Browsing by Author "Neumann, Simon Paul"

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    Analysis of receptor-receptor interactions and their implications on the formation of signaling competent TRAIL receptor clusters
    (2013) Neumann, Simon Paul; Scheurich, Peter (Prof. Dr.)
    The cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its associated receptors constitute an elaborate signaling system fulfilling important functions in regulation of the immune system and in tumor surveillance. Two TRAIL membrane receptors, receptor 1 (TRAILR1) and receptor 2 (TRAILR2), possess an intracellular death domain. Activation of these receptors can lead to apoptosis in TRAIL susceptible cells, but also non-apoptotic signals can be induced. Two additional TRAIL receptors, TRAILR3 and TRAILR4, are generally referred to as decoy receptors. TRAILR3 is linked to the plasma membrane via a glycosylphosphatidylinositol anchor, hence it is devoid of intracellular and transmembrane domains and thus most likely incapable of signal transduction. The intracellular domain of TRAILR4 contains merely a partial death domain consensus motif, therefore it is often referred to as a truncated death domain. Signaling capabilities of TRAILR4 are likely contextual and cell type-dependent, furthermore TRAILR4 can exclusively transduce non-apoptotic signals. Expression of either of the decoy receptors has been shown to reduce sensitivity to TRAIL-induced apoptosis. However, the underlying molecular mechanisms, in particular concerning TRAILR4, remain vaguely defined. Alike other members of the tumor necrosis factor receptor superfamily, TRAIL receptors contain a pre-ligand binding assembly domain (PLAD) in their extracellular domain which mediates receptor-receptor interactions. However, the outcome of TRAIL receptor oligomerization, that is the stoichiometry of the formed complexes as well as the issue whether the PLAD mediates only homotypic or also heterotypic interactions, remained inconclusive until now. Accordingly, different techniques for the investigation of protein-protein interactions were applied in order to study ligand-independent oligomerization of TRAIL receptors 1, 2 and 4. Interactions of these TRAIL receptors in all six possible combinations were demonstrated through fluorescence resonance energy transfer. The stoichiometry of the resulting TRAIL receptor complexes was clarified through chemical crosslinking experiments. Homophilic interaction of the death receptor TRAILR2, as well as heterophilic interactions between the two death receptors or between either of the death receptors and the decoy receptor TRAILR4 resulted in the formation of dimeric complexes. Subsequent biochemical assays and biosensor measurements performed with the soluble extracellular domains of TRAILR1 and TRAILR4 confirmed that ligand-independent dimerization is an intrinsic capability of the extracellular domains of TRAIL receptors. The implications of the demonstrated receptor-receptor interactions on TRAIL-induced signal transduction were investigated in cellular models expressing TRAILR1 and either wild type or a truncated, non-signaling variant of TRAILR4. The apoptotic effects of TRAIL were significantly reduced in presence of TRAILR4 and this reduction was directly correlated to the expression level of receptor 4. Not only inhibition of proapoptotic signaling could be demonstrated, but also reduced activation of the transcription factor NFkappaB, assessed by analyzing the phosphorylation of its inhibitor IkappaBalpha. Accordingly, TRAILR4 exerted a general inhibitory function on signaling by TRAILR1 in the studied cellular model. The inhibitory capacity of TRAILR4 could be clearly attributed to signaling-independent mechanisms. This could be demonstrated through the capacity of the truncated non-signaling receptor variant to interfere with TRAIL-mediated signaling to a comparable extent as the wild type receptor. The reduced cellular sensitivity to the apoptotic effects of TRAIL manifested itself already at the level of procaspase-8 processing, indicating that TRAILR4 impedes proapoptotic signaling early in the extrinsic apoptosis pathway. As a molecular mechanism underlying this, I propose the reduction of signaling competent death receptor dimers through PLAD-mediated formation of mixed receptor complexes. Furthermore, no intracellular signaling pathways emanating from TRAILR4, neither constitutive nor ligand-induced, could be identified in the studied cellular system. The reported experimental findings were integrated into a model explaining the capability of TRAILR4 to interfere with apoptosis induction by TRAIL death receptors through ligand-independent receptor pre-assembly. Moreover, the proposed model affirms the necessity to take account of TRAILR4 expression in matters of TRAIL-based tumor therapy.