The extrinsic pathway of apoptosis, also known as the death receptor mediated pathway of apoptosis is triggered by the binding of specific pro-apoptotic ligands to death receptors. The death receptors are type I transmembrane proteins and are members of the TNF superfamily of receptors. These receptors share homology in their extracellular region, which is characterized by up to 6 cysteine-rich domains (CRDs) that define their ligand specificity. Perhaps the most characteristic feature of these receptors is the presence of an intracellular region known as the “death domain” (DD) that enables the receptors to initiate cytotoxic signals when activated by cognate ligands (Guicciardi et al., 2009; Schmitz, et al., 2000).
1.3.2.1
Death receptors and the death inducing signaling complex
The most extensively studied death receptors are Fas (CD95/APO-1), TNF-receptor 1 (TNF-R1), TNF-related apoptosis-inducing ligand receptor 1 [TRAIL-R1/ Death Receptor 4 (DR4)], and receptor 2 (TRAIL-R2/DR5/APO-2/KILLER). Death receptors are activated by ligands that are part of a group of cytokines belonging to the TNF protein family. These ligands can either be membrane bound (eg. mFasL, mTNFα) or released as soluble cytokines (eg. sFasL, sTNFα) through proteolytic cleavage. Prior to binding of the ligands to the receptors, death receptors form receptor complexes through the interaction of the first cysteine-rich domain of the extracellular tail of the receptor, known as the preligand assembly domain (PLAD). PLAD is not directly involved in ligand binding, however the oligomerization and formation of complexes is a crucial step for receptor- ligand interaction (Guicciardi & Gores, 2009). Ligand binding causes a conformational change in the receptor complex, leading to the recruitment of adaptor proteins to the intracellular region of the receptor. Adaptor proteins such as TRADD and
Figure 1.0.5
Figure 1.6. Pathways of Apoptosis. There are two main pathways of apoptosis; the extrinsic, and intrinsic pathway. The extrinsic pathway is characterized by activation of death receptors, such as TNFR1 and Fas, followed by activation of caspase 8. Caspase 8 can directly activate effector caspases, or can cleave and activate Bid, which translocates to the mitochondria and drives cells through a mitochondrial-dependent apoptotic pathway. The intrinsic pathway involves activation of Bcl-2 family members leading to mitochondrial outer membrane permeabilization, release of pro-apoptotic factors and activation of effector caspases.
FADD are able to associate with receptors through the homotypic interaction of their death domain (DD) to the DD of the receptor. Adaptor molecules may also contain an additional domain, known as a DED (death-effector domain) which mediate the recruitment of caspases and cFLIPL (caspase-8-like inhibitor protein) through their DEDs. This complex is referred to as the death-inducing signaling complex (DISC), which can generate an apoptotic signaling cascade, however the mechanism for this remains a topic of debate.
1.3.2.2
Caspase activation in extrinsic apoptosis
The first proposed model of caspase-activation is known as the ‘induced-proximity model’. In this model, the recruitment of FADD to the receptor complex results in clustering of initiator procaspases (procaspase-8) at the DISC, which leads to their self-processing into the active form which is able to cleave caspase-3 and Bid. The second model, known as the ‘proximity-induced dimerization model’ suggests that accumulation of caspases at the DISC promotes their dimerization which results in their activation. The final model known as the ‘induced-conformation model’ suggests that activation of the initiator caspases occurs through a conformational change in their active site following interaction with the adaptor protein complex. Activation of caspase-8 at the DISC can be regulated by cFLIP, possibly by competing for binding and recruitment to FADD (Kantari & Walczak, 2011; Guicciardi et al., 2009). Following activation, caspase-8 is released into the cytosol as an active heterotetrameric form containing two large and two small subunits. The downstream signaling events initiated by caspase-8 are likely dependent on the amount of active caspase 8 released into the cytosol, whereby large amounts of caspase-8 can directly process and activate effector caspases such as caspase-3 to induce apoptosis. In cases where small amounts of caspase-8 are released, cleavage of the BH3-only protein Bid is required to induce apoptosis (Kantari & Walczak, 2011).
1.3.2.3
The BH3-only protein Bid
The BH3-only protein Bid acts as a molecular bridge between the extrinsic and intrinsic pathway of apoptosis. In its uncleaved, full-length form, Bid is thought to be inactive. Following death receptor stimulation and activation of caspase-8, Bid is cleaved by
caspase-8 to its truncated form tBid. tBid is a 15 kDa protein that is capable of translocating to the mitochondria to induce mitochondrial outer membrane permeabilization (MOMP) through activation of Bax (Yin, 2006) (Figure 1.6). As previously described, permeabilization of the mitochondrial outer membrane results in cytochrome c release and downstream activation of effector caspases, culminating in apoptosis. The requirement for MOMP to elicit apoptosis following engagement of death receptors separates cells into two categories. In type I cells, activation of the extrinsic pathway is sufficient to induce death, and they do not require cleavage of Bid and MOMP. Alternatively, type II cells have weak DISC formation capability and therefore cleavage of Bid and activation of the mitochondrial pathway are required to induce apoptosis (Kantari & Walczak, 2011; Schmitz et al., 2000).