Dopamine (DA) is important in the regulation of mood, cognition, motivation, motor activity, and endocrine function. There are multiple DA receptors (D1, D2, D3, D4, D5) that are typically divided into two groups of receptors, the D1-like receptors (D1 and D5) and the D2- like receptors (D2, D3, D4). Drugs that target Dl-like or D-2 like receptors have been identified, but finding drugs to target only a specific member within the D1-like subfamily (D1 versus D5 receptor) has proven to be much more difficult. Therefore, we are using an alternative site, that of the receptor-G protein interface. A variety of studies have implicated the carboxyl-terminus of G protein a subunits in mediating receptor-G protein interaction. We have shown that peptides corresponding to the carboxy-terminal sequence of Ga can be used to block receptor signaling. For this project, we will identify high affinity peptide analogs based on the carboxyl terminus of Gas subunit (Gs) that bind selectively to activated D5 receptor. The identified peptides will be analyzed for their ability to bind active versus inactive receptor, modulate agonist binding affinity, compete with binding of heterotrimeric Gs, and antagonize Gs signaling to adenylyl cyclase. To characterize the ability of the high affinity peptide analogs to inhibit D5 receptor signaling in whole cells, minigene constructs carrying oligonucleotide sequences corresponding to the high affinity peptide will be ligated into a mammalian expression vector, and the vectors will be transfected into HEK 293 cells. Following agonist stimulation the transiently transfected cells will be tested for changes that occur as a result of blocking the D5-Gs protein interface. We will also test to see if the peptides are selective for D5, or if they also inhibit D1-Gs coupling. In the future, we will exploit the high affinity peptides to identify small molecules that can competitively dissociate the peptides from the D5 receptor. Our approach will potentially lead to the discovery of agents that are D5 receptor specific.