Conformational biosensors reveal adrenoceptor signalling from endosomes
By Roshanak Irannejad, Jin C. Tomshine, Jon R. Tomshine, Michael Chevalier, Jacob P. Mahoney, J. Steyaert, S. Rasmussen, R. Sunahara, H. El-Samad, Bo Huang and 1 more. Nature, 2013
798 citations recorded by the source corpus.
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.
Subfields: Cellular and Molecular Neuroscience, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/7c97890adc67a9822c75405a76506ec575d6751e
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Citations in the corpus
- Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors (2018, methodology): “5 Luminescence-based cAMP assay Experiments were conducted as previously described (23).”
- Endocytosis, Signaling, and Beyond (2014, background): “Many of these receptors are regulated by activation-induced endocytosis, and for GPCRs such as the D1-type dopamine receptor and m- and d- type opioid receptors, this has been clearly shown in vivo (Sternini et al. 1996; Dumartin et al. 1998; Keith et al. 1998; Pradhan et al. 2009).”