The three-dimensional solution structure and dynamic properties of the human FADD death domain

FADD (also known as MORT-1) is an essential adapter protein that couples the transmembrane receptors Fas (CD95) and tumor necrosis factor receptor-1 (TNF-R1) to intracellular cysteine proteases known as caspases, which propagate and execute the programmed cell death-inducing signal triggered by Fas ligand (FasL, CD95L) and TNF. FADD contains 208 amino acid residues, and comprises two functionally and structurally distinct domains: an N-terminal death effector domain (DED) that promotes activation of the downstream proteolytic cascade through binding of the DED domains of procaspase-8; and a C-terminal death domain (DD). FADD-DD provides the site of FADD recruitment to death receptor complexes at the plasma membrane by, for example, interaction with the Fas receptor cytoplasmic death domain (Fas-DD), or binding of the TNF-R1 adapter molecule TRADD. We have determined the three-dimensional solution structure and characterised the internal polypeptide dynamics of human FADD-DD using heteronuclear NMR spectroscopy of (15)N and (13)C,(15)N-labelled samples. The structure comprises six alpha-helices joined by short loops and displays overall similarity to the death domain of the Fas receptor. The analysis of the dynamic properties reveals no evidence of contiguous stretches of polypeptide chain with increased internal motion, except at the extreme chain termini. A pattern of increased rates of amide proton solvent exchange in the alpha3 helix correlates with a higher degree of solvent exposure for this secondary structure element. The properties of the FADD-DD structure are discussed with respect to previously reported mutagenesis data and emerging models for FasL-induced FADD recruitment to Fas and caspase-8 activation.     

The Transmembrane Domains of TNF-Related Apoptosis-Inducing Ligand (TRAIL) Receptors 1 and 2 Co-Regulate Apoptotic Signaling Capacity

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) ligand family that exerts its apoptotic activity in human cells by binding to two transmembrane receptors, TRAILR1 and TRAILR2. In cells co-expressing both receptors the particular contribution of either protein to the overall cellular response is not well defined. Here we have investigated whether differences in the signaling capacities of TRAILR1 and TRAILR2 can be attributed to certain functional molecular subdomains. We generated and characterized various chimeric receptors comprising TRAIL receptor domains fused with parts from other members of the TNF death receptor family. This allowed us to compare the contribution of particular domains of the two TRAIL receptors to the overall apoptotic response and to identify elements that regulate apoptotic signaling. Our results show that the TRAIL receptor death domains are weak apoptosis inducers compared to those of CD95/Fas, because TRAILR-derived constructs containing the CD95/Fas death domain possessed strongly enhanced apoptotic capabilities. Importantly, major differences in the signaling strengths of the two TRAIL receptors were linked to their transmembrane domains in combination with the adjacent extracellular stalk regions. This was evident from receptor chimeras comprising the extracellular part of TNFR1 and the intracellular signaling part of CD95/Fas. Both receptor chimeras showed comparable ligand binding affinities and internalization kinetics. However, the respective TRAILR2-derived molecule more efficiently induced apoptosis. It also activated caspase-8 and caspase-3 more strongly and more quickly, albeit being expressed at lower levels. These results suggest that the transmembrane domains together with their adjacent stalk regions can play a major role in control of death receptor activation thereby contributing to cell type specific differences in TRAILR1 and TRAILR2 signaling.     

Increase of Fas-induced apoptosis by inhibition of extracellular phosphorylation of Fas receptor in Jurkat cell line

Apoptosis signalling through the Fas pathway requires several steps of aggregation of the Fas receptor in the membrane, including aggregation that may occur in the absence of Fas ligand. Association of Fas domains is determinant to signal transmission following Fas ligand binding to a specific domain. The domains involved in Fas aggregation are located in its extracellular region and contain three potential protein kinase C-binding motifs. We therefore studied the possibility that phosphorylation of the extracellular region of Fas might be implicated in the regulation of Fas-mediated apoptosis. Inhibition experiments of extracellular phosphorylation were performed in human Jurkat T leukemia cells with K252b, an impermeant protein-kinase inhibitor. Extracellular phosphorylation of Fas receptor was related to ecto-kinase, as assessed by the [γ-³²P] ATP labelling of Fas-116 kDa aggregates, suppressed by K252b inhibitor which significantly increased the sensitivity to Fas-mediated apoptosis. Ecto-PKC involvement was demonstrated by bisindolylmaleimide VIII, a selective inhibitor of protein kinase C which significantly increased both Fas aggregation in the membrane and Fas-mediated apoptosis and by the addition of the PKC pseudo-substrate 19–36 which inhibited the phosphorylation of 116 kDa Fas aggregates. These data support a role for Fas phosphorylation in the decreased sensitivity to apoptosis in the Jurkat T leukemia cell line. ^*There was an equal contribution from these two authors.     

Single chain TNF derivatives with individually mutated receptor binding sites reveal differential stoichiometry of ligand receptor complex formation for TNFR1 and TNFR2

Most members of the tumor necrosis factor ligand family form noncovalently linked homotrimers, capable to bind up to three molecules of the respective membrane receptors. For several receptors a membrane distal homophilic interaction domain has been identified, called pre-ligand binding assembly domain. Accordingly, affinity values determined by typical equilibrium binding studies are likely to be influenced by avidity effects. Using our recently introduced covalently stabilized TNF (single chain TNF, scTNF), we have here investigated receptor–ligand binding stoichiometry in our well characterized system of TNFR–Fas chimeras. We produced scTNF derivatives with functionally deleted individual receptor binding sites, resulting in TNF mutants capable to only bind to one or two receptor molecules, rather than three. Equilibrium binding affinity studies on ice with these molecules revealed no significant changes after a single receptor binding site had been functionally deleted. In contrast, functional abrogation of two receptor binding sites showed a strong decrease in both, affinity and bioactivity on TNFR2–Fas. In contrast, TNFR1–Fas ligand binding and receptor activation was only affected after functional deletion of all three receptor binding sites. Our data demonstrate pivotal differences in ligand/receptor interactions between TNFR1–Fas and TNFR2–Fas, arguing for avidity effects important for TNF binding and downstream signaling of TNFR2, but to a lesser extent of TNFR1. These results are supported by data revealed from chemical crosslinking experiments suggesting the existence of preformed TNFR–Fas homodimers.     

Imaging of Fas-FasL membrane microdomains during apoptosis in a reconstituted cell-cell junction

The Fas death receptor interacts with its ligand FasL and induces apoptosis. The Fas-FasL interaction occurs at the cell-cell interface in vivo, since both proteins are expressed in cell membranes. However, most studies on the Fas signal pathway have been performed in a nonphysiological manner by using soluble molecules (antibody or crosslinked FasL proteins) to stimulate Fas. The Fas-FasL interaction at the cell-cell contact site has only been studied recently, but the information derived from cell-cell interaction studies is still rather limited and not necessarily consistent with the past results obtained by using soluble Fas-stimulatory molecules. Therefore, we develop a novel reconstituted system that mimics the Fas-FasL interaction at cell-cell contact sites for further examination of the physiological Fas-FasL signaling system. By conjugating FasL extracellular domains to planar lipid bilayers, we created a model cell membrane to activate Fas-expressing cells. Using this system, we generated an image of Fas-FasL interactions at the cell-membrane interface at high resolution. We observed that the Fas-FasL interaction between two membranes creates submicron membrane microdomains. Shortly after microdomain formation, the cells exhibit various features of apoptosis. These results suggest that our reconstituted system provides a useful platform to dissect Fas-FasL apoptosis signaling at near physiological conditions.     

Identification of a lysine-rich region of Fas as a raft nanodomain targeting signal necessary for Fas-mediated cell death

Fas interaction at the plasma membrane with its lipid and protein environment plays a crucial role in the early steps of Fas signalling induced by Fas ligand binding. Particularly, Fas localisation in the raft nanodomains, ezrin-mediated interaction with the actin cytoskeleton and subsequent internalization are critical steps in Fas-mediated cell death. We identified a lysine-rich region (LRR) in the cytoplasmic, membrane-proximal region of Fas as a key determinant modulating these initial events. Through a genetic approach, we demonstrate that Fas LRR represents another signal additional to palmitoylation targeting Fas to the raft nanodomains, and modulates Fas interaction with the cytoskeleton.     

The crystal structure of death receptor 6 (DR6): a potential receptor of the amyloid precursor protein (APP)

Death receptors belong to the tumor necrosis factor receptor (TNFR) super family and are intimately involved in the signal transduction during apoptosis, stress response and cellular survival. Here we present the crystal structure of recombinantly expressed death receptor six (DR6), one family member that was recently shown to bind to the amyloid precursor protein (APP) and hence to be probably involved in the development of Alzheimer's disease. The extracellular cysteine rich region of DR6, the typical ligand binding region of all TNFRs, was refined to 2.2 Å resolution and shows that its four constituting cysteine rich domains (CRDs) are arranged in a rod-like overall structure, which presents DR6-specific surface patches responsible for the exclusive recognition of its ligand(s). Based on the structural data, the general ligand binding modes of TNFRs and molecular modeling experiments we were able to elucidate structural features of the potential DR6-APP signaling complex.     

Heterologous production of death ligands' and death receptors' extracellular domains: structural features and efficient systems

The extracellular domains of death ligands and those of death receptors are closely related to many serious human diseases through the initiation of apoptosis. Recombinant production of the extracellular domains has been investigated due to demand for a large amount of purified samples, which are a prerequisite for their biochemical characterization and constitute the fundamentals of medical applications. This review focuses on the recombinant production of extracellular domains of the major members of death ligand and death receptor families using non-mammalian expression systems with an emphasis on Fas ligand and Fas receptor. In contrast to the efficient production of the functional extracellular domains of TRAIL, TNFα and LTα by intracellular expression systems using Escherichia coli or Pichia pastoris, that of Fas ligand requires the secretory expression systems using P. pastoris or Dictyostelium discoideum, and the productivity in P. pastoris was largely dependent on tag sequence, potential N-glycosylation site and expressed protein region. On the other hand, the exploitation of insect cell systems is generally useful for the preparation of functional extracellular domains of death receptors containing many disulfide bridges in the absence of extended secondary structure, and a Bombyx mori larvae secretion system presented a superior productivity for human Fas receptor extracellular domain. Based on the results obtained so far, further efforts should be devoted to the artificial control of death ligand - death receptor interactions in order to make a contribution to medicine, represented by the development of novel biopharmaceuticals.     

Construction and analysis of a detailed three-dimensional model of the ligand binding domain of the human B cell receptor CD40

The interaction between the human B cell receptor CD40 and its ligand on T cells is critical for B cell proliferation and the regulation of humoral immune responses. CD40 is a member of the tumor necrosis factor receptor (TNFR) family. We report here the construction and analysis of a detailed three-dimensional model of the TNFR-homologous extracellular region of CD40. This study provides an example for structure-based model building in the presence of low sequence similarity. The assessment of model quality and sequence-structure compatibility is emphasized, and limitations of the model are discussed. The current CD40 model predicts structural details beyond the backbone level. Features of the CD40 ligand binding site are discussed in conjunction with the results of a previous mutagenesis study. Proteins 27:59–70 © 1997 Wiley-Liss, Inc.     

Structural characterization and interaction of periostin and bone morphogenetic protein for regulation of collagen cross-linking

Periostin appears to be a unique extracellular protein secreted by fibroblasts that is upregulated following injury to the heart or changes in the environment. Periostin has the ability to associate with other critical extracellular matrix (ECM) regulators such as TGF-β, tenascin, and fibronectin, and is a critical regulator of fibrosis that functions by altering the deposition and attachment of collagen. Periostin is known to be highly expressed in carcinoma cells, but not in normal breast tissues. The protein has a structural similarity to insect fasciclin-1 (Fas 1) and can be induced by transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP)-2. To investigate the molecular interaction of periostin and bone morphogenetic protein, we modeled these three-dimensional structures and their binding sites. We demonstrated direct interaction between periostin and BMP1/2 in vitro using several biochemical and biophysical assays. We found that the structures of the first, second, and fourth Fas1 domains in periostin are similar to that of the fourth Fas 1 domain of TGFBIp. However, the structure of the third Fas 1 domain in periostin is different from those of the first, second, and fourth Fas1 domains, while it is similar to the NMR structure of Fasciclin-like protein from Rhodobacter sphaeroides. These results will useful in further functional analysis of the interaction of periostin and bone morphogenetic protein.     

CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis

CXCR3 is a G-protein-coupled seven-transmembrane domain chemokine receptor that plays an important role in effector T-cell and NK cell trafficking. Three gamma interferon-inducible chemokines activate CXCR3: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Here, we identify extracellular domains of CXCR3 that are required for ligand binding and activation. We found that CXCR3 is sulfated on its N terminus and that sulfation is required for binding and activation by all three ligands. We also found that the proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation but not CXCL9 activation. In addition, we found that residue R216 in the second extracellular loop is required for CXCR3-mediated chemotaxis and calcium mobilization but is not required for ligand binding or ligand-induced CXCR3 internalization. Finally, charged residues in the extracellular loops contribute to the receptor-ligand interaction. These findings demonstrate that chemokine activation of CXCR3 involves both high-affinity ligand-binding interactions with negatively charged residues in the extracellular domains of CXCR3 and a lower-affinity receptor-activating interaction in the second extracellular loop. This lower-affinity interaction is necessary to induce chemotaxis but not ligand-induced CXCR3 internalization, further suggesting that different domains of CXCR3 mediate distinct functions.     

CR1/CR2 interactions modulate the functions of the cell surface epidermal growth factor receptor

Recent crystallographic data on the isolated extracellular domain of the epidermal growth factor receptor (EGFR) have suggested a model for its activation by ligand. We have tested this model in the context of the full-length EGFR displayed at the cell surface, by introducing mutations in two regions (CR1 and CR2) of the extracellular domain thought to be critical for regulation of receptor activation. Mutations in the CR1 and CR2 domains have opposing effects on ligand binding affinity, receptor dimerization, tyrosine kinase activation, and signaling competence. Tyr(246) is a critical residue in the CR1 loop, which is implicated in the positioning and stabilization of the receptor dimer interface after ligand binding; mutations of Tyr(246) impair or abolish receptor function. Mutations in CR2, which weaken the interaction that restricts the receptor to the tethered (inactive) state, enhance responsiveness to EGF by increasing affinity for the ligand. However, weakening of the CR1/CR2 interaction does not result in spontaneous activation of the receptors' kinase. We have used an antibody (mAb 806), which recognizes a transition state of the EGF receptor between the negatively constrained, tethered state and the fully active back-to-back dimer conformation, to follow conformational changes in the wild-type and mutant EGF receptors after ligand binding. Our results suggest that EGFR on the cell surface can be untethered, but this form is inactive; thus, untethering of the receptor is not sufficient for activation, and ligand binding is essential for the correct positioning of the two receptor subunits to achieve kinase activation.     

CD226 Protein Is Involved in Immune Synapse Formation and Triggers Natural Killer (NK) Cell Activation via Its First Extracellular Domain

CD226, an activating receptor that interacts with the ligands CD155 and CD112, activates natural killer (NK) cells via its immunoreceptor tyrosine-based activatory motif (ITAM). There are two extracellular domains of CD226; however, the comparative functional relevance of these domains remains unknown. In this study, two different deletion mutants, rCD226-ECD1 (the first extracellular domain) and rCD226-ECD (full extracellular domains), were recombinantly expressed. We observed that rCD226-ECD1, similar to rCD226-ECD, specifically bound to ligand-positive cell lines and that this interaction could be competitively blocked by an anti-CD226 mAb. In addition, rCD226-ECD1 was able to block the binding of CD112 mAb to tumor cells in a competitive binding assay. Importantly, based on surface plasmon resonance (SPR), we determined that rCD226-ECD1, similar to rCD226-ECD, directly bound to its ligand CD155 on a protein chip. Functionally, NK cell cytotoxicity against K562 or HeLa cells was blocked by rCD226-ECD1 by reducing the expression of CD69 and granzyme B, indicating the critical role of ECD1 in NK cell activation. We also examined the role of rCD226-ECD1 in effector/target interactions by using rCD226-ECD to block these interactions. Using flow cytometry, we found that the number of conjugates between IL-2-dependent NKL cells and HeLa cells was reduced and observed that the formation of immune synapses was also decreased under confocal microscopy. In addition, we prepared two anti-rCD226-ECD1 agonistic antibodies, 2E6 and 3B9. Both 2E6 and 3B9 antibodies could induce the phosphorylation of ERK in NK-92 cells. Taken together, our results show that CD226 functions via its first extracellular domain.     

Solution structure of the third extracellular loop of human thromboxane A2 receptor

The extracellular domains of the thromboxane A2 receptor (TP receptor) were found to be involved in the specific ligand recognition. Determination of the three-dimensional (3D) structure of the extracellular loops would help to explain the mechanism of the ligand binding to its receptor with regard to the tertiary structure. Based on our previous studies on the extracellular loop of the human TP receptor, the synthetic loop peptides, whose termini are constrained to 10 to 14-A separations, are more likely to mimic the native structure of the extracellular loops. In this study, a peptide with the sequence of the third extracellular loop (eLP3, residues 271-289) of the TP receptor was synthesized, and its termini were constrained by the formation of a disulfide bond between the additional homocysteines located at both ends. Fluorescence spectroscopic studies showed that the fluorescence intensity of this constrained loop peptide could be increased by the addition of SQ29,548, a TP receptor antagonist, which indicated the interaction between the peptide and the ligand. The structure of this peptide was then studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. 1H NMR assignments of the peptide were obtained and structure constraints were derived from nuclear Overhauser effects and J-coupling constants. The solution structure of the peptide was then calculated based on these constraints. The overall structure shows a beta turn from residues 278 to 281. It also shows a distance of 9.45A between the ends of the N and C termini of the peptide, which agrees with the distance between the two residues at the ends of the transmembrane helices connecting the eLP3 on the TP receptor working model generated using molecular modeling, based on the crystal structure of bovine rhodopsin. These results provide valuable information for the characterization of the complete 3D structure of the extracellular domains of the human TP receptor.     

Cell surface receptors and their ligands: in vitro analysis of CD6-CD166 interactions

CD6 is a cell surface receptor belonging to the scavenger receptor cysteine-rich (SRCR) protein superfamily (SRCRSF). It specifically binds activated leukocyte cell adhesion molecule (ALCAM, CD166), a member of the immunoglobulin (Ig) superfamily (IgSF). CD166 was among the first molecules identified as a ligand for an SRCRSF receptor, and the CD6-CD166 interaction was the first interaction characterized involving SRCRSF and IgSF proteins. We focus here on what has been learned about the specifics of the CD6-CD166 interaction from in vitro analysis. The studies are thought to provide an instructive example for the analysis of interactions between single-path transmembrane cell surface proteins. Using soluble recombinant forms, the extracellular binding domains of receptor and ligand have been identified and characterized in a variety of assay systems. Both CD6 and CD166 have been subjected to intense mutagenesis and monoclonal antibody (mAb) binding studies and residues critical for their interaction have been identified. The availability of structural prototypes of both superfamilies has made it possible to map the binding site in CD166 and, more recently, in CD6 and compare these regions to epitopes of mAbs that block, or do not block, the interaction. In addition, the molecular basis of observed cross-species receptor-ligand interactions could be rationalized. These studies illustrate the value of structural templates for the interpretation of sequence and mutagenesis analyses. Proteins 2000;40:420-428.     

A membrane-bound Fas decoy receptor expressed by human thymocytes

Human thymocytes at several stages of maturation express Fas, yet resist apoptosis induction through its ligation. A proximal step in apoptotic signaling through Fas is implicated in this resistance, as these cells undergo normal levels of apoptosis induction after exposure to tumor necrosis factor-alpha. We studied the Fas receptors expressed in human thymocytes to search for mechanisms of receptor-mediated inhibition of Fas signaling in these cells. We describe here a unique, membrane-bound form of Fas receptor that contained a complete extracellular domain of Fas but that lacked a death domain due to alternative splicing of exon 7. This Fas decoy receptor (FDR) was shown to have nearly wild-type ability to bind native human Fas ligand and was expressed predominantly at the plasma membrane. Unlike soluble forms of Fas receptor, FDR dominantly inhibited apoptosis induction by Fas ligand in transfected human embryonic kidney cells. Titration of FDR in Fas-expressing cells suggests that FDR may operate through the formation of mixed receptor complexes. FDR also dominantly inhibited Fas-induced apoptosis in Jurkat T cells. In mixing experiments with wild-type Fas, FDR was capable of inhibiting death signaling at molar ratios less than 0.5, and this relative level of FDR:wild type message was observed in at least some thymocytes tested. The data suggest that Fas signal pathways in primary human cells may be regulated by expression of a membrane-bound decoy receptor, analogous to the regulation of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis by decoy receptors.     

Activation of transmembrane cell‐surface receptors via a common mechanism? The “rotation model”

It has long been thought that transmembrane cell‐surface receptors, such as receptor tyrosine kinases and cytokine receptors, among others, are activated by ligand binding through ligand‐induced dimerization of the receptors. However, there is growing evidence that prior to ligand binding, various transmembrane receptors have a preformed, yet inactive, dimeric structure on the cell surface. Various studies also demonstrate that during transmembrane signaling, ligand binding to the extracellular domain of receptor dimers induces a rotation of transmembrane domains, followed by rearrangement and/or activation of intracellular domains. The paper here describes transmembrane cell‐surface receptors that are known or proposed to exist in dimeric form prior to ligand binding, and discusses how these preformed dimers are activated by ligand binding.     

Mechanisms of radio-induced apoptosis

A general overview of the activation mechanisms of programmed cell death or apoptosis following an irradiation is given in this review. First, are summarized the main induction pathways of radiation-induced apoptosis by which extracellular (tumor necrosis factor (TNF), Fas ligand, TNF-related apoptosis-inducing ligand (TRAIL)) and intracellular (mitochondria and caspases) signals are integrated. A second part is then devoted to the importance of p53 and of its regulators (ATR, ATM, DNA-PKcs) in the process of radiation-induced apoptosis. Thereafter, signal transduction pathways and more specially the role of some protein kinases (MEKK, SAPK/JNK, p38-MAPK) is treated. At last, a chapter concerns the clinical interest of radiation-induced apoptosis and the implication of apoptosis in the treatment of certain diseases.     

The autocrine motility factor (AMF) and AMF-receptor combination needs sugar chain recognition ability and interaction using the C-terminal region of AMF

The autocrine motility factor (AMF) promotes cellular locomotion or invasion, and regulates tumor angiogenesis or ascites accumulation. These signals are triggered by binding between AMF and its receptor (AMFR), a glycoprotein on the cell surface. AMF has been identified as phosphohexose isomerase (PHI). Previous reports have suggested that the substrate-recognition of exo-PHI is significant for receptor binding. Crystallographic studies have shown that AMF consists of three domains, and that the substrate or inhibitor of PHI is stored between the large and small domains, corresponding to approximately residues 117-288. Here, site-directed mutagenesis was used to investigate 18 recombinant human AMF point mutants involving critical amino acid residues for substrate or enzyme inhibitor recognition or binding. Mutation of residues that interact with the phosphate group of the PHI substrate significantly reduced the cell motility-stimulating activity. Their binding capacities for AMFR were also lower than wild-type human AMF. Mutants that retained the enzymic activity showed the motility-stimulating effect and receptor binding and had sensitivity to a PHI inhibitor. Mutant AMFR lacking the N-sugar chain was expressed on the cell membrane but did not respond to AMF-stimulation, and N-glycosidase-treated AMFR did not compete with receptor binding of AMF. Furthermore, the AMF domains that contain the substrate storage domain and C-terminal region stimulate cell locomotion. These results suggest that the N-glyco side-chain of AMFR is a trigger and that interaction between the 117-C-terminal part of AMF and the extracellular core protein of AMFR is needed during AMF-AMFR interactions.     

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.