The long-term goal of this research is to understand the molecular and mechanistic basis of G protein activation by an activated receptor. We will investigate the conformational changes that the G protein ? subunit undergoes as it interacts with activated rhodopsin, ultimately leading to GDP release, GTP binding, and activation. We will define these movements using a strategy of local "sensors" of conformational change using site-directed Cys mutagenesis to label particular sites with fluorophores and spin-labels. Additional information will be gained from labeling two sites and measuring the distances between them in different conformations using double electron-electron resonance (DEER). In the last funding cycle, we showed by DEER studies that there is a large opening of the cleft between GTPase and helical domains of G? triggered by the C-terminal helix contact with receptor, but the detailed mechanisms of the trigger of this large conformational change are unknown. In this grant period, we will seek (in specific aim 1) to elucidate how GDP release is coupled to conformational changes in regions involved in nucleotide binding. We will examine the dynamics of conformational changes on G? using an atomic resolution model that incorporates both the energetic as well as the DEER distance distributions. This model provides insight into how the bound GDP stabilizes the closed state as well as how the receptor triggers GDP release and domain opening.
In specific aim 2, we will investigate the receptor-mediated conformational changes in the G? subunit which lead to domain opening. Finally, in specific aim 3, we will determine the mechanisms by which GTP binding leads to dissociation from R* and G protein activation. These studies will lead to a more complete picture of the membrane-bound signaling proteins in their native states, undergoing the conformational changes that lead to G protein activation. Signaling through this class of receptors underlies most aspects of our physiology and behavior, and many pathologies as well, and these studies may provide insights into how we might disrupt signaling through particular GPCR-G protein interactions in a number of disease states.

Public Health Relevance

The goal of this research is to understand the molecular and mechanistic basis of G protein activation by an activated receptor. Signaling through this class of receptors underlies most aspects of our physiology and behavior, and many pathologies as well. These studies may provide insights into how we might disrupt signaling through particular GPCR-G protein interactions in a number of disease states.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY006062-28
Application #
8529533
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Neuhold, Lisa
Project Start
1985-02-01
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
28
Fiscal Year
2013
Total Cost
$370,500
Indirect Cost
$133,000
Name
Vanderbilt University Medical Center
Department
Pharmacology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Alexander, Nathan S; Preininger, Anita M; Kaya, Ali I et al. (2014) Energetic analysis of the rhodopsin-G-protein complex links the ?5 helix to GDP release. Nat Struct Mol Biol 21:56-63
Thaker, Tarjani M; Sarwar, Maruf; Preininger, Anita M et al. (2014) A transient interaction between the phosphate binding loop and switch I contributes to the allosteric network between receptor and nucleotide in G?i1. J Biol Chem 289:11331-41
Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A et al. (2014) A conserved phenylalanine as a relay between the ?5 helix and the GDP binding region of heterotrimeric Gi protein ? subunit. J Biol Chem 289:24475-87
Preininger, Anita M; Meiler, Jens; Hamm, Heidi E (2013) Conformational flexibility and structural dynamics in GPCR-mediated G protein activation: a perspective. J Mol Biol 425:2288-98
Hamm, Heidi E; Kaya, Ali I; Gilbert 3rd, James A et al. (2013) Linking receptor activation to changes in Sw I and II of G* proteins. J Struct Biol 184:63-74
Preininger, Anita M; Kaya, Ali I; Gilbert 3rd, James A et al. (2012) Myristoylation exerts direct and allosteric effects on G* conformation and dynamics in solution. Biochemistry 51:1911-24
Kaya, Ali I; Thaker, Tarjani M; Preininger, Anita M et al. (2011) Coupling efficiency of rhodopsin and transducin in bicelles. Biochemistry 50:3193-203
Van Eps, Ned; Preininger, Anita M; Alexander, Nathan et al. (2011) Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit. Proc Natl Acad Sci U S A 108:9420-4
Caruso, Giovanni; Bisegna, Paolo; Andreucci, Daniele et al. (2011) Identification of key factors that reduce the variability of the single photon response. Proc Natl Acad Sci U S A 108:7804-7
Jernigan, Kristin K; Cselenyi, Christopher S; Thorne, Curtis A et al. (2010) Gbetagamma activates GSK3 to promote LRP6-mediated beta-catenin transcriptional activity. Sci Signal 3:ra37

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