Conformational biosensors reveal GPCR signalling from endosomes
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Conformational biosensors reveal GPCR signalling from endosomes. / Irannejad, R; Tomshine, Jin C; Tomshine, Jon R; Chevalier, Michael; Mahoney, Jacob P; Steyaert, Jan; Rasmussen, Søren Gøgsig Faarup; Sunahara, Roger K; El-Samad, Hana; Huang, Bo; von Zastrow, Mark.
In: Nature, Vol. 495, No. 7442, 28.03.2013, p. 534-8.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Conformational biosensors reveal GPCR signalling from endosomes
AU - Irannejad, R
AU - Tomshine, Jin C
AU - Tomshine, Jon R
AU - Chevalier, Michael
AU - Mahoney, Jacob P
AU - Steyaert, Jan
AU - Rasmussen, Søren Gøgsig Faarup
AU - Sunahara, Roger K
AU - El-Samad, Hana
AU - Huang, Bo
AU - von Zastrow, Mark
PY - 2013/3/28
Y1 - 2013/3/28
N2 - 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.
AB - 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.
U2 - 10.1038/nature12000
DO - 10.1038/nature12000
M3 - Journal article
C2 - 23515162
VL - 495
SP - 534
EP - 538
JO - Nature
JF - Nature
SN - 0028-0836
IS - 7442
ER -
ID: 45425107