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Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches

  • Kyla Bourque
  • Dominic Devost
  • Asuka Inoue
  • Terence E. HébertEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1947)

Abstract

Ligand-biased signaling could have a significant impact on drug discovery programs. As such, many approaches to screening now target a larger section of the signaling responses downstream of an individual G protein-coupled receptor (GPCR). Biosensor-based platforms have been developed to capture signaling signatures. Despite the ability to use such signaling signatures, they may still be particular to an individual cell type and thus such platforms may not be portable from cell to cell, necessitating further cell-specific biosensor development. We have developed a complementary strategy based on capturing receptor-proximal conformational profiles using intra-molecular BRET-based sensors composed of a Renilla luciferase donor engineered into the carboxy-terminus and CCPGCC motifs which bind fluorescent hairpin biarsenical dyes engineered into different positions into the receptor primary structure. Here, we discuss how these experiments can be conducted and combined with CRISPR/Cas9 genome editing to assess specific G protein-dependent and -independent events.

Key words

GPCRs BRET Genome-editing Conformation-sensitive biosensors CRISPR Drug discovery 

Notes

Acknowledgements

The work was funded by a grant to TEH from the Canadian Institutes for Health Research (MOP-130309). KB was supported by an internal Faculty of Medicine Studentship from McGill University.

References

  1. 1.
    Sauliere A et al (2012) Deciphering biased-agonism complexity reveals a new active AT1 receptor entity. Nat Chem Biol 8(7):622–630CrossRefGoogle Scholar
  2. 2.
    Beautrait A et al (2017) A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling. Nat Commun 8:15054CrossRefGoogle Scholar
  3. 3.
    Namkung Y et al (2016) Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET. Nat Commun 7:12178CrossRefGoogle Scholar
  4. 4.
    Besserer-Offroy E et al (2017) The signaling signature of the neurotensin type 1 receptor with endogenous ligands. Eur J Pharmacol 805:1–13CrossRefGoogle Scholar
  5. 5.
    Bourque K et al (2017) Distinct conformational dynamics of three G protein-coupled receptors measured using FlAsH-BRET biosensors. Front Endocrinol (Lausanne) 8:61CrossRefGoogle Scholar
  6. 6.
    Devost D et al (2017) Conformational profiling of the AT1 angiotensin II receptor reflects biased agonism, G protein coupling, and cellular context. J Biol Chem 292(13):5443–5456CrossRefGoogle Scholar
  7. 7.
    Sleno R et al (2017) Conformational biosensors reveal allosteric interactions between heterodimeric AT1 angiotensin and prostaglandin F2α receptors. J Biol Chem 292(29):12139–12152CrossRefGoogle Scholar
  8. 8.
    Sleno R et al (2016) Designing BRET-based conformational biosensors for G protein-coupled receptors. Methods 92:11–18CrossRefGoogle Scholar
  9. 9.
    Machleidt T, Robers M, Hanson GT (2007) Protein labeling with FlAsH and ReAsH. Methods Mol Biol 356:209–220PubMedGoogle Scholar
  10. 10.
    Hoffmann C et al (2010) Fluorescent labeling of tetracysteine-tagged proteins in intact cells. Nat Protoc 5(10):1666–1677CrossRefGoogle Scholar
  11. 11.
    Grundmann M et al (2018) Lack of β-arrestin signaling in the absence of active G proteins. Nat Commun 9(1):341CrossRefGoogle Scholar
  12. 12.
    Stallaert W et al (2017) Purinergic receptor transactivation by the β2-adrenergic receptor increases intracellular Ca(2+) in nonexcitable cells. Mol Pharmacol 91(5):533–544CrossRefGoogle Scholar
  13. 13.
    O’Hayre M et al (2017) Genetic evidence that β-arrestins are dispensable for the initiation of β2-adrenergic receptor signaling to ERK. Sci Signal 10(484).  https://doi.org/10.1126/scisignal.aal3395CrossRefGoogle Scholar
  14. 14.
    Milligan G, Inoue A (2018) Genome editing provides new insights into receptor-controlled signalling pathways. Trends Pharmacol Sci 39(5):481–493CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kyla Bourque
    • 1
  • Dominic Devost
    • 1
  • Asuka Inoue
    • 2
    • 3
  • Terence E. Hébert
    • 1
    Email author
  1. 1.Department of Pharmacology and TherapeuticsMcGill UniversityMontrealCanada
  2. 2.Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan
  3. 3.Japan Science and Technology Agency (JST)Precursory Research for Embryonic Science and Technology (PRESTO)KawaguchiJapan

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