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Using In Vitro Pull-Down and In-Cell Overexpression Assays to Study Protein Interactions with Arrestin.
Perry NA, Zhan X, Gurevich EV, Iverson TM, Gurevich VV
(2019) Methods Mol Biol 1957: 107-120
MeSH Terms: Animals, Arrestin, Biological Assay, COS Cells, Cercopithecus aethiops, HEK293 Cells, Humans, Immobilized Proteins, Mice, Protein Binding, Protein Interaction Mapping, Recombinant Fusion Proteins
Show Abstract · Added April 1, 2019
Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor (GPCR) subtypes and dozens of non-receptor signaling proteins. Here we describe the methods used to identify the interaction sites of arrestin-binding partners on arrestin-3 and the use of monofunctional individual arrestin-3 elements in cells. Our in vitro pull-down assay with purified proteins demonstrates that relatively few elements in arrestin engage each partner, whereas cell-based functional assays indicate that certain arrestin elements devoid of other functionalities can perform individual functions in living cells.
0 Communities
1 Members
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12 MeSH Terms
Myosin IIA drives membrane bleb retraction.
Taneja N, Burnette DT
(2019) Mol Biol Cell 30: 1051-1059
MeSH Terms: Actins, Animals, Blister, COS Cells, Cell Membrane, Cell Membrane Structures, Cell Movement, Cell Surface Extensions, Cercopithecus aethiops, Cytokinesis, Cytoplasm, Cytoskeletal Proteins, HeLa Cells, Humans, Myosin Type II, Nerve Tissue Proteins, Nonmuscle Myosin Type IIA, Nonmuscle Myosin Type IIB
Show Abstract · Added March 27, 2019
Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.
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18 MeSH Terms
Heterologous phosphorylation-induced formation of a stability lock permits regulation of inactive receptors by β-arrestins.
Tóth AD, Prokop S, Gyombolai P, Várnai P, Balla A, Gurevich VV, Hunyady L, Turu G
(2018) J Biol Chem 293: 876-892
MeSH Terms: Angiotensin II, Animals, COS Cells, Cercopithecus aethiops, HEK293 Cells, Humans, Immunoblotting, Microscopy, Confocal, Mitogen-Activated Protein Kinases, Phosphorylation, Receptors, G-Protein-Coupled, beta-Arrestins
Show Abstract · Added March 14, 2018
β-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and β-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether β-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of β-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, G-coupled GPCR, or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to unliganded AT angiotensin receptor (ATR). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and β-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters β-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-β-arrestin interaction, but also governs the structural rearrangements within β-arrestins. Furthermore, we found that β-arrestin2 binds to PKC-phosphorylated ATR in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of β-arrestins and reveal their novel role in receptor cross-talk.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
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12 MeSH Terms
Identification and Characterization of the First Selective Y Receptor Positive Allosteric Modulator.
Schubert M, Stichel J, Du Y, Tough IR, Sliwoski G, Meiler J, Cox HM, Weaver CD, Beck-Sickinger AG
(2017) J Med Chem 60: 7605-7612
MeSH Terms: Allosteric Regulation, Animals, Arrestins, COS Cells, Cercopithecus aethiops, Cyclohexanols, GTP-Binding Proteins, HEK293 Cells, Humans, Models, Molecular, Receptors, Neuropeptide Y, Signal Transduction
Show Abstract · Added March 17, 2018
The human Y receptor (YR) and its cognate ligand, pancreatic polypeptide (PP), are involved in the regulation of energy expenditure, satiety, and food intake. This system represents a potential target for the treatment of metabolic diseases and has been extensively investigated and validated in vivo. Here, we present the compound tBPC (tert-butylphenoxycyclohexanol), a novel and selective YR positive allosteric modulator that potentiates YR activation in G-protein signaling and arrestin3 recruitment experiments. The compound has no effect on the binding of the orthosteric ligands, implying its allosteric mode of action at the YR and evidence for a purely efficacy-driven positive allosteric modulation. Finally, the ability of tBPC to selectively potentiate YR agonism initiated by PP was confirmed in mouse descending colon mucosa preparations expressing native YR, demonstrating YR positive allosteric modulation in vitro.
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12 MeSH Terms
Hepatic β-arrestin 2 is essential for maintaining euglycemia.
Zhu L, Rossi M, Cui Y, Lee RJ, Sakamoto W, Perry NA, Urs NM, Caron MG, Gurevich VV, Godlewski G, Kunos G, Chen M, Chen W, Wess J
(2017) J Clin Invest 127: 2941-2945
MeSH Terms: Animals, Blood Glucose, COS Cells, Cercopithecus aethiops, Diabetes Mellitus, Type 2, Diet, High-Fat, Gene Deletion, Gene Expression Regulation, Glucagon, Hepatocytes, Homeostasis, Insulin, Liver, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Phenotype, Receptors, Glucagon, Signal Transduction, beta-Arrestin 1, beta-Arrestin 2
Show Abstract · Added March 14, 2018
An increase in hepatic glucose production (HGP) represents a key feature of type 2 diabetes. This deficiency in metabolic control of glucose production critically depends on enhanced signaling through hepatic glucagon receptors (GCGRs). Here, we have demonstrated that selective inactivation of the GPCR-associated protein β-arrestin 2 in hepatocytes of adult mice results in greatly increased hepatic GCGR signaling, leading to striking deficits in glucose homeostasis. However, hepatocyte-specific β-arrestin 2 deficiency did not affect hepatic insulin sensitivity or β-adrenergic signaling. Adult mice lacking β-arrestin 1 selectively in hepatocytes did not show any changes in glucose homeostasis. Importantly, hepatocyte-specific overexpression of β-arrestin 2 greatly reduced hepatic GCGR signaling and protected mice against the metabolic deficits caused by the consumption of a high-fat diet. Our data support the concept that strategies aimed at enhancing hepatic β-arrestin 2 activity could prove useful for suppressing HGP for therapeutic purposes.
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22 MeSH Terms
Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors.
Prokop S, Perry NA, Vishnivetskiy SA, Toth AD, Inoue A, Milligan G, Iverson TM, Hunyady L, Gurevich VV
(2017) Cell Signal 36: 98-107
MeSH Terms: Amino Acid Sequence, Animals, Arrestins, COS Cells, Cattle, Cercopithecus aethiops, Conserved Sequence, HEK293 Cells, Humans, Lysine, Mutant Proteins, Mutation, Protein Binding, Protein Structure, Secondary, Receptors, G-Protein-Coupled, Rhodopsin
Show Abstract · Added March 14, 2018
Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M muscarinic receptor, so that agonist activation of the M did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M, whereas its interactions with other receptors, including the β-adrenergic receptor and the D and D dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the β-adrenergic and D dopamine receptors, while reducing its interaction with the D dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes.
Copyright © 2017. Published by Elsevier Inc.
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16 MeSH Terms
A Deep Hydrophobic Binding Cavity is the Main Interaction for Different Y R Antagonists.
Burkert K, Zellmann T, Meier R, Kaiser A, Stichel J, Meiler J, Mittapalli GK, Roberts E, Beck-Sickinger AG
(2017) ChemMedChem 12: 75-85
MeSH Terms: Animals, Arginine, Benzazepines, Binding Sites, COS Cells, Cells, Cultured, Cercopithecus aethiops, Dose-Response Relationship, Drug, HEK293 Cells, Humans, Hydrophobic and Hydrophilic Interactions, Molecular Docking Simulation, Molecular Structure, Receptors, Neuropeptide Y, Structure-Activity Relationship
Show Abstract · Added April 8, 2017
The neuropeptide Y receptor (Y R) is involved in various pathophysiological processes such as epilepsy, mood disorders, angiogenesis, and tumor growth. Therefore, the Y R is an interesting target for drug development. A detailed understanding of the binding pocket could facilitate the development of highly selective antagonists to study the role of Y R in vitro and in vivo. In this study, several residues crucial to the interaction of BIIE0246 and SF-11 derivatives with Y R were investigated by signal transduction assays. Using the experimental results as constraints, the antagonists were docked into a comparative structural model of the Y R. Despite differences in size and structure, all three antagonists display a similar binding site, including a deep hydrophobic cavity formed by transmembrane helices (TM) 4, 5, and 6, as well as a hydrophobic patch at the top of TM2 and 7. Additionally, we suggest that the antagonists block Q , a position that has been shown to be crucial for binding of the amidated C terminus of NPY and thus for receptor activation.
© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
1 Communities
1 Members
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15 MeSH Terms
C-terminal motif of human neuropeptide Y receptor determines internalization and arrestin recruitment.
Wanka L, Babilon S, Burkert K, Mörl K, Gurevich VV, Beck-Sickinger AG
(2017) Cell Signal 29: 233-239
MeSH Terms: Amino Acid Motifs, Amino Acid Sequence, Amino Acids, Animals, COS Cells, Cercopithecus aethiops, Endocytosis, HEK293 Cells, Humans, Mutant Proteins, Receptors, Neuropeptide Y, Reproducibility of Results, Sequence Alignment, Sequence Deletion, Structure-Activity Relationship, beta-Arrestin 2
Show Abstract · Added March 14, 2018
The human neuropeptide Y receptor is a rhodopsin-like G protein-coupled receptor (GPCR), which contributes to anorexigenic signals. Thus, this receptor is a highly interesting target for metabolic diseases. As GPCR internalization and trafficking affect receptor signaling and vice versa, we aimed to investigate the molecular mechanism of hYR desensitization and endocytosis. The role of distinct segments of the hYR carboxyl terminus was investigated by fluorescence microscopy, binding assays, inositol turnover experiments and bioluminescence resonance energy transfer assays to examine the internalization behavior of hYR and its interaction with arrestin-3. Based on results of C-terminal deletion mutants and substitution of single amino acids, the motif EESEHLPLSTVHTEVSKGS was identified, with glutamate, threonine and serine residues playing key roles, based on site-directed mutagenesis. Thus, we identified the internalization motif for the human neuropeptide Y receptor, which regulates arrestin-3 recruitment and receptor endocytosis.
Copyright © 2016 Elsevier Inc. All rights reserved.
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16 MeSH Terms
Impact of the Motor and Tail Domains of Class III Myosins on Regulating the Formation and Elongation of Actin Protrusions.
Raval MH, Quintero OA, Weck ML, Unrath WC, Gallagher JW, Cui R, Kachar B, Tyska MJ, Yengo CM
(2016) J Biol Chem 291: 22781-22792
MeSH Terms: Actins, Animals, COS Cells, Cercopithecus aethiops, Humans, Myosin Heavy Chains, Myosin Type III, Pseudopodia
Show Abstract · Added April 7, 2017
Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30-34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
1 Communities
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8 MeSH Terms
Discovery of Small-Molecule Modulators of the Human Y4 Receptor.
Sliwoski G, Schubert M, Stichel J, Weaver D, Beck-Sickinger AG, Meiler J
(2016) PLoS One 11: e0157146
MeSH Terms: Allosteric Regulation, Animals, COS Cells, Cercopithecus aethiops, Drug Evaluation, Preclinical, Humans, Ligands, Niclosamide, Receptors, Neuropeptide Y, Small Molecule Libraries, Structure-Activity Relationship
Show Abstract · Added April 8, 2017
The human neuropeptide Y4 receptor (Y4R) and its native ligand, pancreatic polypeptide, are critically involved in the regulation of human metabolism by signaling satiety and regulating food intake, as well as increasing energy expenditure. Thus, this receptor represents a putative target for treatment of obesity. With respect to new approaches to treat complex metabolic disorders, especially in multi-receptor systems, small molecule allosteric modulators have been in the focus of research in the last years. However, no positive allosteric modulators or agonists of the Y4R have been described so far. In this study, small molecule compounds derived from the Niclosamide scaffold were identified by high-throughput screening to increase Y4R activity. Compounds were characterized for their potency and their effects at the human Y4R and as well as their selectivity towards Y1R, Y2R and Y5R. These compounds provide a structure-activity relationship profile around this common scaffold and lay the groundwork for hit-to-lead optimization and characterization of positive allosteric modulators of the Y4R.
1 Communities
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11 MeSH Terms