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Robert J Lefkowitz - One of the best experts on this subject based on the ideXlab platform.
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Biased signalling from simple switches to allosteric microprocessors
Nature Reviews Drug Discovery, 2018Co-Authors: Jeffrey S Smith, Robert J Lefkowitz, Sudarshan RajagopalAbstract:A given G protein-coupled receptor can signal through a range of downstream transducers depending on the stimulating ligand, enabling Biased signalling towards different biological outcomes. Lefkowitz and colleagues describe the latest advances in the field, including efforts to harness Biased signalling for improved therapeutic outcomes. G protein-coupled receptors (GPCRs) are the largest class of receptors in the human genome and some of the most common drug targets. It is now well established that GPCRs can signal through multiple transducers, including heterotrimeric G proteins, GPCR kinases and β-arrestins. While these signalling pathways can be activated or blocked by 'balanced' agonists or antagonists, they can also be selectively activated in a 'Biased' response. Biased responses can be induced by Biased Ligands, Biased receptors or system bias, any of which can result in preferential signalling through G proteins or β-arrestins. At many GPCRs, signalling events mediated by G proteins and β-arrestins have been shown to have distinct biochemical and physiological actions from one another, and an accurate evaluation of Biased signalling from pharmacology through physiology is crucial for preclinical drug development. Recent structural studies have provided snapshots of GPCR–transducer complexes, which should aid in the structure-based design of novel Biased therapies. Our understanding of GPCRs has evolved from that of two-state, on-and-off switches to that of multistate allosteric microprocessors, in which Biased Ligands transmit distinct structural information that is processed into distinct biological outputs. The development of Biased Ligands as therapeutics heralds an era of increased drug efficacy with reduced drug side effects.
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allosteric modulation of β arrestin Biased angiotensin ii type 1 receptor signaling by membrane stretch
Journal of Biological Chemistry, 2014Co-Authors: Wei Tang, Ryan T Strachan, Robert J Lefkowitz, Howard A RockmanAbstract:Abstract It has recently been appreciated that the angiotensin-II type-1 receptor (AT1R), a prototypic member of the G protein-coupled receptor (GPCR) superfamily, also functions as a mechanosensor. Specifically, mechanical stretch activates the AT1R to promote downstream signaling mediated exclusively by the multifunctional scaffold protein, β-arrestin, in a manner consistent with previously identified β-arrestin-Biased Ligands. However, the ligand-independent mechanism by which mechanical stretch promotes β-arrestin-Biased signaling remains unknown. Implicit in the concept of Biased agonism (i.e., the ability of an agonist to activate a subset of receptor-mediated signaling pathways) is the notion that distinct active conformations of the receptor mediate differential activation of signaling pathways. Here we determined if mechanical stretch stabilizes distinct β-arrestin-activating conformations of the AT1R by using β-arrestin2-Biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to β-arrestin (AT1R-β-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the β-arrestin-Biased agonist TRV120023, with no effect on the balanced agonist AngII. In addition, the effect of osmotic stretch on ERK activation was markedly augmented in cells expressing the AT1R-β-arrestin2 fusion compared to the wild type AT1R, and completely blocked in cells expressing the AT1R-Gq fusion. Biophysical experiments with an intramolecular BRET β-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize β-arrestin2 active conformations which differ from those stabilized by the AT1R activated by angiotensin II. Together, these data support a novel ligand-independent mechanism whereby mechanical stretch allosterically stabilizes specific β-arrestin-Biased active conformations of the AT1R and have important implications for understanding pathophysiological AT1R signaling.
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allosteric modulation of β arrestin Biased angiotensin ii type 1 receptor signaling by membrane stretch
Journal of Biological Chemistry, 2014Co-Authors: Wei Tang, Ryan T Strachan, Robert J Lefkowitz, Howard A RockmanAbstract:It has recently been appreciated that the angiotensin II type 1 receptor (AT1R), a prototypic member of the G protein-coupled receptor superfamily, also functions as a mechanosensor. Specifically, mechanical stretch activates the AT1R to promote downstream signaling mediated exclusively by the multifunctional scaffold protein, β-arrestin, in a manner consistent with previously identified β-arrestin-Biased Ligands. However, the ligand-independent mechanism by which mechanical stretch promotes β-arrestin-Biased signaling remains unknown. Implicit in the concept of Biased agonism (i.e. the ability of an agonist to activate a subset of receptor-mediated signaling pathways) is the notion that distinct active conformations of the receptor mediate differential activation of signaling pathways. Here we determined whether mechanical stretch stabilizes distinct β-arrestin-activating conformations of the AT1R by using β-arrestin2-Biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to β-arrestin (AT1R-β-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the β-arrestin-Biased agonist TRV120023, with no effect on the balanced agonist AngII. In addition, the effect of osmotic stretch on ERK activation was markedly augmented in cells expressing the AT1R-β-arrestin2 fusion compared with the wild type AT1R and completely blocked in cells expressing the AT1R-Gq fusion. Biophysical experiments with an intramolecular BRET β-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize β-arrestin2 active conformations that differ from those stabilized by the AT1R activated by angiotensin II. Together, these data support a novel ligand-independent mechanism whereby mechanical stretch allosterically stabilizes specific β-arrestin-Biased active conformations of the AT1R and has important implications for understanding pathophysiological AT1R signaling.
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divergent transducer specific molecular efficacies generate Biased agonism at a g protein coupled receptor gpcr
Journal of Biological Chemistry, 2014Co-Authors: Ryan T Strachan, Jonathan D. Violin, David H Rominger, Jinpeng Sun, Seungkirl Ahn, Alex R B Thomsen, Xiao Zhu, Andrew B Kleist, Tommaso Costa, Robert J LefkowitzAbstract:The concept of "Biased agonism" arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. "efficacy") can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of Biased agonism, the molecular mechanism(s) whereby Biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and Biased Ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that Biased AT1R agonists possess Biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate Biased agonism at a GPCR.
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β arrestin Biased agonism at the β2 adrenergic receptor
Journal of Biological Chemistry, 2008Co-Authors: Matthew T Drake, Jonathan D. Violin, Erin J Whalen, James W Wisler, Sudha K Shenoy, Robert J LefkowitzAbstract:Abstract Classically, the β2-adrenergic receptor (β2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including β2AR, can signal via β-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among β2AR agonists there exist Ligands that disproportionately signal via G proteins or β-arrestins and are hence “Biased.” Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known β2AR agonists exhibit relative efficacies for β-arrestin-associated activities (β-arrestin membrane translocation and β2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three βAR Ligands there is a marked bias toward β-arrestin signaling; these Ligands stimulate β-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these Biased Ligands reveals that all three are catecholamines containing an ethyl substitution on the α-carbon, a motif absent on all of the other, unBiased Ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR Ligands with a distinct bias for β-arrestin-mediated signaling.
Jonathan D. Violin - One of the best experts on this subject based on the ideXlab platform.
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Biased mu opioid receptor Ligands a promising new generation of pain therapeutics
Current Opinion in Pharmacology, 2017Co-Authors: Edward R Siuda, David H Rominger, Richard Carr, Jonathan D. ViolinAbstract:Opioid chemistry and biology occupy a pivotal place in the history of pharmacology and medicine. Morphine offers unmatched efficacy in alleviating acute pain, but is also associated with a host of adverse side effects. The advent of Biased agonism at G protein-coupled receptors has expanded our understanding of intracellular signaling and highlighted the concept that certain Ligands are able to differentially modulate downstream pathways. The ability to target one pathway over another has allowed for the development of Biased Ligands with robust clinical efficacy and fewer adverse events. In this review we summarize these concepts with an emphasis on Biased mu opioid receptor pharmacology and highlight how far opioid pharmacology has evolved.
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cardiac myosin light chain phosphorylation and inotropic effects of a Biased ligand trv120023 in a dilated cardiomyopathy model
Cardiovascular Research, 2015Co-Authors: Madhusudhan Tarigopula, Jonathan D. Violin, David M Ryba, Beata M Wolska, Rd Robert T Davis, Paul T Mungai, David F Wieczorek, Conrad L Cowan, John R SolaroAbstract:Aims Therapeutic approaches to treat familial dilated cardiomyopathy (DCM), which is characterized by depressed sarcomeric tension and susceptibility to Ca2+-related arrhythmias, have been generally unsuccessful. Our objective in the present work was to determine the effect of the angiotensin II type 1 receptor (AT1R) Biased ligand, TRV120023, on contractility of hearts of a transgenic mouse model of familial DCM with mutation in tropomyosin at position 54 (TG-E54K). Our rationale is based on previous studies, which have supported the hypothesis that Biased G-protein-coupled receptor Ligands, signalling via β-arrestin, increase cardiac contractility with no effect on Ca2+ transients. Our previous work demonstrated that the Biased ligand TRV120023 is able to block angiotensin-induced hypertrophy, while promoting an increase in sarcomere Ca2+ response. Methods and results We tested the hypothesis that the depression in cardiac function associated with DCM can be offset by infusion of the AT1R Biased ligand, TRV120023. We intravenously infused saline, TRV120023, or the unBiased ligand, losartan, for 15 min in TG-E54K and non-transgenic mice to obtain left ventricular pressure–volume relations. Hearts were analysed for sarcomeric protein phosphorylation. Results showed that the AT1R Biased ligand increases cardiac performance in TG-E54K mice in association with increased myosin light chain-2 phosphorylation. Conclusion Treatment of mice with an AT1R Biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R Biased Ligands may prove to be a novel inotropic approach in familial DCM.
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divergent transducer specific molecular efficacies generate Biased agonism at a g protein coupled receptor gpcr
Journal of Biological Chemistry, 2014Co-Authors: Ryan T Strachan, Jonathan D. Violin, David H Rominger, Jinpeng Sun, Seungkirl Ahn, Alex R B Thomsen, Xiao Zhu, Andrew B Kleist, Tommaso Costa, Robert J LefkowitzAbstract:The concept of "Biased agonism" arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. "efficacy") can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of Biased agonism, the molecular mechanism(s) whereby Biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and Biased Ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that Biased AT1R agonists possess Biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate Biased agonism at a GPCR.
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beta arrestin Biased Ligands at the at1r a novel approach to the treatment of acute heart failure
Drug Discovery Today: Therapeutic Strategies, 2012Co-Authors: Jonathan D. Violin, David G Soergel, Michael W LarkAbstract:The angiotensin II type 1 receptor (AT1R) is an important drug target for hypertension and chronic heart failure. This receptor is also important in the pathophysiology of acute heart failure, stimulating both adverse and beneficial hemodynamic and renal responses. This review will discuss advances in our understanding of the molecular mechanisms of AT1R function, and how these insights have enabled the discovery of β-arrestin-Biased AT1R Ligands, one of which is now in clinical trials for acute heart failure (AHF) treatment.
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Biased Ligands for better cardiovascular drugs dissecting g protein coupled receptor pharmacology
Circulation Research, 2011Co-Authors: Scott M Dewire, Jonathan D. ViolinAbstract:Drug discovery efforts targeting G-protein-coupled receptors (GPCR) have been immensely successful in creating new cardiovascular medicines. Currently marketed GPCR drugs are broadly classified as either agonists that activate receptors or antagonists that prevent receptor activation by endogenous stimuli. However, GPCR couple to a multitude of intracellular signaling pathways beyond classical G-protein signals, and these signals can be independently activated by Biased Ligands to vastly expand the potential for new drugs at these classic targets. By selectively engaging only a subset of a receptor's potential intracellular partners, Biased Ligands may deliver more precise therapeutic benefit with fewer side effects than current GPCR-targeted drugs. In this review, we discuss the history of Biased ligand research, the current understanding of how Biased Ligands exert their unique pharmacology, and how research into GPCR signaling has uncovered previously unappreciated capabilities of receptor pharmacology. We focus on several receptors to illustrate the approaches taken and discoveries made, and how these are steadily illuminating the intricacies of GPCR pharmacology. Discoveries of Biased Ligands targeting the angiotensin II type 1 receptor and of separable pharmacology suggesting the potential value of Biased Ligands targeting the β-adrenergic receptors and nicotinic acid receptor GPR109a highlight the powerful clinical promise of this new category of potential therapeutics.
Bryan L Roth - One of the best experts on this subject based on the ideXlab platform.
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d2 dopamine receptor g protein Biased partial agonists based on cariprazine
Journal of Medicinal Chemistry, 2019Co-Authors: Yudao Shen, John D Mccorvy, Michael L. Martini, Jing Liu, William C. Wetsel, Ramona M. Rodriguiz, Vladimir M. Pogorelov, Karen M. Ward, Bryan L RothAbstract:Functionally selective G protein-coupled receptor Ligands are valuable tools for deciphering the roles of downstream signaling pathways that potentially contribute to therapeutic effects versus side effects. Recently, we discovered both Gi/o-Biased and β-arrestin2-Biased D2 receptor agonists based on the Food and Drug Administration (FDA)-approved drug aripiprazole. In this work, based on another FDA-approved drug, cariprazine, we conducted a structure-functional selectivity relationship study and discovered compound 38 (MS1768) as a potent partial agonist that selectively activates the Gi/o pathway over β-arrestin2. Unlike the dual D2R/D3R partial agonist cariprazine, compound 38 showed selective agonist activity for D2R over D3R. In fact, compound 38 exhibited potent antagonism of dopamine-stimulated β-arrestin2 recruitment. In our docking studies, compound 38 directly interacts with S1935.42 on TM5 but has no interactions with extracellular loop 2, which appears to be in contrast to the binding poses of D2R β-arrestin2-Biased Ligands. In in vivo studies, compound 38 showed high D2R receptor occupancy in mice and effectively inhibited phencyclidine-induced hyperlocomotion.
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Defining Structure-Functional Selectivity Relationships (SFSR) for a Class of Non-Catechol Dopamine D1 Receptor Agonists.
Journal of medicinal chemistry, 2019Co-Authors: Michael L. Martini, Marc G. Caron, Jing Liu, Caroline A. Ray, Aarti N. Urs, Nikhil M. Urs, Bryan L RothAbstract:G protein-coupled receptors (GPCRs) are capable of downstream signaling through distinct noncanonical pathways such as β-arrestins in addition to the canonical G protein-dependent pathways. GPCR Ligands that differentially activate the downstream signaling pathways are termed functionally selective or Biased Ligands. A class of novel non-catechol G protein-Biased agonists of the dopamine D1 receptor (D1R) was recently disclosed. We conducted the first comprehensive structure–functional selectivity relationship study measuring GS and β-arrestin2 recruitment activities focused on four regions of this scaffold, resulting in over 50 analogs with diverse functional selectivity profiles. Some compounds became potent full agonists of β-arrestin2 recruitment, while others displayed enhanced GS bias compared to the starting compound. Pharmacokinetic testing of an analog with an altered functional selectivity profile demonstrated excellent blood–brain barrier penetration. This study provides novel tools for studyin...
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Defining Structure–Functional Selectivity Relationships (SFSR) for a Class of Non-Catechol Dopamine D1 Receptor Agonists
2019Co-Authors: Michael L. Martini, John D Mccorvy, Marc G. Caron, Jing Liu, Caroline Ray, Xi-ping Huang, Aarti Urs, Nikhil Urs, Bryan L RothAbstract:G protein-coupled receptors (GPCRs) are capable of downstream signaling through distinct noncanonical pathways such as β-arrestins in addition to the canonical G protein-dependent pathways. GPCR Ligands that differentially activate the downstream signaling pathways are termed functionally selective or Biased Ligands. A class of novel non-catechol G protein-Biased agonists of the dopamine D1 receptor (D1R) was recently disclosed. We conducted the first comprehensive structure–functional selectivity relationship study measuring GS and β-arrestin2 recruitment activities focused on four regions of this scaffold, resulting in over 50 analogs with diverse functional selectivity profiles. Some compounds became potent full agonists of β-arrestin2 recruitment, while others displayed enhanced GS bias compared to the starting compound. Pharmacokinetic testing of an analog with an altered functional selectivity profile demonstrated excellent blood–brain barrier penetration. This study provides novel tools for studying ligand bias at D1R and paves the way for developing the next generation of Biased D1R Ligands
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D2 Dopamine Receptor G Protein-Biased Partial Agonists Based on Cariprazine
2019Co-Authors: Yudao Shen, John D Mccorvy, Bryan L Roth, Michael L. Martini, Jing Liu, William C. Wetsel, Ramona M. Rodriguiz, Vladimir M. Pogorelov, Karen M. Ward, Jian JinAbstract:Functionally selective G protein-coupled receptor Ligands are valuable tools for deciphering the roles of downstream signaling pathways that potentially contribute to therapeutic effects versus side effects. Recently, we discovered both Gi/o-Biased and β-arrestin2-Biased D2 receptor agonists based on the Food and Drug Administration (FDA)-approved drug aripiprazole. In this work, based on another FDA-approved drug, cariprazine, we conducted a structure–functional selectivity relationship study and discovered compound 38 (MS1768) as a potent partial agonist that selectively activates the Gi/o pathway over β-arrestin2. Unlike the dual D2R/D3R partial agonist cariprazine, compound 38 showed selective agonist activity for D2R over D3R. In fact, compound 38 exhibited potent antagonism of dopamine-stimulated β-arrestin2 recruitment. In our docking studies, compound 38 directly interacts with S1935.42 on TM5 but has no interactions with extracellular loop 2, which appears to be in contrast to the binding poses of D2R β-arrestin2-Biased Ligands. In in vivo studies, compound 38 showed high D2R receptor occupancy in mice and effectively inhibited phencyclidine-induced hyperlocomotion
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discovery of g protein Biased d2 dopamine receptor partial agonists
Journal of Medicinal Chemistry, 2016Co-Authors: Xin Chen, John D Mccorvy, Matthew G Fischer, Kyle V Butler, Yudao Shen, Bryan L Roth, Jian JinAbstract:Biased Ligands (also known as functionally selective Ligands) of G protein-coupled receptors are valuable tools for dissecting the roles of G protein-dependent and independent signaling pathways in health and disease. Biased Ligands have also been increasingly pursued by the biomedical community as promising therapeutics with improved efficacy and reduced side effects compared with unBiased Ligands. We previously discovered first-in-class β-arrestin-Biased agonists of dopamine D2 receptor (D2R) by extensively exploring multiple regions of aripiprazole, a balanced D2R agonist. In our continuing efforts to identify Biased agonists of D2R, we unexpectedly discovered a G protein-Biased agonist of D2R, compound 1, which is the first G protein-Biased D2R agonist from the aripiprazole scaffold. We designed and synthesized novel analogues to explore two regions of 1 and conducted structure–functional selectivity relationship (SFSR) studies. Here we report the discovery of 1, findings from our SFSR studies, and ch...
Ryan T Strachan - One of the best experts on this subject based on the ideXlab platform.
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allosteric modulation of β arrestin Biased angiotensin ii type 1 receptor signaling by membrane stretch
Journal of Biological Chemistry, 2014Co-Authors: Wei Tang, Ryan T Strachan, Robert J Lefkowitz, Howard A RockmanAbstract:Abstract It has recently been appreciated that the angiotensin-II type-1 receptor (AT1R), a prototypic member of the G protein-coupled receptor (GPCR) superfamily, also functions as a mechanosensor. Specifically, mechanical stretch activates the AT1R to promote downstream signaling mediated exclusively by the multifunctional scaffold protein, β-arrestin, in a manner consistent with previously identified β-arrestin-Biased Ligands. However, the ligand-independent mechanism by which mechanical stretch promotes β-arrestin-Biased signaling remains unknown. Implicit in the concept of Biased agonism (i.e., the ability of an agonist to activate a subset of receptor-mediated signaling pathways) is the notion that distinct active conformations of the receptor mediate differential activation of signaling pathways. Here we determined if mechanical stretch stabilizes distinct β-arrestin-activating conformations of the AT1R by using β-arrestin2-Biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to β-arrestin (AT1R-β-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the β-arrestin-Biased agonist TRV120023, with no effect on the balanced agonist AngII. In addition, the effect of osmotic stretch on ERK activation was markedly augmented in cells expressing the AT1R-β-arrestin2 fusion compared to the wild type AT1R, and completely blocked in cells expressing the AT1R-Gq fusion. Biophysical experiments with an intramolecular BRET β-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize β-arrestin2 active conformations which differ from those stabilized by the AT1R activated by angiotensin II. Together, these data support a novel ligand-independent mechanism whereby mechanical stretch allosterically stabilizes specific β-arrestin-Biased active conformations of the AT1R and have important implications for understanding pathophysiological AT1R signaling.
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allosteric modulation of β arrestin Biased angiotensin ii type 1 receptor signaling by membrane stretch
Journal of Biological Chemistry, 2014Co-Authors: Wei Tang, Ryan T Strachan, Robert J Lefkowitz, Howard A RockmanAbstract:It has recently been appreciated that the angiotensin II type 1 receptor (AT1R), a prototypic member of the G protein-coupled receptor superfamily, also functions as a mechanosensor. Specifically, mechanical stretch activates the AT1R to promote downstream signaling mediated exclusively by the multifunctional scaffold protein, β-arrestin, in a manner consistent with previously identified β-arrestin-Biased Ligands. However, the ligand-independent mechanism by which mechanical stretch promotes β-arrestin-Biased signaling remains unknown. Implicit in the concept of Biased agonism (i.e. the ability of an agonist to activate a subset of receptor-mediated signaling pathways) is the notion that distinct active conformations of the receptor mediate differential activation of signaling pathways. Here we determined whether mechanical stretch stabilizes distinct β-arrestin-activating conformations of the AT1R by using β-arrestin2-Biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to β-arrestin (AT1R-β-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the β-arrestin-Biased agonist TRV120023, with no effect on the balanced agonist AngII. In addition, the effect of osmotic stretch on ERK activation was markedly augmented in cells expressing the AT1R-β-arrestin2 fusion compared with the wild type AT1R and completely blocked in cells expressing the AT1R-Gq fusion. Biophysical experiments with an intramolecular BRET β-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize β-arrestin2 active conformations that differ from those stabilized by the AT1R activated by angiotensin II. Together, these data support a novel ligand-independent mechanism whereby mechanical stretch allosterically stabilizes specific β-arrestin-Biased active conformations of the AT1R and has important implications for understanding pathophysiological AT1R signaling.
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divergent transducer specific molecular efficacies generate Biased agonism at a g protein coupled receptor gpcr
Journal of Biological Chemistry, 2014Co-Authors: Ryan T Strachan, Jonathan D. Violin, David H Rominger, Jinpeng Sun, Seungkirl Ahn, Alex R B Thomsen, Xiao Zhu, Andrew B Kleist, Tommaso Costa, Robert J LefkowitzAbstract:The concept of "Biased agonism" arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. "efficacy") can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of Biased agonism, the molecular mechanism(s) whereby Biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and Biased Ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that Biased AT1R agonists possess Biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate Biased agonism at a GPCR.
Howard A Rockman - One of the best experts on this subject based on the ideXlab platform.
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allosteric modulation of β arrestin Biased angiotensin ii type 1 receptor signaling by membrane stretch
Journal of Biological Chemistry, 2014Co-Authors: Wei Tang, Ryan T Strachan, Robert J Lefkowitz, Howard A RockmanAbstract:Abstract It has recently been appreciated that the angiotensin-II type-1 receptor (AT1R), a prototypic member of the G protein-coupled receptor (GPCR) superfamily, also functions as a mechanosensor. Specifically, mechanical stretch activates the AT1R to promote downstream signaling mediated exclusively by the multifunctional scaffold protein, β-arrestin, in a manner consistent with previously identified β-arrestin-Biased Ligands. However, the ligand-independent mechanism by which mechanical stretch promotes β-arrestin-Biased signaling remains unknown. Implicit in the concept of Biased agonism (i.e., the ability of an agonist to activate a subset of receptor-mediated signaling pathways) is the notion that distinct active conformations of the receptor mediate differential activation of signaling pathways. Here we determined if mechanical stretch stabilizes distinct β-arrestin-activating conformations of the AT1R by using β-arrestin2-Biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to β-arrestin (AT1R-β-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the β-arrestin-Biased agonist TRV120023, with no effect on the balanced agonist AngII. In addition, the effect of osmotic stretch on ERK activation was markedly augmented in cells expressing the AT1R-β-arrestin2 fusion compared to the wild type AT1R, and completely blocked in cells expressing the AT1R-Gq fusion. Biophysical experiments with an intramolecular BRET β-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize β-arrestin2 active conformations which differ from those stabilized by the AT1R activated by angiotensin II. Together, these data support a novel ligand-independent mechanism whereby mechanical stretch allosterically stabilizes specific β-arrestin-Biased active conformations of the AT1R and have important implications for understanding pathophysiological AT1R signaling.
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allosteric modulation of β arrestin Biased angiotensin ii type 1 receptor signaling by membrane stretch
Journal of Biological Chemistry, 2014Co-Authors: Wei Tang, Ryan T Strachan, Robert J Lefkowitz, Howard A RockmanAbstract:It has recently been appreciated that the angiotensin II type 1 receptor (AT1R), a prototypic member of the G protein-coupled receptor superfamily, also functions as a mechanosensor. Specifically, mechanical stretch activates the AT1R to promote downstream signaling mediated exclusively by the multifunctional scaffold protein, β-arrestin, in a manner consistent with previously identified β-arrestin-Biased Ligands. However, the ligand-independent mechanism by which mechanical stretch promotes β-arrestin-Biased signaling remains unknown. Implicit in the concept of Biased agonism (i.e. the ability of an agonist to activate a subset of receptor-mediated signaling pathways) is the notion that distinct active conformations of the receptor mediate differential activation of signaling pathways. Here we determined whether mechanical stretch stabilizes distinct β-arrestin-activating conformations of the AT1R by using β-arrestin2-Biased agonists as conformational probes in pharmacological and biophysical assays. When tested at cells expressing the AT1R fused to β-arrestin (AT1R-β-arrestin2), we found that osmotic stretch increased the binding affinity and potency of the β-arrestin-Biased agonist TRV120023, with no effect on the balanced agonist AngII. In addition, the effect of osmotic stretch on ERK activation was markedly augmented in cells expressing the AT1R-β-arrestin2 fusion compared with the wild type AT1R and completely blocked in cells expressing the AT1R-Gq fusion. Biophysical experiments with an intramolecular BRET β-arrestin2 biosensor revealed that osmotic stretch and TRV120023 activate AT1Rs to stabilize β-arrestin2 active conformations that differ from those stabilized by the AT1R activated by angiotensin II. Together, these data support a novel ligand-independent mechanism whereby mechanical stretch allosterically stabilizes specific β-arrestin-Biased active conformations of the AT1R and has important implications for understanding pathophysiological AT1R signaling.