PROTEIN INTERACTIONS IN CATECHOLAMINE REGULATION

描述（申请人提供）： 建议的工作建立在以前的研究建立（1）GTP， 环化水解酶I，辅因子合成中的限速酶 四氢生物蝶呤，直接与蛋白质酪氨酸羟化酶相互作用 复合物和（2）跨膜蛋白，儿茶酚胺-up（或Catsup）， 作为三磷酸鸟苷环化水解酶和酪氨酸羟化酶的负调节剂， 也与这些酶形成复合物。番茄酱被假设为调节 通过隔离这些关键酶，直到信号 这两种酶的释放和它们的激活， 磷酸化本提案的具体目标是测试本提案的各个方面， 模型，是： 确定Catsup与TSH和GTPCH是否直接相互作用。 GST融合下拉法和酵母双杂交法被提出来解决 这个问题 为了确定磷酸化是否在调节 使用相互作用蛋白质的体外操作与Catsup形成复合物。 使用酵母反向双杂交相互作用测定来定位结合结构域， 相互作用是直接的，或者使用酵母双杂交cDNA文库筛选， 寻找相互作用的蛋白质，如果番茄酱和这两个之间的相互作用 酶是间接的。 进行突变筛选，以分离与 番茄酱 儿茶酚胺表达的调节是正常神经细胞中的一个关键特征， 功能，这一途径中的缺陷与各种 神经系统疾病，包括帕金森病和遗传性进行性 肌张力障碍这些关键基因之间高度的序列保守性 在果蝇和哺乳动物中， 这表明，这些研究将有广泛的适用性，在理解 儿茶酚胺调节机制。

DESCRIPTION(Provided by Applicant): 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.