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 dopamine 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 specific D1 receptor residues can be identified that interact with arrestin in a phosphorylation-independent manner. The binding of arrestin-2 and -3 to intracellular fragments of the dopamine D1 receptor will be evaluated using an in vitro pull-down assay. Selected residues in the intracellular domains will be deleted or mutated to identify arrestin binding sites. Interaction sites will be verified by characterization of full-length mutant receptors in human embryonic kidney 293 cells and in neurons. The second specific aim is based on the hypothesis that mutating two binding sites for arrestin will additively decrease binding, and will seek to optimize an arrestin-insensitive D2 receptor, characterized by expression in 293 cells and in neurons. The third specific aim will test the hypotheses that arrestin mediates D1 and D2 receptor internalization and desensitization, and participates in D2 receptor signaling. The consequences of dopamine receptor:arrestin interactions on D1 and D2 receptor desensitization and internalization, D2 receptor activation of mitogen-activated protein kinases, and D2 receptor-induced heterologous sensitization of adenylate cyclase will be determined in vitro and in vivo using dopamine receptor mutants deficient in arrestin binding. The fourth specific aim is driven by the hypotheses that one mechanism of D2 receptor- stimulated heterologous sensitization of adenylate cyclase is phosphorylation of type 5 adenylate cyclase by the mitogen-activated protein kinase kinase kinase Raf-1, and that arrestin also contributes to heterologous sensitization. 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, and the role of arrestin will be assessed using arrestin-insensitive mutant receptors.
The study of mechanisms of regulation of dopamine receptor responsiveness will contribute both to general theories of receptor regulation and to our knowledge of the mechanisms that are unique to dopamine receptors, which is particularly important because of the central role that dopamine receptors play in the treatment of neuropsychiatric disorders such as schizophrenia, Parkinson's disease, and drug abuse. Analysis of wild type and mutant dopamine receptors expressed in mammalian cell lines, in neurons, and in vivo offers powerful model systems with which to perform basic research with important clinical implications.
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