Mechanisms and principles of tumor necrosis factor (TNF)-receptor activation

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Tumor necrosis factor (TNF) receptors become activated by ligands of the TNF family. The latter are typically expressed as trimeric membrane-bound molecules but often occur also in a soluble form, e.g. due to proteolytic processing. Binding of membrane-bound TNF ligands invariably results in strong TNF receptor activation. TNF receptors, however, differently respond to soluble ligands. Some TNF receptor types are strongly activated by binding of soluble ligand molecules (e.g. TNFR1), while others bind soluble ligands but fail to trigger intracellular signaling (e.g. TNFR2). The lack of responsiveness of some TNF receptor types towards soluble ligands mirrors a limitation of the receptor and not a missing quality of the ligand molecule. For example, soluble TNF strongly triggers TNFR1 signaling but is practical inactive on TNFR2 despite good binding.A basal observation in our previous work was that secondary aggregation of initially formed trimeric TNF ligand receptor complexes represents an important if not even obligate step in the activation of various TNF receptor-associated pathways. This concept is supported for those types of TNF receptors that become not or only poorly activated upon binding of soluble ligand trimers by various experimental data. However, it is unclear whether TNF receptor types that become quite well activated by soluble ligands also form and utilize supramolecular ligand receptor clusters for signal transduction. Using the example of TNFR1, this basic aspect will be addressed in the project by a bimolecular luminescence complementation assay, coimmunoprecipitation experiments and single-molecule tracking fluorescence microscopy.We found in unpublished work that the monomeric soluble ectodomain of TNFR1, thus the shedded form of the receptor, already interacts with high affinity with TNF. We will thus clarify whether such soluble receptor molecules are incorporated into trimeric and oligomeric TNF ligand receptor complexes and act there in a dominant negative fashion.Based on own data and initial evidence from the literature, we will further evaluate for CD95 and TNFR2 the possibility that distinct TNF receptor-associated signaling pathways are differentially activated by trimeric and oligomeric ligand receptor complexes. For this purpose, we will comparatively analyze the identity, proportion and modifications of signaling proteins associated with trimeric and oligomeric TNFR2 and CD95 signaling complexes by mass spectrometry and western blotting. The findings obtained will then be evaluated with respect to their functional relevance for TNFR2 and CD95 signaling by established biochemical approaches.