The long term goal of this proposal is to understand the role of the disk membranes and the complexes formed at their surfaces in visual signaling. The focus is on those complexes involving subunits of the G protein, transducin (Gt), because it plays a pivotal role in visual signaling, because its protein partners in these complexes have been implicated in retinal disease, and because proper regulation of G protein pathways throughout the body is critical to human health. Two broad areas are addressed by the current proposal. The first area is understanding the structure and dynamics of membrane-bound signal transduction complexes whose components have already been identified at a molecular level. The emphasis will be on holo-Gt, its effector enzyme, cGMP PDE, and complexes containing one or more subunits of each. These complexes will be analyzed using biochemical reconstitution studies, time-resolved and steady state spectroscopy, and high resolution electron microscopy. The second area is understanding the membrane-mediated regulation of Gt's GTP hydrolysis kinetics. The rate at which Gt hydrolyzes GTP after activation by photo-excited rhodopsin plays an important role in regulating the kinetics of recovery from a light response. Work will be continued on isolating and characterizing at a molecular level a tightly membrane- associated GAP, that works together with one or more PDE subunits to accelerate Gt's hydrolysis of GTP. An important element of this work will be determination of the primary structure of GAP by peptide sequencing and molecular cloning. The mechanism by which light regulates GAP activity will be explored in isolated rod outer segments and in reconstituted systems.
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