PROTEIN INTERACTIONS IN CATECHOLAMINE REGULATION

描述(由申请人提供)： 拟议的工作建立在建立(1)GTP的先前研究的基础上 辅因子合成中的限速酶--环水解酶I 四氢生物蝶呤与蛋白质中的酪氨酸羟基酶直接相互作用 复合体和(2)跨膜蛋白，儿茶酚胺-UP(或番茄酱)， 作为GTP环水解酶和酪氨酸羟基酶的负调节因子， 也与这些酶形成复合体。番茄酱被认为是为了调节 通过隔离这些关键酶直到信号被释放来表达儿茶酚胺 收到，导致两种酶的释放和它们的激活 磷酸化。这项提案的具体目标，考验了这一点的各个方面 型号，包括： 确定番茄酱与TSH和GTPCH的相互作用是否为直接作用。 提出了GST融合下拉试验和酵母双杂交试验 这个问题。 确定磷酸化是否在调节血管形成中起作用 与番茄酱的络合物使用相互作用的蛋白质的体外操作。 利用酵母反向双杂交技术定位IF结合域 相互作用是直接的，还是用酵母双杂交文库筛选 如果番茄酱与这两种物质相互作用，则寻找相互作用的蛋白质 酶是间接的。 进行突变筛选以分离与之相互作用的基因的新突变 番茄酱。 儿茶酚胺表达的调节是正常神经的一个重要特征 功能，该通路中的缺陷与多种 包括帕金森氏病和遗传性进行性疾病在内的神经系统疾病 肌张力障碍。这些关键基因之间高度的序列保守性 在果蝇和哺乳动物的儿茶酚胺表达中， 表明这些研究将在理解 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.