Single-molecule imaging reveals the oligomeric state of functional TNFα-induced plasma membrane TNFR1 clusters in cells.

Abstract

Ligand-induced tumor necrosis factor receptor 1 (TNFR1) activation controls nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling, cell proliferation, programmed cell death, and survival and is crucially involved in inflammation, autoimmune disorders, and cancer progression. Despite the relevance of TNFR1 clustering for signaling, oligomerization of ligand-free and ligand-activated TNFR1 remains controversial. At present, models range from ligand-independent receptor predimerization to ligand-induced oligomerization. Here, we used quantitative, single-molecule superresolution microscopy to study TNFR1 assembly directly in native cellular settings and at physiological cell surface abundance. In the absence of its ligand TNFα, TNFR1 assembled into monomeric and dimeric receptor units. Upon binding of TNFα, TNFR1 clustered predominantly not only into trimers but also into higher-order oligomers. A functional mutation in the preligand assembly domain of TNFR1 resulted in only monomeric TNFR1, which exhibited impaired ligand binding. In contrast, a form of TNFR1 with a mutation in the ligand-binding CRD2 subdomain retained the monomer-to-dimer ratio of the unliganded wild-type TNFR1 but exhibited no ligand binding. These results underscore the importance of ligand-independent TNFR1 dimerization in NF-κB signaling.

• Authors: Christos Karathanasis, Juliane Medler, Franziska Fricke, Sonja Smith, Sebastian Malkusch, Darius Widera, Simone Fulda, Harald Wajant, Sjoerd J L van Wijk, Ivan Dikic, Mike Heilemann
• Journal: Science signaling (Sci Signal) Vol. 13 Issue 614 (01 14 2020) ISSN: 1937-9145 [Electronic] United States
• PMID: 31937565 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Chemical References

• NF-kappa B
• Receptors, Tumor Necrosis Factor, Type I
• Tumor Necrosis Factor-alpha

Topics

• Animals
• Apoptosis (drug effects)
• Cell Membrane (drug effects, metabolism)
• Cells, Cultured
• Embryo, Mammalian (cytology)
• Fibroblasts (cytology, drug effects, metabolism)
• HeLa Cells
• Humans
• Mice, Knockout
• Mice, Transgenic
• Models, Molecular
• Mutation
• NF-kappa B (metabolism)
• Protein Binding
• Protein Multimerization
• Protein Transport (drug effects)
• Receptors, Tumor Necrosis Factor, Type I (chemistry, genetics, metabolism)
• Signal Transduction (drug effects)
• Single Molecule Imaging (methods)
• Tumor Necrosis Factor-alpha (metabolism, pharmacology)