Intra-cluster receptor density (IRD) dictates TNFR1 clusters’ signaling efficacy

Tumor Necrosis Factor Receptor 1 (TNFR1) signaling determines cell fate during inflammation, immunopathogenesis, and tumorigenesis. TNFR1 proteins homo-oligomerize into clusters on the plasma membrane. The potential impact of TNFR1 clustering on downstream signaling remains unexplored. Homo-FRET measurements elucidate that alterations in intra-cluster receptor density (IRD) dictate the outcomes of downstream TNFR1 signaling. Soluble TNF-α (sTNF-α) elevates IRD within the TNFR1 clusters’ core while diminishing it in the rim, through intra-cluster dynamic reorganization of TNFR1. Decreasing TNFR1 IRD through increasing membrane tension, administering TNFR1 antagonist zafirlukast, actin depolymerization, or depleting cholesterol impedes sTNF-α-mediated stimulation. Conversely, increasing IRD by reducing membrane tension or exposing cells to 3D gel-like microenvironment induces ligand-independent TNFR1 signaling. These findings suggest a broader applicability of IRD in modulating signaling pathways across other receptor families, offering insights for innovative strategies in TNFR1 signaling modulation.