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Proceedings of the National Academy of... Oct 2023β-arrestins are multivalent adaptor proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) to inhibit G protein signaling, mediate receptor...
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.
Topics: beta-Arrestins; beta-Arrestin 1; Arrestins; Allosteric Regulation; Signal Transduction; Receptors, G-Protein-Coupled; Phosphorylation; beta-Arrestin 2
PubMed: 37844230
DOI: 10.1073/pnas.2303794120 -
Proceedings of the National Academy of... Aug 2023Catecholamine-stimulated β-adrenergic receptor (βAR) signaling via the canonical G-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions,...
Catecholamine-stimulated β-adrenergic receptor (βAR) signaling via the canonical G-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous β-agonists in the treatment of airway disease. βAR signaling is tightly regulated by GRKs and β-arrestins, which together promote βAR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to βAR signaling toward the G pathway while avoiding β-arrestin-mediated effects may provide a strategy to improve the functional consequences of βAR activation. Since attempts to develop G-biased agonists and allosteric modulators for the βAR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit β-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of β-arrestin recruitment to the βAR while having no effect on βAR coupling to G. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the βAR and protects against the functional desensitization of β-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the βAR with minimal effects on the βAR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the βAR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the βAR.
Topics: beta-Arrestins; Arrestin; Signal Transduction; beta-Arrestin 1; Receptors, Adrenergic; Receptors, Adrenergic, beta-2
PubMed: 37490535
DOI: 10.1073/pnas.2302668120 -
Nature Communications Jul 2023Representing the most attractive and successful druggable receptors of the proteome, GPCRs regulate a myriad of physiological and pathophysiological functions. Although...
Representing the most attractive and successful druggable receptors of the proteome, GPCRs regulate a myriad of physiological and pathophysiological functions. Although over half of present pharmaceuticals target GPCRs, the advancement of drug discovery is hampered by a lack of adequate screening tools, the majority of which are limited to probing agonist-induced G-protein and β-arrestin-2-mediated events as a measure of receptor activation. Here, we develop Tango-Trio, a comprehensive cell-based high-throughput platform comprising cumate-inducible expression of transducers, capable of the parallelized profiling of both basal and agonist-dependent GPCR activities. We capture the functional diversity of GPCRs, reporting β-arrestin-1/2 couplings, selectivities, and receptor internalization signatures across the GPCRome. Moreover, we present the construction of cumate-induced basal activation curves at approximately 200 receptors, including over 50 orphans. Overall, Tango-Trio's robustness is well-suited for the functional characterization and screening of GPCRs, especially for parallel interrogation, and is a valuable addition to the pharmacological toolbox.
Topics: Receptors, G-Protein-Coupled; Ligands; Signal Transduction; beta-Arrestin 2; Drug Discovery; beta-Arrestins
PubMed: 37407564
DOI: 10.1038/s41467-023-39132-x -
British Journal of Pharmacology Mar 2024More than 30 years after their discovery, arrestins are recognised multiprotein scaffolds that play essential roles in G protein-coupled receptor (GPCR) regulation and... (Review)
Review
More than 30 years after their discovery, arrestins are recognised multiprotein scaffolds that play essential roles in G protein-coupled receptor (GPCR) regulation and signalling. Originally named for their capacity to hinder GPCR coupling to G proteins and facilitate receptor desensitisation, arrestins have emerged as key hubs for a myriad of other functions, including receptor internalisation and scaffolding of signalling complexes. Recent structural studies have started to provide snapshots of the complexes formed by GPCRs and arrestins, supporting a wealth of biochemical data delineating the molecular determinants of such interactions. Furthermore, biophysical techniques have also provided key information with regards to the basal and active conformations of arrestins, and how these are affected upon GPCR activation. Here, we review the most recent advances on our understanding of GPCR-arrestin complexes, from structure to interactions of arrestins with the lipid bilayer and other proteins. We also present an updated view on the development of tools to study the conformational flexibility of arrestins, with the potential to provide experimental data to describe the dynamic models of arrestin activation.
PubMed: 38479842
DOI: 10.1111/bph.16331 -
Science Signaling Aug 2023G protein-coupled receptors engage both G proteins and β-arrestins, and their coupling can be biased by ligands and mutations. Here, to resolve structural elements and...
G protein-coupled receptors engage both G proteins and β-arrestins, and their coupling can be biased by ligands and mutations. Here, to resolve structural elements and mechanisms underlying effector coupling to the angiotensin II (AngII) type 1 receptor (AT1R), we combined alanine scanning mutagenesis of the entire sequence of the receptor with pharmacological profiling of Gα and β-arrestin engagement to mutant receptors and molecular dynamics simulations. We showed that Gα coupling to AT1R involved a large number of residues spread across the receptor, whereas fewer structural regions of the receptor contributed to β-arrestin coupling regulation. Residue stretches in transmembrane domain 4 conferred β-arrestin bias and represented an important structural element in AT1R for functional selectivity. Furthermore, we identified allosteric small-molecule binding sites that were enclosed by communities of residues that produced biased signaling when mutated. Last, we showed that allosteric communication within AT1R emanating from the Gα coupling site spread beyond the orthosteric AngII-binding site and across different regions of the receptor, including currently unresolved structural regions. Our findings reveal structural elements and mechanisms within AT1R that bias Gα and β-arrestin coupling and that could be harnessed to design biased receptors for research purposes and to develop allosteric modulators.
Topics: beta-Arrestins; Receptor, Angiotensin, Type 1; beta-Arrestin 1; Signal Transduction; GTP-Binding Proteins; Angiotensin II
PubMed: 37552769
DOI: 10.1126/scisignal.adf2173 -
Molecular Pharmacology Oct 2023Atypical chemokine receptor 3 (ACKR3) is an arrestin-biased receptor that regulates extracellular chemokine levels through scavenging. The scavenging process restricts...
Atypical chemokine receptor 3 (ACKR3) is an arrestin-biased receptor that regulates extracellular chemokine levels through scavenging. The scavenging process restricts the availability of the chemokine agonist CXCL12 for the G protein-coupled receptor (GPCR) CXCR4 and requires phosphorylation of the ACKR3 C-terminus by GPCR kinases (GRKs). ACKR3 is phosphorylated by GRK2 and GRK5, but the mechanisms by which these kinases regulate the receptor are unresolved. Here we determined that GRK5 phosphorylation of ACKR3 results in more efficient chemokine scavenging and -arrestin recruitment than phosphorylation by GRK2 in HEK293 cells. However, co-activation of CXCR4-enhanced ACKR3 phosphorylation by GRK2 through the liberation of G, an accessory protein required for efficient GRK2 activity. The results suggest that ACKR3 "senses" CXCR4 activation through a GRK2-dependent crosstalk mechanism, which enables CXCR4 to influence the efficiency of CXCL12 scavenging and -arrestin recruitment to ACKR3. Surprisingly, we also found that despite the requirement for phosphorylation and the fact that most ligands promote -arrestin recruitment, -arrestins are dispensable for ACKR3 internalization and scavenging, suggesting a yet-to-be-determined function for these adapter proteins. Since ACKR3 is also a receptor for CXCL11 and opioid peptides, these data suggest that such crosstalk may also be operative in cells with CXCR3 and opioid receptor co-expression. Additionally, kinase-mediated receptor cross-regulation may be relevant to other atypical and G protein-coupled receptors that share common ligands. SIGNIFICANCE STATEMENT: The atypical receptor ACKR3 indirectly regulates CXCR4-mediated cell migration by scavenging their shared agonist CXCL12. Here, we show that scavenging and -arrestin recruitment by ACKR3 are primarily dependent on phosphorylation by GRK5. However, we also show that CXCR4 co-activation enhances the contribution of GRK2 by liberating G. This phosphorylation crosstalk may represent a common feedback mechanism between atypical and G protein-coupled receptors with shared ligands for regulating the efficiency of scavenging or other atypical receptor functions.
Topics: Humans; beta-Arrestins; Chemokine CXCL12; G-Protein-Coupled Receptor Kinases; HEK293 Cells; Ligands; Phosphorylation; Protein Binding; Receptors, CXCR4
PubMed: 37474305
DOI: 10.1124/molpharm.123.000710 -
Pharmacological Research Nov 2023Drugs acting at the opioid receptor family are clinically used to treat chronic and acute pain, though they represent the second line of treatment behind GABA analogs,... (Review)
Review
Drugs acting at the opioid receptor family are clinically used to treat chronic and acute pain, though they represent the second line of treatment behind GABA analogs, antidepressants and SSRI's. Within the opioid family mu and kappa opioid receptor are commonly targeted. However, activation of the mu opioid receptor has side effects of constipation, tolerance, dependence, euphoria, and respiratory depression; activation of the kappa opioid receptor leads to dysphoria and sedation. The side effects of mu opioid receptor activation have led to mu receptor drugs being widely abused with great overdose risk. For these reasons, newer safer opioid analgesics are in high demand. For many years a focus within the opioid field was finding drugs that activated the G protein pathway at mu opioid receptor, without activating the β-arrestin pathway, known as biased agonism. Recent advances have shown that this may not be the way forward to develop safer analgesics at mu opioid receptor, though there is still some promise at the kappa opioid receptor. Here we discuss recent novel approaches to develop safer opioid drugs including efficacy vs bias and fine-tuning receptor activation by targeting sub-pockets in the orthosteric site, we explore recent works on the structural basis of bias, and we put forward the suggestion that Gα subtype selectivity may be an exciting new area of interest.
Topics: Humans; Analgesics, Opioid; Receptors, Opioid, mu; Receptors, Opioid, kappa; Receptors, Opioid; Acute Pain
PubMed: 37844653
DOI: 10.1016/j.phrs.2023.106961 -
Assessment of the potential of novel and classical opioids to induce respiratory depression in mice.British Journal of Pharmacology Dec 2023Opioid-induced respiratory depression limits the use of μ-opioid receptor agonists in clinical settings and is the main cause of opioid overdose fatalities. The...
BACKGROUND AND PURPOSE
Opioid-induced respiratory depression limits the use of μ-opioid receptor agonists in clinical settings and is the main cause of opioid overdose fatalities. The relative potential of different opioid agonists to induce respiratory depression at doses exceeding those producing analgesia is understudied despite its relevance to assessments of opioid safety. Here we evaluated the respiratory depressant and anti-nociceptive effects of three novel opioids and relate these measurements to their in vitro efficacy.
EXPERIMENTAL APPROACH
Respiration was measured in awake, freely moving male CD-1 mice using whole body plethysmography. Anti-nociception was measured using the hot plate test. Morphine, oliceridine and tianeptine were administered intraperitoneally, whereas methadone, oxycodone and SR-17018 were administered orally. Receptor activation and arrestin-3 recruitment were measured in HEK293 cells using BRET assays.
KEY RESULTS
Across the dose ranges examined, all opioids studied depressed respiration in a dose-dependent manner, with similar effects at the highest doses, and with tianeptine and oliceridine showing reduced duration of effect, when compared with morphine, oxycodone, methadone and SR-17018. When administered at doses that induced similar respiratory depression, all opioids induced similar anti-nociception, with tianeptine and oliceridine again showing reduced duration of effect. These data were consistent with the in vitro agonist activity of the tested compounds.
CONCLUSION AND IMPLICATIONS
In addition to providing effective anti-nociception, the novel opioids, oliceridine, tianeptine and SR-17018 depress respiration in male mice. However, the different potencies and kinetics of effect between these novel opioids may be relevant to their therapeutic application in different clinical settings.
Topics: Male; Humans; Animals; Mice; Analgesics, Opioid; Oxycodone; HEK293 Cells; Morphine; Respiratory Insufficiency; Methadone
PubMed: 37489013
DOI: 10.1111/bph.16199 -
The Journal of Biological Chemistry Nov 2023β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G...
β-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether β-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., β-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate β-arrestins, thereby limiting the ability to distinguish G protein from β-arrestin-mediated signaling events. We used β2-adrenergic receptor (β2AR) and its β2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gα, β-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gα and β-arrestins in controlling gene expression. We found that Gα is not required for β2AR and β-arrestin conformational changes, β-arrestin recruitment, and receptor internalization, but that Gα dictates the GPCR kinase isoforms involved in β-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of β2AR in wildtype and β-arrestin1/2-KO cells but absent in Gα-KO cells. These results were validated by re-expressing Gα in the corresponding KO cells and silencing β-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of β2AR. Overall, our results support that Gs is essential for β2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas β-arrestins initiate signaling events modulating Gα-driven nuclear transcriptional activity.
Topics: Humans; beta-Arrestin 1; beta-Arrestin 2; beta-Arrestins; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation; GTP-Binding Proteins; Mitogen-Activated Protein Kinases; Phosphorylation; Receptors, Adrenergic, beta-2; HEK293 Cells; GTP-Binding Protein alpha Subunits; Protein Structure, Tertiary; Protein Isoforms; Enzyme Activation
PubMed: 37774973
DOI: 10.1016/j.jbc.2023.105293 -
Pharmacological Reviews May 2024G-protein coupled receptors (GPCRs) transduce a wide range of extracellular signals. They are key players in the majority of biologic functions including vision,... (Review)
Review
G-protein coupled receptors (GPCRs) transduce a wide range of extracellular signals. They are key players in the majority of biologic functions including vision, olfaction, chemotaxis, and immunity. However, as essential as most of them are to body function and homeostasis, overactivation of GPCRs has been implicated in many pathologic diseases such as cancer, asthma, and heart failure (HF). Therefore, an important feature of G protein signaling systems is the ability to control GPCR responsiveness, and one key process to control overstimulation involves initiating receptor desensitization. A number of steps are appreciated in the desensitization process, including cell surface receptor phosphorylation, internalization, and downregulation. Rapid or short-term desensitization occurs within minutes and involves receptor phosphorylation via the action of intracellular protein kinases, the binding of -arrestins, and the consequent uncoupling of GPCRs from their cognate heterotrimeric G proteins. On the other hand, long-term desensitization occurs over hours to days and involves receptor downregulation or a decrease in cell surface receptor protein level. Of the proteins involved in this biologic phenomenon, -arrestins play a particularly significant role in both short- and long-term desensitization mechanisms. In addition, -arrestins are involved in the phenomenon of biased agonism, where the biased ligand preferentially activates one of several downstream signaling pathways, leading to altered cellular responses. In this context, this review discusses the different patterns of desensitization of the -, - and the adrenoceptors and highlights the role of -arrestins in regulating physiologic responsiveness through desensitization and biased agonism. SIGNIFICANCE STATEMENT: A sophisticated network of proteins orchestrates the molecular regulation of GPCR activity. Adrenoceptors are GPCRs that play vast roles in many physiological processes. Without tightly controlled desensitization of these receptors, homeostatic imbalance may ensue, thus precipitating various diseases. Here, we critically appraise the mechanisms implicated in adrenoceptor desensitization. A better understanding of these mechanisms helps identify new druggable targets within the GPCR desensitization machinery and opens exciting therapeutic fronts in the treatment of several pathologies.
Topics: Humans; Animals; Signal Transduction; Receptors, Adrenergic; Receptors, G-Protein-Coupled; beta-Arrestins
PubMed: 38697858
DOI: 10.1124/pharmrev.123.000831