The proposed work builds on previous studies establishing (1) that GTP cyclohydrolase I, the rate-limiting enzyme in the synthesis of the cofactor tetrahydrobiopterin, interacts directly with tyrosine hydroxylase in protein complexes and (2) that a transmembrane protein, Catecholamines-up (or Catsup), acts as a negative regulator of GTP cyclohydrolase and tyrosine hydroxylase, also forming complexes with these enzymes. Catsup is hypothesized to regulate catecholamine expression by sequestering these key enzymes until a signal is received, resulting in the release of the two enzymes and their activation by phosphorylation.
The specific aims of this proposal, which test aspects of this model, are: To determine whether the interaction of Catsup with TSH and GTPCH is direct. GST-fusion pull-down assays and yeast two-hybrid assays are proposed to address this question. To determine whether phosphorylation has a role in regulating the formation of complexes with Catsup using in vitro manipulation of the interacting proteins. To use yeast reverse two-hybrid interaction assays to map binding domains if interactions are direct, or to use yeast two-hybrid cDNA library screens to search for interacting proteins if interactions between Catsup and these two enzymes are indirect. To conduct mutant screens to isolate new mutations in genes that interact with Catsup. Regulation of catecholamine expression is a key feature in normal neural function, and defects in this pathway are associated with a variety of neurological disorders including Parkinson's disease and hereditary progressive dystonia. The very high degree of sequence conservation between these key genes in catecholamine expression in Drosophila and their counterparts in mammals, suggest that these studies will have broad applicability in understanding catecholamine regulatory mechanisms.
