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 transduction machine and serves as a model system for understanding the molecular mechanisms of signal transduction through G protein coupled receptors (GPCR) in general. In this competing renewal we will continue to probe the mechanisms of protein-protein interaction and activation in visual transduction.
In specific aim 1, we will seek to determine the structural details of how activated rhodopsin causes G protein activation, and the conformational changes in Galpha that lead to GDP release and formation of a high-affinity complex between Metarhodopsin II and empty-pocket Gt. A model of the rhodopsin Gt complex will be constructed based on the high-resolution crystal structures of rhodopsin and Gt to aid in interpretation of the results and planning of additional experiments.
In specific aim 2, the conformational changes needed for GTP to induce dissociation of Gt from rhodopsin and subunit dissociation will be determined. Finally, in specific aim 3, the sites on Galpha.GTP of interaction with PDE and the RGS9/Gbeta5 complex will be elucidated. Site-directed cysteine mutagenesis will be used to label Galpha with environment-sensitive probes such as fluorophores and spin labels. These labels will report local structure, conformational changes, and interaction with other proteins in the visual transduction cascade. We hope to define the structures of parts of the G protein that were disordered in the crystal structures. The strengths of these studies are that they will lead to a more complete picture of the membrane-bound 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 to disrupt signaling in a number of disease states.
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