Tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine biosynthesis, is regulated by factors that increase its rate of synthesis (induction) and its intrinsic enzyme activity by post-transnational phosphorylation (activation). This proposal addresses both aspects of regulation. The mechanism by which various known inducers (cAMP, glucocorticoids, epidermal growth factor (EGF)) and environmental factors such as stress (transsynaptic induction) or trophic molecules induce TH RNA in vivo or in cultured cells will be studied. In particular, experiments will determine whether inductive factors responsible for the phenotypic switch in transmitter synthesis increase TH transcription rates or changes TH mRNA levels by post-transcriptional mechanism. Experiments will define cis-acting DNA sequences responsible for hormone or tissue-specific expression. These combine analyses of the functional DNA sties by transfection into cell lines and the ability of trans-acting factors to bind these sites. To correlate whether putative sites affect in vivo function, transient transfections will be performed in the presence of excess competing DNA encompassing the potential site, which would be expected to block hormone induction or tissue-specificity is blocked. Tissue specificity will also be assessed in vivo using transgenic mice bearing putative tissue-specific enhancers directing bacterial chloramphenicol acetyltransferase (CAT). The possibility that alternative splicing generates distinct TH molecules that can be differentially activated by phosphorylation will be explored by cloning cDNAs for various putative isoforms and determining their tissue distribution and inducibility by various manipulations. These isoforms will be individually expressed and their ability to be phosphorylated and activated by purified kinases in vitro will be assessed.
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