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Super-Resolution Imaging of G Protein-Coupled Receptors Using Ground State Depletion Microscopy

  • Fabiana A. Caetano Crowley
  • Bryan Heit
  • Stephen S. G. FergusonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1947)

Abstract

G protein-coupled receptors (GPCRs) comprise the largest family of integral membrane proteins, which are coupled to heterotrimeric G proteins to influence cell signaling. Subsequent to G protein activation, agonist-stimulated G protein-coupled receptor kinase (GRK) phosphorylation results in the recruitment of β-arrestin proteins, which form both stable and unstable complexes with GPCRs. β-Arrestins when bound to GPCRs not only contribute to the uncoupling of G protein signaling but also to the redistribution of GPCRs to clathrin-coated pits via their association with both clathrin and β2-adaptin facilitating GPCR endocytosis. This allows β-arrestins to couple GPCRs to additional cell signaling proteins allowing a second wave of receptor signaling. Importantly, the β-arrestin-regulated subcellular localization of these complexes also plays a critical role in regulating how these signals are transduced and which proteins are recruited. Here, we describe a methodology for assessing the GPCR subcellular localization by super-resolution microscopy and suggest that this methodology can be extended to the study of GPCR/protein complexes.

Key words

β-Arrestin RalGDS Green fluorescent protein G protein-coupled receptor Ground state depletion microscopy Translocation Endocytosis Agonist 

References

  1. 1.
    Ferguson SSG (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53:1–24PubMedGoogle Scholar
  2. 2.
    Laporte SA, Oakley RH, Zhang J, Ferguson SSG, Caron MG, Barak LS (1999) Recruitment of adaptin AP-2 to β2-adrenergic receptor/βarrestin complexes during endocytosis. Proc Natl Acad Sci U SA 96:3712–3717CrossRefGoogle Scholar
  3. 3.
    Goodman OB Jr, Krupnick JG, Santini F, Gurevich VV, Penn RB, Gagnon AW, Keen JH, Benovic JL (1996) β-Arrestin acts as a clathrin adaptor in endocytosis of the β2-adrenergic receptor. Nature 383:447–450CrossRefGoogle Scholar
  4. 4.
    Peterson YK, Luttrell LM (2017) The diverse roles of Arrestin scaffolds in G protein-coupled receptor signaling. Pharmacol Rev 69:256–297CrossRefGoogle Scholar
  5. 5.
    Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG (1999) Association of β-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization. J Biol Chem 274:32248–32257CrossRefGoogle Scholar
  6. 6.
    Anborgh PH, Seachrist J, Dale L, Ferguson SSG (2000) Receptor/β-arrestin complex formation and the differential trafficking and resensitization of β2-adrenergic and angiotensin II type 1A receptors. Mol Endocrinol 14:2040–2053PubMedGoogle Scholar
  7. 7.
    Seachrist JL, Laporte SA, Dale LB, Babwah AV, Caron MG, Anborgh PH, Ferguson SSG (2002) Rab5 association with the angiotensin II type 1A receptor promotes Rab5 GTP-binding and vesicular fusion. J Biol Chem 277:679–685CrossRefGoogle Scholar
  8. 8.
    Rajagopal S, Shenoy SK (2018) GPCR desensitization: acute and prolonged phases. Cell Signal 41:9–16CrossRefGoogle Scholar
  9. 9.
    Barak LS, Ferguson SSG, Zhang J, Caron MG (1997) A β-arrestin/green fluorescent protein biosensor for identifying G protein-coupled receptor activation. J Biol Chem 272:27497–27500CrossRefGoogle Scholar
  10. 10.
    Marullo S, Bouvier M (2007) Resonance energy transfer approaches in molecular pharmacology and beyond. Trends Pharmacol Sci 28:362–365CrossRefGoogle Scholar
  11. 11.
    Caetano FA, Cavanagh PC, Tam JHK, Dirk BS, Ferguson SSG, Pasternak SH, Dikeakos JD, DeBruyn J, Heit B (2016) Quantitative analysis of protein interactions, dynamics and formation of multi-protein structures by super-resolution imaging. PLoS Comp Biol 11(12):e1004634CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Fabiana A. Caetano Crowley
    • 1
  • Bryan Heit
    • 2
  • Stephen S. G. Ferguson
    • 3
    Email author
  1. 1.Department of Physiology and PharmacologyWestern UniversityLondonCanada
  2. 2.Department of Microbiology and ImmunologyWestern UniversityLondonCanada
  3. 3.Department of Cellular and Molecular Medicine, University of Ottawa Brain and Mind Research InstituteUniversity of OttawaOttawaCanada

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