Our overall interest is to understand the molecular basis of signal transduction in the vertebrate visual system. G proteins are essential elements in diverse signal transducing systems, including the visual system. Indeed, the transducin-rhodopsin couple remains the most well understood of all the G protein mediated signal transducing systems. Recent studies have shown that these G proteins are post-translationally modified by the covalent linkage of a hydrophobic isoprenoid moiety to a carboxyl terminal cysteine residue followed by the reversible carboxymethylation of the same residue in a process termed prenylation. It appears that these modifications are critical for the physiological function of the G proteins, although the mechanisms by which these modifications might affect function is unknown. We have found that the visual system heterotrimeric G proteins transducin is prenylated and methylated, as are the rod outer segment (ROS) 'small' G proteins. We propose to study the biochemical basis and the role of the G protein methylation, which is reversible, and prenylation in ROS at the molecular level. In other signal transducing systems, such as in bacterial chemotaxis, reversible methylation reactions have key regulatory significance. The proposed investigation will initially entail studying the interactions between methylated and demethylated transducin and activated rhodopsin to understand the biochemical role of the reversible methylation step. We will also quantitatively study the membrane binding properties of methylated and demethylated G proteins and model compounds, including synthetic peptides, and the possible role that specific lipid-lipid interactions might play in membrane targeting. Finally, the ROS associated methylase and demethylase enzymes will be purified and characterized. Specific inhibitors will be designed for these enzymes, based on their novel mechanistic properties. These inhibitors will be used to further elucidate the physiologic role(s) of G protein methylation and demethylation in signal transduction in the visual system. It is expected that the studies reported herein on G protein processing and regulation in the visual system will serve as a general model.
Showing the most recent 10 out of 48 publications
