The long-term goal of this research is to understand the molecular and mechanistic basis of visual excitation by rhodopsin. Visual transduction by rod photoreceptors is an elegantly sensitive trasduction machine and serves as a model system for understanding the molecular mechanisms of signal transduction through G protein coupled receptors (GPCR) in general. 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 fluorescence resonance energy homotransfer and spin-spin interactions. In this application period, we will seek (in Specific Aim 1) to determine the structural details of how activated rhodopsin causes the conformational changes in G? that lead to GDP release and formation of high-affinity complex between Metarhodopsin II and empty-pocket Gt.
In Specific Aim 2, the conformational changes needed for GTP to induce dissociation of Gt from rhodopsin and will be determined. Finally, in Specific Aim 3, we will investigate the point-to-point interaction between rhodopsin and the heterotrimeric Gt using dual labeling of sites within both G? and rhodopsin. These studies will lead to a more complete picture of these membrane bound signaling proteins in their native states, undergoing the conformational changes that lead to visual excitation. Such detailed structural studies cannot be easily done with other GPCR systems and so they will serve as a useful guide to the mechanism by which the large family of G protein-coupled receptors interact with and activate their cognate G proteins. 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.

National Institute of Health (NIH)
National Eye Institute (NEI)
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Biophysics of Neural Systems Study Section (BPNS)
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Mariani, Andrew P
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Vanderbilt University Medical Center
Schools of Medicine
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Kaya, Ali I; Lokits, Alyssa D; Gilbert, James A et al. (2016) A Conserved Hydrophobic Core in G?i1 Regulates G Protein Activation and Release from Activated Receptor. J Biol Chem 291:19674-86
Kaya, Ali I; Iverson, T M; Hamm, Heidi E (2015) Functional stability of rhodopsin in a bicelle system: evaluating G protein activation by rhodopsin in bicelles. Methods Mol Biol 1271:67-76
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
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
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
Natarajan, Chandramohan; Hata, Aaron N; Hamm, Heidi E et al. (2013) Extracellular loop II modulates GTP sensitivity of the prostaglandin EP3 receptor. Mol Pharmacol 83:206-16
Thaker, Tarjani M; Kaya, Ali I; Preininger, Anita M et al. (2012) Allosteric mechanisms of G protein-Coupled Receptor signaling: a structural perspective. Methods Mol Biol 796:133-74
Makino, Clint L; Wen, Xiao-Hong; Michaud, Norman A et al. (2012) Rhodopsin expression level affects rod outer segment morphology and photoresponse kinetics. PLoS One 7:e37832

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