Compartmentalization of TNF Receptor 1 SignalingInternalized TNF Receptosomes as Death Signaling Vesicles

Abstract

The molecular regulation of the recruitment of initial signaling complexes at the TNF-R1 is poorly defined. We demonstrate here that within minutes internalized TNF-R1 (TNF receptosomes) recruits TRADD, FADD, and caspase-8 to establish the "death-inducing signaling complex" (DISC). In addition, we identified the TNF-R1 internalization domain (TRID) required for receptor endocytosis and provide evidence that TNF-R1 internalization, DISC formation, and apoptosis are inseparable events. Analyzing cell lines expressing an internalization-deficient receptor (TNF-R1 DeltaTRID) revealed that recruitment of RIP-1 and TRAF-2 to TNF-R1 occurred at the level of the plasma membrane. In contrast, aggregation of TRADD, FADD, and caspase-8 to establish the TNF-R1-associated DISC is critically dependent on receptor endocytosis. Furthermore, fusion of TNF receptosomes with trans-Golgi vesicles results in activation of acid sphingomyelinase and cathepsin D. Thus, TNF receptosomes establish the different TNF signaling pathways by compartmentalization of plasma membrane-derived endocytic vesicles harboring the TNF-R1-associated DISC.

The adaptor protein TRADD activates distinct mechanisms of apoptosis from the nucleus and the cytoplasm

“…In the first, which occurs at the cell membrane in association with the activated TNFR1 receptor, TRADD interacts with TNFR1, TRAF proteins, and RIP, and this leads primarily to antiapoptotic signaling. After TNFR1 stimulation, a cytoplasmic complex 16 or a complex in endocytic vesicles 17 involving TRADD interactions with FADD leads to caspase-8-dependent apoptosis. Finally a third, nuclear complex that does not involve FADD or caspase-8 can also lead to apoptosis, but this occurs through caspase-9 and leads to both caspase-dependent and caspase-independent effects.…”

“…17 However, we previously showed that N-Benzyloxcarbonyl-Val-Ala-Asp(o-Me) fluoromethyl ketone (zVAD.fmk), which inhibits caspase-8-dependent apoptosis, was unable to block cell death induced by a nuclear TRADD molecule, 18 suggesting that nuclear TRADD works independently of these proteins. To test this hypothesis, we expressed a truncated TRADD death domain molecule that retains the nuclear import sequence but has no nuclear export sequence and is exclusively nuclear 18 (nuclear TRADD) or a TRADD mutant that is exclusively in the cytoplasm (cytoplasmic TRADD) through the addition of a myristolation signal that anchors it to the cell membrane preventing nuclear shuttling ( Figure 1a).…”

“…TRADD is comprised of two distinct functional domains including an N-terminal domain that mediates binding to TRAF2 and promotes NFkB activation, and a C-terminal death domain that is required for binding to death domain containing proteins such as FADD and Rip. TRADD binding to FADD, which has been suggested to occur in the cytoplasm after dissociation from the receptor complex 16 and more recently proposed to occur in endocytic vesicles, 17 activates caspase-8-dependent apoptosis. Surprisingly, for a protein that binds to a cell surface receptor and signals from the cytoplasm or endocytic vesicles, TRADD also contains a nuclear import sequence and a nuclear export sequence that together promote nuclear/cytoplasmic shuttling.…”

New insights into apoptosis signaling by Apo2L/TRAIL

“…Clathrin-mediated endocytosis of TNFR1 and Fas has been shown to be required for apoptosis stimulation, but not for activation of the NF-kB and MAPK pathways. It has been reported that in response to TNF or FasL, DISC components are directly recruited to internalized receptors residing in endosomal compartments, dubbed receptosomes (Schneider-Brachert et al, 2004Lee et al, 2006). By contrast, recruitment of the signaling complexes that drive NF-kB and MAPK activation in response to TNFR1 or Fas ligation occurs at the cell surface and does not rely on receptor internalization (Schneider-Brachert et al, 2004Lee et al, 2006).…”

“…It has been reported that in response to TNF or FasL, DISC components are directly recruited to internalized receptors residing in endosomal compartments, dubbed receptosomes (Schneider-Brachert et al, 2004Lee et al, 2006). By contrast, recruitment of the signaling complexes that drive NF-kB and MAPK activation in response to TNFR1 or Fas ligation occurs at the cell surface and does not rely on receptor internalization (Schneider-Brachert et al, 2004Lee et al, 2006). Recent work suggests that actin polymerization and internalization events are required for Fas activation by agonistic antibody but not FasL (Chaigne-Delalande et al, 2009).…”