Gαi is required for carvedilol-induced β1 adrenergic receptor β-arrestin biased signaling

Wang, Jialu; Hanada, Kenji; Staus, Dean P.; Makara, Michael; Dahal, Giri Raj; Chen, Qiang; Ahles, Andrea; Engelhardt, Stefan; Rockman, Howard A.

doi:10.1038/s41467-017-01855-z

Cited by 96 publications

(79 citation statements)

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“…In apparent contrast, other studies have demonstrated an essential role for β-arrestin in some pathways regulating ERK activation 16 . Interestingly, two decades of work has demonstrated that for G αi coupled receptors, β-arrestin-mediated ERK activation is invariably pertussis toxin sensitive 22,23,24 . However, how the G αi and β-arrestin transducers cooperate in this pathway has remained obscure.…”

Section: Introductionmentioning

confidence: 99%

Noncanonical scaffolding of G_αiand β-arrestin by G protein-coupled receptors

Smith

Pack

Inoue

et al. 2019

Preprint

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47G-protein-coupled receptors (GPCRs) enable cells to sense and respond appropriately 48 to hormonal and environmental signals, and are a target of ~30% of all FDA-approved 49 medications. Canonically, each GPCR couples to distinct Gα proteins, such as Gαs, Gαi, Gαq or 50 Gα12/13, as well as b-arrestins. These transducer proteins translate and integrate 51 extracellular stimuli sensed by GPCRs into intracellular signals through what are broadly 52 considered separable signalling pathways. However, the ability of Gα proteins to directly interact 53 with b-arrestins to integrate signalling has not previously been appreciated. Here we show a 54 novel interaction between Gαi protein family members and β-arrestin. Gαi:β-arrestin complexes 55 were formed by all GPCRs tested, regardless of their canonical G protein isoform coupling, and 56 could bind both GPCRs as well as the extracellular signal-regulated kinase (ERK). This novel 57 paradigm of Gαi:β-arrestin scaffolds enhances our understanding of GPCR signalling. 58 93 protein:protein interactions 9 ) by complementing a small peptide (smBiT) fused to one protein to 94 a large protein fragment (LgBiT) fused to another protein of interest. The signal generated by 95 complementation of this split luciferase can then transfer to a third protein tagged with a 96 fluorescent protein acceptor, monomeric Kusabira Orange (mKO), generating a BRET 97 response. Thus, this technique enables real-time quantification of interactions between a two-98 protein complex and a third protein in living cells. 99Using this technology, we were surprised to discover that the canonically Gas-coupled V2R 100 also formed a 'megaplex' with Gai and β-arrestin following agonist treatment ( Fig. 1d,e). To further 101 interrogate the Gai:β-arrestin:V2R megaplex, we proceeded to swap the location of dipole donor 102 and acceptor components ( Fig. 1f). Altering the location of complex BRET components increased 103 the observed signal of the Gai-containing megaplex following agonist treatment, and further 104 confirmed that Gai:β-arrestin complexes can associate with the V2R (Fig 1g,h, Extended Data Fig. 105 1a). Agonist treatment of the canonically Gas-coupled β2AR also formed Gai:β-arrestin:β2AR 106 megaplexes (Fig 1i,j, Extended Data Fig. 1b), although less robustly than the V2R. We further 107 validated the specificity of megaplex formation by simultaneously transfecting both mKO-tagged 108 and untagged V2R and β2AR and treating with agonist. Only treating a mKO-tagged receptor with 109 its cognate agonist formed an observable Gai:β-arrestin:GPCR megaplex (Extended Data Fig 1c-110 h), indicating a specific interaction and not a bystander effect. 111 112 Figure 1: Formation of G protein:b-arrestin:GPCR megaplexes. a, Arrangement of luciferase fragments and mKO 113 acceptor fluorophore for complex BRET on G protein (LgBiT), b-arrestin (mKO), and V2R (smBiT). HEK 293T cells were 114 transiently transfected with the indicated receptor and assay components and stimulated with the indicated agonist or 115...

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Section: Introductionmentioning

confidence: 99%

Noncanonical scaffolding of G_αiand β-arrestin by G protein-coupled receptors

Smith

Pack

Inoue

et al. 2019

Preprint

Self Cite

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“…Specifically, carvedilol can promote β-arrestin signaling while acting as an inverse agonist towards G protein signaling (Wang et al, 2017). This type of β-arrestin signaling has been shown to have anti-apoptotic effects (Ahn et al, 2009;DeFea et al, 2000;Povsic et al, 2003) that may prevent Q344X rod death.…”

Section: Discussionmentioning

confidence: 99%

Drug Screening with Zebrafish Visual Behavior Identifies Carvedilol as a Potential Treatment for Retinitis Pigmentosa

Ganzen

Zhang

et al. 2020

Preprint

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Retinitis Pigmentosa (RP) is an incurable inherited retinal degeneration affecting approximately 1 in 4,000 individuals globally. The goal of this work was to identify drugs that can help patients suffering from the disease. To accomplish this, we screened drugs on a zebrafish RP model. This model expresses a truncated human rhodopsin transgene (Tg(rho:Hsa.RH1_Q344X)) causing significant rod degeneration by 7 days post-fertilization (dpf). Consequently, the larvae displayed a deficit in visual motor response (VMR) under scotopic condition. The diminished VMR was leveraged to screen an ENZO SCREEN-WELL® REDOX library since oxidative stress is postulated to play a role in RP progression. Our screening identified a beta-blocker, carvedilol, that ameliorated the deficient VMR of the RP larvae and increased their rod number. Carvedilol can act directly on rods as it affected the adrenergic pathway in a rod-like human Y79 cell line. Since carvedilol is an FDA-approved drug, our findings suggest that carvedilol can potentially be repurposed to treat RP patients.Summary StatementThis paper presents the utilization of zebrafish visual behavior, a novel paradigm to screen and identify drugs to treat retinitis pigmentosa, an incurable retinal-degenerative disease.

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“…Exogenous application of biased ligands to replace the missing peptides attenuates the development of the disease (Yang et al 2017b). Conversely, the β-agonist carvedilol has been proposed as a β-arrestin biased ligand, beneficially and selectively activating this pathway compared to a balanced compound (Wang et al 2017), although it should be noted there may also be a significant contribution from α1-adrenoreceptor antagonism. While both ET receptors are internalized by β-arrestin and dynamin/clathrin-dependent mechanisms, ET A are recycled to the plasma membrane for further signaling while ET B are targeted to lysosomes and degraded (Bremnes et al 2000).…”

Section: Biased Signaling In the Et Pathwaymentioning

confidence: 99%

New Drugs and Emerging Therapeutic Targets in the Endothelin Signaling Pathway and Prospects for Personalized Precision Medicine

Davenport

Kuc²,

Southan³

et al. 2018

Physiol Res

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During the last thirty years since the discovery of endothelin-1, the therapeutic strategy that has evolved in the clinic, mainly in the treatment of pulmonary arterial hypertension, is to block the action of the peptide either at the ETA subtype or both receptors using orally active small molecule antagonists. Recently, there has been a rapid expansion in research targeting ET receptors using chemical entities other than small molecules, particularly monoclonal antibody antagonists and selective peptide agonists and antagonists. While usually sacrificing oral bio-availability, these compounds have other therapeutic advantages with the potential to considerably expand drug targets in the endothelin pathway and extend treatment to other pathophysiological conditions. Where the small molecule approach has been retained, a novel strategy to combine two vasoconstrictor targets, the angiotensin AT1 receptor as well as the ETA receptor in the dual antagonist sparsentan has been developed. A second emerging strategy is to combine drugs that have two different targets, the ETA antagonist ambrisentan with the phosphodiesterase inhibitor tadalafil, to improve the treatment of pulmonary arterial hypertension. The solving of the crystal structure of the ETB receptor has the potential to identify allosteric binding sites for novel ligands. A further key advance is the experimental validation of a single nucleotide polymorphism that has genome wide significance in five vascular diseases and that significantly increases the amount of big endothelin-1 precursor in the plasma. This observation provides a rationale for testing this single nucleotide polymorphism to stratify patients for allocation to treatment with endothelin agents and highlights the potential to use personalized precision medicine in the endothelin field.

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Gαi is required for carvedilol-induced β1 adrenergic receptor β-arrestin biased signaling

Cited by 96 publications

References 50 publications

Noncanonical scaffolding of G_αiand β-arrestin by G protein-coupled receptors

Noncanonical scaffolding of G_αiand β-arrestin by G protein-coupled receptors

Drug Screening with Zebrafish Visual Behavior Identifies Carvedilol as a Potential Treatment for Retinitis Pigmentosa

New Drugs and Emerging Therapeutic Targets in the Endothelin Signaling Pathway and Prospects for Personalized Precision Medicine

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Gαi is required for carvedilol-induced β1 adrenergic receptor β-arrestin biased signaling

Cited by 96 publications

References 50 publications

Noncanonical scaffolding of Gαiand β-arrestin by G protein-coupled receptors

Noncanonical scaffolding of Gαiand β-arrestin by G protein-coupled receptors

Drug Screening with Zebrafish Visual Behavior Identifies Carvedilol as a Potential Treatment for Retinitis Pigmentosa

New Drugs and Emerging Therapeutic Targets in the Endothelin Signaling Pathway and Prospects for Personalized Precision Medicine

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Noncanonical scaffolding of G_αiand β-arrestin by G protein-coupled receptors

Noncanonical scaffolding of G_αiand β-arrestin by G protein-coupled receptors