Apoptosis is the physiological form of cell death involved in homeostasis of tissues and is dysregulated in numerous diseases. The death receptor CD95 (APO-1/Fas) induces apoptosis by forming a death-inducing signaling complex (DISC) comprised of CD95, the adaptor molecule FADD, caspases 8 and 10, and c-FLIP. In Type I cells (cells that die in a mitochondria-independent manner such as T cells) triggering CD95 with either CD95 ligand (CD95L) or an anti-CD95 antibody induces the receptor to form clusters at the cell surface. Formation of these clusters is dependent on a small amount of DISC-generated active caspase-8. We hypothesize that active caspase-8 generated at the DISC is part of a positive feedback loop and that activation of caspase-8 by other means such as by other death receptors or certain antitumor drugs can induce the clustering of CD95. We recently showed for the first time that after activation, the CD951CD95L complex is internalized through an endosomal pathway in an actin-dependent fashion. Inhibition of filamentous actin with latrunculin A in Type I cells strongly reduces DISC formation and sensitivity to CD95 apoptosis whereas Type II cells which do not internalize CD95 form very little DISC. Depending on the situation, CD95 can also activate prosurvival pathways which depend on activation of caspase-8. Induction of these protective pathways is completely abrogated when actin filaments and internalization of CD95 is blocked. We hypothesize that the act independent internalization of CD95 is required for efficient formation of the DISC and signaling through CD95 and that internalization of CD95 transports and directs signaling molecules to specific intracellular compartments activating apoptosis or prosurvival pathways depending on the cell. To test these hypotheses we propose to study the following Specific Aims using biochemical, cell biological, and molecular biological techniques: 1) Characterize the function of caspase-8 in the initiation of CD95 signaling. We will identify the caspase-8 target that regulates CD95 signal initiation and characterize its function. 2) Determine the role of the actin cytoskeleton in CD95 signaling. We will identify how CD95 is linked to the actin cytoskeleton and study the mechanism of actin regulation of CD95 signaling and internalization. 3) Determine the significance of receptor internalization for CD95 signaling. We will identify the route of internalization of the CD95/CD95L complex in apoptosing and nonapoptosing cells. We will test whether internalized DISC generates different apoptosis dependent and independent signals when compared to surface DISC. The results of this study will provide key insights into understanding how death receptors initiate death and prosurvival signals which could be the basis to interfere with death receptor mediated signaling in diseases with dysregulated apoptosis.
