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The Journal of Neuroscience : the... Oct 2020Members of the arrestin superfamily have great propensity of self-association, but the physiological significance of this phenomenon is unclear. To determine the...
Members of the arrestin superfamily have great propensity of self-association, but the physiological significance of this phenomenon is unclear. To determine the biological role of visual arrestin-1 oligomerization in rod photoreceptors, we expressed mutant arrestin-1 with severely impaired self-association in mouse rods and analyzed mice of both sexes. We show that the oligomerization-deficient mutant is capable of quenching rhodopsin signaling normally, as judged by electroretinography and single-cell recording. Like wild type, mutant arrestin-1 is largely excluded from the outer segments in the dark, proving that the normal intracellular localization is not due the size exclusion of arrestin-1 oligomers. In contrast to wild type, supraphysiological expression of the mutant causes shortening of the outer segments and photoreceptor death. Thus, oligomerization reduces the cytotoxicity of arrestin-1 monomer, ensuring long-term photoreceptor survival. Visual arrestin-1 forms dimers and tetramers. The biological role of its oligomerization is unclear. To test the role of arrestin-1 self-association, we expressed oligomerization-deficient mutant in arrestin-1 knock-out mice. The mutant quenches light-induced rhodopsin signaling like wild type, demonstrating that monomeric arrestin-1 is necessary and sufficient for this function. In rods, arrestin-1 moves from the inner segments and cell bodies in the dark to the outer segments in the light. Nonoligomerizing mutant undergoes the same translocation, demonstrating that the size of the oligomers is not the reason for arrestin-1 exclusion from the outer segments in the dark. High expression of oligomerization-deficient arrestin-1 resulted in rod death. Thus, oligomerization reduces the cytotoxicity of high levels of arrestin-1 monomer.
Topics: Adaptation, Ocular; Animals; Arrestins; Cell Survival; Electroretinography; Female; Light Signal Transduction; Male; Mice; Mice, Knockout; Mice, Transgenic; Mutation; Retina; Retinal Rod Photoreceptor Cells; Rhodopsin
PubMed: 32948676
DOI: 10.1523/JNEUROSCI.0749-20.2020 -
Nature Communications Sep 2022β-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of...
β-arrestins mediate regulatory processes for over 800 different G protein-coupled receptors (GPCRs) by adopting specific conformations that result from the geometry of the GPCR-β-arrestin complex. However, whether β-arrestin1 and 2 respond differently for binding to the same GPCR is still unknown. Employing GRK knockout cells and β-arrestins lacking the finger-loop-region, we show that the two isoforms prefer to associate with the active parathyroid hormone 1 receptor (PTH1R) in different complex configurations ("hanging" and "core"). Furthermore, the utilisation of advanced NanoLuc/FlAsH-based biosensors reveals distinct conformational signatures of β-arrestin1 and 2 when bound to active PTH1R (P-R*). Moreover, we assess β-arrestin conformational changes that are induced specifically by proximal and distal C-terminal phosphorylation and in the absence of GPCR kinases (GRKs) (R*). Here, we show differences between conformational changes that are induced by P-R* or R* receptor states and further disclose the impact of site-specific GPCR phosphorylation on arrestin-coupling and function.
Topics: Arrestins; G-Protein-Coupled Receptor Kinases; Luciferases; Parathyroid Hormone; Phosphorylation; Protein Isoforms; Receptors, G-Protein-Coupled; Signal Transduction; beta-Arrestin 1; beta-Arrestin 2; beta-Arrestins
PubMed: 36163356
DOI: 10.1038/s41467-022-33307-8 -
International Journal of Molecular... Apr 2022α-Arrestins, also called arrestin-related trafficking adaptors (ARTs), constitute a large family of proteins conserved from yeast to humans. Despite their evolutionary... (Review)
Review
α-Arrestins, also called arrestin-related trafficking adaptors (ARTs), constitute a large family of proteins conserved from yeast to humans. Despite their evolutionary precedence over their extensively studied relatives of the β-arrestin family, α-arrestins have been discovered relatively recently, and thus their properties are mostly unexplored. The predominant function of α-arrestins is the selective identification of membrane proteins for ubiquitination and degradation, which is an important element in maintaining membrane protein homeostasis as well as global cellular metabolisms. Among members of the arrestin clan, only α-arrestins possess PY motifs that allow canonical binding to WW domains of Rsp5/NEDD4 ubiquitin ligases and the subsequent ubiquitination of membrane proteins leading to their vacuolar/lysosomal degradation. The molecular mechanisms of the selective substrate's targeting, function, and regulation of α-arrestins in response to different stimuli remain incompletely understood. Several functions of α-arrestins in animal models have been recently characterized, including redox homeostasis regulation, innate immune response regulation, and tumor suppression. However, the molecular mechanisms of α-arrestin regulation and substrate interactions are mainly based on observations from the yeast model. Nonetheless, α-arrestins have been implicated in health disorders such as diabetes, cardiovascular diseases, neurodegenerative disorders, and tumor progression, placing them in the group of potential therapeutic targets.
Topics: Animals; Arrestin; Arrestins; Endocytosis; Humans; Neoplasms; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitination
PubMed: 35563378
DOI: 10.3390/ijms23094988 -
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 -
Antioxidants & Redox Signaling May 2022Adaptor proteins control the spatiotemporal dynamics of cellular signaling. Dysregulation of adaptor protein function can cause aberrant cell signaling and promote... (Review)
Review
Adaptor proteins control the spatiotemporal dynamics of cellular signaling. Dysregulation of adaptor protein function can cause aberrant cell signaling and promote cancer. The arrestin family of adaptor proteins are known to regulate signaling by the superfamily of G protein-coupled receptors (GPCRs). The GPCRs are highly druggable and implicated in cancer progression. However, the molecular mechanisms responsible for arrestin dysregulation and the impact on GPCR function in cancer have yet to be fully elucidated. A new family of mammalian arrestins, termed the α-arrestins, was recently discovered. The α-arrestin, arrestin domain-containing protein 3 (ARRDC3), in particular, has been identified as a tumor suppressor and is reported to control cellular signaling of GPCRs in cancer. Compared with the extensively studied mammalian β-arrestins, there is limited information regarding the regulatory mechanisms that control α-arrestin activation and function. Here, we discuss the molecular mechanisms that regulate ARRDC3, which include post-translational modifications such as phosphorylation and ubiquitination. We also provide evidence that ARRDC3 can interact with a wide array of proteins that control diverse biological functions. ARRDC3 interacts with numerous proteins and is likely to display diverse functions in cancer, metabolic disease, and other syndromes. Thus, understanding the regulatory mechanisms of ARRDC3 activity in various cellular contexts is critically important. Recent studies suggest that α-arrestins may be regulated through post-translational modification, which is known to impact adaptor protein function. However, additional studies are needed to determine how these regulatory mechanisms affect ARRDC3 tumor suppressor function. 36, 1066-1079.
Topics: Adaptor Proteins, Signal Transducing; Animals; Arrestin; Arrestins; Mammals; Neoplasms; Receptors, G-Protein-Coupled; Ubiquitination
PubMed: 34465145
DOI: 10.1089/ars.2021.0193 -
British Journal of Pharmacology May 2024GPR84 is an understudied rhodopsin-like class A G protein-coupled receptor, which is arousing particular interest from a therapeutic perspective. Not least this reflects... (Review)
Review
GPR84 is an understudied rhodopsin-like class A G protein-coupled receptor, which is arousing particular interest from a therapeutic perspective. Not least this reflects that gpr84 expression is significantly up-regulated following acute inflammatory stimuli and in inflammatory diseases, and that receptor activation plays a role in regulating pro-inflammatory responses and migration of cells of the innate immune system such as neutrophils, monocytes, macrophages and microglia. Although most physiological responses of GPR84 reflect receptor coupling to G-proteins, several studies indicate that agonist-activated GPR84 can recruit arrestin adaptor proteins and this regulates receptor internalisation and desensitisation. To date, little is known on the patterns of either basal or ligand regulated GPR84 phosphorylation and how these might control these processes. Here, we consider what is known about the regulation of GPR84 signalling with a focus on how G protein receptor kinase-mediated phosphorylation regulates arrestin protein recruitment and receptor function. LINKED ARTICLES: This article is part of a themed issue GPR84 Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.10/issuetoc.
Topics: Arrestin; Macrophages; Phosphorylation; Receptors, G-Protein-Coupled; Signal Transduction; Humans
PubMed: 37085331
DOI: 10.1111/bph.16098 -
Progress in Molecular Biology and... 2018Arrestins were discovered as proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) and block their interactions with G proteins, i.e., for their... (Review)
Review
Arrestins were discovered as proteins that bind active phosphorylated G protein-coupled receptors (GPCRs) and block their interactions with G proteins, i.e., for their role in homologous desensitization of GPCRs. Mammals express only four arrestin subtypes, two of which are largely restricted to the retina. Two nonvisual arrestins are ubiquitous and interact with hundreds of different GPCRs and dozens of other binding partners. Changes of just a few residues on the receptor-binding surface were shown to dramatically affect GPCR preference of inherently promiscuous nonvisual arrestins. Mutations on the cytosol-facing side of arrestins modulate their interactions with individual downstream signaling molecules. Thus, it appears feasible to construct arrestin mutants specifically linking particular GPCRs with signaling pathways of choice or mutants that sever the links between selected GPCRs and unwanted pathways. Signaling-biased "designer arrestins" have the potential to become valuable molecular tools for research and therapy.
Topics: Animals; Arrestins; Humans; Models, Molecular; Phosphorylation; Signal Transduction
PubMed: 30470292
DOI: 10.1016/bs.pmbts.2018.07.007 -
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 Nov 2017A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP) is...
A unique aspect of arrestin-3 is its ability to support both receptor-dependent and receptor-independent signaling. Here, we show that inositol hexakisphosphate (IP) is a non-receptor activator of arrestin-3 and report the structure of IP-activated arrestin-3 at 2.4-Å resolution. IP-activated arrestin-3 exhibits an inter-domain twist and a displaced C-tail, hallmarks of active arrestin. IP binds to the arrestin phosphate sensor, and is stabilized by trimerization. Analysis of the trimerization surface, which is also the receptor-binding surface, suggests a feature called the finger loop as a key region of the activation sensor. We show that finger loop helicity and flexibility may underlie coupling to hundreds of diverse receptors and also promote arrestin-3 activation by IP. Importantly, we show that effector-binding sites on arrestins have distinct conformations in the basal and activated states, acting as switch regions. These switch regions may work with the inter-domain twist to initiate and direct arrestin-mediated signaling.
Topics: Amino Acid Sequence; Animals; Arrestins; Binding Sites; Cattle; Crystallography, X-Ray; Humans; Mitogen-Activated Protein Kinase 10; Models, Molecular; Phytic Acid; Protein Conformation; Protein Structure, Quaternary; Recombinant Proteins; Signal Transduction
PubMed: 29127291
DOI: 10.1038/s41467-017-01218-8