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Signal profiling of the β₁AR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by β₁AR and β₂AR.

ABSTRACT: A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β₁-adrenergic receptor (β₁AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the βAR. These include coupling to G_z and G₁₂ pathways. The signalling cascade linking the β₁AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both G_s and G₁₂ are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.

SUBMITTER: Lukasheva V

PROVIDER: S-EPMC7260363 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Publications

Signal profiling of the β1AR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by β1AR and β2AR.

Lukasheva Viktoriya V Devost Dominic D Le Gouill Christian C Namkung Yoon Y Martin Ryan D RD Longpré Jean-Michel JM Amraei Mohammad M Shinjo Yuji Y Hogue Mireille M Lagacé Monique M Breton Billy B Aoki Junken J Tanny Jason C JC Laporte Stéphane A SA Pineyro Graciela G Inoue Asuka A Bouvier Michel M Hébert Terence E TE

Scientific reports 20200529 1

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β1-adrenergic receptor (β1AR). In addition to generating signalling signatures and capturing functional selectivity for t ...[more]

PMID: 32471984

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Project description:Heart failure is the terminal stage of many cardiac diseases, in which β1-adrenoceptor (β1-AR) autoantibody (β1-AA) has a causative role. By continuously activating β1-AR, β1-AA can induce cytotoxicity, leading to cardiomyocyte apoptosis and heart dysfunction. However, the mechanism underlying the persistent activation of β1-AR by β1-AA is not fully understood. Receptor endocytosis has a critical role in terminating signals over time. β2-adrenoceptor (β2-AR) is involved in the regulation of β1-AR signaling. This research aimed to clarify the mechanism of the β1-AA-induced sustained activation of β1-AR and explore the role of the β2-AR/Gi-signaling pathway in this process. The beating frequency of neonatal rat cardiomyocytes, cyclic adenosine monophosphate content, and intracellular Ca2+ levels were examined to detect the activation of β1-AA. Total internal reflection fluorescence microscopy was used to detect the endocytosis of β1-AR. ICI118551 was used to assess β2-AR/Gi function in β1-AR sustained activation induced by β1-AA in vitro and in vivo. Monoclonal β1-AA derived from a mouse hybridoma could continuously activate β1-AR. β1-AA-restricted β1-AR endocytosis, which was reversed by overexpressing the endocytosis scaffold protein β-arrestin1/2, resulting in the cessation of β1-AR signaling. β2-AR could promote β1-AR endocytosis, as demonstrated by overexpressing/interfering with β2-AR in HL-1 cells, whereas β1-AA inhibited the binding of β2-AR to β1-AR, as determined by surface plasmon resonance. ICI118551 biasedly activated the β2-AR/Gi/G protein-coupled receptor kinase 2 (GRK2) pathway, leading to the arrest of limited endocytosis and continuous activation of β1-AR by β1-AA in vitro. In vivo, ICI118551 treatment attenuated myocardial fiber rupture and left ventricular dysfunction in β1-AA-positive mice. This study showed that β1-AA continuously activated β1-AR by inhibiting receptor endocytosis. Biased activation of the β2-AR/Gi/GRK2 signaling pathway could promote β1-AR endocytosis restricted by β1-AA, terminate signal transduction, and alleviate heart damage.

Dataset Information

Signal profiling of the β₁AR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by β₁AR and β₂AR.

Publications

Signal profiling of the β<sub>1</sub>AR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by β<sub>1</sub>AR and β<sub>2</sub>AR.

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