Activation of receptors of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) by heteromeric ligands of the TNF superfam,ily (TNFSF)

• Applicant: Professor Dr. Harald Günther Wajant
• Subject Area: Cell Biology ; Biochemistry
• Term: from 2020 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 436843377

The ligands of the TNF superfamily (TNFLs) form homotrimers, which are very well studied, and occur as transmembrane and soluble molecules. There are also a very few heterotrimeric TNFLs, which with the exception of the LTaß2 heterotrimer have been poorly understood with respect to biology and function. Due to their asymmetric nature, heterotrimeric TNFLs trigger receptor complexes that differ in stoichiometry and composition from those of homotrimeric TNFLs. We identified LTa2ß and LTa2LIGHT heterotrimers as novel ligands of TNFR1 and TNFR2. In particular, we could show that the membrane, but not the soluble forms of these heteromers activate TNFR1 and TNFR2. We found that LTa2ß binds TNFR1 and TNFR2 only with its LTa-LTa but not with its two LTa-LTß contact zones. The current model of TNFR activation is that TNFLs recruits three receptor molecules and that the resulting trimeric TNFL-TNFR complexes have to cluster further for strong receptor activation. The activating clustering step is driven by the pre-ligand assembly domain (PLAD) of the TNFRs mediating weak-affinity ligand-independent receptor self-association. The fact that monovalent membrane LTa2ß is strongly agonistic for TNFR1 and TNFR2 is unexpected and suggests that PLAD-mediated clustering of the TNF receptors in the cell-to-cell contact zone is so efficient that it even overcomes the need for intermediate formation of trimeric liganded TNFR complexes. We also analyzed heteromer formation between LTa, LTß, LIGHT, TNF and TL1A and indeed found LTa-LIGHT, LTß-LIGHT, LIGHT-TL1A and LTß-TL1A heteromer formation which was comparable effective as those of the corresponding homotypic TNFLs. The central question raised by the identification of the novel heteromeric TNFLs is to what extent heteromeric molecules arise and, more importantly, how they contribute to the already known biology of their homotrimeric sister TNFLs and their receptors. To answer this question, the renewal proposal pursues four experimental goals: i) Systematic evaluation of all 19 TNFL protomers for heteromer formation. ii) Investigation of the TNFR-stimulating properties of the new heteromeric TNFLs. Since the formation of TNFL heteromers is associated with a decrease in TNFL homotrimer formation, the possibility that the induced expression of a heteromerizing TNFL reduces the formation of homomeric TNFLs and thus their activity is also studied. iii) The development of TNFL heteromer-specific antibodies. This would allow for simplified detection of heteromeric TNFLs, enables their detection in vivo and most importantly, if these antibodies compete for TNFR binding, would enable functional analysis of heteromeric TNFLs in vivo. iv) The function of LTa2ß heteromers and the newly identified LIGHT-LTa, LIGHT-TL1A, LIGHT-LTß and LTß-TL1A heterotrimers will be investigated in primary immune cells.

• DFG Programme: Research Grants