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Real-time monitoring of ubiquitination in living cells by BRET

Real-time monitoring of ubiquitination in living cells by BRET. studied by Julie Perroy, Stephanie Pontier, Pascale G Charest, et al. Presented by Jesse Yongyu Wang. Nature Methods  1, 203 - 208 (2004). The ubiquitin-proteosome pathway. Consequences of protein ubiquitination.

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Real-time monitoring of ubiquitination in living cells by BRET

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  1. Real-time monitoring of ubiquitination in living cells by BRET studied byJulie Perroy, Stephanie Pontier, Pascale G Charest, et al. Presented by Jesse Yongyu Wang Nature Methods  1, 203 - 208 (2004)

  2. The ubiquitin-proteosome pathway

  3. Consequences of protein ubiquitination

  4. Application of BRET to monitoring protein ubiquitination

  5. β-arrestin is involved in desensitization and endocytosis of 7MSR

  6. β-arrestin-mediated endocytosis of the other receptors

  7. transduction of receptor signals by β-arrestin Other signaling: Tyrosine kinases AKT PI3 Kinases NF-KB Pathway

  8. β-Arrestin2 Regulates NF-κB pathway

  9. β-arrestin regulated Histone acetylation and gene transcription

  10. Arrestins and distribution

  11. ß-Arrestin 2: Structural model and receptor-dependent conformational changes

  12. A list of β-arrestin–interacting proteins

  13. Roles of ubiquitination in 7TM receptor trafficking and signaling

  14. The β-arrestin unbiquitination BRET assay

  15. Specific BRET2 signal between Rluc−β-arrestin and GFP2-Ubi BRET result reflects the ubiqitination of B-arrestin

  16. Comparison of BRET signals between GFP2-Ubi or GFP2-UbiWT GFP2-Ubi WT: Wide type GFP2-Ubi: mutant by changing lys48,36 to ala

  17. Detection of GPCR-induced β-arrestin ubiquitination EC50:1.4nM EC50:140nM

  18. Concomitant detection of β-arrestin ubiquitination and its recruitment to the activated receptor

  19. Real-time assessment of receptor stimulated β-arrestin ubiquitination B2-AR V2-R The nature of the receptor determines the dynamics of B-arrestin Ubiquitination

  20. Summary and comments • Ubiquitination of arrestin is very important to GPCR signaling • Ubiquitination of arrestin is very dynamic and its important to the receptor’s physical function • Prolong ubiquitination of arrestin could stable the receptor complex and related to the MAPK signaling

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