Mechanism of beta-arrestin 1 mediated Src activation via Src SH3 domain revealed by cryo-electron microscopy

Nature Communications · 2026-02-20

Beta-arrestins (βarrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how βarrs communicate with their downstream effectors. Here, we delineate structural mechanisms underlying βarr-mediated signal transduction. Using cryo-electron microscopy, we elucidate how βarr1 recruits and activates the non-receptor tyrosine kinase Src, a well-established signaling partner of βarrs. βarr1 engages Src SH3 through two distinct sites, each employing a different recognition mechanism: a polyproline motif in the N-domain and a non-proline-based interaction in the central crest region. At both sites βarr1 interacts with the aromatic surface of SH3, disrupting the autoinhibited conformation of Src and directly triggering its allosteric activation. This structural evidence establishes βarr1 as an active regulatory protein rather than a passive scaffold and suggests a potentially general mechanism for βarr-mediated signaling across diverse effectors.

A positive allosteric modulator of the β1AR with antagonist activity for catecholaminergic polymorphic ventricular tachycardia

Journal of Clinical Investigation · 2025-10-16 · 1 citations

Orthosteric beta blockers represent the leading pharmacological intervention for managing heart diseases owing to their ability to competitively antagonize β-adrenergic receptors (βARs). However, their use is often limited by adverse effects such as fatigue, hypotension, and reduced exercise capacity, due in part to nonselective inhibition of multiple βAR subtypes. These challenges are particularly problematic in treating catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease characterized by lethal tachyarrhythmias directly triggered by cardiac β1AR activation. To identify small-molecule allosteric modulators of the β1AR with enhanced subtype specificity and robust functional antagonism of β1AR-mediated signaling, we conducted a DNA-encoded small-molecule library screen and discovered Compound 11 (C11). C11 selectively potentiates the binding affinity of orthosteric agonists to the β1AR while potently inhibiting downstream signaling after β1AR activation. C11 prevents agonist-induced spontaneous contractile activity, Ca2+ release events, and exercise-induced ventricular tachycardia in the CSQ2-/- murine model of CPVT. Our studies demonstrate that C11 belongs to an emerging class of allosteric modulators termed positive allosteric modulator antagonists that positively modulate agonist binding but block downstream function. Its pharmacological properties and selective functional antagonism of β1AR-mediated signaling make C11 a promising therapeutic candidate for the treatment of CPVT and other forms of cardiac disease associated with excessive β1AR activation.

Small-molecule modulation of β-arrestins

bioRxiv (Cold Spring Harbor Laboratory) · 2024-12-27 · 3 citations

β-arrestins are multifunctional regulators of G protein-coupled receptor (GPCR) signaling, orchestrating diverse downstream signaling events and physiological responses across the vast GPCR superfamily. While GPCR pharmacology has advanced to target orthosteric and allosteric sites, as well as G proteins and GRKs, comparable chemical tools to study β-arrestins remain lacking. Here, we report the discovery of small-molecule inhibitors that selectively target β-arrestins and delineate their mechanism of action through integrated pharmacological, biochemical, biophysical, and structural analyses. These inhibitors disrupt β-arrestin-engagement with agonist-activated GPCRs, impairing desensitization, internalization, and β-arrestin-dependent functions while sparing G protein-receptor coupling. Cryo-EM, MD simulations, and structure-guided mutagenesis reveal that one modulator, Cmpd-5, engages a cryptic pocket formed by the middle, C-, and lariat loops of β-arrestin1-a critical receptor-binding interface-stabilizing a distinct conformation incompatible with GPCR engagement. Together, these findings provide a mechanistic framework for β-arrestin modulation, introducing transducer-targeted strategies to fine-tune GPCR signaling and guide the development of pathway-specific therapeutics.

Loss of biased signaling at a G protein-coupled receptor in overexpressed systems

PLoS ONE · 2023-03-24 · 34 citations

G protein-coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β-arrestins. [Sarcosine1Ile4Ile8]-angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT1R), fails to activate G protein in physiologically relevant models. Despite this, SII and several derivatives induce cellular signaling outcomes through β-arrestin-2-dependent mechanisms. However, studies reliant on exogenous AT1R overexpression indicate that SII is a partial agonist for G protein signaling and lacks β-arrestin-exclusive functional specificity. We investigated this apparent discrepancy by profiling changes in functional specificity at increasing expression levels of AT1R using a stably integrated tetracycline-titratable expression system stimulated with AngII, SII, and four other AngII analogs displaying different signaling biases. Unbiased and G protein-biased ligands activated dose-dependent calcium responses at all tested receptor concentrations. In contrast, β-arrestin-biased ligands induced dose-dependent calcium signaling only at higher AT1R overexpression levels. Using inhibitors of G proteins, we demonstrated that both Gi and Gq/11 mediated overexpression-dependent calcium signaling by β-arrestin-biased ligands. Regarding β-arrestin-mediated cellular events, the β-arrestin-biased ligand TRV026 induced receptor internalization at low physiological receptor levels insufficient for it to initiate calcium signaling. In contrast, unbiased AngII exhibited no relative preference between these outcomes under such low receptor conditions. However, with high receptor overexpression, TRV026 lost its functional selectivity. These results suggest receptor overexpression misleadingly distorts the bias of AT1R ligands and highlight the risks of using overexpressed systems to infer the signaling bias of GPCR ligands in physiologically relevant contexts.

Signal transduction at GPCRs: Allosteric activation of the ERK MAPK by β-arrestin

Proceedings of the National Academy of Sciences · 2023-10-16 · 57 citations

β-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor internalization, and initiate alternative signaling events. β-arrestins link agonist-stimulated GPCRs to downstream signaling partners, such as the c-Raf-MEK1-ERK1/2 cascade leading to ERK1/2 activation. β-arrestins have been thought to transduce signals solely via passive scaffolding by facilitating the assembly of multiprotein signaling complexes. Recently, however, β-arrestin 1 and 2 were shown to activate two downstream signaling effectors, c-Src and c-Raf, allosterically. Over the last two decades, ERK1/2 have been the most intensely studied signaling proteins scaffolded by β-arrestins. Here, we demonstrate that β-arrestins play an active role in allosterically modulating ERK kinase activity in vitro and within intact cells. Specifically, we show that β-arrestins and their GPCR-mediated active states allosterically enhance ERK2 autophosphorylation and phosphorylation of a downstream ERK2 substrate, and we elucidate the mechanism by which β-arrestins do so. Furthermore, we find that allosteric stimulation of dually phosphorylated ERK2 by active-state β-arrestin 2 is more robust than by active-state β-arrestin 1, highlighting differential capacities of β-arrestin isoforms to regulate effector signaling pathways downstream of GPCRs. In summary, our study provides strong evidence for a new paradigm in which β-arrestins function as active "catalytic" scaffolds to allosterically unlock the enzymatic activity of signaling components downstream of GPCR activation.

Abstract P2154: A Novel Allosteric Modulator Of The β ₁ AR Identified By DNA-encoded Small Molecule Library Screening Demonstrates Unique Pharmacology And Function

Circulation Research · 2023-08-04

While traditional β-blockers (i.e. competitive orthosteric antagonists of β-adrenergic receptors; βARs) are widely used as cardiovascular therapeutics, adverse effects such as fatigue and the nonselective inhibition of multiple receptor subtypes often limit maximal effectiveness. An emerging approach to enhance therapeutic targeting is to identify allosteric modulators that act cooperatively with orthosteric ligands. In contrast to orthosteric ligands which bind the endogenous ligand binding site, allosteric modulators bind to regions that are topographically distinct from the orthosteric pocket and can enhance (positive allosteric modulator; PAM) or reduce (negative allosteric modulator; NAM) the activities of orthosteric agonists/antagonists. Since allosteric regions exhibit greater sequence and structural diversity among receptor subtypes relative to the more highly conserved orthosteric pocket, allosteric modulators are more likely to be subtype specific and/or generate less adverse effects. We therefore embarked on a DNA-encoded small molecule library screen to identify novel allosteric modulators of the β 1 AR. Following multiple rounds of affinity selection using purified, functional, β 1 ARs reconstituted in lipid nanodiscs and HitGen’s OpenDEL® small molecule library containing more than 1 billion unique compounds, we identified Compound 11 (C11) as an allosteric modulator with unique pharmacological properties. Notably, C11 binds to the β 1 AR with micromolar affinity and enhances the binding affinity of orthosteric agonists and certain antagonists to the β 1 AR. In contrast to its positive cooperative effect on ligand binding, cell signaling assays showed C11 potently inhibits G protein and β-arrestin signaling downstream of the β 1 AR. Importantly, C11 showed high β 1 AR specificity with no effect on β 2 AR or AT 1 R signaling. These results suggest that C11 is a β 1 AR-specific PAM in terms of ligand binding but a NAM in terms of agonist efficacy, belonging to a largely under-characterized class of allosteric modulators termed PAM-antagonists. With an extremely unique pharmacological profile, C11 is a promising potential therapeutic and experiments evaluating its ability to modulate β 1 AR signaling in vivo are ongoing.

Allosteric modulator potentiates β2AR agonist–promoted bronchoprotection in asthma models

Journal of Clinical Investigation · 2023-07-12 · 13 citations

Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled β2-adrenergic receptor (β2AR) agonists (β2-agonists) promote - with limited efficacy - bronchodilation in asthma. All β2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a β2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of G-protein coupled receptor allosteric ligands, we investigated the impact of Cmpd-6 on β2AR-mediated bronchoprotection. Consistent with our findings using human β2ARs, Cmpd-6 allosterically potentiated β2-agonist binding to guinea pig β2ARs and downstream signaling of β2ARs. In contrast, Cmpd-6 had no such effect on murine β2ARs, which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced β2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but - in line with the binding studies - not in mice. Moreover, Cmpd-6 robustly potentiated β2 agonist-mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced β2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of β2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.

Loss of biased signaling at a G protein-coupled receptor in overexpressed systems

Zenodo (CERN European Organization for Nuclear Research) · 2023-01-11 · 1 citations

Minimal data set associated with the manuscript: Li, A., Liu, S., Huang, R., Ahn, S., & Lefkowitz, R. J. (2023). Loss of biased signaling at a G protein-coupled receptor in overexpressed systems.

How carvedilol does not activate β2-adrenoceptors

Nature Communications · 2023-11-30 · 12 citations

Loss of Biased Signaling Specificity of the Angiotensin II Type 1 Receptor in Overexpressed Systems

The FASEB Journal · 2022-05-01

G protein‐coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β‐arrestins. [Sarcosine 1 Ile 4 Ile 8 ]‐angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT 1 R), fails to activate G protein in multiple primary cell lines. Despite this, SII induces phosphorylation of the extracellular signal‐regulated kinases 1 and 2 (ERK1/2) through β‐arrestin‐2‐dependent mechanisms. However, studies reliant on exogenous overexpression of the AT 1 R indicate that SII is a partial agonist for G protein signaling and lacks β‐arrestin‐exclusive signaling specificity. We investigated this apparent discrepancy by profiling changes in signaling specificity at the AT­­ 1 R with increasing levels of receptor expression. We hypothesized that overexpression of the AT 1 R causes loss of signaling pathway specificity, such that ligands capable of inducing only β‐arrestin‐mediated responses at lower physiologic receptor expression levels gain the ability to activate G proteins upon receptor overexpression. We established a tetracycline‐inducible cellular system for titratable expression of the AT 1 R to assess signaling responses at varying levels of receptor expression when stimulated with AngII, SII, and four other AngII analogs with different signaling biases. Unbiased and G protein‐biased ligands activated dose‐dependent calcium responses at all levels of AT 1 R expression. In contrast, β‐arrestin‐biased ligands induced dose‐dependent calcium signaling only at higher levels of receptor overexpression. Using inhibitors of different G proteins, we demonstrated that both G i and G q mediated this overexpression‐dependent calcium signaling by β‐arrestin‐biased ligands. All ligands induced ERK1/2 phosphorylation at receptor levels below those required for calcium signaling by β‐arrestin‐biased ligands. Thus, receptor overexpression causes loss of biased signaling specificity of AT 1 R ligands, highlighting the potential risks of using such systems to simulate GPCR ligand behavior in physiologically relevant contexts.