Signaling through the Death Receptor CD95 (APO-1/FAS)

INTRODUCTION AND METHOD. Using classical immunoprecipitation protocols in combination with high-resolution 2D gels, we identified a complex of proteins that formed around the death domain of the stimulated death receptor CD95 (APO-1/Fas) receptor which we called the death-inducing signaling complex (DISC)(1). We went on to characterize the three major components of the DISC: First, the adaptor FADD/Mort1 was identified (1) then caspase-8 and the caspase-8-like apoptosis inhibitor c-FLIP were cloned (2,3). Formation of the DISC upon apoptosis induction results in activation of caspase-8 by proteolytical cleavage into the prodomain containing two death effector domains (DED) and two active subunits, p18 and p10 (4). Thus, caspase-8 plays an essential role in the execution of death receptor-mediated apoptosis. To determine the localization of endogenous caspase-8 we used a panel of subunitspecific anti-caspase-8 monoclonal antibodies in confocal immunofluorescence microscopy.