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.

Piecing it together: Unraveling the elusive structure-function relationship in single-pass membrane receptors

“…Results by Song et al showed that TRAIL-induced activation of DR5, measured by activation of Caspase-8, is sensitive to the presence of cholesterol in the membrane [116]. Our recent results demonstrate that plasma membrane physical and biochemical properties facilitate DR5 dimer formation [85]. We first showed in cells that DR5 agonists induce migration of the receptor to cholesterol-rich membrane regions, and depletion of membrane cholesterol using methyl-β-cyclodextrin (MβCD) reduces ligand-induced activity.…”

“…In the case of TNFR1, structures demonstrate pre-ligand dimerization via the PLAD [73], however other experiments suggest pre-ligand trimerization [76]. Despite the early focus on the PLAD, we and others have recently demonstrated a clear role for the TM domain and intracellular contacts in dimerization and activity of TNFRs [3,83–85]. However, whether these non-PLAD interactions are conserved and how essential they are among all TNFRs remains to be seen.…”

“…The cholesterol extraction does, on the other hand, significantly diminish the level of disulfide-linked receptor dimers. This suggests that TM domain dimers are a critical component in activation of DR5 [85]. …”

“…those containing cholesterol) increase the length of the TM domain helix by adding helical turns at the N-terminal region of the TM domain. This increases the likelihood that C209 sits within a stable helical turn [85]. We hypothesized that this results in an increased propensity for dimerization by stabilizing a potential Cys-Cys interaction interface.…”

“…This shift in the distribution of receptor mobilities is accompanied by an overall decrease in the percentage of nearly-immobile receptors upon the addition of ligand, and the change is exacerbated when cells are pre-treated with MβCD [137]. This raises an interesting question: If membrane cholesterol facilitates interactions between TNFR TM helices, as described above [85], does this increase in TNFR1 mobility reflect a change in the oligomeric state of the receptor? Looking forward, single molecule tracking experiments should be performed to quantify the stoichiometry and kinetics of TNFR interactions.…”