Ligand-induced internalization of TNF receptor 2 mediated by a di-leucin motif is dispensable for activation of the NFκB pathway.
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.
Fischer
Roman
R
Institute of Cell Biology and Immunology, University Stuttgart, Allmandring 31, Stuttgart, Germany.
Maier
Olaf
O
Naumer
Matthias
M
Krippner-Heidenreich
Anja
A
Scheurich
Peter
P
Pfizenmaier
Klaus
K
D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000737:chemistry*; D008024:Ligands; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
D002966:Clathrin; D008024:Ligands; D016328:NF-kappa B; D047889:Receptors, Tumor Necrosis Factor, Type II; D014409:Tumor Necrosis Factor-alpha; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; C000716465:rab7 GTP-binding proteins, mouse; D020691:rab GTP-Binding Proteins; D007930:Leucine