Kim A. Neve, Ph.D. - US grants

The objective of this research program is to determine the characteristics and mechanisms of the regulation of dopamine D-2 receptors. It is crucial that we understand the regulation of D-2 receptors, since idiopathic or drug-induced changes in the density of dopamine receptors are thought to be involved in the pathophysiology or treatment of psychiatric and movement disorders such as schizophrenia, parkinsonism, and tardive dyskinesia. The cloning of a cDNA for the D-2 receptor makes it possible to develop tools that have not been available for the study of D-2 receptor regulation. In particular, cell lines expressing a high density of D-2 receptors are being created by transfecting cells with the cDNA clone, RGB-2. The aim of this proposal is to develop several cell lines derived from various tissues and to use these cells to determine the mechanisms of desensitization and down-regulation of D-2 receptors. To achieve these goals, the following specific objectives will be met. 1) Four new cell lines expressing D-2 receptors will be created by transfecting cells derived from various tissues with either cDNA or genomic DNA encoding the D-2 receptor. 2) Receptors on the cells will be characterized by radioligand binding, by biochemical assays of adenylate cyclase activity, and by photoaffinity labeling and gel electrophoresis. 3) Conditions for treating cells will be optimized by assessing the possibility of growth-dependent changes in D-2 receptor density. 4) Desensitization of D-2 receptors will be evaluated using four measures of rapid desensitization that assess coupling of receptors to G proteins and subcellular localization of the receptors. 5) The effect on the density of D-2 receptors of treating cells with D-2 receptor agonists will be determined. The time course of agonist-induced loss of receptors will be compared to that of desensitization of adenylate cyclase and to the efficacy of the agonist for inhibition of adenylate cyclase. 6) Agonist-induced changes in levels and transcription of D-2 receptor messenger RNA will be quantified by Northern blot analysis and nuclear runoff experiments.

The overall objective of this research program is to identify the molecular mechanisms of the agonist-dependent regulation of responsiveness of dopamine (DA) receptors. The molecular cloning of many subtypes of DA receptors makes it possible to compare the regulation of defined populations of receptors on identical cell backgrounds. Furthermore, the structural features of the receptors that specify the types of regulatory responses observed can be determined by construction of mutant or chimeric receptors. 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 psychiatric and movement disorders such as schizophrenia, parkinsonism, and tardive dyskinesia. The specific aims of the project are as follows: 1a) The effect of prolonged exposure to agonists on the coupling of DA D1 receptors to adenylyl cyclase will be assessed. Both homologous and heterologous components of regulation will be assessed. In addition, regulatory responses of recombinant and endogenous receptors will be compared. 1b) The structural determinants of the sensitivity of coupling of DA receptors to adenylyl cyclase will be assessed by the construction of mutant Dl receptors in which potential sites of phosphorylation are eliminated. A special emphasis of this aim will be to distinguish structural features of receptors necessary for homologous regulation from those necessary for heterologous regulation. 2a) Agonist-dependent regulation of the density of Dl, D2, and D3 receptors will be characterized. Regulation of endogenous and recombinant Dl and D2 receptors will be compared. A key objective is to determine if agonist-induced proliferation of D2 and D3 receptors results from altered synthesis or degradation of receptors. 2b) The structural determinants of agonist-dependent regulation of Dl, D2, and D3 receptor density will be identified by the characterization of Dl/D2 and Dl/D3 chimeric receptors and receptors with point mutations.

The overall objective of this research program is to identify proteins that regulate the activity or subcellular localization of the alpha subunit (G- alpha) of heterotrimeric G proteins. Regulatory proteins may be proteins whose interaction with G-alpha was not previously appreciated, or they may be novel proteins or protein families. The Regulators of G Protein Signaling (RGS proteins) are a recent example; RGS proteins enhance the intrinsic GTPase activity of many G-alpha subunits, thus tending to decrease the activity of the G proteins. Four proteins that interact with G- alpha-s have been identified in the yeast two-hybrid assay. Two of the four proteins are NEFA and the mu subunit of the clathrin-associated protein AP2. The other two are essentially unknown, one being homologous to a predicted protein of unknown function identified in the C. elegans genome project, and the other described only as an anonymous brain mRNA encoding a 1405 amino acid protein. Subsequent to the identification of NEFA in the two-hybrid assay, we confirmed a physical interaction between NEFA and G-alpha-s. The objective of the present application is to determine the functional significance of this interaction, and to initiate experiments to confirm and characterize the interactions between the other three proteins and G-alpha-s. The first specific aim is driven by the hypothesis that the identification of the proteins NEFA, AP2-mu, C16C10.10, and AB002373 in the two-hybrid assay is due to a physical interaction between each of these proteins and G-alpha-s. 1) The physical interaction between G-alpha-s and proteins identified in the initial yeast two-hybrid screen will be confirmed. The second specific aim is based on the hypothesis that the physical interaction between NEFA and G protein alpha subunits is physiologically relevant. 2) The subcellular and regional distribution of NEFA and its cognate mRNA will be characterized, and the possibility of a functional interaction between NEFA and certain subtypes of G-alpha will be evaluated. The third specific aim is to assess the hypothesis that the existence of a physical interaction between a given target protein and G-alpha-s, as determined in aim l, reflects a functional interaction between the protein and some subtype of G-alpha. 3) Functional consequences of physical interactions, confirmed in aim l, between G-alpha and any of the other three target proteins will be determined.

DESCRIPTION: (provided by applicant) DOPAMINE 2002 will take place in Portland, Oregon from July 10 to July 14, 2002. The meeting will be held at the Benson Hotel in downtown Portland. DOPAMINE 2002 is the next in a series of approximately quadrennial international meetings that began in 1977 at the University of Southampton, England. The goal of the DOPAMINE meeting is to provide broad coverage of the most cutting-edge areas of research related to the neurotransmitter dopamine, and to do this in an informal setting that maximizes interactions among meeting attendants. The characteristics of the meeting that promote interaction include its relatively small size, the scheduling of all events in series (i.e., there are no symposia or poster sessions taking place simultaneously), and the inclusion of a majority of meals as part of the conference schedule. The scientific program includes 12 symposia and 1 workshop organized within 4 themes. Theme I, "Molecular Biology of Dopamine Signaling," includes symposia on presynaptic mechanisms, receptor structure and function, and receptor trafficking, as well as a workshop on knockout and transgenic mice. Theme II, "Physiology and Pharmacology of Dopamine Systems," includes symposia on dopamine regulation of non-neuronal systems, interactions between dopamine and other neurotransmitters, and the development and differentiation of dopamine systems. Theme III, "Cognition and Behavior," includes symposia on the roles of dopamine in reward and substance abuse, in psychiatric disorders, and in cortical function. Theme IV, "Dopamine and Neurological Disorders," includes symposia on dopamine and mechanisms of neurotoxicity, Parkinson's disease, and hyperkinetic movement disorders. The symposia will consist of formal presentations from senior leaders in the field as well as junior investigators engaged in particularly exciting research related to dopamine. Poster sessions presenting work by a diverse mix of participants from senior investigators to pre- and post-doctoral fellows will accompany each theme. The banquet will feature informal talks by 2000 Nobel Prize Laureates, Arvid Carlsson and Paul Greengard, and the opening lecture will be given by Marc Caron, the organizer of Dopamine '94 in Quebec City.

DESCRIPTION (provided by applicant): Trace amines are biogenic non-catecholamine monoamines that are present at low concentrations in the vertebrate nervous system. Some trace amines, such as octopamine and tyramine, are more abundant in invertebrate nervous systems, where their function as neurotransmitters is fairly well established. The heterogeneous CNS distribution and rapid turnover of trace amines, together with the existence of trace amine receptors, suggest that some trace amines may also be neurotransmitters or co-transmitters in the mammalian CNS. This proposal takes advantage of the recent identification and molecular cloning of the DNA for a family of trace amine receptors (TARs). Interestingly, the TARs are clustered in a region of human chromosome 6 (6q23) where linkage to schizophrenia has been suggested by a number of studies. A role for trace amines in neuropsychiatric disorders has been proposed, and the TARS are also sites of action for many abused drugs. These findings suggest the hypothesis that a polymorphism in one of the TARs changes the function of the protein and thus contributes to the symptoms of schizophrenia and/or to drug abuse. As an initial step towards evaluating this hypothesis we propose the following specific aims: 1. Each TAR will be stably expressed in mammalian cells under conditions that promote its expression on the cell surface. Three strategies to be tested are genetic modification of the receptors, the use of potential ligands as "pharmacological chaperones", and inhibition of receptor internalization. 2. Agonists and antagonists for the TARs will be identified by testing the ability of potential monoamine ligands to stimulate or inhibit cyclic AMP accumulation and to activate mitogen-activated protein kinases in mammalian cells stably expressing the receptor subtypes. 3. TAR variants with non-synonymous SNPs will be stably expressed in mammalian cells to determine the effect of the polymorphism on cell surface expression, ligand binding, and function of the receptor. The ideal outcome of these studies would be to identify conditions under which all TARs encoding full-length receptors are expressed on the cell surface, to identify agonists and antagonists for each TAR subtype, and to identify allelic variants that alter the function or ligand binding of a TAR subtype

DESCRIPTION (provided by applicant): 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.