The goal is to understand the molecular mechanisms underlying visual excitation and adaptation in photoreceptors. During the next period of this work, several different approaches will be used to define the role of cGMP and the c6MP enzymatic cascade in visual transduction and adaptation. 1. The light-activated cGMP enzyme cascade may play a pivotal role in visual transduction. The details of molecular interactions between rhodopsin, the G-protein, and cGMP phosphodiesterase are thus of general interest. We propose to continue ongoing immunological studies of the structure-function relationships of the G-protein Alpha subunit using a series of monoclonal antibodies to the G-protein that disrupt particular functions, and extend these studies to functional sites of the G-protein Beta subunit. 2. We propose to address the question, can the physiological behavior of rod outer segments be explained by the behavior of cGMP? Recently, it has been shown that cGMP can directly regulate light-sensitive channels, lending strong support to the hypothesis that cGMP is the """"""""internal transmitter"""""""" in visual transduction. However, proof of this awaits the demonstration that free cGMP levels in the rod cytoplasm show light-dependent changes with the time course of electrophysiological changes. We will attempt to measure free and bound cGMP, and in addition study the regulation of free cGMP in the cytoplasm by cGMP binding proteins and guanylate cyclase. 3. We will also examine whether cGMP dependent protein phosphorylations play a role in visual adaptation. 4. The finding that cGMP binds to the light-sensitive channel provides a biochemical handle to identify the channel and study its properties in isolation. The cGMP-binding component of the light-sensitive channel will be identified by photoaffinity labelling and purified by cGMP affinity chromatography. Monoclonal antibodies to this protein that block channel function will confirm the identity of the channel protein. The understanding of visual transduction at a molecular level may lead to approaches to treatment of retinal diseases, as well as diseases of signal transduction pathways in other systems.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Visual Sciences A Study Section (VISA)
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University of Illinois at Chicago
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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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