The transmembrane domain structure of TNFR1 suppresses ligand-independent autoactivation but is not required for TNF-induced signaling

Tumor necrosis factor (TNF) is a pivotal inflammatory cytokine, and it predominantly signals by binding to TNF receptor 1 (TNFR1), a type I single-spanning transmembrane protein that is thought to exist primarily as monomers and dimers. The binding of trimeric ligands induces the formation of signaling-competent trimers and higher-order oligomers that lead to full activation of downstream NF-κB and MAPK signaling pathways. Several TNFR superfamily members, including TNFR1, can form trimeric structures through their transmembrane domains (TMDs). For Fas and DR5, these structures support ligand-induced activation, and ectodomain (ECD) interactions prevent ligand-independent signaling. To explore the structures' role in TNFR1 activation, we replaced the native TNFR1 TMDs with natural and de novo designed versions that formed stable and specific structures of defined monomeric or oligomeric states. We found that none of these TMD variant receptors displayed defects in TNF-induced signaling in mouse fibroblasts, but some showed increased autoactivation in the absence of ligand, particularly where the engineered TMD sequence prevented self-association. Autoactivation depended on intracellular death domain interactions and was exacerbated by a disease-associated mutation but was unaffected by mutation in the preligand assembly domain. Our results demonstrate that, unlike for other TNFR family members, no specific oligomeric TMD structure is required for normal, ligand-induced activation of TNFR1, but self-association through the native TMD may instead act together with ECD interactions to help suppress ligand-free autoactivation.