| The Fas ligand (FasL), a member of the tumor necrosis factor family of proteins, is a potent inducer of apoptosis. This ligand acts via cognate interactions with its receptor, the Fas receptor (also called APO-1 or CD95), to engage the cellular death machinery. Members of the tumor necrosis factor family are type II transmembrane proteins which share homology over a region of about 150 amino acid residues in their extracellular domains. Soluble fragments containing this region have been shown to form trimers under physiological conditions. X-ray crystallographic data of TNF-alpha, LT-alpha and CD40L alone and LT-alpha bound to TNFR I demonstrated that these ligands are composed primarily of beta-strands and intervening loop regions. Using the transmembrane human FasL, mutagenesis was performed by deletion and single amino acid residue substitution analysis. This approach has defined the very C-terminus as absolutely essential for Fas receptor binding and biological activity. A putative self-association domain has also been defined in a distinct region of the ligand. Finally, N-glycosylation of the FasL was demonstrated to be important for efficient steady-state expression of this protein, but not receptor binding or biological activity.; Using a series of extracellular domain chimeras between the Fas receptor and TNFR I, it was demonstrated that all three cysteine-rich domains (CRDs) from the Fas receptor are absolutely required for binding to its ligand. It was also shown that TNFR I CRD1 could partially substitute for Fas CRD1.; Data are presented which demonstrate that the intracellular domain Canale-Smith syndrome mutants are biologically inactive because they are unable to engage the proximal Fas signal transducing molecule FADD/MORT1. It was also shown that expression of increasing amounts of mutant Fas receptor with a fixed amount of wild-type receptor resulted in a dose-dependent inhibition of FADD/MORT1 recruitment to the activated receptor complex. Finally, two extracellular domain Fas receptor mutants found in patients have been studied. In vitro results suggest that these mutations do not directly contribute to Fas resistance. Thus, Canale-Smith syndrome patients with these mutations may have other defects which contribute to their disease.; Finally, data will be presented from my initial laboratory project, which involved the identification of Fas receptor-associated cytoplasmic proteins. (Abstract shortened by UMI.)... |
