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Hedgehog Signaling Protocols pp 61–70Cite as

Investigation of Protein–Protein Interactions and Conformational Changes in Hedgehog Signaling Pathway by FRET

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1322))

Abstract

Protein–protein interactions and signal-induced protein conformational changes are fundamental molecular events that are considered as essential in modern life sciences. Among various techniques developed to study such phenomena, fluorescence resonance energy transfer (FRET) is a widely used method with many advantages in detecting these molecular events. Here, we describe the application of FRET in the mechanistic investigation of cell signal transduction, taking the example of the Hh signaling pathway, which plays a critical role in embryonic development and tissue homeostasis. A number of general guidelines as well as some key notes have been summarized as a protocol for reader’s reference.

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References

  1. Förster T (1948) Intermolecular energy migration and fluorescence. Ann Phys 2:55–75

    Article  Google Scholar 

  2. Stryer L (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47:819–846

    Article  CAS  PubMed  Google Scholar 

  3. Ingham PW, McMahon AP (2001) Hedgehog signaling in animal development: paradigms and principles. Genes Dev 15:3059–3087

    Article  CAS  PubMed  Google Scholar 

  4. Rubin LL, De Sauvage FJ (2006) Targeting the Hedgehog pathway in cancer. Nat Rev Drug Discov 5:1026–1033

    Article  CAS  PubMed  Google Scholar 

  5. Jiang J, Hui CC (2008) Hedgehog signaling in development and cancer. Dev Cell 15:801–812

    Article  CAS  PubMed  Google Scholar 

  6. Hui CC, Angers S (2011) Gli proteins in development and disease. Annu Rev Cell Dev Biol 27:513–537

    Article  CAS  PubMed  Google Scholar 

  7. Vyas N, Goswami D, Manonmani A, Sharma P, Ranganath HA, VijayRaghavan K et al (2008) Nanoscale organization of hedgehog is essential for long-range signaling. Cell 133:1214–1227

    Article  CAS  PubMed  Google Scholar 

  8. Lu X, Liu S, Kornberg TB (2006) The C-terminal tail of the Hedgehog receptor Patched regulates both localization and turnover. Genes Dev 20:2539–2551

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Zhao Y, Tong C, Jiang J (2007) Hedgehog regulates smoothened activity by inducing a conformational switch. Nature 450:252–258

    Article  CAS  PubMed  Google Scholar 

  10. Chen Y, Sasai N, Ma G, Yue T, Jia J, Briscoe J, Jiang J (2011) Sonic Hedgehog dependent phosphorylation by CK1alpha and GRK2 is required for ciliary accumulation and activation of smoothened. PLoS Biol 9:e1001083

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Shi D, Lv X, Zhang Z, Yang X, Zhou Z, Zhang L, Zhao Y (2013) Smoothened oligomerization/higher order clustering in lipid rafts is essential for high Hedgehog activity transduction. J Biol Chem 288:12605–12614

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Shi Q, Li S, Jia J, Jiang J (2011) The Hedgehog-induced Smoothened conformational switch assembles a signaling complex that activates Fused by promoting its dimerization and phosphorylation. Development 138:4219–4231

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Tang JY, Xiao TZ, Oda Y, Chang KS, Shpall E, Wu A et al (2011) Vitamin D3 inhibits hedgehog signaling and proliferation in murine Basal cell carcinomas. Cancer Prev Res (Phila) 4:744–751

    Article  CAS  Google Scholar 

  14. Zhang Y, Mao F, Lu Y, Wu W, Zhang L, Zhao Y (2011) Transduction of the Hedgehog signal through the dimerization of Fused and the nuclear translocation of Cubitus interruptus. Cell Res 21:1436–1451

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Zhang Y, Fu L, Qi X, Zhang Z, Xia Y, Jia J et al (2013) Structural insight into the mutual recognition and regulation between Suppressor of Fused and Gli/Ci. Nat Commun 4:2608

    PubMed  Google Scholar 

  16. Wang C, Wu H, Katritch V, Han GW, Huang XP, Liu W et al (2013) Structure of the human smoothened receptor bound to an antitumour agent. Nature 497:338–343

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Rana R, Carroll CE, Lee HJ, Bao J, Marada S, Grace CR et al (2013) Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling. Nat Commun 4:2965

    Article  PubMed Central  PubMed  Google Scholar 

  18. Lv X, Fu L, Zhao Y (2013) aPKC iota/lambda: a potential target for the therapy of Hh-dependent and Smo-inhibitor-resistant advanced BCC. Acta Biochim Biophys Sin 45:610–611

    Article  PubMed  Google Scholar 

  19. Lum L, Zhang C, Oh S, Mann RK, von Kessler DP, Taipale J et al (2003) Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2. Mol Cell 12:1261–1274

    Article  CAS  PubMed  Google Scholar 

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Acknowledgment

This work was supported by grants from the National Basic Research Program of China (973 Program: 2011CB943902), the “Strategic Priority Research Program” of the Chinese Academy of Sciences (XDA01010405).

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Authors and Affiliations

  1. State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai, 200031, People’s Republic of China

    Lin Fu, Xiangdong Lv, Yue Xiong & Yun Zhao

Authors
  1. Lin Fu
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  2. Xiangdong Lv
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  3. Yue Xiong
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  4. Yun Zhao
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Corresponding author

Correspondence to Yun Zhao .

Editor information

Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

    Natalia A. Riobo

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Fu, L., Lv, X., Xiong, Y., Zhao, Y. (2015). Investigation of Protein–Protein Interactions and Conformational Changes in Hedgehog Signaling Pathway by FRET. In: Riobo, N. (eds) Hedgehog Signaling Protocols. Methods in Molecular Biology, vol 1322. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2772-2_6

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  • DOI: https://doi.org/10.1007/978-1-4939-2772-2_6

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2771-5

  • Online ISBN: 978-1-4939-2772-2

  • eBook Packages: Springer Protocols

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