Identification and Characterization of a Ligand-independent Oligomerization Domain in the Extracellular Region of the CD95 Death Receptor

Abstract

The CD95 death receptor plays an important role in several physiological and pathological apoptotic processes involving in particular the immune system. CD95 ligation leads to clustering of the receptor cytoplasmic "death domains" and recruitment of the zymogen form of caspase-8 to the cell surface. Activation of this protease through self-cleavage, followed by activation of downstream effector caspases, culminates in cleavage of a set of cellular proteins resulting in apoptosis with disassembly of the cell. It is very well known that the extracellular region of the CD95 receptor is required for CD95L interaction and that the death domain is necessary for the induction of the apoptotic signaling. Here, we identified and characterized a novel CD95 ligandand death domain-independent oligomerization domain mapping to the NH 2 -terminal extracellular region of the CD95 receptor. In vitro and in vivo studies indicated that this domain, conserved among all soluble CD95 variants, mediates homo-oligomerization of the CD95 receptor and of the soluble CD95 proteins, as well as heterooligomerization of the receptor with the soluble variants. These results offer new insight into the mechanism of apoptosis inhibition mediated by the soluble CD95 proteins and suggest a role of the extracellular oligomerization domain in the regulation of the non-signaling state of the CD95 receptor.Apoptosis is a cell death process that can be elicited by a variety of stimuli, such as growth factor deprivation, ionizing radiation, and other DNA-damaging agents, or by triggering specific cell surface receptors, the death receptors. Death receptors belong to the tumor necrosis factor (TNF) 1 receptor gene superfamily, which is characterized by similar cysteinerich extracellular domains (1). The death receptors TNF-R1 (also called p55 or CD120a), CD95 (also called Fas or Apo-1), DR3 (also called Wsl-1, Apo-3, LARD, or TRAMP), DR4 (also called TRAIL-R1), DR5 (also called Apo-2, TRAIL-R2, TRICK2, or KILLER) (2), and DR6 (3) are also characterized by the presence of a death domain within the cytoplasmic region critical for the initiation of apoptotic signaling (4, 5). Notably, CD95 ligand (CD95L) and CD95 receptor interactions play an important role in the regulation of the immune response and in the control of peripheral B and T cell survival that is critical to the maintenance of immune cell homeostasis (6). This system has also been implicated in maintaining immunoprivilege in the eye and testis (7, 8), attenuating immunosurveillance against certain types of tumors, such as melanoma and hepatocellular carcinoma (9). Moreover, alterations in the control of apoptosis, also mediated by the CD95/CD95L system, are involved in the pathogenesis of several diseases such as Hashimoto thyroiditis (10), fulminant hepatitis (11, 12), and toxic epidermal necrolysis (Lyell's syndrome) (13). Thus, it is particularly interesting to elucidate the mechanisms that regulate CD95 and CD95L interactions.A common feature of the death receptors is that, upon binding to...

The CD95(APO-1/Fas) DISC and beyond

“…2 CD95 receptors are expressed on the surface of cells as preassociated homotrimers. 3,4 A similar association was described for several members of the TNF receptor superfamily, including the TNF receptor itself, CD40 and the TNFrelated apoptosis-inducing ligand (TRAIL) receptor I. 5 These interactions were found to be mediated by a domain in the Nterminus, within the first of the cysteine-rich domains.…”

“…5 These interactions were found to be mediated by a domain in the Nterminus, within the first of the cysteine-rich domains. 3,4 This association was described by binding of in vitro generated proteins to cells transfected with CD95, and by the use of chemical crosslinking reagents that allowed visualization of the associated homotrimers present on the cell surface. 3,4 Additionally, the use of the fluorescence resonance energy transfer technique, in which the emission wavelength of one spectral variant of the green fluorescent protein (GFP) is used to excite a second spectral variant of GFP when the two are in close proximity (approximately 100 Å ), supported these observations.…”

Death receptor 4 (DR4) efficiently kills breast cancer cells irrespective of their sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

“…Ligand-independent oligomerization was previously demonstrated for another death receptor, Fas. 30,31 For cancer gene therapy applications, strong cytotoxicity, exhibited by DR4 overexpression, required targeting. For this purpose, we used the promoter of the hTERT gene, which has been shown to be selectively active in cancer cells, [32][33][34][35] to drive DR4 expression.…”

New insights into apoptosis signaling by Apo2L/TRAIL

“…However, substantial evidence now indicates that certain TNFRs are preassembled into oligomers before ligation (Chan, 2007). Preligand association was first detected for Fas, TNFR1 and TNFR2 (Papoff et al, 1999;Chan et al, 2000;Siegel et al, 2000), which appear to form homotypic trimers in the absence of ligand. A preligand assembly domain (PLAD), located in the first CRD of the receptor, mediates the association between monomers.…”