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.

Public Health Relevance

Visual arrestins are key players in photoreceptor signaling, governing the rate of signal shutoff and photoresponse recovery. Here we propose to elucidate molecular mechanisms of arrestin function, focusing on the arrestin interaction with rhodopsin, the conformation of rhodopsin-bound arrestin and the stoichiometry and shape of the arrestin-rhodopsin complex, as well as on the mechanism and biological role of arrestin self- association in photoreceptor cells. We propose to use this structural information to construct custom- designed arrestin proteins with high affinity for light-activated unphosphorylated rhodopsin and test the ability of these enhanced mutants to compensate for the defects of rhodopsin phosphorylation in mouse models of congenital visual disorders.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY011500-18
Application #
8515412
Study Section
Special Emphasis Panel (ZRG1-CB-G (90))
Program Officer
Neuhold, Lisa
Project Start
1997-04-01
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
18
Fiscal Year
2013
Total Cost
$534,906
Indirect Cost
$191,944
Name
Vanderbilt University Medical Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Gimenez, Luis E; Babilon, Stefanie; Wanka, Lizzy et al. (2014) Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes. Cell Signal 26:1523-31
Zhan, Xuanzhi; Perez, Alejandro; Gimenez, Luis E et al. (2014) Arrestin-3 binds the MAP kinase JNK3?2 via multiple sites on both domains. Cell Signal 26:766-76
Zhuo, Ya; Vishnivetskiy, Sergey A; Zhan, Xuanzhi et al. (2014) Identification of receptor binding-induced conformational changes in non-visual arrestins. J Biol Chem 289:20991-1002
Gurevich, Vsevolod V; Gurevich, Eugenia V (2014) Extensive shape shifting underlies functional versatility of arrestins. Curr Opin Cell Biol 27:1-9
Vishnivetskiy, Sergey A; Zhan, Xuanzhi; Chen, Qiuyan et al. (2014) Arrestin expression in E. coli and purification. Curr Protoc Pharmacol 67:Unit 2.11.
Gurevich, Vsevolod V; Gurevich, Eugenia V (2014) Arrestin makes T cells stop and become active. EMBO J 33:531-3
Kook, S; Zhan, X; Cleghorn, W M et al. (2014) Caspase-cleaved arrestin-2 and BID cooperatively facilitate cytochrome C release and cell death. Cell Death Differ 21:172-84
Gurevich, Vsevolod V; Gurevich, Eugenia V (2014) Overview of different mechanisms of arrestin-mediated signaling. Curr Protoc Pharmacol 67:Unit 2.10.
Moaven, Hormoz; Koike, Yukihiro; Jao, Christine C et al. (2013) Visual arrestin interaction with clathrin adaptor AP-2 regulates photoreceptor survival in the vertebrate retina. Proc Natl Acad Sci U S A 110:9463-8
Gurevich, Vsevolod V; Gurevich, Eugenia V (2013) Structural determinants of arrestin functions. Prog Mol Biol Transl Sci 118:57-92

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