Editors' ChoiceGPCR SIGNALING

Remaining Active in Endosomes

Sci. Signal.  02 Apr 2013:
Vol. 6, Issue 269, pp. ec78
DOI: 10.1126/scisignal.2004197

The binding of ligands to the β2-adrenergic receptor leads to activation of the associated heterotrimeric G protein subunit Gαs, which stimulates generation of cyclic adenosine monophosphate (cAMP) by adenylyl cyclases. Phosphorylation of activated β2-adrenergic receptors recruits β-arrestin and induces endocytosis of the receptors by clathrin-coated pits, a process that is thought to prevent further signaling from the receptors. Irannejad et al. (see also Lohse and Calebiro) tagged the single-domain antibody (nanobody) Nb80, which recognizes and stabilizes agonist-bound β2-adrenergic receptors, with green fluorescent protein (Nb80-GFP). Application of the β-adrenergic agonist isoprenaline resulted in recruitment of Nb80-GFP to β2-adrenergic receptors at the plasma membrane. Upon internalization of β2-adrenergic receptors, Nb80-GFP did not initially colocalize with β2-adrenergic receptors in endosomes but subsequently was recruited to β2-adrenergic receptor–containing endosomes, which required phosphorylation and internalization of the β2-adrenergic receptor and was reversed by a competitive antagonist. Nb80-GFP did not colocalize with β2-adrenergic receptors in clathrin-coated pits. Activated Gαs was also localized to the plasma membrane after isoprenaline treatment and to β2-adrenergic receptor–containing endosomes, as detected by N37-GFP, a nanobody that recognizes a catalytic intermediate of Gαs. Inhibiting endocytosis with a dynamin inhibitor did not reduce the initial increase in cAMP production caused by isoprenaline stimulation but rather the later accumulation of cAMP. These results suggest that agonist-bound β2-adrenergic receptors continue after internalization (after a short delay) to activate downstream signaling pathways.

R. Irannejad, J. C. Tomshine, J. R. Tomshine, M. Chevalier, J. P. Mahoney, J. Steyaert, S. G. F. Rasmussen, R. K. Sunahara, H. El-Samad, B. Huang, M. von Zastrow, Conformational biosensors reveal GPCR signalling from endosomes. Nature 495, 534–538 (2013). [PubMed]

M. J. Lohse, D. Calebiro, Receptor signals come in waves. Nature 495, 457–458 (2013). [PubMed]