The rod visual signal transduction cascade is a model system for understanding the transduction of signals such as neurotransmitters, hormones, and sensory stimuli into cellular responses. These signal transduction mechanisms are essential for maintenance of cellular homeostasis in the face of changing external conditions, and as such the are key targets for toxins and disease processes. Recent studies have implicated a growing number of inherited diseases, including retinal diseases, cancer and endocrine diseases, as well as the common killers diabetes and cardiovascular diseases, which target G protein cascades and disrupt normal signalling. Understanding the basic mechanisms of receptor- mediated cellular signalling is thus important as a starting point for gene targeting and drug interventions in a variety of disease states. The goal of this proposal is to understand mechanisms of visual excitation in rod photoreceptors in mechanistic and structural detail. Some of the critical questions that require further experimentation are """"""""How do activated receptors promote GDP release and G protein activation? What is the mechanism of interaction between alpha and beta-gamma subunits and how does GTP regulate subunit interaction? How do GTP-liganded G proteins activate effectors? What is the mechanism of GTPase activity and how do effectors regulate the GTPase rate?."""""""" To approach these questions a variety of biochemical and biophysical approaches are being used. Synthetic peptides corresponding to sites of protein-protein interaction act as competitive inhibitors and serve to probe mechanisms of interaction. Proteolytic digestion, photoaffinity cross-linking, and immunological probes will also be used in these studies. The three-dimensional structures of the alpha and beta-gamma subunits of G(t) as well as the heterotrimeric G(t), solved in collaboration with Paul Sigler's laboratory, provide a structural framework for the studies. Inactivation and adaptation mechanisms that impact the excitation cascade will also be addressed in this proposal. The effector enzyme cGMP phosphodiesterase, in concert with a membrane-bound factor, can accelerate the turnoff of excitation by speeding the GTP hydrolysis reaction, and the mechanism of this effect will be studied. In addition, cyclic nucleotide- dependent phosphorylation mechanisms also have been proposed to impact the G protein activation rate, and this will be investigated further. These biochemical and mechanistic studies will increase understanding of the mechanisms of visual transduction. In addition, they will allow us to design specific blockers or stimulants of G protein-mediated signalling cascades that will be useful for drug development and gene targeting approaches to multiple disease states.
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