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 the DARPP-32/protein phosphatase-1 signaling cascade. 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 these molecules in mice. This project represents a Scientific Core facility to provide technical support to all members of the Program Project. The responsibilities of the Core will include the production and supply of animals and key reagents, and the performance of routine yet critical tasks that will be required to accomplish the studies described in 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. The Core will maintain the colonies of knockout and transgenic mice that are already in hand or will be produced by Project IV. These include knockouts of DARPP-32, spinophilin, neurabin, conditional knockouts of phosphatase-1 and isoforms, and transgenic animals harboring phosphorylation site mutations in DARPP-32. Animal husbandry will include the setting up of appropriate matings, weaning backcrossing, and genotyping the offspring, using PCR and Southern blot strategies. Stocks purified protein kinases, protein phosphatases, affinity- purified antibodies, and substrate proteins will maintained. 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 supervise the production and testing of new polyclonal anti-peptide enzymological and structural studies. The Core will supervise will supervise the production and testing of new polyclonal anti-peptide antibodies and phosphorylation state-specific antibodies for neurabin, spinophilin and their novel binding partners, and the B56 subunit of PP-2A. 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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