Arrestins interact with G protein-coupled receptors (GCPRs) and numerous downstream scaffolding and signaling molecules. These interactions promote the termination of signaling events initiated by activated GPCRs and result in GPCR internalization via calthrin-coated pits. Arrestins also associate with the Src-family kinases Src and Hck to initiate G protein-independent signaling pathways. Arrestins are therefore involved in virtually every aspect of normal and abnormal human physiology from the visual system via rhodopsin to the cardiovascular system via adrenergic receptors. Understanding arrestin activation mechanisms and the commensurate structural changes is essential to modulating a wide variety of disease states. Current models suggest that structural changes within arrestins are critical for proper function. The objectives of this application are to define these structural changes. To probe structural changes in arrestins during their functional cycle, we will use hydrogen exchange coupled with mass spectrometry (HX MS). By monitoring the mass changes that result from deuterium incorporation, HX MS can be used to probe protein structure and dynamics on a wide time-scale and with small quantities (pmol) of large (>40 kDa) proteins and protein complexes. The following specific aims will be accomplished: (1). We wilt probe the conformation of wild type arrestins in solution using HX MS and compare the results to similar HX MS studies of numerous mutant forms of arrestin that appear """"""""pre-activated"""""""" and display higher affinity for GPCRs. (2). We will use HX MS to determine the structural alterations within arrestins following binding to the intracellular domains of GPCRs such as the N-formyl peptide receptor. (3). We will investigate binding-induced structural alterations during the interactions between arrestins and the Src-family kinase Hck, both with full-length Hck protein and the Hck SH3 domain which appears critical for binding. (4). We will design and examine the functional properties of novel site-directed mutants to test current structural/function hypotheses and newly acquired structural insights from the first three aims. By accomplishing these specific aims, we will definitively identify the structural changes that are involved in arrestin activation and those that accompany association with both upstream and downstream signaling partners. Our results will contribute to our basic knowledge of this key signaling molecule and provide the basis for the design of therapeutic agents related to GPCR signaling.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068901-03
Application #
7018514
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Dunsmore, Sarah
Project Start
2004-04-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
3
Fiscal Year
2006
Total Cost
$276,838
Indirect Cost
Name
University of New Mexico
Department
Physiology
Type
Schools of Medicine
DUNS #
868853094
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
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Wagener, Brant M; Marjon, Nicole A; Revankar, Chetana M et al. (2009) Adaptor protein-2 interaction with arrestin regulates GPCR recycling and apoptosis. Traffic 10:1286-300
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Carter, Jennifer M; Gurevich, Vsevolod V; Prossnitz, Eric R et al. (2005) Conformational differences between arrestin2 and pre-activated mutants as revealed by hydrogen exchange mass spectrometry. J Mol Biol 351:865-78
Prossnitz, Eric R (2004) Novel roles for arrestins in the post-endocytic trafficking of G protein-coupled receptors. Life Sci 75:893-9
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