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TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation
Quan Lu1, Lila Weiqiao Hope, Michael Brasch
1Department of Genetics, Program in Cancer Biology, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
Abstract:
Down-regulation of mitogenic signaling in mammalian cells relies in part on endosomal trafficking of activated receptors into lysosomes, where the receptors are degraded. These events are mediated by ubiquitination of the endosomal cargo and its consequent sorting into multivesicular bodies that form at the surfaces of late endosomes. Tumor susceptibility gene 101 (tsg101) recently was found to be centrally involved in this process. Here we report that TSG101 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), an early endosomal protein, and that disruption of this interaction impedes endosomal trafficking and endocytosis-mediated degradation of mitogenic receptors. TSG101/HRS interaction occurs between a ubiquitin-binding domain of TSG101 and two distinct proline-rich regions of HRS, and is modulated by a C-terminal TSG101 sequence that resembles a motif targeted in HRS. Mutational perturbation of TSG101/HRS interaction prevented delivery of epidermal growth factor receptor (EGFR) to late endosomes, resulted in the cellular accumulation of ubiquitinated EGFR in early endosomes, and inhibited ligand-induced down-regulation of EGFR. Our results reveal the TSG101 interaction with HRS as a crucial step in endocytic down-regulation of mitogenic signaling and suggest a role for this interaction in linking the functions of early and late endosomes.
Insights
Tumor susceptibility gene 101 (TSG101) and hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) interaction is vital for receptor degradation. Disrupting this link halts endosomal trafficking, preventing mitogenic receptor down-regulation.
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Down-regulation of mitogenic signaling involves receptor degradation in lysosomes via endosomal trafficking.
- Ubiquitination and sorting into multivesicular bodies are key steps in this process.
- Tumor susceptibility gene 101 (TSG101) has been identified as a critical factor in endosomal sorting.
Purpose of the Study:
- To investigate the interaction between TSG101 and hepatocyte growth factor-regulated tyrosine kinase substrate (HRS).
- To determine the role of the TSG101/HRS interaction in endosomal trafficking and receptor degradation.
- To elucidate the molecular mechanism of TSG101/HRS involvement in mitogenic signaling down-regulation.
Main Methods:
- Co-immunoprecipitation to assess TSG101/HRS interaction.
- Mutational analysis to disrupt the TSG101/HRS binding interface.
- Confocal microscopy to track epidermal growth factor receptor (EGFR) localization.
- Western blotting to evaluate EGFR ubiquitination and degradation.
Main Results:
- TSG101 directly interacts with HRS through its ubiquitin-binding domain and HRS's proline-rich regions.
- Disruption of the TSG101/HRS interaction impairs endosomal trafficking of ubiquitinated cargo.
- Perturbation of this interaction prevents EGFR delivery to late endosomes and inhibits its degradation, leading to EGFR accumulation in early endosomes.
Conclusions:
- The interaction between TSG101 and HRS is essential for the endocytic down-regulation of mitogenic signaling.
- This interaction serves as a critical link between early and late endosomal functions in receptor trafficking.
- Targeting the TSG101/HRS interaction could offer therapeutic strategies for diseases involving aberrant signaling.