Conformational biosensors reveal adrenoceptor signalling from endosomes

• Published in: Nature (London) 2013-03, Vol.495 (7442)
• Main Authors: Irannejad, Roshanak, Tomshine, Jin C., Tomshine, Jon R., Chevalier, Michael, Mahoney, Jacob P., Steyaert, Jan, Rasmussen, Søren G. F., Sunahara, Roger K., El-Samad, Hana, Huang, Bo, von Zastrow, Mark
• Format: Article
• Language: English
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature12000

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 non-subcellular resolution 1 . It has been subsequently proposed that signalling by internalized GPCR is restricted to G-protein-independent mechanisms such as scaffolding by arrestins 2 , 3 , or GPCR activation elicits a discrete form of persistent G protein activation 4 – 9 , or that internalized GPCR can indeed contribute to the acute G protein-mediated response 10 . Evidence supporting these various latter hypotheses is indirect or subject to alternate interpretation, and it remains unknown if endosome-localized GPCR 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 β 2 -adrenoceptor, a prototypical GPCR 11 , and its cognate G protein, G s (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 .