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Deciphering biased-agonism complexity reveals a new active AT1 receptor entity

Nature Chemical Biology volume 8, pages 622–630 (2012)Cite this article

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Abstract

Functional selectivity of G protein–coupled receptor (GPCR) ligands toward different downstream signals has recently emerged as a general hallmark of this receptor class. However, pleiotropic and crosstalk signaling of GPCRs makes functional selectivity difficult to decode. To look from the initial active receptor point of view, we developed new, highly sensitive and direct bioluminescence resonance energy transfer–based G protein activation probes specific for all G protein isoforms, and we used them to evaluate the G protein–coupling activity of [1Sar4Ile8Ile]-angiotensin II (SII), previously described as an angiotensin II type 1 (AT1) receptor–biased agonist that is G protein independent but β-arrestin selective. By multiplexing assays sensing sequential signaling events, from receptor conformations to downstream signaling, we decoded SII as an agonist stabilizing a G protein–dependent AT1A receptor signaling module different from that of the physiological agonist angiotensin II, both in recombinant and primary cells. Thus, a biased agonist does not necessarily select effects from the physiological agonist but may instead stabilize and create a new distinct active pharmacological receptor entity.

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Figure 1: Selectivity of Gαβ1γ2 activation of BRET probes in HEK293T cells.
Figure 2: Gαβ1γ2 activation after AT1A-R stimulation with Ang II or SII.
Figure 3: AT1A-R–mediated β-arrestin 2 recruitment after Ang II or SII stimulation.
Figure 4: Calcium production after Ang II or SII stimulation in cells expressing AT1A-R.
Figure 5: Adenylyl cyclase activity after Ang II or SII stimulation in cells expressing AT1A-R.
Figure 6: BRET measurements of AT1A-R and Gα interactions in living cells.
Figure 7: AT1A-R–mediated ERK phosphorylation after Ang II or SII stimulation.
Figure 8: Schematic representation of G protein–dependent signaling pathways activated by Ang II or SII AT1A-R agonists.

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Acknowledgements

This paper is dedicated to the memory of Dr. Hervé Paris, who passed away suddenly during the process of this work. We thank T. Hébert, J. Javitch and M. Lohse for their critical reading of the manuscript and R. D'Angelo for excellent technical support and advice regarding imaging (Cellular Imaging Facility I2MC, TRI Platform). Wild-type, β-arrestin 1 knockout, and β-arrestin 1 and 2 double-knockout MEF cells were generously provided by R. Lefkowitz (Duke University Medical Center). This work was supported by a grant from the French National Research Agency (ANR-06-BLAN-0400-02 to C.G.) J.L.H. is sponsored by the Danish National Research Foundation, the Danish Council for Independent Research Medical Sciences and the Novo Nordisk Foundation.

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Author notes

  1. Jakob L Hansen

    Present address: Present address: Novo Nordisk A/S, Novo Nordisk Park, GLP-1 & Obesity Biology Section, Måløv, Denmark.,

  2. Hervé Paris: Deceased.

Authors and Affiliations

  1. Institut des Maladies Métaboliques et Cardiovasculaires, Institut National de la Santé et de la Recherche Médicale, Université Toulouse III Paul Sabatier, Toulouse, France

    Aude Saulière, Morgane Bellot, Hervé Paris, Colette Denis, Marie-Françoise Altié, Marie-Hélène Seguelas, Atul Pathak, Jean-Michel Sénard & Céline Galés

  2. Department of Cellular and Molecular Biology, Pierre Fabre Research Institute, Castres, France

    Frédéric Finana

  3. Department of Diabetes NBEs and Obesity Biology, Laboratory for Molecular Cardiology, Danish National Research Foundation Centre for Cardiac Arrhythmia, University of Copenhagen, Copenhagen, Denmark

    Jonas T Hansen

  4. Department of Pharmacology, Centre Hospitalier Universitaire de Toulouse, Toulouse, France

    Atul Pathak & Jean-Michel Sénard

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Contributions

A.S. did most of the experiments, analyzed and interpreted data and cowrote the manuscript. M.B. did and interpreted all of the ERK experiments. H.P. helped construct and characterize the G protein BRET probes. C.D. did and analyzed the aequorin-based Ca2+ measurements in HEK293T cells. F.F. constructed and characterized the BRET-based PKA biosensor. M.-F.A. helped construct the G protein probes and M.-H.S. helped with cardiac-fibroblast primary cell culture. J.L.H. and J.T.H. provided the YM-254890 Gq inhibitor and several AT1-R–encoding plasmid vectors, and they helped with data interpretation and manuscript preparation. A.P. and J.-M.S. assisted with data processing and analysis and with manuscript preparation. C.G. designed and managed the overall project, analyzed and interpreted data and cowrote the manuscript.

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Correspondence to Céline Galés.

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Saulière, A., Bellot, M., Paris, H. et al. Deciphering biased-agonism complexity reveals a new active AT1 receptor entity. Nat Chem Biol 8, 622–630 (2012). https://doi.org/10.1038/nchembio.961

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