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PloS One 2014In mammals, the expression of the unusual visual pigment, melanopsin, is restricted to a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs),...
In mammals, the expression of the unusual visual pigment, melanopsin, is restricted to a small subset of intrinsically photosensitive retinal ganglion cells (ipRGCs), whose signaling regulate numerous non-visual functions including sleep, circadian photoentrainment and pupillary constriction. IpRGCs exhibit attenuated electrical responses following sequential and prolonged light exposures indicative of an adaptational response. The molecular mechanisms underlying deactivation and adaptation in ipRGCs however, have yet to be fully elucidated. The role of melanopsin phosphorylation and β-arrestin binding in this adaptive process is suggested by the phosphorylation-dependent reduction of melanopsin signaling in vitro and the ubiquitous expression of β-arrestin in the retina. These observations, along with the conspicuous absence of visual arrestin in ipRGCs, suggest that a β-arrestin terminates melanopsin signaling. Here, we describe a light- and phosphorylation- dependent reduction in melanopsin signaling mediated by both β-arrestin 1 and β-arrestin 2. Using an in vitro calcium imaging assay, we demonstrate that increasing the cellular concentration of β-arrestin 1 and β-arrestin 2 significantly increases the rate of deactivation of light-activated melanopsin in HEK293 cells. Furthermore, we show that this response is dependent on melanopsin carboxyl-tail phosphorylation. Crosslinking and co-immunoprecipitation experiments confirm β-arrestin 1 and β-arrestin 2 bind to melanopsin in a light- and phosphorylation- dependent manner. These data are further supported by proximity ligation assays (PLA), which demonstrate a melanopsin/β-arrestin interaction in HEK293 cells and ipRGCs. Together, these results suggest that melanopsin signaling is terminated in a light- and phosphorylation-dependent manner through the binding of a β-arrestin within the retina.
Topics: Animals; Arrestins; Blotting, Western; Cells, Cultured; HEK293 Cells; Humans; Immunoprecipitation; Light; Mice; Mice, Inbred C57BL; Phosphorylation; Photic Stimulation; Polymerase Chain Reaction; Retinal Ganglion Cells; Rod Opsins; Signal Transduction; beta-Arrestin 1; beta-Arrestin 2; beta-Arrestins
PubMed: 25401926
DOI: 10.1371/journal.pone.0113138 -
Cell Dec 2020Binding of arrestin to phosphorylated G-protein-coupled receptors (GPCRs) controls many aspects of cell signaling. The number and arrangement of phosphates may vary...
Binding of arrestin to phosphorylated G-protein-coupled receptors (GPCRs) controls many aspects of cell signaling. The number and arrangement of phosphates may vary substantially for a given GPCR, and different phosphorylation patterns trigger different arrestin-mediated effects. Here, we determine how GPCR phosphorylation influences arrestin behavior by using atomic-level simulations and site-directed spectroscopy to reveal the effects of phosphorylation patterns on arrestin binding and conformation. We find that patterns favoring binding differ from those favoring activation-associated conformational change. Both binding and conformation depend more on arrangement of phosphates than on their total number, with phosphorylation at different positions sometimes exerting opposite effects. Phosphorylation patterns selectively favor a wide variety of arrestin conformations, differently affecting arrestin sites implicated in scaffolding distinct signaling proteins. We also reveal molecular mechanisms of these phenomena. Our work reveals the structural basis for the long-standing "barcode" hypothesis and has important implications for design of functionally selective GPCR-targeted drugs.
Topics: Arrestin; Computer Simulation; HEK293 Cells; Humans; Phosphates; Phosphopeptides; Phosphorylation; Protein Binding; Protein Conformation; Receptors, G-Protein-Coupled; Signal Transduction; Spectrum Analysis
PubMed: 33296703
DOI: 10.1016/j.cell.2020.11.014 -
Angewandte Chemie (International Ed. in... Jun 2022The μ-opioid receptor (μOR) is the major target for opioid analgesics. Activation of μOR initiates signaling through G protein pathways as well as through β-arrestin...
The μ-opioid receptor (μOR) is the major target for opioid analgesics. Activation of μOR initiates signaling through G protein pathways as well as through β-arrestin recruitment. μOR agonists that are biased towards G protein signaling pathways demonstrate diminished side effects. PZM21, discovered by computational docking, is a G protein biased μOR agonist. Here we report the cryoEM structure of PZM21 bound μOR in complex with G protein. Structure-based evolution led to multiple PZM21 analogs with more pronounced G protein bias and increased lipophilicity to improve CNS penetration. Among them, FH210 shows extremely low potency and efficacy for arrestin recruitment. We further determined the cryoEM structure of FH210 bound to μOR in complex with G protein and confirmed its expected binding pose. The structural and pharmacological studies reveal a potential mechanism to reduce β-arrestin recruitment by the μOR, and hold promise for developing next-generation analgesics with fewer adverse effects.
Topics: Analgesics, Opioid; GTP-Binding Proteins; Receptors, Opioid, mu; Signal Transduction; beta-Arrestins
PubMed: 35385593
DOI: 10.1002/anie.202200269 -
Structure (London, England : 1993) Mar 2020Arrestins desensitize and/or internalize G-protein-coupled receptors by interacting with phosphorylated receptors. A few studies have reported that arrestins themselves...
Arrestins desensitize and/or internalize G-protein-coupled receptors by interacting with phosphorylated receptors. A few studies have reported that arrestins themselves can be phosphorylated, and the phosphorylation status modulates their cellular functions. However, the effects of phosphorylation on arrestin structure have not been studied. Here, we investigated the conformational changes in β-arrestin-1 and -2 upon incorporation of phospho-mimetic mutations into the known phosphorylation sites (i.e., S412D for β-arrestin-1 and S14D, T276D, S14D/T276D, S361D, T383D, and S361D/T383D for β-arrestin-2) by using hydrogen/deuterium-exchange mass spectrometry (HDX-MS). HDX-MS analysis suggested that β-arrestin-2 S14D/T276D shows an HDX profile similar to the pre-active states, resulting in increased interaction with receptors. Phospho-mimetic mutation at corresponding residues of β-arrestin-1 (i.e., S13D/T275D) induced similar conformational and functional consequences, and the detailed structural changes related to β-arrestin-1 S13D/T275D were investigated further by X-ray crystallography.
Topics: Amino Acid Sequence; Animals; Crystallography, X-Ray; Hydrogen Deuterium Exchange-Mass Spectrometry; Models, Molecular; Mutation; Phosphorylation; Protein Binding; Protein Conformation; Rats; beta-Arrestin 1; beta-Arrestin 2
PubMed: 31948726
DOI: 10.1016/j.str.2019.12.008 -
International Journal of Molecular... Aug 2022Arrestins were first discovered as suppressors of G protein-mediated signaling by G protein-coupled receptors. It was later demonstrated that arrestins also initiate...
Arrestins were first discovered as suppressors of G protein-mediated signaling by G protein-coupled receptors. It was later demonstrated that arrestins also initiate several signaling branches, including mitogen-activated protein kinase cascades. Arrestin-3-dependent activation of the JNK family can be recapitulated with peptide fragments, which are monofunctional elements distilled from this multi-functional arrestin protein. Here, we use maltose-binding protein fusions of arrestin-3-derived peptides to identify arrestin elements that bind kinases of the ASK1-MKK4/7-JNK3 cascade and the shortest peptide facilitating JNK signaling. We identified a 16-residue arrestin-3-derived peptide expressed as a Venus fusion that leads to activation of JNK3α2 in cells. The strength of the binding to the kinases does not correlate with peptide activity. The ASK1-MKK4/7-JNK3 cascade has been implicated in neuronal apoptosis. While inhibitors of MAP kinases exist, short peptides are the first small molecule tools that can activate MAP kinases.
Topics: Arrestin; Arrestins; Mitogen-Activated Protein Kinase 10; Peptides; Phosphorylation; Protein Binding; beta-Arrestin 2; beta-Arrestins
PubMed: 35955810
DOI: 10.3390/ijms23158679 -
American Journal of Physiology. Cell... May 2022G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and are the target of approximately one-third of all Food and Drug Administration... (Review)
Review
G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors and are the target of approximately one-third of all Food and Drug Administration (FDA)-approved pharmaceutical drugs. GPCRs interact with many transducers, such as heterotrimeric G proteins, GPCR kinases (GRKs), and β-arrestins. Recent experiments have demonstrated that some ligands can activate distinct effector proteins over others, a phenomenon termed "biased agonism." These discoveries have raised the potential of developing drugs which preferentially activate therapeutic signaling pathways over those that lead to deleterious side effects. However, to date, only one biased GPCR therapeutic has received FDA approval and many others have either failed to meet their specified primary end points and or demonstrate superiority over currently available treatments. In addition, there is a lack of understanding regarding how biased agonism measured at a GPCR leads to specific downstream physiological responses. Here, we briefly summarize the history and current status of biased agonism at GPCRs and suggest adoption of a "systems pharmacology" approach upon which to develop GPCR-targeted drugs that demonstrate heightened therapeutic efficacy with improved side effect profiles.
Topics: Ligands; Network Pharmacology; Receptors, G-Protein-Coupled; Signal Transduction; beta-Arrestins
PubMed: 35196164
DOI: 10.1152/ajpcell.00449.2021 -
Scientific Reports Dec 2017G protein-coupled receptors (GPCRs) constitute a large family of membrane proteins that plays a key role in transmembrane signal transduction and draw wide attention...
G protein-coupled receptors (GPCRs) constitute a large family of membrane proteins that plays a key role in transmembrane signal transduction and draw wide attention since it was discovered. Arrestin is a small family of proteins which can bind to GPCRs, block G protein interactions and redirect signaling to G-protein-independent pathways. The detailed mechanism of how arrestin interacts with GPCR remains elusive. Here, we conducted molecular dynamics simulations with coarse-grained (CG) and all-atom (AA) models to study the complex structure formed by arrestin and rhodopsin, a prototypical GPCR, in a POPC bilayer. Our results indicate that the formation of the complex has a significant impact on arrestin which is tightly anchored onto the bilayer surface, while has a minor effect on the orientation of rhodopsin in the lipid bilayer. The formation of the complex induces an internal change of conformation and flexibility in both rhodopsin and arrestin, mainly at the binding interface. Further investigation on the interaction interface identified the hydrogen bond network, especially the long-lived hydrogen bonds, and the key residues at the contact interface, which are responsible for stabilizing the complex. These results help us to better understand how rhodopsin interacts with arrestin on membranes, and thereby shed lights on arrestin-mediated signal transduction through GPCRs.
Topics: Arrestin; Hydrogen Bonding; Lipid Bilayers; Molecular Dynamics Simulation; Multiprotein Complexes; Mutation; Phosphatidylcholines; Protein Conformation; Protein Stability; Rhodopsin
PubMed: 29209002
DOI: 10.1038/s41598-017-17243-y -
Cells Jun 2023Arrestins bind active phosphorylated G protein-coupled receptors (GPCRs). Among the four mammalian subtypes, only arrestin-3 facilitates the activation of JNK3 in cells....
Arrestins bind active phosphorylated G protein-coupled receptors (GPCRs). Among the four mammalian subtypes, only arrestin-3 facilitates the activation of JNK3 in cells. In available structures, Lys-295 in the lariat loop of arrestin-3 and its homologue Lys-294 in arrestin-2 directly interact with the activator-attached phosphates. We compared the roles of arrestin-3 conformational equilibrium and Lys-295 in GPCR binding and JNK3 activation. Several mutants with enhanced ability to bind GPCRs showed much lower activity towards JNK3, whereas a mutant that does not bind GPCRs was more active. The subcellular distribution of mutants did not correlate with GPCR recruitment or JNK3 activation. Charge neutralization and reversal mutations of Lys-295 differentially affected receptor binding on different backgrounds but had virtually no effect on JNK3 activation. Thus, GPCR binding and arrestin-3-assisted JNK3 activation have distinct structural requirements, suggesting that facilitation of JNK3 activation is the function of arrestin-3 that is not bound to a GPCR.
Topics: Animals; beta-Arrestin 2; Phosphorylation; Arrestins; Receptors, G-Protein-Coupled; Protein Binding; Mammals
PubMed: 37371033
DOI: 10.3390/cells12121563 -
Scientific Reports Jan 2019Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin-arrestin-1 complexes that eventually decay to opsin,...
Arrestin-1 desensitizes the activated and phosphorylated photoreceptor rhodopsin by forming transient rhodopsin-arrestin-1 complexes that eventually decay to opsin, retinal and arrestin-1. Via a multi-dimensional screening setup, we identified and combined arrestin-1 mutants that form lasting complexes with light-activated and phosphorylated rhodopsin in harsh conditions, such as high ionic salt concentration. Two quadruple mutants, D303A + T304A + E341A + F375A and R171A + T304A + E341A + F375A share similar heterologous expression and thermo-stability levels with wild type (WT) arrestin-1, but are able to stabilize complexes with rhodopsin with more than seven times higher half-maximal inhibitory concentration (IC) values for NaCl compared to the WT arrestin-1 protein. These quadruple mutants are also characterized by higher binding affinities to phosphorylated rhodopsin, light-activated rhodopsin and phosphorylated opsin, as compared with WT arrestin-1. Furthermore, the assessed arrestin-1 mutants are still specifically associating with phosphorylated or light-activated receptor states only, while binding to the inactive ground state of the receptor is not significantly altered. Additionally, we propose a novel functionality for R171 in stabilizing the inactive arrestin-1 conformation as well as the rhodopsin-arrestin-1 complex. The achieved stabilization of the active rhodopsin-arrestin-1 complex might be of great interest for future structure determination, antibody development studies as well as drug-screening efforts targeting G protein-coupled receptors (GPCRs).
Topics: Animals; Arrestins; Cattle; HEK293 Cells; Humans; Models, Molecular; Multiprotein Complexes; Mutation; Opsins; Phosphorylation; Protein Binding; Protein Conformation; Protein Engineering; Protein Stability; Rhodopsin
PubMed: 30679635
DOI: 10.1038/s41598-018-36881-4 -
Molecular Pharmacology Sep 2015The fact that over 30% of current pharmaceuticals target heptahelical G protein-coupled receptors (GPCRs) attests to their tractability as drug targets. Although GPCR... (Review)
Review
The fact that over 30% of current pharmaceuticals target heptahelical G protein-coupled receptors (GPCRs) attests to their tractability as drug targets. Although GPCR drug development has traditionally focused on conventional agonists and antagonists, the growing appreciation that GPCRs mediate physiologically relevant effects via both G protein and non-G protein effectors has prompted the search for ligands that can "bias" downstream signaling in favor of one or the other process. Biased ligands are novel entities with distinct signaling profiles dictated by ligand structure, and the potential prospect of biased ligands as better drugs has been pleonastically proclaimed. Indeed, preclinical proof-of-concept studies have demonstrated that both G protein and arrestin pathway-selective ligands can promote beneficial effects in vivo while simultaneously antagonizing deleterious ones. But along with opportunity comes added complexity and new challenges for drug discovery. If ligands can be biased, then ligand classification becomes assay dependent, and more nuanced screening approaches are needed to capture ligand efficacy across several dimensions of signaling. Moreover, because the signaling repertoire of biased ligands differs from that of the native agonist, unpredicted responses may arise in vivo as these unbalanced signals propagate. For any given GPCR target, establishing a framework relating in vitro efficacy to in vivo biologic response is crucial to biased drug discovery. This review discusses approaches to describing ligand efficacy in vitro, translating ligand bias into biologic response, and developing a systems-level understanding of biased agonism in vivo, with the overall goal of overcoming current barriers to developing biased GPCR therapeutics.
Topics: Animals; Arrestins; Humans; Kinetics; Protein Binding; Receptors, G-Protein-Coupled; Signal Transduction
PubMed: 26134495
DOI: 10.1124/mol.115.099630