The betagamma subunits from heterotrimeric G proteins occupy a central position in the G protein coupled receptor system. They are multifaceted proteins that have determinants that allow for interaction with G protein coupled receptors, G protein alpha subunits and multiple downstream target effector molecules. While the three dimensional structure for betagamma subunits has been solved, the structural and biochemical features that are responsible for functional interactions with protein partners are not entirely understood. No definitive common sequence motif(s) has been identified that specifies betagamma subunit interactions with a diverse array of targets. Some possible reasons for this are: 1) There are consensus motifs for different groups of effectors but the specific sequence requirements are subtle, making sequence motifs difficult to identify by simple homology analysis. 2) Interactions with betagamma subunits are specified by common structural motifs that can be adopted by many sequences. 3) Each protein has a unique mode of interaction with the betagamma subunits. A combination of these is also possible if the targets have multiple interactions with betagamma subunits and only some in common. This proposal is designed to test these hypotheses by identifying structural determinants required for interactions with betagamma subunits. In the course of these experiments we will develop concepts and tools that will be useful for studying betagamma mediated signal transduction. More specifically, we propose to: I. Determine peptide sequence requirements for interaction with betagamma subunits by screening random peptides libraries displayed on filamentous phage for interactions with betagamma subunits. II. Screen phage display libraries to develop peptide antagonists for different betagamma- effector interactions. III. Confirm the selectivity and utility of the peptide antagonists in assays of receptor mediated signal transduction. IV. Determine the structural basis for these interactions using NMR spectroscopy to solve the 3 dimensional structure of peptides that bind to different surfaces of betagamma subunits.
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