Combining RNAi and Immunofluorescence Approaches to Investigate Post-endocytic Sorting of GPCRs into Multivesicular Bodies

  • Xuezhi Li
  • Stéphanie Rosciglione
  • Andréanne Laniel
  • Christine LavoieEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1947)


Following stimulation, G protein-coupled receptors (GPCRs) are internalized and transported to early endosomes where they are either recycled back to the plasma membrane for another round of activation or targeted to the lysosomes for degradation and long-term signal termination. This latter requires internalization of receptors into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs) for complete degradation following fusion with lysosomes. This endosomal sorting step is highly regulated and has profound functional consequences. This chapter describes how RNAi and confocal microscopy methods can be combined to evaluate whether a protein of interest (herein Gαs) is involved in GPCR sorting into ILVs of MVBs.

Key words

GPCR Immunofluorescence Gαs Confocal microscopy Endosome Intraluminal vesicles Multivesicular bodies siRNA 



This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to C.L.


  1. 1.
    Hanyaloglu AC, von Zastrow M (2008) Regulation of GPCRs by membrane trafficking and its potential implications. Annu Rev Pharmacol Toxicol 48:537–568CrossRefGoogle Scholar
  2. 2.
    Piper RC, Katzmann DJ (2007) Biogenesis and function of multivesicular bodies. Annu Rev Cell Dev Biol 23:519–547. Scholar
  3. 3.
    Hasdemir B, Bunnett NW, Cottrell GS (2007) Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor. J Biol Chem 282(40):29646–29657CrossRefGoogle Scholar
  4. 4.
    Marchese A, Raiborg C, Santini F, Keen JH, Stenmark H, Benovic JL (2003) The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4. Dev Cell 5(5):709–722CrossRefGoogle Scholar
  5. 5.
    Henry AG, White IJ, Marsh M, von Zastrow M, Hislop JN (2011) The role of ubiquitination in lysosomal trafficking of delta-opioid receptors. Traffic 12(2):170–184. Scholar
  6. 6.
    Hislop JN, Henry AG, von Zastrow M (2011) Ubiquitination in the first cytoplasmic loop of mu-opioid receptors reveals a hierarchical mechanism of lysosomal down-regulation. J Biol Chem 286(46):40193–40204. Scholar
  7. 7.
    Eichel K, von Zastrow M (2018) Subcellular organization of GPCR signaling. Trends Pharmacol Sci 39(2):200–208. Scholar
  8. 8.
    Irannejad R, Tomshine JC, Tomshine JR, Chevalier M, Mahoney JP, Steyaert J, Rasmussen SG, Sunahara RK, El-Samad H, Huang B, von Zastrow M (2013) Conformational biosensors reveal GPCR signalling from endosomes. Nature 495(7442):534–538. Scholar
  9. 9.
    Shenoy SK, Lefkowitz RJ (2011) Beta-Arrestin-mediated receptor trafficking and signal transduction. Trends Pharmacol Sci 32(9):521–533. Scholar
  10. 10.
    Irannejad R, von Zastrow M (2014) GPCR signaling along the endocytic pathway. Curr Opin Cell Biol 27:109–116. Scholar
  11. 11.
    Tsvetanova NG, Irannejad R, von Zastrow M (2015) G Protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes. J Biol Chem 290(11):6689–6696. Scholar
  12. 12.
    Booden MA, Eckert LB, Der CJ, Trejo J (2004) Persistent signaling by dysregulated thrombin receptor trafficking promotes breast carcinoma cell invasion. Mol Cell Biol 24(5):1990–1999CrossRefGoogle Scholar
  13. 13.
    Li YM, Pan Y, Wei Y, Cheng X, Zhou BP, Tan M, Zhou X, Xia W, Hortobagyi GN, Yu D, Hung MC (2004) Upregulation of CXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell 6(5):459–469. Scholar
  14. 14.
    Rosciglione S, Theriault C, Boily MO, Paquette M, Lavoie C (2014) Galphas regulates the post-endocytic sorting of G protein-coupled receptors. Nat Commun 5:4556. Scholar
  15. 15.
    Zheng B, Lavoie C, Tang TD, Ma P, Meerloo T, Beas A, Farquhar MG (2004) Regulation of epidermal growth factor receptor degradation by heterotrimeric Galphas protein. Mol Biol Cell 15(12):5538–5550CrossRefGoogle Scholar
  16. 16.
    Génier S, Degrandmaison J, Lavoie C, Gendron L, Parent J-L (2018) Monitoring the aggregation of GPCRs by fluorescence microscopy. Methods Mol Biol. this issueGoogle Scholar
  17. 17.
    Li X, Letourneau D, Holleran B, Leduc R, Lavigne P, Lavoie C (2017) Galphas protein binds ubiquitin to regulate epidermal growth factor receptor endosomal sorting. Proc Natl Acad Sci U S A 114(51):13477–13482. Scholar

Copyright information

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

Authors and Affiliations

  • Xuezhi Li
    • 1
  • Stéphanie Rosciglione
    • 1
  • Andréanne Laniel
    • 1
  • Christine Lavoie
    • 1
    Email author
  1. 1.Département de Pharmacologie-Physiologie, Faculté de Médecine et des Sciences de la Santé, Institut de Pharmacologie de SherbrookeUniversité de SherbrookeSherbrookeCanada

Personalised recommendations