Supplemental Results

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1 Supplemental Results TRAIL induces disulfide linked dimers of DR5 within high molecular weight networks We have shown that both TRAIL and a DR5-specific antibody agonist induce network formation that involves dimerization of receptor monomer units. To conclusively determine whether the observed dimer is formed via disulfide bond, Jurkat cells were treated with and without TRAIL and cell surface cross-linker, and lysates were analyzed by SDS-PAGE and Western (Figure S1A). In the absence of reducing agents the addition of TRAIL results in the formation of receptor dimers, and cross-linking causes the dimer band to disappear, presumably because it is embedded in high molecular weight clusters (> 260 kd), as described in the main text. Under reducing conditions, on the other hand, the dimer band is absent under all conditions, indicating that ligand-induced dimerization is mediated by disulfide bond formation. The clusters are still observed in cross-linked samples run under reducing conditions, indicating that disulfide bonds are not necessary the detection of networks via SDS-PAGE and Western blot. Cross-linking with BS3 does not cause non-specific aggregation of TRAIL and non-dr5 proteins The addition of cross-linker potentially could result in non-specific cross-linking of TRAIL and DR5 to nearby, but unrelated membrane proteins. While we expect that any cross-linker promiscuity would likely be insensitive to the addition of TRAIL (Figure 3C, compare lanes 4 and 5), we wanted to rule out the possibility that the addition of TRAIL is increasing the overall level of high molecular weight protein species in the presence of cross-linker. The same cell lysates used in Figure S1A were separated under reducing conditions and the gel Coomassie stained (Figure S1B). Staining of all proteins in the lysate shows no detectable change in the band pattern upon TRAIL addition, indicating that the observed increase in DR5 at high molecular weights (Figures 3C-E and S1A) are not a result of random crosslinking upon the addition of TRAIL. TRAIL ligand causes disulfide linked dimers in the presence and absence of ligand enhancers It has been suggested that agonist antibody cross-linking, or enhancement of ligand prior to treating cells increases its activity by clustering the ligand into multimers of the trimeric ligand complex (1). We tested whether Flag-TRAIL ligand in the absence or presence of an enhancing anti-flag antibody has any effect on DR5 dimerization. Flag-TRAIL was pre-incubated with an enhancing agent (anti-flag antibody) at room temperature for 15 minutes. TRAIL was incubated with Jurkat cells as before. The addition of a ligand enhancer has no affect on the ability of TRAIL to produce DR5 dimers and high molecular weight clusters, suggesting that ligand-induced dimerization of DR5 is not dependent on antibody-mediated clustering of TRAIL ligand (Figure S2). The addition of an antibody agonist also produces dimeric DR5, at an identical weight, suggesting that the addition of ligand either TRAIL or antibody agonist induces dimerization and correlates with receptor function. Pre-ligand assembly of DR5 results in receptor trimers Pre-ligand assembly is critical for ligand binding and function of DR5 (2, 3). In determining the molecular architecture of ligand-induced DR5-networks, we found that cross-linking of surface proteins results in the formation of DR5 trimers at approximately 120kd (see Figure 3 C-E). To confirm that this band is indeed DR5, we knocked down DR5 in Jurkat cells by transfection of shrna and selection (see Supplemental Methods). DR5 knockdown cells and the parental (wild type) Jurkat cells were cross- 1

2 linked, or not (as described in the Experimental Procedures), and lysates were separated via SDS-PAGE and analyzed by Western blot. Cells stably expressing shrna targeting DR5 show a reduced band at ~120kd, confirming that cross-linking of Jurkat cells in the absence of ligand produces a distinct band corresponding to pre-ligand, trimeric DR5 (Figure S3). Further, we have noted that this trimer band is not diminished in the presence of TRAIL or agonist. Based on the model presented in Figure 1, it would seem likely that pre-assembled DR5 trimers, upon binding bind TRAIL (or agonist), would decrease the amount of trimeric DR5, perhaps shifting this band upwards by the molecular weight of the trimer. We have offered two interpretations for our observation that the 120kd trimeric DR5 band persists in the presence of TRAIL or agonist. First, it is conceivable that the chemistry and/or linker-length of the cross-linker is such that networks are more readily cross-linked than individual trimeric ligand-receptor (i.e. crystal structure) units. That is, perhaps the crystal structure unit is not chemically cross-linkable (at least not with BS3). Alternatively, it is possible that ligand binding to pre-assembled trimers, resulting in the formation of high molecular weight networks, drives further pre-assembly of DR5, replenishing the pool of pre-assembled complexes. Full-length, fluorophore-tagged DR5 is functional Death receptors, as other proteins, are often tagged with GFP and variants to study localization, distribution and structure (4-7). Here, we use XFP-tagged DR5 to determine molecular-level structure and binding kinetics as well as cellular localization and distribution of both truncated and full length DR5. We evaluated the function of these receptors, including truncated (lacking the cytosolic domain) and full length, tagged and untagged as shown in Figure S4, top. Briefly, each construct was transiently transfected into BJAB DR5-deficient cells, and activity was assessed by a fluorescent caspase-8 marker in the absence and presence of a DR5-specific agonist ligand. Activity results are shown in Figure S4, bottom. Control transfections (empty pires2-egfp or peyfp-n1 vectors) show no difference in caspase-8 activity compared to untransfected cells, nor do these cells have any sensitivity to ligand. As a positive control, cells were transfected with DR5-S-IRES-GFP, resulting in co-expression of untagged DR5-S and GFP. DR5-S-IRES-GFP transfected cells have an increased basal level of caspase-8 activity, and the addition of ligand augments the level of caspase-8 activation, showing that receptor expression increases ligand sensitivity. Transfection of truncated DR5-S-YFP (residues 1-211), where YFP is located just downstream of the predicted transmembrane domain, results in no caspase-8 activity. Further addition of agonist does not increase caspase-8 activity, confirming the role of the intracellular domain (including the functional death domain) in receptor function. Transfection of the full length DR5-S-YFP (residues 1-411), where YFP is covalently linked to the C-terminus of DR5-S, results in comparable levels of ligand-independent and ligand-induced caspase-8 activation. Collectively, these results show clearly that full length DR5-S is equally functional both in the untagged and XFP-tagged (C-terminus) form. Truncation of DR5-S lacking the cytosolic domain (and thus the death domain) results in no measurable activity, either in the absence or presence of ligand. 2

3 Supplemental Methods Reagents and antibodies antibodies against DR5 were purchased from Cell Signaling and ProSci. We note that the antibody obtained from ProSci shows a strong, unknown band at ~48kd, and since this band does not shift under reducing/non-reducing conditions (as is observed in transiently expressing cells), we conclude that this band is in fact not DR5. The caspase-8 activity kit was purchased from Biovision (CaspGLOW red Caspase-8 activity kit). Flag-TRAIL and an enhancer (typically an anti-flag antibody) were purchased from Enzo Life Sciences. Cloning CFP and YFP, in the pecfp-n1 and peyfp-n1 vectors (Clontech), respectively, were mutated to form the monomeric form of the fluorescent protein, the A206K mutation. DR5 was cloned into these vectors and sequence. Additionally, DR5 (full length) was cloned into the pires2-egfp vector (Clontech) for co-expression of untagged DR5 with EGFP. Transfection Jurkat cells were transfected via electroporation at 200V and 975uF. Shortly after transfection, live cells were isolated by ficoll gradient centrifugation. Cells were analyzed as described approximately 48 hours after transfection DR5 knockdown Jurkat cells were transfected with each of four different shrna constructs targeting DR5. Cells were selected with puromycin (2ug/ml), and after selection live cells were isolated by ficoll gradient centrifugation. Live, puromycin-resistant cells were cultured for several weeks, and the resulting non-homogenous cell population was used to test whether the ~120kd band in the presence of cross-linker is indeed DR5. TRAIL enhancing TRAIL was enhanced using an enhancing agent purchased from Enzo Life Sciences to determine if antibody-mediated ligand cross-linking has any bearing on dimer formation. TRAIL was incubated with the enhancing agent for minutes at room temperature, per the manufacturer s instructions, immediately prior to incubation with cells. Caspase-8 activity Cells were transfected by electroporation with the indicated plasmid, and shortly after electroporation live cells (i.e. cells that survived the electroporation process) were isolated by ficoll gradient centrifugation. Twenty-four to forty-eight hours after transfection, cells were treated with a DR5- specific antibody agonist and analyzed for active caspase-8. Briefly, live cells (based on FSC, SSC patterns) were analyzed for their expression of fluorescent protein (either GFP or YFP). Live, GFP/YFP + cells were analyzed for caspase-8 activity 3

4 REFERENCES 1. Holler N, et al. (2003) Two adjacent trimeric Fas ligands are required for Fas signaling and formation of a death-inducing signaling complex. Molecular and cellular biology 23(4): Chan FK, et al. (2000) A domain in TNF receptors that mediates ligand-independent receptor assembly and signaling. Science (New York, N.Y 288(5475): Clancy L, et al. (2005) Preligand assembly domain-mediated ligand-independent association between TRAIL receptor 4 (TR4) and TR2 regulates TRAIL-induced apoptosis. Proceedings of the National Academy of Sciences of the United States of America 102(50): Chan FK, et al. (2001) Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein. Cytometry 44(4): Ozsoy HZ, Sivasubramanian N, Wieder ED, Pedersen S, & Mann DL (2008) Oxidative stress promotes ligand-independent and enhanced ligand-dependent tumor necrosis factor receptor signaling. The Journal of biological chemistry 283(34): Siegel RM, et al. (2000) Measurement of molecular interactions in living cells by fluorescence resonance energy transfer between variants of the green fluorescent protein. Sci STKE 2000(38):pl1. 7. Vilar M, et al. (2009) Activation of the p75 neurotrophin receptor through conformational rearrangement of disulphide-linked receptor dimers. Neuron 62(1):

5 Figure S1 Figure S1 Jurkat cells were treated with TRAIL or control (PBS) and subsequently cross-linked as indicated with a cell surface cross-linker. (A) Whole cell lysates were run in the absence or presence of reducing agents, transferred to nitrocellulose, and analyzed for DR5 oligomerization. (B) Cross-linked, whole cell lysates were reduced, separated on a 4-12% SDS-PAGE gel, and Coomassie stained. 5

6 Figure S2 Figure S2 Jurkat cells were treated with Flag-TRAIL (with and without an anti-flag enhancer, see Supplemental Methods) or a DR5-specific antibody agonist (α-dr5) as indicated. Whole cell lysates were run under non-reducing or reducing (with βme and DTT) conditions, transferred to nitrocellulose, and analyzed for DR5 dimerization. Closed arrowheads indicate DR5 under reducing conditions and open arrowheads indicate DR5 under non-reducing conditions, including the dimer species at approximately 80kd. The asterisk indicates a strong unknown band associated with this particular antibody (ProSci anti- DR5 antibody). 6

7 Figure S3 Figure S3 DR5 was knocked down in Jurkat cells via shrna transfection and selections (see Supplemental Methods). The DR5-knockdown cells (and parental Jurkat cells as a control) were crosslinked using BS3 surface cross-linker, and whole cell lysates were analyzed by Western blot using an antibody against DR5. 7

8 Figure S4 Figure S4 (Top) Untagged and tagged DR5 constructs are shown. The truncated DR5-S includes residues and the full length includes residues The full length, untagged DR5-S was cloned into an IRES-EGFP vector for co-expression of DR5-S and GFP. (Bottom) BJAB DR5-deficient cells were transfected with the indicated plasmid: GFP, DR5-S + GFP, YFP, DR5-S-YFP (1-211 truncated) and DR5-S-YFP (1-411 full length). Twenty-four to forty-eight hours post-transfection, cells were treated for 4 hours with either DR5-antibody agonist (black bars) or control (gray bars), and caspase-8 activity was measured by flow cytometry using a red fluorescent marker for active caspase-8. The results represent the percentage of live, GFP- or YFP-positive cells with active caspase-8, compared to an untreated control. 8