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Boster Pathways ➔ Apoptosis: Cell Death

Death Receptor Signaling Pathway

Apoptosis or programmed cell death can be activated through the intrinsic death receptor pathway and the extrinsic death receptor pathway.

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Apoptosis & the death receptor pathway

Apoptosis or programmed cell death can be activated through two distinct but related pathways. The first pathway, called the intrinsic death receptor pathway (aka intrinsic apoptosis pathway or mitochondrial pathway), is activated upon significant cell stress through the mitochondria while the second pathway, called the extrinsic death receptor pathway, is signaled by extracellular death ligands that bind to death receptors, triggering cell suicide. These death receptors are embedded in the cellular membrane and activate the apoptotic pathway when bound by their cognate death ligands. Following this, the death domains of the death receptors bind the TNFR-associated death domain (TRADD) which in turn recruits and interacts with the Fas-associated death domain (FADD). FADD then couples with procaspase 8 or 10, which are subsequently activated and cleaved to caspase 8 or 10, to form death-inducing signaling complexes (DISCs). These DISCs trigger the signal cascades implicated in apoptosis. Among the most well-known death receptors are Fas, TNFR1, TRAILR1, TRAILR2, DR3, and NGFR, which are all members of the tumor necrosis factor (TNF) receptor superfamily of proteins.

Apoptosis


Apoptosis is a cellular process that has garnered significant interest in different biological research areas due to its role in development, immune response, and pathology of some human diseases, such as neurodegenerative diseases, autoimmune disorders, and most notably cancer. These disorders are all commonly characterized by defective apoptotic mechanisms in cells, which can be either impaired, as in the case of cancer, or excessive, as in the case of neurodegenerative diseases (Elmore, 2007). Thus, the proper signaling and modulation of the apoptosis pathway is necessary for normal physiological function, especially since apoptosis is a constantly occurring event in multicellular organisms that can be triggered by different stresses such as chemotherapy and irradiation (Lavrik, Golks, & Crammer, 2005). It is implicated in the elimination of undesired cells in the early development of organisms, while in adults it is involved in the removal of damaged cells (National Human Genome Research Institute, n.d.).

The term apoptosis was coined by Kerr, Wyllie, and Currie (1972) to refer to a form of cell death that differs in many ways to necrotic cell death. Changes in the morphological and biochemical makeup of cells precede apoptosis, including membrane blebbing, cell shrinkage, chromatin condensation, nuclear disruption, and DNA fragmentation. The cell cycle is also heavily involved in apoptosis, as cell cycle arrest events are required for apoptosis to initiate (Elmore 2007). While the linkage between cell cycle arrest and the death receptor pathway is faint, the pathway is heavily implicated in all the other morphological and biochemical cellular changes mentioned.

Death receptors and death ligands


The elucidation of the death receptor pathway responsible for apoptosis have generated possible therapeutic targets in both the death receptors and their ligands (Kumar, Herbert, & Warrens, 2005). Each death receptor has a unique ligand that activates it and triggers the downstream signaling upon binding.

TNFR1/DR1 has TNF as its activating ligand, while Fas/DR2 has FasL as its cognate ligand. DR3/Apo3 has Apo3L/TWEAK as its activating ligand. The death ligand TRAIL/Apo2L is the cognate ligand of both TRAIL-R1/DR4 and TRAIL-R2/DR5. Lastly, DR6 has TRADD as its activating ligand that binds to its death domain instead of its extracellular domain. This is the case for all the other death receptors (Kumar, Herbert, & Warrens, 2005).

The death receptors are composed of extracellular cysteine-rich domains (CRDs) that bind the death ligands and a cytoplasmic death domain which recruits adapter molecules such as TRADD and FADD (Gurney, Muralidhar, & Linton, 2013). While all death receptors have a single intracellular death domain, the number of their extracellular CRDs differ.

TNFR1 or death receptor 1 (DR1), Apo3/DR3, and DR6 all have four CRDs in their extracellular domain. Fas/DR2 has three CRDs in its extracellular domain, while TRAIL-R1/DR4 and TRAIL-R2/DR5 both have two CRDs (Guicciardi & Gores, 2003). The death ligands bind to their cognate death receptors at the most distal CRD to the cellular membrane. Ultimately, the death receptor pathway is initiated by the activation of death receptors upon the binding of their cognate ligands.

Genomic location

In terms of genomic location, the genes coding for the death receptors are located on different chromosomes. TNFR1/DR1 can be found in chromosome 12p13, while Fas/DR2 is located in chromosome 10q23. Apo3/DR3 is located in chromosome 1p36 while both TRAIL-R1/DR4 and TRAIL-R2/DR5 are found in chromosome 8p21. Lastly, DR6 can be found in chromosome 6p12. Like their cognate receptors, the genomic locations of the death ligands also vary widely. TNF is situated in chromosome 6p21 while FasL can be found in chromosome 1q24. Apo3L/TWEAK is located in chromosome 17p13 while TRAIL/Apo2L can be found in chromosome 3q26. Lastly, TRADD is located in chromosome 16q22. Interestingly, none of the death receptors are located in the same chromosome, as is true for the death ligands as well.

Signal transduction and modulation of the death receptor pathway

  • General 4 Steps

    The cytotoxic death receptor pathway can be generalized into four steps.

    1. The death ligand binds to the death receptor and activates it.
    2. The activated death receptor recruits adaptor proteins.
    3. The adaptor protein complex then associates with caspases, forming the DISC which dissociates from the death receptor.
    4. These DISCs activate effector caspases which trigger various signaling pathways leading to cell death (Guicciardi & Gores, 2009).

  • Death-inducing signaling complexes (DISCs)

    Fas/DR2, TRAIL-R1/DR4, and TRAIL-R2/DR5 share the same DISC structure, consisting of oligomerized receptors, FADD, the FLICE-inhibitory protein (FLIP), procaspase 10, and isoforms a and b of procaspase 8. Upon FasL binding into Fas/DR2, the DISC is formed. Procaspases 8 and 10 are proteolytically activated in the DISC into caspases 8 and 10, which signal effector caspases like caspase 3 to signal target proteins and trigger apoptotic effects. Some of the target proteins like actin, GAS2, and α-fodrin are implicated in cell shrinkage, while ROCK1 is involved in membrane blebbing.

    DFF45 and DFF40, on the other hand, are targeted for DNA fragmentation and acinus is targeted for chromatin condensation. Caspase 3 also activates caspase 7 which in turn targets PARP, another protein involved in apoptosis. Boster offers antibodies that can be used to study proteins in the Fas death receptor pathway, including the anti-Fas, anti-FasL, anti-FADD, anti-FLIP, anti-caspase 3, anti-caspase 8, anti-caspase 10, anti-actin, anti-ROCK1, and anti-PARP antibodies.

  • Mitochondria

    While Apo3/DR3, TRAIL-R1/DR4, and TRAIL-R2/DR5 form a similar DISC as Fas upon cognate ligand binding, they have another signal transduction pathway that involves the mitochondria. When there are insufficient amounts of active caspase 8, the Bid protein is cleaved by caspase 8 to become truncated Bid (tBid), which in turn signals the mitochondria to release cytochrome c (Cyt C). Cyt C signals the formation of the apoptotic complex involving APAF1 and procaspase 9, where procaspase 9 is activated to become caspase 9. Caspase 9 proteolytically activates downstream caspases like caspase 3 and caspase 6, which targets lamin A resulting in membrane blebbing.

    The proteins involved in the TRAIL-R death receptor pathway can be analyzed using Boster’s line of antibodies, including the anti-TWEAK, anti-Apo3/DR3, anti-TRAIL-R1/DR4, anti-TRAIL-R2/DR5, anti-Bid, anti-caspase 9, anti-Cyt C, anti-APAF1, and anti-lamin A antibodies.

  • TNFR signaling

    TNFR signaling, on the other hand, is different in some ways. Upon TNF-α binding to TNFR-1, TRAF2, TRADD, cIAP1/2, and RIP1, the DISC is formed and released into the cytosol, consisting of FADD, RIP1, and procaspase 8 which is subsequently activated. Caspase 8 activates caspase 3 which transduces downstream apoptotic signaling. However, another complex may be formed with the addition of RIP3, triggering necroptosis or programmed inflammatory cell death.

    TNFR1 signaling can also result in non-cytotoxic effects, through NF-кB signaling which is implicated in cell survival. TNFR2, another receptor of TNF-α but lacks a death domain, also initiates NF-кB signaling upon TRAF1, TRAF2, and cIAP1/2 complex formation at its cytoplasmic domain. NF-кB and RelB forms a complex that transduces cell survival processes such as transcription. Boster has a selection of antibodies that can be used for research in proteins involved in TNFR signaling, including anti-TNFR2, anti-RIP1, anti-RIP3, anti-TRADD, anti-ciAP1, anti-TRAF1, and anti-TRAF2.

  • Modulation of death receptor signaling

    Modulation of death receptor signaling happens at many levels. At the level of ligand binding, decoy death receptors (DcRs) can compete for death ligands by binding them without triggering the downstream effects. Procaspase 8 activation can be inhibited by FLIP at the DISC formation level. Inhibitors of apoptosis (IAPs) such as XIAP and cIAP1/2 can also block caspase activation at different steps of the pathway. Smac serves as an inhibitor of both XIAP and cIAP1/2 in the pathway. Some of the antibodies offered by Boster that can be used to investigate signal modulation of the death receptor pathway are anti-FLIP, anti-XIAP, and anti-Smac.

Therapeutic applications of the death receptor pathway


Proteins in the death receptor pathway, especially the death receptors themselves, serve as valuable therapeutic targets as a result of both their cytotoxic and non-cytotoxic effects. The specificity of death ligands to their cognate death receptors have made them valuable molecular targets for the targeted therapy of diseases involving the death receptor pathway (Sheikh & Huang, 2004).

Fas, the most studied of the death receptors, has also emerged as one of the top drug targets for therapies involving the death receptor pathway. Known anticancer drugs such as doxorubicin, cisplatin, cytarabine, and methotrexate have been shown by Friesen, Fulsa, & Debatin (1999) to induce FasL and increase its binding to Fas, resulting in increased cell death events. Thus, one of the mechanisms by which these drugs promote tumor elimination is by promoting apoptosis mediated by Fas signaling. Tumor cells resistant to chemotherapies have also been studied to have defective Fas signaling. Antibodies that bind the death receptors have been shown to reduce apoptosis by blocking the binding of death ligands as well. This is in turn helpful in pathologies where there may be excessive apoptosis, such as neurodegenerative diseases.

The death receptor pathway influences many diseases and pathologies, rendering death receptors, death ligands, and other proteins in the pathway as valuable therapeutic targets that can be investigated through immunoassays such as ELISA. Boster helps with research on death receptor signaling by offering a wide range of antibodies that reliably target proteins in the death receptor pathway.

References


  • Elmore, S. (2007). Apoptosis: a review of programmed cell death. Toxicologic pathology, 35(4), 495-516.
  • Friesen, C., Fulda, S., & Debatin, K. M. (1999). Cytotoxic drugs and the CD95 pathway. Leukemia, 13(11), 1854-1858.
  • Guicciardi, M. E., & Gores, G. J. (2003). The death receptor family and the extrinsic pathway. In Essentials of apoptosis (pp. 67-84). Humana Press, Totowa, NJ.
  • Guicciardi, M. E., & Gores, G. J. (2009). Life and death by death receptors. The FASEB Journal, 23(6), 1625-1637.
  • Gurney, M., Muralidhar, G., & Linton, P. J. (2013). Autophagy in the Immune System. In Autophagy in Health and Disease (pp. 41-55). Academic Press.
  • Kerr, J. F., Wyllie, A. H., & Currie, A. R. (1972). Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. British Journal of Cancer, 26(4), 239-257.
  • Kumar, R., Herbert, P. E., & Warrens, A. N. (2005). An introduction to death receptors in apoptosis. International Journal of Surgery, 3(4), 268-277.
  • Lavrik, I., Golks, A., & Krammer, P. H. (2005). Death receptor signaling. Journal of cell science, 118(2), 265-267.
  • National Human Genome Research Institute. (n.d.). Apoptosis. Retrieved June 17, 2021 from https://www.genome.gov/genetics-glossary/apoptosis.
  • Sheikh, M. S., & Huang, Y. (2004). Death receptors as targets of cancer therapeutics. Current cancer drug targets, 4(1), 97-104.