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

Author(s):Show less -

Wright, Shane C. ^{[1, 2]} ; Lukasheva, Viktoriya ^[2] ; Le Gouill, Christian ^[2] ; Kobayashi, Hiroyuki ^[2] ; Breton, Billy ^[2] ; Mailhot-Larouche, Samuel ^{[2, 3]} ; Blondel-Tepaz, Elodie ^{[1, 2]} ; Vieira, Nichelle Antunes ^[4] ; Costa-Neto, Claudio ^[4] ; Heroux, Madeleine ^[2] ; Lambert, Nevin A. ^[5] ; Parreiras-e-Silva, Lucas Tabajara ^{[4, 2]} ; Bouvier, Michel ^{[1, 2, 3]}

Affiliation

^[1] Univ Montreal, Dept Biochem & Mol Med, Montreal, PQ H3T 1J4 - Canada

^[2] Univ Montreal, Inst Res Immunol & Canc, Montreal, PQ H3T 1J4 - Canada

^[3] Univ Montreal, Mol Biol Program, Montreal, PQ H3T 1J4 - Canada

^[4] Univ Sao Paulo, Ribeirao Preto Med Sch, Dept Biochem & Immunol, BR-14049900 Sao Paulo, SP - Brazil

^[5] Augusta Univ, Med Coll Georgia, Dept Pharmacol & Toxicol, Augusta, GA 30912 - USA

• Document type: Journal article
• Source: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA; v. 118, n. 20 MAY 18 2021.
• Web of Science Citations: 0

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 Gq trafficking and activity following activation of Gq-coupled receptors, including the angiotensin II type 1, bradykinin B2, oxytocin, thromboxane A2 alpha isoform, and muscarinic acetylcholine M3 receptors. Our data reveal that GPCR-promoted activation of Gq at the plasma membrane induces its translocation to endosomes independently of beta-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 Gq signaling, 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. (AU)

• FAPESP's process: 16/24120-3 - Study of non-canonical roles of G proteins as new signaling mechanisms with impact on discovery and development of new drugs
• Grantee: Lucas Tabajara Parreiras e Silva
• Support Opportunities: Research Grants - Young Investigators Grants