Heterotrimeric and small G (ras-family) proteins are post- translationally modified by isoprenylation/methylation, a modification important for membrane association and function of these important signal transducing proteins. This grant request is focused on the molecular enzymology and functional importance of isoprenylation/methylation as it relates to signal transduction in the visual system. With respect to the enzymology, two novel endoproteases are essential for the maturation and degradation of transducin, the photoreceptor heterotrimeric G protein which interacts with activated rhodopsin. The two endoproteases are to be purified, chemically labeled, cloned, and sequenced. The biochemical roles of these proteases with respect to transducin and the ras family of G proteins will be explored. Deleterious mutations in either of these proteases would be expected to have serious consequences in vision. Inhibitors of one of the proteases blocks oncogenic ras function, suggesting the possibility that these inhlbitors may be candidate anti-cancer agents. While isoprenylation/methylation operates by a hydrophobic mechanism in allowing transducin to associate with photoreceptor disk membranes, the situation with respect to the small G protein family is unclear. Here specific ligand-receptor interactions may be mediated via the isoprenylated/methylated cysteine moiety. Functional studies on isoprenylation/methylation will be extended to the ras family of small G proteins where the importance of the individual enzymatic steps of isoprenylation/methylation will be determined. The specific effects of small molecule farnesylcysteine analogs on small G protein function will be addressed in this context. Biochemical assays will include determining the effects of isoprenylation/methylation and the farnesylcysteine analogs on small G protein membrane docking, and on activation of effectors, such as the PAK and MAP kinases. These studies will be extended to an investigation of the nature of the small O proteins in photoreceptors, and their possible roles in visual signal transduction.A subset of farnesylcysteine analogs have been found to activate protein kinase C (PKC) specifically, an important signal transducing enzyme found in photoreceptors and many other cell types. Structure-activity studies on these analogs have uncovered novel specific purine analogs which are effector-site inhibitors of PKC. These new antagonists will be used to uncover the physiological importance of protein PKC in visual signal transduction. The possibility that endogenous purines are antagonists of PKC will be explored.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Visual Sciences C Study Section (VISC)
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Harvard University
Schools of Medicine
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