The overall objective of this research program is to identify the molecular mechanisms of agonist-dependent regulation of the sensitivity of dopamine (DA) receptors. For G protein-coupled receptors (GPCRs), the study of how the sensitivity of receptors is regulated has become chiefly the study of agonist-induced receptor trafficking and of agonist-induced GPCR interaction with other proteins. It is crucial that we understand the mechanisms of regulation of DA receptors, since idiopathic or drug-induced changes in the responsiveness of DA receptors are thought to be involved in the pathophysiology or treatment of neuropsychiatric disorders such as schizophrenia, parkinsonism, and drug abuse. The first specific aim is driven by the hypothesis that agonist binding to the D1 receptor induces rapid receptor internalization and translocation of endogenous arrestin3 to the membrane. To test this hypothesis, the. effect of D1-like receptor stimulation on the subcellular localization of the D1 receptor and of arrestins in neostriatal neurons and NS20Y neuroblastoma cells will be determined using immunoblotting, immunoprecipitation, and immunocytochemical approaches. The second specific aim is based on the hypothesis that specific D1 and D2 receptor residues can be identified that interact with arrestin in a phosphorylation-independent manner. Interaction of arrestin2 and -3 with second, third, and fourth intracellular domains of the DA D1 and D2 receptors will be evaluated using the GST pull-down assay. Selected residues will be deleted or mutated to identify arrestin binding sites on the receptors. Potential sites of phosphorylation will be mutated to aspartic acid to mimic phosphorylation. Interaction sites will be verified by characterization of full-length mutant receptors in NS20Y cells. The third specific aim will test the hypotheses that arrestin mediates D1 and D2 receptor internalization and some pathways for D2 receptor signaling. The consequences of DA receptor:arrestin interactions on D1 and D2 receptor internalization, D2 receptor activation of ERK MAP kinases, and D2 receptor-induced heterologous sensitization of adenylate cyclase will be determined using DA receptor mutants deficient in arrestin binding and by manipulating the expression of arrestin. The fourth specific aim is driven by the hypothesis that one mechanism of D2 receptor-stimulated heterologous sensitization of adenylate cyclase is phosphorylation of type 5 adenylate cyclase by the MAP kinase kinase kinase Raf-1. Raf-1-catalyzed phosphorylation of type 5 adenylate cyclase and the role of that phosphorylation in D2 receptor-mediated heterologous sensitization of adenylate cyclase will be determined.