Signal transduction in vertebrate photoreceptors occurs at the interface between the membrane bilayer and the cytoplasm. The long term goal of this research program is to understand the events that occur at that interface in terms of structures, mechanisms, and dynamics of the proteins, lipids and small molecules and ions that mediate the phototransduction cascade and that assemble and disassemble into multi-partner complexes as the cells respond to light. It is essential to understand these events at the molecular and biophysical levels both in order to develop a full understanding of the visual process, and to serve as a model for understanding the most common class of signaling pathways, those mediated by heterotrimeric G proteins.
Specific Aim 1 is to use high-resolution cryo-electron microscopy and energy transfer measurements to determine the structures of the effector enzyme of vision, PDE6 (a peripheral membrane protein) and its membrane-dependent complex with the activated G protein, Gat.
Specific Aim 2 is to use Fluorescence Recovery After Photobleaching (FRAP) and Fluctuation Correlation Spectroscopy (FCS) in transgenic frogs to study the diffusion of phototransduction proteins on and in disk membranes of rod outer segments and to probe the microdomain structure of the disk membranes.
Specific Aim 3 is to test possible roles for phosphoinositides, sphingolipids and cholesterol in regulating phototransduction and in forming microdomains within disk membranes. Because G protein signaling pathways are the most common targets of drugs, and because neurodegenerative disorders such as retinal degeneration can result from defects in membrane structure and function, these studies will contribute to improving human health by identifying the key features of membrane structure and function for the phototransduction cascade, and by identifying many common features needed for G protein signaling.
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