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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM063097-02
Application #
6531979
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Cole, Alison E
Project Start
2001-04-01
Project End
2005-03-31
Budget Start
2001-10-01
Budget End
2002-03-31
Support Year
2
Fiscal Year
2001
Total Cost
$177,251
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Cleghorn, Whitney M; Bulus, Nada; Kook, Seunghyi et al. (2018) Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs. Cell Signal 42:259-269
Chen, Qiuyan; Iverson, Tina M; Gurevich, Vsevolod V (2018) Structural Basis of Arrestin-Dependent Signal Transduction. Trends Biochem Sci 43:412-423
Bychkov, E R; Gurevich, V V; Joyce, J N et al. (2008) Arrestins and two receptor kinases are upregulated in Parkinson's disease with dementia. Neurobiol Aging 29:379-96
Ahmed, Mohamed Rafiuddin; Gurevich, Vsevolod V; Dalby, Kevin N et al. (2008) Haloperidol and clozapine differentially affect the expression of arrestins, receptor kinases, and extracellular signal-regulated kinase activation. J Pharmacol Exp Ther 325:276-83
Hanson, Susan M; Francis, Derek J; Vishnivetskiy, Sergey A et al. (2006) Visual arrestin binding to microtubules involves a distinct conformational change. J Biol Chem 281:9765-72
Hanson, Susan M; Francis, Derek J; Vishnivetskiy, Sergey A et al. (2006) Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin. Proc Natl Acad Sci U S A 103:4900-5
Wu, Nan; Macion-Dazard, Rosemarie; Nithianantham, Stanley et al. (2006) Soluble mimics of the cytoplasmic face of the human V1-vascular vasopressin receptor bind arrestin2 and calmodulin. Mol Pharmacol 70:249-58
Nair, K Saidas; Hanson, Susan M; Mendez, Ana et al. (2005) Light-dependent redistribution of arrestin in vertebrate rods is an energy-independent process governed by protein-protein interactions. Neuron 46:555-67
Sutton, R Bryan; Vishnivetskiy, Sergey A; Robert, Justin et al. (2005) Crystal structure of cone arrestin at 2.3A: evolution of receptor specificity. J Mol Biol 354:1069-80
Key, T Alexander; Vines, Charlotte M; Wagener, Brant M et al. (2005) Inhibition of chemoattractant N-formyl peptide receptor trafficking by active arrestins. Traffic 6:87-99

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