The long-term goal of this research program is to elucidate molecular mechanisms of function and regulation of the rod and cone cGMP-PDEs that serve as key effector enzymes in the visual transduction cascade of vertebrate photoreceptor cells. In the visual cascade, the GTP-bound form of G protein, transducin, activates PDE by relieving the inhibition imposed by the PDE-gamma subunits (PDE-gamma) on the enzyme catalytic subunits. Insights into the interaction between PDE-gamma and the catalytic subunits are critical for understanding the mechanisms of PDE activity inhibition by PDE-gamma and PDE activation by transducin. One of the objectives is to develop a detailed map of the PDE-gamma binding sites on PDE catalytic subunits using synthetic peptides as probes, and employing a crosslinking of specifically designed PDE-gamma mutants to the PDE catalytic subunits. In depth investigation of the photoreceptor PDE structure and function requires an extensive mutational analysis of the enzyme catalytic subunits. Mutagenesis of the PDE catalytic site, noncatalytic cGMP-binding sites and the PDE-gamma binding sites will be carried out. Specific amino-acid residues involved in the PDE functions will be identified. These studies will help to elucidate molecular details of cGMP hydrolysis by PDE, and of PDE activity inhibition by PDE-gamma. A better understanding of the role of noncatalytic cGMP-binding sites and their reciprocal relationships with the PDE-gamma binding sites is to be achieved. As a tool to develop an efficient expression system for mutagenesis of PDE, chimeric proteins with a maximal structural and functional resemblance to native PDEs will be made between photoreceptor PDEs and homologous cGMP-binding PDE. Transcriptional regulation of PDE genes is likely to participate in controlling proper stoichiometry and assembly of PDE subunits. The hypothesis that PDE-gamma may be involved in transcriptional regulation of the PDE catalytic subunit genes will be investigated. Clinical relevance of structure-function studies of PDE is underscored by the findings that link certain forms of human retinal degeneration to mutations of PDE catalytic subunits. These studies will aid in understanding the mechanisms of retinal degeneration caused by mutations of PDE genes.
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