Conformational biosensors reveal GPCR signalling from endosomes.

TitleConformational biosensors reveal GPCR signalling from endosomes.
Publication TypeJournal Article
Year of Publication2013
AuthorsIrannejad, R., Tomshine J. C., Tomshine J. R., Chevalier M., Mahoney J. P., Steyaert J., Rasmussen S. G. F., Sunahara R. K., El-Samad H., Huang B., and von Zastrow M.
JournalNature
Volume495
Issue7442
Pagination534-8
Date Published2013 Mar 28
ISSN1476-4687
KeywordsAdrenergic beta-2 Receptor Agonists, Biosensing Techniques, Cell Membrane, Clathrin-Coated Vesicles, Cyclic AMP, Endocytosis, Endosomes, Green Fluorescent Proteins, GTP-Binding Protein alpha Subunits, Gs, HEK293 Cells, Humans, Isoproterenol, Models, Biological, Protein Conformation, Receptors, Adrenergic, beta-2, Signal Transduction, Single-Domain Antibodies
Abstract

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the β2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.

DOI10.1038/nature12000
Alternate JournalNature
PubMed ID23515162
PubMed Central IDPMC3835555
Grant ListDA010711 / DA / NIDA NIH HHS / United States
DA012864 / DA / NIDA NIH HHS / United States
F32 DA029993 / DA / NIDA NIH HHS / United States
F32 DA029993 / DA / NIDA NIH HHS / United States
GM083118 / GM / NIGMS NIH HHS / United States
P01 NS053709 / NS / NINDS NIH HHS / United States
R01 DA012864 / DA / NIDA NIH HHS / United States
R01 GM083118 / GM / NIGMS NIH HHS / United States
R29 DA010711 / DA / NIDA NIH HHS / United States
R37 DA010711 / DA / NIDA NIH HHS / United States
T32 GM007767 / GM / NIGMS NIH HHS / United States
subject_category: 
Research group: