2001 — 2004 |
Gurevich, Vsevolod V. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Arrestin Function
DESCRIPTION (Verbatim from the Applicant's Abstract): The decrease of cell responsiveness to a persistent stimulus, usually termed desensitization, is a widespread biological phenomenon. Signaling by a wide variety of G protein-coupled receptors (GPCRs) is attenuated by a two-step mechanism: receptor phosphorylation by a specific kinase, followed by tight binding of an arrestin protein to activated phosphorylated receptor. Arrestin binding terminates the signaling via G protein, tags receptor for internalization, and in some cases initiates an additional signaling cascade via Src kinase. Internalized receptor is either recycled back to the plasma membrane (resensitization) or transported to lysosomes and degraded (down-regulation). It is well established that arrestins play a key role in desensitization and trafficking of various GPCRs. However, the molecular mechanisms that dictate arrestins' remarkable selectivity toward activated phosphorylated receptors, determine receptor specificity of different arrestin proteins, and regulate arrestins' interaction with a variety of other partners in the cell remain to be elucidated. The objectives of this proposal are to identify the elements of b-arrestin and arrestin3 involved in their interaction with receptors and determine which of these elements dictate arrestins' receptor specificity. We propose to elucidate the mechanism of arrestin transition from its basal inactive state into high-affinity receptor binding state. A combination of mutagenesis, site-directed spin labeling, and X-ray crystallography will be used for this purpose. Various arrestin mutants will be tested in vitro, in cell culture, and in Xenopus oocytes. Arrestin mutants with special functional characteristics will be constructed, such as "constitutively active" arrestins that bind to phosphorylated and unphosphorylated receptors, and arrestins with enhanced specificity for certain receptors. These mutants will be used to study the mechanisms of receptor trafficking in cells. Excessive signaling by certain GPCRs causes a variety of disorders, including several forms of cancer. Arrestin mutants with enhanced specificity for these receptors and enhanced capability to attenuate such faulty signaling promise to become useful tools for gene therapy of these disorders.
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2001 — 2021 |
Gurevich, Vsevolod V. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure-Function Studies of Visual Arrestin
PROJECT SUMMARY Visual arrestins are key players in photoreceptor signaling, governing the rate of signal shutoff and photoresponse recovery. Closely related non-visual arrestins orchestrate signaling and trafficking of hundreds of different G protein-coupled receptors expressed in virtually every eukaryotic cell. Here we propose to elucidate molecular mechanisms of arrestin function in photoreceptors, focusing on arrestin1 (a.k.a. rod arrestin), which is expressed at high level in both rods and cones. Using a combination of biochemical and biophysical methods we propose to determine the conformation of rhodopsin-bound arrestin and the shape of the arrestin-rhodopsin complex. This will allow us to understand how arrestin complexes with hyper- phosphorylated rhodopsin contribute to photoreceptor death in cases of retinitis pigmentosa associated with constitutive formation of these aberrant complexes. We propose to test whether arrestin preferentially interacts with monomeric or dimeric rhodopsin, thereby defining the stoichiometry of the biologically relevant arrestin-rhodopsin complex. Based on our studies of the mechanism of arrestin self-association, we propose to elucidate the biological role of this process in photoreceptor cells in mice by replacing wild type arrestin with self-association-impaired mutants that retain all other arrestin functions. Based on the success of our initial proof-of-principle experiments, where we showed that arrestin mutants with high affinity for light-activated unphosphorylated rhodopsin improve the survival and facilitate photoresponse recovery in rhodopsin phosphorylation-deficient rods, we propose to design new enhanced arrestins with better ability to compensate for the defects of rhodopsin phosphorylation in mouse models of congenital visual disorders. We believe that this compensational approach will have high therapeutic value in all inherited disorders caused by gain-of-function receptor mutants, where traditional gene replacement approaches, that cannot silence excessive signaling by a mutant receptor, are ineffective.
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2007 — 2015 |
Gurevich, Vsevolod V. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Arrestin Interactions With Non-Receptor Binding Partners
DESCRIPTION (provided by applicant): Arrestins were first described as negative regulators of G protein-coupled receptor (GPCR) signaling via G proteins. New data show that the free and receptor-bound arrestins initiate signaling through MAP kinases, which regulate cell death, survival, and proliferation. In particular, both free and receptor- associated arrestin-3 scaffolds ASK1-MKK-JNK cascade, promoting JNK activation. Here we propose to elucidate the structural basis of arrestin-dependent activation of pro-apoptotic JNK family kinases and their activators MKK4/7 using biochemical and biophysical methods. The molecular mechanisms of the assembly of multi-protein signaling complexes (signalosomes) organized by arrestin-3 will be established, and arrestin-3 residues critical for JNK activation will be identified. Based on ths info, arrestin-3 mutants that bind ASK1, MKK and JNK, but do not promote JNK activation will be constructed. The potential of arrestin mutants with dramatically reduced ability to activate JNKs, several of which we already have, to protect cells against insults and prolong their survival will be tested. We have also constructed arrestin-3 mutants that activate JNKs more efficiently than parental wild type arrestin-3. We established the paradigm where arrestin-3-dependent JNK activation plays key role in cell survival. We will test the ability of hyperactive arrestin-3 mutants to facilitate cell death. We showed that inactive mutants tie up JNKs and upstream kinases in unproductive complexes, thereby acting in dominant- negative manner. We will test the potential of these mutants to protect cells and prolong their survival. Molecular toos that specifically increase or block pro-apoptotic signaling have therapeutic potential in disorders associated with excessive cell proliferation (e.g., cancer) or death (e.g., neurodegenerative diseases). PUBLIC HEALTH RELEVANCE: This proposal focuses on the elucidation of the structural basis of arrestin-dependent activation of pro- apoptotic JNK family kinases and their activators MKK4/7 using biochemical and biophysical methods. The potential of arrestin mutants with dramatically reduced ability to activate JNKs, that were constructed based on this info, to protect cells against insults and prolong their survival will be tested, as well as the ability of the mutats that activate JNKs more efficiently than wild type arrestins to facilitate cell death will be also tested. Molecular tools that specifically increase or block pro-apoptotic signaling have therapeutic potential in disorders associated with excessive cell proliferation (e.g., cancer) or death (e.g., neurodegenerative diseases).
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2008 — 2011 |
Gurevich, Vsevolod V. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Conformational Regulation of Arrestin-Mediated Signaling
DESCRIPTION (provided by applicant): The decrease of cell responsiveness to a persistent stimulus, usually termed desensitization, is a widespread biological phenomenon. The signaling by G protein-coupled receptors (GPCRs) is attenuated by a two-step mechanism: receptor phosphorylation by a specific kinase followed by arrestin binding to active phosphoreceptor. Arrestin binding terminates G protein-mediated signaling, tags GPCRs for internalization, and redirects signaling to other pathways via non-receptor binding partners (c-Src and MAP kinases, ubiquitin ligases, etc). Arrestins also interact with microtubules via the same interface that is involved in receptor binding and mobilize several signaling molecules to the cytoskeleton with different functional consequences. The main objective of this proposal is to elucidate the structural basis of arrestin function as an organizer of multi-protein signaling complexes in the cell. We hypothesize that the signaling capability of arrestin molecule is determined by its conformation. We propose to identify arrestin elements involved in receptor and microtubule binding and the nature of receptor- and microtubule-induced conformational changes in both non- visual arrestins that regulate their interactions with non-receptor partners: kinases c-Src, ERK2, JNK3, ubiquitin ligase Mdm2, etc. To this end, we propose to use site-directed mutagenesis, direct binding assay, site-directed spin labeling of arrestins and EPR spectroscopy, as well as functional assays in living cells. We also propose to use double spin-labeled arrestins and spin-labeled arrestins with spin-labeled model receptor, rhodopsin, to measure inter-spin distances in arrestin-receptor complex by conventional EPR and double electron-electron resonance (DEER) to obtain a working structural model of the complex. This information will set the stage for designing arrestin-based molecular tools for targeted manipulation of cellular signaling that can be used for experimental and therapeutic purposes. PUBLIC HEALTH RELEVANCE: Arrestins are multi-functional adaptors that mobilize various signaling molecules to G protein-coupled receptors and microtubules with different functional consequences. The goal of this proposal is to elucidate the conformations of receptor-bound and microtubule-bound arrestins to understand how arrestin conformation affects its interactions with signaling proteins and the consequences of their binding. This information will set the stage for designing arrestin-based molecular tools for targeted manipulation of cellular signaling that can be used for experimental and therapeutic purposes.
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2015 — 2017 |
Gurevich, Vsevolod V. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Gpcr Signaling With Receptor-Specific Arrestins
DESCRIPTION (provided by applicant): Arrestins were originally discovered as negative regulators of G protein-coupled receptor (GPCR) signaling via G proteins. Recent discoveries show that the arrestin-receptor complex initiates signaling through distinct G protein-independent pathways, including those that regulate cell death, survival, and proliferation via MAP kinases. Faulty regulation of GPCR signaling induced by mutations or environmental insults underlies many human diseases. Unfortunately, therapeutic targeting receptor signaling via arrestins, which are natural GPCR regulators, is hampered by lack of selectivity of the non- visual subtypes, both of which interact with dozens of GPCRs. Here we propose to construct and functionally characterize non-visual arrestins with dramatically enhanced specificity for individual GPCRs. Receptor-specific mutants, as well as their enhanced phosphorylation-independent variants, will be tested for their ability to selectively regulate signaling by particuar GPCR subtypes via G proteins, arrestins, and receptor trafficking. Mutants that preferentially interact with particular receptors will be tested for their ability to selectively disrupt receptor coupling to cognate G proteins. Mutants with high preference for D1 and D2 dopamine receptors will be used to determine which receptor subtype plays key role in the development of dyskinesia, the most common devastating side effect of current anti- parkinsonian therapy. Receptor-specific regulation of GPCR signaling has therapeutic potential in multiple disorders associated with congenital or acquired imbalances in cell signaling.
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2017 — 2018 |
Gurevich, Vsevolod V. Iverson, Tina M |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Mechanisms of Signal Bias in Arrestins
PROJECT SUMMARY Arrestin proteins are master regulators of G protein coupled receptor (GPCR) signaling, and act in two ways. First, arrestins terminate the coupling of G proteins to cognate receptor by physically blocking G protein coupling. Second, arrestins can support G protein independent signaling. Here, there are many mysteries in the field ? arrestin can potentially activate one of over 150 signaling proteins, how does it select the correct one? The best studied arrestin-mediated signaling cascades involving mitogen activated protein (MAP) kinases. Recently we determined the structure of activated arrestin-3 in a conformation that is biased toward activating the MAP kinase Jun N-terminal Kinase-3 (JNK3). This JNK3-biased arrestin-3 structure showed two types of conformational change: (1) the expected inter-domain twist, and (2) localized, previously unrecognized conformational changes in the effector binding regions of activated arrestin. Combining structural analysis with functional measurements, we leverage these results to propose how arrestin biases toward one particular pathway. We propose that these different twists and localized conformational changes in arrestin work together to form specific binding sites. The magnitude of these conformational changes would depend upon the identity and phosphorylation pattern of the receptor. Here, we test an extension of the ?bar code hypothesis?, which would suggest that the phosphorylation pattern of receptor can push the arrestin structure toward slightly different conformations. In Aim 1, we develop synthetic phosphopeptides that contain a site-specific photoactivatable crosslinker and different receptor phosphorylation patterns. We will crosslink these peptides to arrestin-3, monitor activation via DEER spectroscopy, and assess how the phosphorylation pattern affects binding to MAP kinases in vitro. We will further crystallize these irreversibly activated arrestins to identify how the conformations vary with the changes in phosphorylation pattern. In Aim2, we propose site-specific mutagenesis and chimeragenesis to systematically assess the contribution of two key points of contact between arrestin and receptor in biased signaling: the phosphate sensor and the activation sensor. These studies will use receptor coupling assays performed in cells.
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2017 — 2021 |
Gurevich, Vsevolod V. |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Targeted Engineering of Designer Arrestins to Regulate Cell Signaling
PROJECT SUMMARY Arrestins were discovered as negative regulators of G protein-coupled receptor (GPCR) signaling via G proteins. New data show that the free and receptor-bound arrestins initiate signaling through MAP kinases, which regulate cell death, survival, and proliferation. For example, free and receptor-bound arrestin-3 scaffolds ASK1-MKK4/7-JNK1/2/3 cascades, promoting the activation of JNK family kinases. The two non-visual arrestins interact with ~800 different GPCRs in humans. We propose to identify arrestin elements responsible for receptor specificity, and elucidate the structural basis of the assembly of multi-protein signaling complexes (signalosomes) organized by arrestins. Our new crystal structure of arrestin-3 in the presence of an abundant cytoplasmic molecule IP6 revealed receptor-bound-like (active) conformation of arrestin-3. The ability of arrestin-3 to assume this conformation in the absence of GPCRs likely explains receptor-independent scaffolding activity of arrestin-3. We identified arrestin-3 elements critical for JNK and ERK activation, as well as conformational requirements of distinct branches of arrestin-mediated signaling. We constructed arrestin-3 mutants that bind ASK1, MKK and JNK, but do not promote JNK activation and identified short arrestin-3 peptides that enhance or suppress JNK activity in cells. We will test the potential of signaling-biased arrestins and arrestin-derived molecular tools to facilitate cell death or survival. Molecular tools that specifically increase or block pro-apoptotic signaling have therapeutic potential in disorders associated with excessive cell proliferation (e.g., cancer) or death (e.g., neurodegenerative diseases). Using arrestin-3 mutant specific for dopamine D1 receptor we showed that arrestin-mediated signaling from D1 plays a role in the behavioral sensitization to L-DOPA and development of L-DOPA-induced diskinesia in mouse model of Parkinson?s disease, but other GPCRs also contribute to these phenomena. We believe that signaling-biased arrestins and arrestinbased molecular tools with specific functional capability will help elucidate the intricacies of cellular signaling and yield novel potent therapeutic tools.
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