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Cellular Signalling Jan 2018It is increasingly apparent that ligand structure influences both the efficiency with which G protein-coupled receptors (GPCRs) engage their downstream effectors and the... (Review)
Review
It is increasingly apparent that ligand structure influences both the efficiency with which G protein-coupled receptors (GPCRs) engage their downstream effectors and the manner in which they are activated. Thus, 'biased' agonists, synthetic ligands whose intrinsic efficacy differs from the native ligand, afford a strategy for manipulating GPCR signaling in ways that promote beneficial signals while blocking potentially deleterious ones. Still, there are significant challenges in relating in vitro ligand efficacy, which is typically measured in heterologous expression systems, to the biological response in vivo, where the ligand is acting on natively expressed receptors and in the presence of the endogenous ligand. This is particularly true of arrestin pathway-selective 'biased' agonists. The type 1 parathyroid hormone receptor (PTHR) is a case in point. Parathyroid hormone (PTH) is the principal physiological regulator of calcium homeostasis, and PTHR expressed on cells of the osteoblast lineage are an established therapeutic target in osteoporosis. In vitro, PTHR signaling is highly sensitive to ligand structure, and PTH analogs that affect the selectivity/kinetics of G protein coupling or that engage arrestin-dependent signaling mechanisms without activating heterotrimeric G proteins have been identified. In vivo, intermittent administration of conventional PTH analogs accelerates the rate of osteoblastic bone formation, largely through known cAMP-dependent mechanisms. Paradoxically, both intermittent and continuous administration of an arrestin pathway-selective PTH analog, which in vivo would be expected to antagonize endogenous PTHR-cAMP signaling, also increases bone mass. Transcriptomic analysis of tissue from treated animals suggests that conventional and arrestin pathway-selective PTH1R ligands act in largely different ways, with the latter principally affecting pathways involved in the regulation of cell cycle, survival, and migration/cytoskeletal dynamics. Such multi-dimensional in vitro and in vivo analyses of ligand bias may provide insights into the physiological roles of non-canonical arrestin-mediated signaling pathways in vivo, and provide a conceptual framework for translating arrestin pathway-selective ligands into viable therapeutics.
Topics: Animals; Arrestins; Drug Design; Humans; In Vitro Techniques; Ligands; Models, Animal; Osteogenesis; Osteoporosis; Parathyroid Hormone; Receptor, Parathyroid Hormone, Type 1; Receptors, G-Protein-Coupled
PubMed: 28495495
DOI: 10.1016/j.cellsig.2017.05.002 -
Current Drug Targets 2020Behçet's disease (BD) is presumably an autoinflammatory disease of unknown etiology for which several animal models have been described over the years. Agents and... (Review)
Review
Behçet's disease (BD) is presumably an autoinflammatory disease of unknown etiology for which several animal models have been described over the years. Agents and methods used for the development of these models have ranged from the herpes simplex type one virus (hsv-1) pathogen to the use of transgenic mice. Other models have also been used to investigate a possible autoimmune component. Each model possesses its own unique set of benefits and shortcomings, with no one model fully being able to recapitulate the disease phenotype. Here, we review the proposed models and provide commentary on their effectiveness and usefulness in studying the disease.
Topics: Animals; Antibodies; Arrestin; Behcet Syndrome; Disease Models, Animal; HLA-B51 Antigen; Heat-Shock Proteins; Herpesvirus 1, Human; Humans; Tropomyosin
PubMed: 32682369
DOI: 10.2174/1389450121666200719010425 -
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 -
Frontiers in Endocrinology 2023β-arrestins, which have multiple cellular functions, were initially described as proteins that desensitize rhodopsin and other G protein-coupled receptors. The... (Review)
Review
β-arrestins, which have multiple cellular functions, were initially described as proteins that desensitize rhodopsin and other G protein-coupled receptors. The cytoskeletal system plays a role in various cellular processes, including intracellular transport, cell division, organization of organelles, and cell cycle. The interactome of β-arrestins includes the major proteins of the three main cytoskeletal systems: tubulins for microtubules, actins for the actin filaments, and vimentin for intermediate filaments. β-arrestins bind to microtubules and regulate their activity by recruiting signaling proteins and interacting with assembly proteins that regulate the actin cytoskeleton and the intermediate filaments. Altered regulation of the cytoskeletal system plays an essential role in the development of Alzheimer's, Parkinson's and other neurodegenerative diseases. Thus, β-arrestins, which interact with the cytoskeleton, were implicated in the pathogenesis progression of these diseases and are potential targets for the treatment of neurodegenerative disorders in the future.
Topics: Humans; beta-Arrestins; Cytoskeleton; Microtubules; Actins; beta-Arrestin 1; Neurodegenerative Diseases
PubMed: 36843600
DOI: 10.3389/fendo.2023.957981 -
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 -
Current Opinion in Structural Biology Aug 2022Agonist-induced recruitment of β-arrestins (βarrs) to G protein-coupled receptors (GPCRs) plays a central role in regulating the spatio-temporal aspects of GPCR... (Review)
Review
Agonist-induced recruitment of β-arrestins (βarrs) to G protein-coupled receptors (GPCRs) plays a central role in regulating the spatio-temporal aspects of GPCR signaling. Several recent studies have provided novel structural and functional insights into our understanding of GPCR-βarr interaction, subsequent βarr activation and resulting functional outcomes. In this review, we discuss these recent advances with a particular emphasis on recognition of receptor-bound phosphates by βarrs, the emerging concept of spatial positioning of key phosphorylation sites, the conformational transition in βarrs during partial to full-engagement, and structural differences driving functional outcomes of βarr isoforms. We also highlight the key directions that require further investigation going forward to fully understand the structural mechanisms driving βarr activation and functional responses.
Topics: Phosphorylation; Protein Isoforms; Receptors, G-Protein-Coupled; Signal Transduction; beta-Arrestins
PubMed: 35738165
DOI: 10.1016/j.sbi.2022.102406 -
Nature Communications Jan 2022G protein-coupled receptors (GPCRs) activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and the formation of receptor-arrestin...
G protein-coupled receptors (GPCRs) activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and the formation of receptor-arrestin complexes. However, the impact of individual GRKs on arrestin binding is not clear. We report the creation of eleven combinatorial HEK293 knockout cell clones lacking GRK2/3/5/6, including single, double, triple and the quadruple GRK knockout. Analysis of β-arrestin1/2 interactions for twelve GPCRs in our GRK knockout cells enables the differentiation of two main receptor subsets: GRK2/3-regulated and GRK2/3/5/6-regulated receptors. Furthermore, we identify GPCRs that interact with β-arrestins via the overexpression of specific GRKs even in the absence of agonists. Finally, using GRK knockout cells, PKC inhibitors and β-arrestin mutants, we present evidence for differential receptor-β-arrestin1/2 complex configurations mediated by selective engagement of kinases. We anticipate our GRK knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and β-arrestin complex formation.
Topics: Arrestin; G-Protein-Coupled Receptor Kinases; GTP-Binding Proteins; HEK293 Cells; Humans; Phosphorylation; Receptors, G-Protein-Coupled; Signal Transduction; beta-Arrestin 1; beta-Arrestin 2
PubMed: 35087057
DOI: 10.1038/s41467-022-28152-8 -
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 -
Pharmacology, Biochemistry, and Behavior May 2022The kappa opioid receptor is a known regulator of ethanol consumption, but the molecular mechanisms behind its actions have been underexplored. The scaffolding protein...
The kappa opioid receptor is a known regulator of ethanol consumption, but the molecular mechanisms behind its actions have been underexplored. The scaffolding protein β-arrestin 2 has previously been implicated in driving ethanol consumption at the related delta opioid receptor and has also been suggested to be a driver behind other negative kappa opioid receptor mediated effects. Here, we used kappa opioid agonists with different efficacies for recruiting β-arrestin 2 and knockout animals to determine whether there is a role for β-arrestin 2 in the modulation of voluntary ethanol consumption by the kappa opioid receptor. We find that an agonist with low β-arrestin 2 efficacy more consistently lowers ethanol consumption than agonists with high efficacy for β-arrestin 2. However, knockdown of β-arrestin 2 amplifies the ethanol consumption-promoting effects of the arrestin-recruiting kappa agonists U50,488 and nalfurafine. We control for potentially confounding sedative effects at the kappa opioid receptor and find that β-arrestin 2 is not necessary for kappa opioid receptor-mediated sedation, and that sedation does not correlate with effects on ethanol consumption. Overall, the results suggest a complex relationship between agonist profile, sex, and kappa opioid receptor modulation of ethanol consumption, with little role for kappa opioid receptor-mediated sedation.
Topics: Alcohol Drinking; Analgesics, Opioid; Animals; Ethanol; Receptors, Opioid, kappa; beta-Arrestin 2; beta-Arrestins
PubMed: 35364122
DOI: 10.1016/j.pbb.2022.173377 -
Trends in Endocrinology and Metabolism:... Feb 2021β-Arrestin-1 and -2 are intracellular proteins that are able to inhibit signaling via G protein-coupled receptors (GPCRs). However, both proteins can also modulate... (Review)
Review
β-Arrestin-1 and -2 are intracellular proteins that are able to inhibit signaling via G protein-coupled receptors (GPCRs). However, both proteins can also modulate cellular functions in a G protein-independent fashion. During the past few years, studies with mutant mice selectivity lacking β-arrestin-1 and/or -2 in metabolically important cell types have led to novel insights into the mechanisms through which β-arrestins regulate key metabolic processes in vivo, including whole-body glucose and energy homeostasis. The novel information gained from these studies should inform the development of novel drugs, including β-arrestin- or G protein-biased GPCR ligands, that could prove useful for the therapy of several important pathophysiological conditions, including type 2 diabetes and obesity.
Topics: Animals; Humans; Mice; Protein Binding; Receptors, G-Protein-Coupled; Signal Transduction; beta-Arrestins
PubMed: 33358450
DOI: 10.1016/j.tem.2020.11.008