Ligand-induced internalization of TNF receptor 2 mediated by a di-leucin motif is dispensable for activation of the NFκB pathway

• Published in: Cellular signalling 2011, Vol.23 (1), p.161-170
• Main Authors: Fischer, Roman, Maier, Olaf, Naumer, Matthias, Krippner-Heidenreich, Anja, Scheurich, Peter, Pfizenmaier, Klaus
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Animals; CD120b; Cell Line; Clathrin; Clathrin - metabolism; Endocytosis; Fibroblasts - metabolism; Humans; Leucine - chemistry; Ligands; Lysosome; Mice; NF-kappa B - metabolism; Protein degradation; R2 MEF; rab GTP-Binding Proteins - metabolism; Receptors, Tumor Necrosis Factor, Type II - chemistry; Receptors, Tumor Necrosis Factor, Type II - genetics; Receptors, Tumor Necrosis Factor, Type II - metabolism; Signal Transduction; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2010.08.016

Endocytosis is an important mechanism to regulate tumor necrosis factor (TNF) signaling. In contrast to TNF receptor 1 (TNFR1; CD120a), the relevance of receptor internalization for signaling as well as the fate and route of internalized TNF receptor 2 (TNFR2; CD120b) is poorly understood. To analyze the dynamics of TNFR2 signaling and turnover at the plasma membrane we established a human TNFR2 expressing mouse embryonic fibroblast cell line in a TNFR1 −/−/TNFR2 −/− background. TNF stimulation resulted in a decrease of constitutive TNFR2 ectodomain shedding. We hypothesized that reduced ectodomain release is a result of TNF/TNFR2 complex internalization. Indeed, we could demonstrate that TNFR2 was internalized together with its ligand and cytoplasmic binding partners. Upon endocytosis the TNFR2 signaling complex colocalized with late endosome/lysosome marker Rab7 and entered the lysosomal degradation pathway. Furthermore, we identified a di-leucin motif in the cytoplasmic part of TNFR2 suggesting clathrin-dependent internalization of TNFR2. Internalization defective TNFR2 mutants are capable to signal, i.e. activate NFκB, demonstrating that the di-leucin motif dependent internalization is dispensable for this response. We therefore propose that receptor internalization primarily serves as a negative feed-back to limit TNF responses via TNFR2.