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Conformational biosensors reveal GPCR signalling from endosomes

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 resolution1. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins2,3, or GPCR activation elicits a discrete form of persistent G protein signalling4,5,6,7,8,9, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response10. 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 GPCR11, 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.

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Figure 1: Nb80–GFP detects activated β2-ARs in the plasma membrane and endosomes.
Figure 2: Nb80–GFP accumulates on β2-AR-containing endosomes after their formation.
Figure 3: Nb80–GFP does not accumulate in clathrin-coated pits or vesicles.
Figure 4: Internalized β2-ARs contribute to the acute cAMP response.

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Acknowledgements

We thank B. Kobilka, P. Robinson, A. Kruse, E. Pardon, P. Temkin, M. Puthenveedu, A. Henry, A. Marley and K. Thorn for assistance, advice and discussion. These studies were supported by the National Institute on Drug Abuse of the US National Institutes of Health (DA010711 and DA012864 to M.v.Z. and F32 DA029993 to J.C.T.). R.I. is supported by the American Heart Association. R.K.S. and J.P.M. are supported by the National Institute of General Medical Sciences (GM083118 to R.K.S. and T32 GM007767 to J.P.M.). S.G.F.R. is supported by the Lundbeck Foundation. J.S. is supported by FWO-Vlaanderen grants (FWO551 and FWO646) and Innoviris-Brussels (BRGEOZ132). B.H. is supported by a Packard Fellowship for Science and Engineering.

Author information

Authors and Affiliations

Authors

Contributions

R.I. constructed and validated the nanobody biosensors, carried out most of the cell biological experiments and analysis, contributed to overall experimental strategy and took a lead role in writing the manuscript. J.C.T. carried out early experiments identifying endocytic inhibitor effects on cellular cAMP signalling, and contributed to initial project planning. J.R.T. built the luminometer system, developed software for analysis of luminometry data, and contributed to early experiments on cellular cAMP signalling. M.C. contributed to experimental design and data analysis, and modelled effects of endocytic inhibitors on the cellular cAMP response. J.P.M. contributed to the production of receptor-containing rHDL particles and carried out in vitro studies of Nb80 binding and dissociation. J.S. developed the nanobody reagents used as the basis for the biosensors described in this study and advised on biosensor design and expression. S.G.F.R. contributed to developing and screening the initial nanobody reagents, and carried out in vitro studies of Nb80 binding and dissociation in rHDL particles reconstituted with bimane-labeled receptors. R.K.S. contributed to overall experimental interpretation, supervised J.P.M. in carrying out in vitro studies of Nb80 binding to receptors, and performed in vitro experiments evaluating Nb37 effects on G protein activation. H.E.-S. contributed to experimental design and data interpretation, and supervised efforts to model endocytic effects on the cellular cAMP response. B.H. contributed to overall experimental design and interpretation, implementation of biosensors and advised on image analysis. M.v.Z. was responsible for overall project strategy, carried out some of the imaging experiments, and drafted the manuscript together with R.I.

Corresponding author

Correspondence to Mark von Zastrow.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-7. (PDF 3845 kb)

Nb80-GFP accumulates on β2-AR-containing endosomes after their formation

This video shows confocal image series of β2-AR (red) and Nb80-GFP (green) from a cell incubated in the presence of 10μM isoproterenol. β2-AR-containing endosomes are devoid of Nb80-GFP (arrow) and subsequently acquire it (arrowhead). (MOV 1309 kb)

Nb80-GFP accumulates on β2-AR-containing endosomes after their formation.

The video shows numerous examples of β2-AR-containing endosomes that later recruit Nb80-GFP. (MOV 4472 kb)

Nb80-GFP membrane recruitment is reversible

This video shows the reversal of Nb80-GFP membrane recruitment after agonist washout and the complete time series corresponding to Figure 1e. (MOV 3865 kb)

Nb80-GFP accumulates on β2-AR-containing endosomes after their formation

This video shows Nb80-GFP (green) and β2-AR (red) imaged by TIRF and the complete time series corresponding to Figure 2a. (MOV 10334 kb)

Nb80-GFP accumulates on β2-AR-containing endosomes after their formation

This video shows TIRF image series showing an individual β2-AR-containing endosome accumulating Nb80-GFP. It corresponds to the experiment shown in Figure 2c. (MOV 125 kb)

Nb80-GFP does not accumulate in clathrin-coated pits or vesicles

This video show TIRF image series of Nb80-GFP (green) and β-arrestin2-mCherry (red). It corresponds to Figure 3a. (MOV 4616 kb)

Nb80-GFP does not accumulate in clathrin-coated pits or vesicles

This video shows TIRF image series of Nb80-GFP (green) and clathrin light chain-dsRed (red). It corresponds to Figure 3b. (MOV 7251 kb)

Nb37-GFP accumulates on β2-AR-containing endosomes after their formation

This video shows confocal image series showing Nb37-GFP (green) and β2-AR (red). It corresponds to the experiment shown in Figure 4b. (MOV 935 kb)

Nb37-GFP accumulation on β2-AR-containing endosomes

This video shows confocal image series of Nb37-GFP (green) and β2-AR (red). It corresponds to the experiment shown in Figure 4c. (MOV 3302 kb)

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Irannejad, R., Tomshine, J., Tomshine, J. et al. Conformational biosensors reveal GPCR signalling from endosomes. Nature 495, 534–538 (2013). https://doi.org/10.1038/nature12000

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