Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor 5 networks that are highly organized

Abstract

Recent evidence suggests that TNF-related apoptosis-inducing ligand (TRAIL), a death-inducing cytokine with anti-tumor potential, initiates apoptosis by re-organizing TRAIL receptors into large clusters, although the structure of these clusters and the mechanism by which they assemble are unknown. Here, we demonstrate that TRAIL receptor 2 (DR5) forms receptor dimers in a ligand-dependent manner at endogenous receptor levels, and these receptor dimers exist within high molecular weight networks. Using mutational analysis, FRET, fluorescence microscopy, synthetic biochemistry, and molecular modeling, we find that receptor dimerization relies upon covalent and noncovalent interactions between membrane-proximal residues. Additionally, by using FRET, we show that the oligomeric structure of two functional isoforms of DR5 is indistinguishable. The resulting model of DR5 activation should revise the accepted architecture of the functioning units of DR5 and the structurally homologous TNF receptor superfamily members.

FIGURE 1.

A current model for organized ligand receptor networks. A, TRAIL-DR5 crystal structure (center, Protein Data Bank code 1d0g, top view), a 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.

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 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 (α-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 ± 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).

FIGURE 3.

Ligand-induced DR5 dimers within high molecular weight networks. A, Jurkat cells were treated with an agonistic antibody specific to DR5 and 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 μm. 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 (α-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 +DR5-S or +DR5-L using α-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 +DR5-S, and DR5-deficient cells +DR5-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.

FIGURE 4.

DR5 dimerization and network formation via covalent and non-covalent interactions. A, HEK293 cells were transiently transfected with DR5-S 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).

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, 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-oligomerization 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 < 0.05; ** indicates statistically significant with p < 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 < 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 K_d2 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).

FIGURE 6.

Transmembrane domain clustering of DR5. A, synthetic DR5-L TM peptide was reconstituted in DPC micelles in the absence and presence of 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 α-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 α-helical TM structure that includes a GG4 (GXXXG) interaction at the dimer interface.