A hallmark of many neuropsychiatric and neurodegenerative diseases is an apparent brain imbalance or abnormality involving the neurotransmitter dopamine. This project will employ pharmacological and genetic tools to dissect the complex signaling cascades that carry out dopamine's actions in the basal ganglia. Prior research has shown that dopamine, acting on D1-type dopamine receptors, increases the activity of cAMP-dependent protein kinases (PKA), leading to phosphorylation of DARPP-32 at Thr34. Phospho-Thr34 inhibits protein phosphatase 1, a major phosphatase in basal ganglia neurons, thereby regulating the state of phosphorylation of many cellular proteins. Previous studies have documented the ability of dopamine to increase the phosphorylation of many cellular proteins. Previous studies have documented the ability of dopamine to increase the phosphorylation of NMDA- and AMPA- type glutamate receptors and receptors for the major inhibitory neurotransmitter, GABA, resulting in changes in their physiological activity. The proposed studies will use several types of gene-knockout mice to identify the contributions of selected isoforms of protein phosphatase 1 (PP1) and the PP1 targeting proteins, spinophilin and neurabin, to the regulation of receptor phosphorylation. In parallel with these experiments, studies performed in Project III will utilizes the same genetic models to identify the precise effects of PP1 isoforms and regulatory proteins on the activity of these receptors in basal ganglia neurons. The role of protein phosphorylation in dictating the cellular localization and spinophilin and neurabin and in directing the respective interactions of these proteins with PP1 and with other effector proteins will be evaluated. Considerable attention will be focused on characterizing a phosphorylated at Thr34, it is an inhibitor of PP1, whereas DARPP-32 phosphorylated at Thr 75 is an inhibitor of PKA. The factors that control the levels of phosphorylated Thr75 in basal ganglia neurons, either by regulating the activity of a cyclin-dependent protein kinases that phosphorylates this site (cdk5), or by controlling the activity of the protein phosphatase (PP2A) that dephosphorylates this site will be identified. In addition, mice bearing selective genetic deletions of either Thr34 or Thr75 will be used to determine the relative contributions of these sites to the actions of mediating the effects of dopamine on the phosphorylation state (this project) and activity (Project III) of glutamate and GABA receptors. It is hoped that, collectively, these studies will elucidate the fundamental circuitry responsible for normal and neuropathic effects of dopamine on neurons of the basal ganglia.
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