A greater understanding of the intracellular signal transduction mechanisms which underlie dopaminergic neurotransmission in the basal ganglia can lead to novel therapeutic targets for the treatment of schizophrenia, and degenerative neurological disorders such as Parkinson's disease and Huntington's disease. A multi-disciplinary approach will be undertaken in the current proposal to investigate the phosphorylation-dependent functional regulation of key molecules in these signaling pathways. Studies will be performed at several levels of organizational complexity, ranging from in vitro biochemical studies with purified molecules to the characterization of phenotypes resulting from targeted deletion of key signal transduction molecules in whole animals. The overall goal of this project is to provide a Scientific Core facility for use by all the members of the Program Project. The responsibilities of the Core will include the production and supply of key reagents and the performance of routine, yet critical tasks that will be required to accomplish the studies described in the other sections of this Program Project. The centralization of routine tasks will facilitate an efficient, cost-effective means to ensure an adequate supply of essential materials and reagents to all the Projects. The Core will maintain a tissue culture facility to provide a continuous supply of cultured cells, including primary neuronal cultures, reaggregated cultures and immortalized cell lines. It will also maintain stocks of purified protein kinases, protein phosphatases, antibodies, and substrate proteins. Standard purification protocols will be used to obtain these enzymes from native sources, and recombinant technologies will also be employed to produce specific proteins in quantities sufficient to carry out detailed enzymological and structural studies.. The Core will be responsible for the expansion and maintenance of the colonies of """"""""knockout"""""""" mice that are deficient in DARPP-32, inhibitor-1, protein phosphatase-1 alpha1 and gamma isoforms, and protein phosphatase- 2B, and transgenic animals harboring phosphorylation site mutations in DARPP-32.. Animal husbandry will include the setting up of appropriate matings and weaning of animals. The Core will also be responsible for the determination of the genotype of the offspring, using PCR and Southern blot strategies. The Core will be responsible for the production of phosphorylation state- specific antibodies to specific sites on DARPP-32, inhibitor-1, protein phosphatase-1, Na+,K+-ATPase alpha subunit isoforms and Na+ and Ca2+ channels. The ability to detect and quantitate changes in the state of phosphorylation of these key substrate proteins will be of great utility in the studies proposed to elucidate their functional significance.
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