Death Receptor 5 Networks Require Membrane Cholesterol for Proper Structure and Function

Abstract

Death receptor 5 (DR5) is an apoptosis-inducing member of the tumor necrosis factor receptor superfamily, whose activity has been linked to membrane cholesterol content. Upon ligand binding, DR5 forms large clusters within the plasma membrane that have often been assumed to be manifestations of receptor co-localization in cholesterol-rich membrane domains. However, we have recently shown that DR5 clusters are more than just randomly aggregated receptors. Instead, these are highly structured networks held together by receptor dimers. These dimers are stabilized by specific transmembrane helix-helix interactions, including a disulfide bond in the long isoform of the receptor. The complex relationships among DR5 network formation, transmembrane helix dimerization, membrane cholesterol, and receptor activity has not been established. It is unknown whether the membrane itself plays an active role in driving DR5 transmembrane helix interactions or in the formation of the networks. We show that cholesterol depletion in cells does not inhibit the formation of DR5 networks. However, the networks that form in cholesterol-depleted cells fail to induce caspase cleavage. These results suggest a potential structural difference between active and inactive networks. As evidence, we show that cholesterol is necessary for the covalent dimerization of DR5 transmembrane domains. Molecular simulations and experiments in synthetic vesicles on the DR5 transmembrane dimer suggest that dimerization is facilitated by increased helicity in a thicker bilayer.

Keywords: cholesterol-rich membrane domains; disulfide bond; ligand/receptor clustering; replica exchange molecular dynamics; transmembrane domain.

Fig. 1

Membrane cholesterol is required for the efficient activation of caspase-8 by DR5. Jurkat cells were pretreated with either (a) control or (b) MβCD, followed by no treatment or anti-DR5 agonist antibody (α-DR5; gray distribution and black line, respectively), and caspase-8 activity was measured using Red-IETD-FMK and flow cytometry. Plotted is a histogram of caspase-8 activity level showing the ligand-dependent activation of caspase-8 and the reduced activity in cells pretreated with MβCD. (c) The activation of caspase-8 is quantified using identical gating schemes on each population, and the results are plotted as the fold activation of caspase-8.

Fig. 2

Ligand–receptor cluster formation does not require membrane cholesterol. Jurkat cells were treated with mAb631 and labeled with fluorescent secondary antibody. Shown are (a) Jurkat cells under transmitted luminescence, (b) ligand–receptor clusters illuminated by fluorescently labeled agonist antibody, and (c) the angular fluorescence intensity trace for the cell shown. The raw trace is shown in black and the Gaussian fit in blue. (d) The total ligand bound, (e) number of clusters per cell, and (f, g) distribution of cluster sizes are invariant between untreated and MβCD-treated cells.

Fig. 3

Extraction of membrane cholesterol inhibits ligand-induced DR5 dimer formation. Jurkat cells were pretreated with either control or MβCD, as indicated, to extract membrane cholesterol. Cells were subsequently washed and treated with α-DR5 agonist antibody and lysed. Lysates were analyzed via non-reducing SDS-PAGE and Western blot using an antibody against DR5. (a) In lanes 4 and 8, agonist-bound DR5 formed oligomeric networks in both untreated and MβCD-treated cells. Asterisks indicate non-specific bands, which do not appear at the known molecular weight of DR5 or any multiple of it and are invariant with ligand treatment. (a) In lane 2, disulfide-linked dimerization of DR5 via Cys209 occurred only in untreated cells. (b) Shown is the dimeric form of DR5 at ~85 kDa from a separate experiment. (c) Quantification of DR5 dimer bands shows a significant increase in DR5 dimerization upon treatment with ligand, and pretreatment with MβCD diminishes DR5 dimerization. (d) The dimer fraction of DR5-L TM peptide increases with increasing cholesterol in DLPC vesicles. DMPC vesicles also favor a larger dimer fraction than DLPC vesicles in the absence of cholesterol.

Fig. 4

Membrane thickness affects DR5-L TM domain helical stability and Cys209 partition depth in molecular dynamics simulations. The DR5-L TM dimer was simulated using REMD. (a) The average GxxxG distance was plotted to identify the likely dimer conformation (<6 Å; dashed box). This dimer conformation was sampled in both the 40-Å (black) and 32-Å (blue) implicit bilayers representing cholesterol-rich and -poor domains, respectively. Subsequent analysis is conducted on these frames only. (b) The fraction of frames with helical secondary structure shows increased helix stability in the 40-Å bilayer. (c) A snapshot of the simulated peptide in its GxxxG dimer configuration is shown with C209 in red, the G(213)xxxG(217) motif as blue spheres, and W237 as a red sphere. (d) Trp237 (Cα atom) partitions directly outside the switching region in both the 40-Å (black) and 32-Å (blue) bilayers. Cys209 (sulfur atom) partitions at the switching region in the 32 Å-bilayer (blue) but deep within the low-dielectric region in the 40-Å bilayer (black). The positions of the switching regions of each bilayer are shown as dashed horizontal lines.

Fig. 5

Updated activation model for DR5 incorporating the bilayer structure. (a) Instability in the TM helix inhibits covalent dimerization of DR5 via Cys209 in thin bilayer fractions. (b) Translocation to thicker, cholesterol-rich membrane domains draws the cysteines into the bilayer interior, stabilizing the TM helices and driving disulfide bond formation.