TRAIL (tumor necrosis factor-related apoptosis inducing ligand) is a cytokine that triggers apoptotic cell death in many cancer cells, but not normal untransformed cells. Because of this selective toxicity to cancer cells, TRAIL is an attractive anti-cancer therapeutic agent. The sensitivity of different cancer cells and normal cells to TRAIL is partly regulated by expression of inhibitory receptors that bind TRAIL but cannot signal for apoptosis. However, little is known about the mechanism by which the inhibition/ receptors controls TRAIL-induced cell death in cancer cells and normal cells. The long term goal of this project is to elucidate the molecular mechanism by which the TRAIL inhibitory receptors control cell death in cancer cells and normal cells. In our preliminary studies, we found that the death receptor TRAIL-R2 and the inhibitory receptor TRAIL-R4 interact with each other in a ligand-independent manner and that this interaction is central to the inhibition of apoptosis by TRAIL-R4. Based on these results, we propose to examine whether pre-ligand interaction between TRAIL-R2 and TRAIL-R4 in different cancer cells correlates with their resistance to TRAIL-induced apoptosis. A second goal of the proposal is to examine the role of "lipid rafts" in regulating pre-ligand TRAIL-R2/TRAIL-R4 assembly. Finally, we will examine whether pre-assembled TRAIL-R2/TRAIL-R4 complex inhibits apoptosis by activating the pro-survival transcription factor NF kappa B. To achieve these goals, we will examine TRAIL-R2 and TRAIL-R4 interaction in cancer cells and normal cells by biochemical co-immunoprecipitations, chemical crosslinking and flow cytometric fluorescence resonance energy transfer (FRET) assays. In addition, we will examine these interactions in different membrane microdomains. Furthermore, we will examine modulate TRAIL-R4 expression in different cancer cells and normal cells by RNA interference and transfection to evaluate the role of TRAIL-R4 in regulating NF-kappa B activation. Lay summary: TRAIL is a promising anti-cancer agent that selectively kills cancer cells, but not normal cells. Moreover, TRAIL can synergize with radiotherapy or chemotherapy to enhance killing of cancer cells. Our experiments may also provide a useful diagnostic tool to determine response of cancers to treatments involving TRAIL or agonist TRAIL antibodies.