Divergent Transducer-specific Molecular Efficacies Generate Biased Agonism at a G Protein-coupled Receptor (GPCR)

Strachan, Ryan T.; Sun, Jin Peng; Rominger, David H.; Violin, Jonathan D.; Ahn, Seungkirl; Thomsen, A.; Zhu, Xiao; Kleist, Andrew B.; Costa, Tommaso; Lefkowitz, Robert J.

doi:10.1074/jbc.m114.548131

Cited by 115 publications

(143 citation statements)

References 62 publications

(71 reference statements)

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“…It has been well appreciated that allosteric transducers such as heterotrimeric G proteins or β-arrestins promote high-affinity agonist binding (10,23,24). We also observed that 15 inhibited the high-affinity binding of a radiolabeled (25) that is promoted by either heterotrimeric Gs protein or β-arrestin1 (Fig.…”

Section: Resultssupporting

confidence: 57%

Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

Ahn

Kahsai

Pani

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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133

119

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The β2-adrenergic receptor (β2AR) has been a model system for understanding regulatory mechanisms of G-protein–coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β2AR. In cell-signaling studies, 15 inhibits cAMP production through the β2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β2AR. This study presents an allosteric small-molecule ligand for the β2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

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Section: Resultssupporting

confidence: 57%

Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

Ahn

Kahsai

Pani

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

133

119

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“…In how far the two binding modes described here are connected to different conformations of holo-arrestin remains to be elucidated.Our findings correlate well with a biochemical study of the type-1 angiotensin receptor, which has shown that receptor binding to β-arrestin is less specific than its interaction with the G q α-subunit. β-arrestin binding occurred in the presence of all peptide agonists, while efficient G q binding was limited to a small subset of the ligands studied (49). Rhodopsin activation has been described extensively, facilitated by natural abundance and its photochemical properties, which provide an ideal system for biophysical and biochemical characterization.…”

Section: Discussionmentioning

confidence: 99%

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin

Elgeti

Kazmin

Rose

et al. 2018

Journal of Biological Chemistry

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Signaling of the prototypical G protein coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (G t ) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin-arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/ peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C-terminus of the G t α-subunit (GαCT), which stabilizes only one specific substate out of the conformational ensemble. However, GαCT binding and both ArrFL-1 binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling.Unlike other class A GPCRs the photoreceptor rhodopsin contains the light-sensitive cofactor retinal as a ligand, which is covalently bound to the opsin apoprotein. Upon light-induced cis/trans isomerization of retinal and subsequent alterations within the binding pocket, the signal propagates via GPCR-conserved interhelical networks towards the cytoplasmic surface of the receptor, where major structural rearrangements take place. The most prominent structural change is the rotational outward tilt of transmembrane (TM) helix 6, which opens a cytoplasmic binding crevice and thus constitutes the main prerequisite for binding and activation of signaling via the G protein transducin (1, 2). Even though first identified in rhodopsin, for many other GPCRs homologous structural changes have been shown to exist, and a common structural and functional framework for GPCRs is being developed (3).

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“…17,18 However; recent findings have revealed a higher mechanistic complexity of this biased compound that involves both β-arr and Gq effectors 14,19 but leads to signaling pathways different from those promoted by Ang II. 14 Interestingly, SII has demonstrated β-arr-dependent pharmacological properties, such as inotropic responses on isolated cardiomyocytes 20 ; however, its low affinity for AT1-R has prevented further in vivo characterization of its biased activity.…”

Section: Ilementioning

confidence: 99%

Cardioprotective Angiotensin-(1–7) Peptide Acts as a Natural-Biased Ligand at the Angiotensin II Type 1 Receptor

et al. 2016

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Hyperactivity of the renin–angiotensin–aldosterone system through the angiotensin II (Ang II)/Ang II type 1 receptor (AT1-R) axis constitutes a hallmark of hypertension. Recent findings indicate that only a subset of AT1-R signaling pathways is cardiodeleterious, and their selective inhibition by biased ligands promotes therapeutic benefit. To date, only synthetic biased ligands have been described, and whether natural renin–angiotensin–aldosterone system peptides exhibit functional selectivity at AT1-R remains unknown. In this study, we systematically determined efficacy and potency of Ang II, Ang III, Ang IV, and Ang-(1–7) in AT1-R–expressing HEK293T cells on the activation of cardiodeleterious G-proteins and cardioprotective β-arrestin2. Ang III and Ang IV fully activate similar G-proteins than Ang II, the prototypical AT1-R agonist, despite weaker potency of Ang IV. Interestingly, Ang-(1–7) that binds AT1-R fails to promote G-protein activation but behaves as a competitive antagonist for Ang II/Gi and Ang II/Gq pathways. Conversely, all renin–angiotensin–aldosterone system peptides act as agonists on the AT1-R/β-arrestin2 axis but display biased activities relative to Ang II as indicated by their differences in potency and AT1-R/β-arrestin2 intracellular routing. Importantly, we reveal Ang-(1–7) a known Mas receptor-specific ligand, as an AT1-R–biased agonist, selectively promoting β-arrestin activation while blocking the detrimental Ang II/AT1-R/Gq axis. This original pharmacological profile of Ang-(1–7) at AT1-R, similar to that of synthetic AT1-R–biased agonists, could, in part, contribute to its cardiovascular benefits. Accordingly, in vivo, Ang-(1–7) counteracts the phenylephrine-induced aorta contraction, which was blunted in AT1-R knockout mice. Collectively, these data suggest that Ang-(1–7) natural-biased agonism at AT1-R could fine-tune the physiology of the renin–angiotensin–aldosterone system.

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Divergent Transducer-specific Molecular Efficacies Generate Biased Agonism at a G Protein-coupled Receptor (GPCR)

Cited by 115 publications

References 62 publications

Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

The arrestin-1 finger loop interacts with two distinct conformations of active rhodopsin

Cardioprotective Angiotensin-(1–7) Peptide Acts as a Natural-Biased Ligand at the Angiotensin II Type 1 Receptor

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