For the last 30 years, the dopamine hypothesis of schizophrenia has been the dominant theoretical model which has guided clinicians in their treatment strategies and researchers in their experimental paradigms. In brief, it proposes an alteration in central dopaminergic pathways within the brains of schizophrenic patients. Particular attention has been given to dopaminergic and dopaminoceptive neurons of the basal ganglia and related structures as a possible area of pathology and/or site of action for neuroleptic treatment. The long-term objectives of may laboratory has been the isolation and characterization of genes specifically enriched within regions of the basal ganglia. A putamen-enriched cDNA library was constructed using subtractive hybridization techniques. This methodology has led to the isolation of a novel, intracellular protein tyrosine phosphatase (PTP) (Lombroso et al., 1991) which is highly enriched within the CNS and is localized within dopaminoceptive neurons of the basal ganglia and related structures (Lombroso et al., 1992). As the cDNA message was originally found by Northern analysis to be highly enriched within rat striatum, it was termed STEP for STriatal Enriched Phosphatase. Further characterization of this and other related members of this PTP family forms the basis of this FIRST (R29) application.
The specific aims of this application include the identification of additional members of the STEP family of PTPs. We have evidence that the originally cloned STEP cDNA is one of several alternatively spliced variants. We now propose to isolate full-length PTPs related to STEP. This will be accomplished by screening brain cDNA libraries with the original STEP cDNA probe, and sequencing the isolated clones and demonstrating tyrosine phosphatase activity. We will generate a panel of monoclonal antibodies which recognize individual family members, which can then be used to further localize STEP isoforms. Potential substrates of STEP will be investigated using a number of strategies. In addition, we propose to investigate the regulation of STEP in vivo after chronic administration of dopamine receptor antagonists and to demonstrate a change in STEP immunoreactivity within the striatum and nucleus accumbens. STEP expression will also be investigated in PC12 cells. STEP message and protein are not present until these cells are exposed to nerve growth factor (NGF). We now propose to identify the STEP-related PTP in PC12 cells that is induced after NGF treatment. We have recently cloned the mouse genomic clone. We now propose to sequence and characterize the promoter region. This work will lead to the identification of regions within the promoter region that controls the high degree of tissue specificity that is found with expression of STEP. This work will lead directly to the generation of transgenic mice in which STEP protein has been knocked out, and these mice will be then investigated for neurological or behavioral abnormalities.
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