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Live Cell Bioluminescence Imaging in Temporal Reaction of G Protein-Coupled Receptor for High-Throughput Screening and Analysis

  • Mitsuru Hattori
  • Takeaki OzawaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1461)

Abstract

G protein-coupled receptors (GPCRs) are notable targets of basic therapeutics. Many screening methods have been established to identify novel agents for GPCR signaling in a high-throughput manner. However, information related to the temporal reaction of GPCR with specific ligands remains poor. We recently developed a bioluminescence method for the quantitative detection of the interaction between GPCR and β-arrestin using split luciferase complementation. To monitor time-course variation of the interactions, a new imaging system contributes to the accurate evaluation of drugs for GPCRs in a high-throughput manner.

Key words

G protein-coupled receptor Bioluminescence Complementary technology Protein–protein interaction High-throughput screening EM-CCD camera 

Notes

Acknowledgements

We thank Molecular Devices Corp. for active support related to the technical aspects of imaging. This work was supported by the Japan Society for the Promotion of Science (JSPS) and MEXT, Japan.

References

  1. 1.
    Grisshammer R (2013) Why we need many more G protein-coupled receptor structures. Expert Rev Proteomics 10:1–3CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Conn PJ, Christopoulos A, Lindsley CW (2009) Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders. Nat Rev Drug Discov 8:41–54CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Musnier A, Blanchot B, Reiter E, Crepieux P (2010) GPCR signalling to the translation machinery. Cell Signal 22:707–716CrossRefPubMedGoogle Scholar
  4. 4.
    Stevens RC, Cherezov V, Katritch V, Abagyan R, Kuhn P, Rosen H, Wuthrich K (2013) The GPCR network: a large-scale collaboration to determine human GPCR structure and function. Nat Rev Drug Discov 12:25–34CrossRefPubMedGoogle Scholar
  5. 5.
    Hopkins AL, Groom CR (2002) The druggable genome. Nat Rev Drug Discov 1:727–730CrossRefPubMedGoogle Scholar
  6. 6.
    Foord SM, Bonner TI, Neubig RR, Rosser EM, Pin JP, Davenport AP, Spedding M, Harmar AJ (2005) International union of pharmacology. XLVI. G protein-coupled receptor list. Pharmacol Rev 57:279–288CrossRefPubMedGoogle Scholar
  7. 7.
    Allen JA, Roth BL (2011) Strategies to discover unexpected targets for drugs active at G protein-coupled receptors. Annu Rev Pharmacol Toxicol 51:117–144CrossRefPubMedGoogle Scholar
  8. 8.
    Zhang R, Xie X (2012) Tools for GPCR drug discovery. Acta Pharmacol Sin 33:372–384CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hattori M, Tanaka M, Takakura H, Aoki K, Miura K, Anzai T, Ozawa T (2013) Analysis of temporal patterns of GPCR–β-arrestin interactions using split luciferase-fragment complementation. Mol Biosyst 9:957–964CrossRefPubMedGoogle Scholar
  10. 10.
    Misawa N, Kafi AKM, Hattori M, Miura K, Ozawa T (2010) Rapid and high-sensitivity cell-based assays of protein–protein interactions using split click beetle luciferase complementation: an approach to the study of G-protein-coupled receptors. Anal Chem 82:2552–2560CrossRefPubMedGoogle Scholar
  11. 11.
    Hida N, Awais M, Takeuchi M, Ueno N, Tashiro M, Takagi C, Singh T, Hayashi M, Ohmiya Y, Ozawa T (2009) High-sensitivity real-time imaging of dual protein–protein interactions in living subjects using multicolor luciferases. PLoS One 4:e5868CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Takakura H, Hattori M, Takeuchi M, Ozawa T (2012) Visualization and quantitative analysis of G protein-coupled receptor–β-arrestin interaction in single cells and specific organs of living mice using split luciferase complementation. ACS Chem Biol 7:901–910CrossRefPubMedGoogle Scholar
  13. 13.
    Hatori M, Ozawa T (2015) Bioluminescent tools for the analysis of G-protein-coupled receptor and arrestin interactions. RSC Adv 5:12655–12663CrossRefGoogle Scholar
  14. 14.
    Takakura H, Hattori M, Tanaka M, Ozawa T (2015) Cell-based assays and animal models for GPCR drug screening. Methods Mol Biol 1272:257–270CrossRefPubMedGoogle Scholar
  15. 15.
    Hattori M, Ozawa T (2015) High-throughput live cell imaging and analysis for temporal reaction of G protein-coupled receptor based on split luciferase fragment complementation. Anal Sci 31:327–330CrossRefPubMedGoogle Scholar
  16. 16.
    Kafi AKM, Hattori M, Misawa N, Ozawa T (2011) Dual-color bioluminescence analysis for quantitatively monitoring G-protein-coupled receptor and β-arrestin interactions. Pharmaceuticals 4:457–469Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Chemistry, School of ScienceThe University of TokyoTokyoJapan

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