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

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

Abstract

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 

Notes

Acknowledgments

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

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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

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