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Molecular Defects of the Disease-Causing Human Arrestin-1 C147F Mutant.
Vishnivetskiy SA, Sullivan LS, Bowne SJ, Daiger SP, Gurevich EV, Gurevich VV
(2018) Invest Ophthalmol Vis Sci 59: 13-20
MeSH Terms: Arrestin, Cells, Cultured, DNA, DNA Mutational Analysis, Humans, Mutant Proteins, Mutation, Phosphorylation, Retinitis Pigmentosa
Show Abstract · Added March 14, 2018
Purpose - The purpose of this study was to identify the molecular defect in the disease-causing human arrestin-1 C147F mutant.
Methods - The binding of wild-type (WT) human arrestin-1 and several mutants with substitutions in position 147 (including C147F, which causes dominant retinitis pigmentosa in humans) to phosphorylated and unphosphorylated light-activated rhodopsin was determined. Thermal stability of WT and mutant human arrestin-1, as well as unfolded protein response in 661W cells, were also evaluated.
Results - WT human arrestin-1 was selective for phosphorylated light-activated rhodopsin. Substitutions of Cys-147 with smaller side chain residues, Ala or Val, did not substantially affect binding selectivity, whereas residues with bulky side chains in the position 147 (Ile, Leu, and disease-causing Phe) greatly increased the binding to unphosphorylated rhodopsin. Functional survival of mutant proteins with bulky substitutions at physiological and elevated temperature was also compromised. C147F mutant induced unfolded protein response in cultured cells.
Conclusions - Bulky Phe substitution of Cys-147 in human arrestin-1 likely causes rod degeneration due to reduced stability of the protein, which induces unfolded protein response in expressing cells.
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9 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
Implications of the differing roles of the β1 and β3 transmembrane and cytoplasmic domains for integrin function.
Lu Z, Mathew S, Chen J, Hadziselimovic A, Palamuttam R, Hudson BG, Fässler R, Pozzi A, Sanders CR, Zent R
(2016) Elife 5:
MeSH Terms: Amino Acid Substitution, Cell Adhesion, Cells, Cultured, Epithelial Cells, Humans, Integrin alpha1, Integrin beta1, Integrin beta3, Mutagenesis, Site-Directed, Mutant Proteins, Platelet Membrane Glycoprotein IIb, Protein Binding, Protein Multimerization
Show Abstract · Added March 26, 2017
Integrins are transmembrane receptors composed of α and β subunits. Although most integrins contain β1, canonical activation mechanisms are based on studies of the platelet integrin, αIIbβ3. Its inactive conformation is characterized by the association of the αIIb transmembrane and cytosolic domain (TM/CT) with a tilted β3 TM/CT that leads to activation when disrupted. We show significant structural differences between β1 and β3 TM/CT in bicelles. Moreover, the 'snorkeling' lysine at the TM/CT interface of β subunits, previously proposed to regulate αIIbβ3 activation by ion pairing with nearby lipids, plays opposite roles in β1 and β3 integrin function and in neither case is responsible for TM tilt. A range of affinities from almost no interaction to the relatively high avidity that characterizes αIIbβ3 is seen between various α subunits and β1 TM/CTs. The αIIbβ3-based canonical model for the roles of the TM/CT in integrin activation and function clearly does not extend to all mammalian integrins.
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13 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
Contribution of Organic Anion-Transporting Polypeptides 1A/1B to Doxorubicin Uptake and Clearance.
Lee HH, Leake BF, Kim RB, Ho RH
(2017) Mol Pharmacol 91: 14-24
MeSH Terms: ATP Binding Cassette Transporter, Subfamily B, Member 1, Animals, Biological Transport, Cell Membrane, Dogs, Doxorubicin, HeLa Cells, Humans, Kinetics, Liver, Liver-Specific Organic Anion Transporter 1, Madin Darby Canine Kidney Cells, Male, Mice, Models, Biological, Mutant Proteins, Organic Anion Transporters, Organic Anion Transporters, Sodium-Independent, Organic Cation Transport Proteins, Rats, Transfection
Show Abstract · Added November 10, 2016
The organic anion-transporting polypeptides represent an important family of drug uptake transporters that mediate the cellular uptake of a broad range of substrates including numerous drugs. Doxorubicin is a highly efficacious and well-established anthracycline chemotherapeutic agent commonly used in the treatment of a wide range of cancers. Although doxorubicin is a known substrate for efflux transporters such as P-glycoprotein (P-gp; MDR1, ABCB1), significantly less is known regarding its interactions with drug uptake transporters. Here, we investigated the role of organic anion transporting polypeptide (OATP) transporters to the disposition of doxorubicin. A recombinant vaccinia-based method for expressing uptake transporters in HeLa cells revealed that OATP1A2, but not OATP1B1 or OATP1B3, and the rat ortholog Oatp1a4 were capable of significant doxorubicin uptake. Interestingly, transwell assays using Madin-Darby canine kidney II cell line cells stably expressing specific uptake and/or efflux transporters revealed that OATP1B1, OATP1B3, and OATP1A2, either alone or in combination with MDR1, significantly transported doxorubicin. An assessment of polymorphisms in SLCO1A2 revealed that four variants were associated with significantly impaired doxorubicin transport in vitro. In vivo doxorubicin disposition studies revealed that doxorubicin plasma area under the curve was significantly higher (1.7-fold) in Slco1a/1b versus wild-type mice. The liver-to-plasma ratio of doxorubicin was significantly decreased (2.3-fold) in Slco1a/1b2 mice and clearance was reduced by 40% compared with wild-type mice, suggesting Oatp1b transporters are important for doxorubicin hepatic uptake. In conclusion, we demonstrate important roles for OATP1A/1B in transporter-mediated uptake and disposition of doxorubicin.
Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
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21 MeSH Terms
KRAS-MEK Signaling Controls Ago2 Sorting into Exosomes.
McKenzie AJ, Hoshino D, Hong NH, Cha DJ, Franklin JL, Coffey RJ, Patton JG, Weaver AM
(2016) Cell Rep 15: 978-987
MeSH Terms: Argonaute Proteins, Cell Line, Tumor, Exosomes, Humans, MicroRNAs, Mitogen-Activated Protein Kinase Kinases, Multivesicular Bodies, Mutant Proteins, Phosphorylation, Phosphoserine, Protein Transport, Proto-Oncogene Proteins p21(ras), Signal Transduction, Subcellular Fractions
Show Abstract · Added April 29, 2016
Secretion of RNAs in extracellular vesicles is a newly recognized form of intercellular communication. A potential regulatory protein for microRNA (miRNA) secretion is the critical RNA-induced silencing complex (RISC) component Argonaute 2 (Ago2). Here, we use isogenic colon cancer cell lines to show that overactivity of KRAS due to mutation inhibits localization of Ago2 to multivesicular endosomes (MVEs) and decreases Ago2 secretion in exosomes. Mechanistically, inhibition of mitogen-activated protein kinase kinases (MEKs) I and II, but not Akt, reverses the effect of the activating KRAS mutation and leads to increased Ago2-MVE association and increased exosomal secretion of Ago2. Analysis of cells expressing mutant Ago2 constructs revealed that phosphorylation of Ago2 on serine 387 prevents Ago2-MVE interactions and reduces Ago2 secretion into exosomes. Furthermore, regulation of Ago2 exosomal sorting controls the levels of three candidate miRNAs in exosomes. These data identify a key regulatory signaling event that controls Ago2 secretion in exosomes.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
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14 MeSH Terms
Analyzing the roles of multi-functional proteins in cells: The case of arrestins and GRKs.
Gurevich VV, Gurevich EV
(2015) Crit Rev Biochem Mol Biol 50: 440-52
MeSH Terms: Animals, Arrestins, Enzyme Activation, G-Protein-Coupled Receptor Kinases, Gene Knockdown Techniques, Gene Knockout Techniques, Humans, Ligands, Models, Molecular, Mutant Proteins, Protein Conformation, Recombinant Proteins, Signal Transduction
Show Abstract · Added February 3, 2016
Most proteins have multiple functions. Obviously, conventional methods of manipulating the level of the protein of interest in the cell, such as over-expression, knockout or knockdown, affect all of its functions simultaneously. The key advantage of these methods is that over-expression, knockout or knockdown does not require any knowledge of the molecular mechanisms of the function(s) of the protein of interest. The disadvantage is that these approaches are inadequate to elucidate the role of an individual function of the protein in a particular cellular process. An alternative is the use of re-engineered proteins, in which a single function is eliminated or enhanced. The use of mono-functional elements of a multi-functional protein can also yield cleaner answers. This approach requires detailed knowledge of the structural basis of each function of the protein in question. Thus, a lot of preliminary structure-function work is necessary to make it possible. However, when this information is available, replacing the protein of interest with a mutant in which individual functions are modified can shed light on the biological role of those particular functions. Here, we illustrate this point using the example of protein kinases, most of which have additional non-enzymatic functions, as well as arrestins, known multi-functional signaling regulators in the cell.
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13 MeSH Terms
Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8.
Shin Y, Du Y, Collier SE, Ohi MD, Lang MJ, Ohi R
(2015) Proc Natl Acad Sci U S A 112: E3826-35
MeSH Terms: Biophysical Phenomena, Cell Tracking, Diffusion, HeLa Cells, Humans, Kinesin, Kinetics, Microtubules, Motion, Mutant Proteins, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Video Recording
Show Abstract · Added April 18, 2017
Kinesin-8s are plus-end-directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are "ultraprocessive," a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end-directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.
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14 MeSH Terms
Pharmacoperones: targeting therapeutics toward diseases caused by protein misfolding.
Ulloa-Aguirre A, Zariñán T, Conn PM
(2015) Rev Invest Clin 67: 15-9
MeSH Terms: Animals, Drug Design, High-Throughput Screening Assays, Humans, Hydrophobic and Hydrophilic Interactions, Molecular Chaperones, Mutant Proteins, Protein Folding, Proteins, Proteostasis Deficiencies
Show Abstract · Added February 18, 2016
Pharmacoperones are hydrophobic molecule drugs that enter cells and serve as a molecular framework to cause misfolded mutant proteins to fold properly and adopt a stable conformation compatible with proper intracellular trafficking. Pharmacoperones have successfully been used experimentally to rescue function of some misfolded proteins (enzymes, receptors, channels) that lead to disease. Identification of pharmacoperones by high-throughput screens of drug libraries will likely provide new molecules that may be potentially useful to treat diseases caused by protein misfolding.
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10 MeSH Terms
Influenza viruses with receptor-binding N1 neuraminidases occur sporadically in several lineages and show no attenuation in cell culture or mice.
Hooper KA, Crowe JE, Bloom JD
(2015) J Virol 89: 3737-45
MeSH Terms: Animals, Cell Line, Disease Models, Animal, Dogs, Female, Humans, Influenza A Virus, H1N1 Subtype, Influenza A Virus, H5N1 Subtype, Mice, Inbred BALB C, Mutant Proteins, Mutation, Missense, Neuraminidase, Orthomyxoviridae Infections, Protein Binding, Receptors, Virus, Reverse Genetics, Viral Proteins, Virus Attachment, Virus Cultivation
Show Abstract · Added February 2, 2015
UNLABELLED - In nearly all characterized influenza viruses, hemagglutinin (HA) is the receptor-binding protein while neuraminidase (NA) is a receptor-cleaving protein that aids in viral release. However, in recent years, several groups have described point mutations that confer receptor-binding activity on NA, albeit in laboratory rather than natural settings. One of these mutations, D151G, appears to arise in the NA of recent human H3N2 viruses upon passage in tissue culture. We inadvertently isolated the second of these mutations, G147R, in the NA of the lab-adapted A/WSN/33 (H1N1) strain while we were passaging a heavily engineered virus in the lab. G147R also occurs at low frequencies in the reported sequences of viruses from three different lineages: human 2009 pandemic H1N1 (pdmH1N1), human seasonal H1N1, and chicken H5N1. Here we reconstructed a representative G147R NA from each of these lineages and found that all of the proteins have acquired the ability to bind an unknown cellular receptor while retaining substantial sialidase activity. We then reconstructed a virus with the HA and NA of a reported G147R pdmH1N1 variant and found no attenuation of viral replication in cell culture or change in pathogenesis in mice. Furthermore, the G147R virus had modestly enhanced resistance to neutralization by the Fab of an antibody against the receptor-binding pocket of HA, although it remained completely sensitive to the full-length IgG. Overall, our results suggest that circulating N1 viruses occasionally may acquire the G147R NA receptor-binding mutation without impairment of replicative capacity.
IMPORTANCE - Influenza viruses have two main proteins on their surface: one (hemagglutinin) binds incoming viruses to cells, while the other (neuraminidase) helps release newly formed viruses from these same cells. Here we characterize unusual mutant neuraminidases that have acquired the ability to bind to cells. We show that the mutation that allows neuraminidase to bind cells has no apparent adverse effect on viral replication but does make the virus modestly more resistant to a fragment of an antibody that blocks the normal hemagglutinin-mediated mode of viral attachment. Our results suggest that viruses with receptor-binding neuraminidases may occur at low levels in circulating influenza virus lineages.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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19 MeSH Terms