TNF ligand-receptor interactions serve diverse functions in immune system regulation. Fas, a member of the TNF receptor family is an important regulator of lymphocyte homeostasis and can cause systemic autoimmunity when genetically mutated. Althuogh a number of signaling proteins mediating TNF receptor signals have been identified, the cell biology of receptor signaling: where signaling events take place within the cell and whether subcellular localization plays a role in regulating TNF receptor signaling, remains relatively unknown. Understanding this process is important for devising better ways to modulate signaling through these receptors for therapeutic goals. In previous work, we established that TNF receptors must pre-associate into mutimeric complexes to signal efficiently. In the past year we have made a number of significant advances in understanding the cell biology of TNF receptors. We showed that localization of the Fas receptor to specialized plasma membrane subdomains termed ?lipid rafts? significantly enhance the efficiency of Fas apoptosis signaling. Triggering the T cell antigen receptor provoked rapid protein-synthesis independent translocation of Fas into lipid rafts in primary T cells, sensitizing them to apoptosis triggered by Fas Ligand. This mechanism may account for the long sought-after ?competancy? signal by which activated T cells are sensitized to undergo apoptosis by T-cell receptor engagement. In addition, we have defined a novel protein-protein interaction and microscopic structures that amplify the Fas-induced apoptosis signal at the plasma membrane. We have found that the adaptor protein FADD, a key mediator of Fas-induced apoptosis, can interact with other FADD molecules through a conserved short sequence motif in its death effector domain (DED). Mutation of this motif abrogates the ability of FADD to function in the Fas apoptosis pathway, and can transform FADD into a dominant-negative inhibitor of apoptosis. We have also observed microscopically visible Fas receptor aggregates at the plasma membrane termed SPOTS (Signaling Protein Oligomeric Transduction Structures) that form within minutes after Fas ligand or anti-Fas antibody stimulation of living cells. These structures require intact Fas and FADD and form in the absence of active caspases, suggesting that they are early mediators of Fas-induced apoptosis. Indeed, in cells with defective Fas-FADD interactions because of genetic deficiency or pathogenic mutations in Fas, the formation of SPOTS is greatly impaired. SPOTS may perform the important function of concentrating pro-caspase-8 to the point where the enzyme autoactivates itself to begin the apoptotic cascade. In collaboration with Dan Kastner in NIAMS, we have also begun studies to address the subcellular trafficking and ultimate fate of p55 TNFR1 receptors harboring point mutations in the extracellular domain linked to the TNF-receptor associated periodic fever syndrome (TRAPS). We are examining possible defects in the ability of these mutant receptors to traffic in the biosynthetic pathway that leads to cell surface expression and secretion in small membrane bound vesicles termed exosomes. It is thought that secreted TNFR1 formed through proteolysis or secreted in exosomes can negatively regulate the pro-inflammatory effects of TNF. These studies may help us better unerstand the pathophysiology of the genetic syndrome TRAPS and design more effective therapeutics against TNF overactivity states.
| Siegel, Richard M; Muppidi, Jagan R; Sarker, Malabika et al. (2004) SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane. J Cell Biol 167:735-44 |
| Muppidi, Jagan R; Tschopp, Jurg; Siegel, Richard M (2004) Life and death decisions: secondary complexes and lipid rafts in TNF receptor family signal transduction. Immunity 21:461-5 |
| Siegel, Richard M; Muppidi, Jagan; Roberts, Margaret et al. (2003) Death receptor signaling and autoimmunity. Immunol Res 27:499-512 |
| Garvey, Tara L; Bertin, John; Siegel, Richard M et al. (2002) Binding of FADD and caspase-8 to molluscum contagiosum virus MC159 v-FLIP is not sufficient for its antiapoptotic function. J Virol 76:697-706 |
