Detection of Fas-associated death domain and its variants' self-association by fluorescence resonance energy transfer in living cells.

Fas-associated death domain (FADD) is an adaptor molecule for the death receptor subfamily of the tumor necrosis factor receptor superfamily, but it is also required for cell proliferation. FADD protein has the potential to highly oligomerize. FADD self-aggregates in vitro and transfected FADD in mammalian cells induce apoptosis by forming large, filamentous structures termed death effector filaments independent of receptor cross-linking at the plasma membrane. We used fluorescence spectroscopy and three-channel fluorescence resonance energy transfer (FRET) microscopy to qualitatively and quantitatively analyze self-association of FADD and its variants in living cells. Our results demonstrate that FADD self-association not only is determined by its N-terminal death effector domain (h-FADD 1-79) but is also obviously regulated by its C-terminal phosphorylatable tail (h-FADD 183-208).