BRET-based effector membrane translocation assay monitors GPCR-promoted and endocytosis-mediated Gqactivation at early endosomes

Shane C. Wright, Viktoriya Lukasheva, Christian Le Gouill, Hiroyuki Kobayashi, Billy Breton, Samuel Mailhot-Larouche, Élodie Blondel-Tepaz, Nichelle Antunes Vieira, Claudio Costa-Neto, Madeleine Héroux, Nevin A. Lambert, Lucas Tabajara Parreiras-E-Silva, Michel Bouvier

Research output: Contribution to journalArticlepeer-review

Abstract

G protein-coupled receptors (GPCRs) are gatekeepers of cellular homeostasis and the targets of a large proportion of drugs. In addition to their signaling activity at the plasma membrane, it has been proposed that their actions may result from translocation and activation of G proteins at endomembranes-namely endosomes. This could have a significant impact on our understanding of how signals from GPCR-targeting drugs are propagated within the cell. However, little is known about the mechanisms that drive G protein movement and activation in subcellular compartments. Using bioluminescence resonance energy transfer (BRET)-based effector membrane translocation assays, we dissected the mechanisms underlying endosomal Gqtrafficking and activity following activation of Gq-coupled receptors, including the angiotensin II type 1, bradykinin B2, oxytocin, thromboxane A2alpha isoform, and muscarinic acetylcholine M3receptors. Our data reveal that GPCR-promoted activation of Gqat the plasma membrane induces its translocation to endosomes independently of β-arrestin engagement and receptor endocytosis. In contrast, Gq activity at endosomes was found to rely on both receptor endocytosisdependent and -independent mechanisms. In addition to shedding light on the molecular processes controlling subcellular Gqsignaling, our study provides a set of tools that will be generally applicable to the study of G protein translocation and activation at endosomes and other subcellular organelles, as well as the contribution of signal propagation to drug action.

Original languageEnglish (US)
Article numbere2025846118
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number20
DOIs
StatePublished - May 18 2021

Keywords

  • Arrestin
  • Endosomal signaling
  • GPCR
  • Gq/11

ASJC Scopus subject areas

  • General

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