complex fide-linked TM domain using REMD simulations (67). As
(which do not include any of the additional residues in the expected from sequence-based ␣-helical TM prediction
DR5L isoform), the last resolved residues place the three TM tools (see Fig. 2A), several of the residues in the long isoform
␣-helices ϳ50 Å apart. This has led to the conclusion that the form a contiguous helix and insert in the membrane (Fig.
TM helices of DR5 are independent and noninteracting, a pre- 6B). Additionally, the structural prediction of the DR5 TM
sumption that is propagated in all molecular schematics of TNF monomer suggests a potential role for the widely conserved
receptors. Conversely, the pre-ligand dimeric structure of GG4 (GXXXG) helix dimerization motif (68), which has
TNFR1 places the TM helices less than 20 Å apart, suggesting approximately the same circumferential location on the

21274 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 25 • JUNE 15, 2012

TRAIL-induced DR5 Networks Are Highly Organized

␣-helix as Cys-209. REMD simulation of the disulfide-linked vide the first substantive support for these types of models, Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012
DR5 TM dimer further shows a noncovalent GG4 interac- shown in Fig. 1E, in which TRAIL-induced networks display an
tion in the disulfide-linked state (Fig. 6C). In the symmetric organization via DR5 dimerization through membrane-proxi-
dimer, this dimerization motif lies on the same helical inter- mal residues. In the case of DR5, our data suggest that pre-
face as the Cys residue forming a CXXXGXXXG sequence in formed receptor trimers, different from the pre-assembled
the long isoform. It is unclear how often a cysteine residue dimers observed in the case of TNFR1, are engaged by tri-
occurs in proximity to a GG4 motif, although a brief bioin- merized TRAIL (24) to form a TRAIL-DR5 trimer complex
formatics search reveals that several membrane proteins during which the receptors undergo a conformational change
contain the CXXXGXXXG motif within the TM domain, that exposes the dimeric interaction motifs. We have shown
including LST1, a protein that spontaneously forms disul- that this motif includes interactions between TM domain
fide-linked homodimers (69), and death receptor 4, a related ␣-helices (e.g. the Cys-209 in the case of DR5-L and possibly
TRAIL receptor for which no known TM interactions exist GXXXG in the case of both DR5-L and DR5-S), but we do not
(although we note that the motif is located in a different rule out the likely possibility that the interaction is further sta-
region of the predicted TM helix than in DR5). Collectively, bilized by interactions in the ECD and intracellular domains as
these results show a strong role for TM ␣-helix dimers in the well (for example those in or near the PLAD and death domain).
overall architecture of activated DR5. This ligand-induced structural rearrangement promotes for-
mation of receptor dimers that tether the trimeric ligand-re-
DISCUSSION ceptor complex (Fig. 1D) thus driving the formation of orga-
nized receptor networks (Fig. 1E). Therefore, formation of the
The current understanding of the structure-function rela- network results serves two general purposes. First, network for-
tionship in the TRAIL-DR5 complex is based not on native mation stabilizes the receptor dimer species that may in turn
full-length protein expressed at endogenous levels but rather stabilize the dimerization of intracellular death domains as well
on crystals of fragmented soluble domains, the behavior of as caspase-8, as suggested by structural and functional data to
overexpressed receptors in model cell lines, and fluorescence be the active conformation. Also, network formation may pro-
imaging at the whole-cell level. These studies have provided vide a high, local concentration of active dimeric intracellular
highly useful information about the fundamental role of the death domains able to overcome inhibitory pathways.
PLAD, ligand-binding, and death domains and suggested a role
for supramolecular clustering. However, little has been done to In the context of the hypothetical network models (Fig. 1E),
elucidate the structural and molecular mechanisms in early one interpretation of our data is that ligand-induced dimeriza-
events of DR5 signaling. It is therefore of great interest and tion via TM residues (including both Cys-209 disulfide bond
difficulty to understand the nuanced role of not just the specific formation and noncovalent association) occurs between neigh-
interactions between TRAIL and DR5 but also between DR5 boring trimeric structures of ligand and receptor. An alterna-
monomers and exactly how the sum of such interactions result tive and equally valid interpretation of the data is that ligand
in the orchestration of macromolecular aggregates visible by induces a dimeric association (via Cys-209 disulfide bond or
fluorescence microscopy (39). noncovalent interactions) within a trimeric ligand-receptor
structure. i.e. TM association occurs between two receptor
Supramolecular clustering of receptors, including TNFR1, monomers within a crystal structure unit. This intra-trimeric
Fas, and DR5 has emerged as a potentially powerful paradigm TM association would likely tend to oppose network formation,
shift in the field of apoptotic signaling. The canonical view of a as intra-trimeric association would leave only one free TM
single trimeric ligand-receptor structure is giving way to a domain to associate with an adjacent trimeric unit. Thus, in the
revised picture of highly organized networks of ligands and extreme case where every ligand-independent, pre-assembled
receptors driven through stable receptor-receptor interactions trimer forms an intra-trimeric association upon ligand binding,
in multiple domains that vary in their stoichiometry to generate one would expect a maximum of two trimeric ligand-receptor
large aggregates of ligand-receptor trimeric structures (41, 42). units per cluster. In the more realistic case, where intra-tri-
The first network model was put forth by Chan (41) and sug- meric association occurs in a percentage of these complexes,
gests that ligand causes aggregation of pre-assembled dimeric network size would tend to be limited due to a lack of free TM
receptors via extensive receptor-receptor interactions in a tri- helices. In the context of DR5-L, if irreversible intra-trimeric
fold symmetry. A second network model was put forth by Cys-209 disulfide bond formation occurs in a fraction of all
Ozsoy et al. (42) suggesting that trimeric TNF ligands engage a trimeric ligand-receptor complexes, we would expect less net-
pre-formed receptor dimer complex and cause a 120° rotational work formation and a potentially less functional isoform of
conformational change resulting in dimeric activation and for- DR5. Interestingly, all of our data are consistent with this inter-
mation of a network in the form of a hexagonal mosaic of pretation that DR5-L tends to form networks to a lesser extent
ligand-receptor trimers. Although both models are intrigu- than DR5-S (see Figs. 3, D and E, and 4C) and is less functional
ing, they lack the necessary diverse experimentation and evi- (see Fig. 2B). Therefore, we cannot rule out the possibility for
dence to support their foundation, which is a ligand-induced intra-trimeric TM association, which may be irreversible in the
dynamic reorganization of pre-ligand assembled dimers that case of DR5-L. Additionally, we cannot rule out the possibility
enables specific receptor-receptor interactions that stabilize that receptor networks may be stabilized via trimeric receptor
the network. bundles (Fig. 5E, compare top (42) and bottom (41) models) or
potentially even by tetrameric receptor bundles via combina-
Our results from an endogenous system (Jurkat cells) and
corroborated in model systems (BJAB and HEK293 cells) pro-

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TRAIL-induced DR5 Networks Are Highly Organized

tions of covalent (Cys-209 disulfide) and noncovalent (GG4) biophysics as has been attempted for DR5, as well as in the study Downloaded from www.jbc.org at University of Minnesota, on September 3, 2012
interactions, as predicted by studies with the purified TM pep- of a separate class of receptor networks (36, 73, 74).
tide (see Fig. 6A). Ongoing efforts include quantification of
cluster size and differences in cluster size between DR5-S and CONCLUSION
DR5-L isoforms.
We have shown that TRAIL-induced cell death via DR5
A second important facet of this study, in particular for the involves structural re-organization and receptor dimerization
ongoing efforts to target DR5 in the treatment of cancer, is the within large protein complexes and that dimerization is medi-
validation and further understanding of two DR5 isoforms ated by membrane-proximal residues. This is the first evidence
expressed on the protein level in human cancer cells. To date, to suggest that ligand-induced receptor clusters are highly
no pattern of DR5-S or DR5-L expression in normal versus organized and that they are likely regular arrays or regularly
tumor cells is known, although it is intriguing to postulate that structured networks. Moreover, given the structural and func-
the presence of DR5-S and/or DR5-L isoforms in tumor cells tional evidence that dimerization of intracellular domains as
may serve as a viable predictive biomarker for TRAIL-based well as downstream signaling proteins is critical for their acti-
targeted therapy and/or any agent that may rely on engaging vation, the ligand-induced receptor dimerization event appears
this pathway. Although the expression of two DR5 isoforms (at to be a crucial step in DR5 activation. Here, we took advantage
the mRNA level) was first noticed relatively early after the dis- of a free cysteine residue in DR5-L to identify a novel protein-
covery of DR5 (57, 70), no studies have investigated the differ- protein interaction motif and show that the fluorophore sepa-
ence, structural or functional, between the two, and only a few ration in the DR5-L disulfide-linked structure is indistinguish-
publications have acknowledged the expression of the two iso- able from that of DR5-S. Thus, at endogenous receptor levels,
forms at the protein level (58, 71–72). The functional role of the ligand-induced dimerization occurs, at least in part, via the TM
29 amino acid insertion in DR5-L is yet unknown, although the domain, a domain in the TNF receptor superfamily that has
sequence is rich in structurally active residues, such as proline, remained largely unstudied. More broadly, given their struc-
as well as chemically active residues, such serine or threonine tural homology, both monomeric and ligand-bound, many
residues that may be modified by phosphorylation or glycosyl- other TNF receptor superfamily members likely undergo a sim-
ation as shown previously (58). It is possible that, with the addi- ilar dimerization event that is critical for signal propagation
tion of 29 amino acids, the cysteine residue evolved to hold the through the membrane. Ongoing efforts to understand the
TM helices in an active conformation that is otherwise less functional relevance of TNF receptor networks should clarify
favorable due to the increased flexibility from the additional whether they exist as a mechanism to concentrate intracellular
residues upstream. signals (to overcome biochemical thresholds), whether the net-
work formation itself causes the key conformational change
Whether there is functional significance to our finding that associated with signaling, or both.
DR5-S and DR5-L preferentially form homo-oligomers over
hetero-oligomers remains unknown. Mutation of C209A does Acknowledgments—We thank Ameeta Kelekar and James Sturgis for
not increase the ability of DR5-L to interact with DR5-S (data invaluable insight and discussion, Andrew Thorburn for providing
not shown), suggesting that the reduced propensity for hetero- DR5-deficient BJAB cells, Suzanne Haydon for help with FRET and
oligomerization is not solely due to the disulfide bonds between analysis, and also the University of Minnesota-University Imaging
DR5-L monomers. That DR5-S and DR5-L have a low propen- Center and the Minnesota Supercomputing Institute for imaging and
sity to form hetero-oligomers begs the question of whether one computational resources.
has a higher affinity for decoy receptors (which do not have free
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