The long term goals of this research are to investigate the structural mechanisms underlying the regulation of glycogen synthase by phosphorylation. The working hypothesis is that there exists a globular core with an inherent synthase D type of activity, and that its interaction with the hydrophilic C-terminus (containing most of the phosphorylation sites) directs a conformational change to the active I form; phosphorylation then serves to uncouple this interaction. Experiments are designed lo test this idea and to delineate the structural elements within the C-terminus that are crucial to the process. The foundation for the study is our recent cloning of the cDNA for rat liver glycogen synthase. The proposed aims are as follows. 1. An expression system for the preparation of recombinant rat liver glycogen synthase will be developed. This will done using E. coli expression vectors. 2. Deletion mutagenesis will be used to investigate the roles of specific subregions of the C-terminus in maintaining the I-state of synthase. The properties of the C-terminal deletion mutants should provide direct evidence for the hypothesis that the catalytic core has the properties of a D-like enzyme and that the C-terminus is involved in maintaining the I state. 3. The effects of synthetic peptides representing the C-terminal subdomains on the native synthase and its deletion mutants will be studied to investigate their interactions with the catalytic core, and to determine how they affect the maintenance of the I-state. These studies are directly complementary to those proposed in 2, and will also allow for testing of """"""""mutant peptides"""""""" that will further define important structural elements to the level of individual residues. 4. Site-directed mutagenesis will used to modify individual phosphorylation sites, so that their contribution to the I to D conversion of synthase can be assessed. Modification of lys4O will be performed to evaluate its function in at the catalytic site. The effects of the restoration of sites la and lb to the liver synthase structure will be studied. As individual residues involved in the C-terminal interaction with the core are identified, their functions will be further investigated by site directed mutagenesis.
