Regulation of eNOS by Subcellular Targeting

描述（由申请人提供）：心血管疾病是不分种族的男性和女性死亡的主要原因，高达25%的人口患有某种形式的慢性心血管疾病。内皮细胞通过内皮型一氧化氮合酶（eNOS）产生一氧化氮（NO）的能力降低是许多心血管疾病的标志，但调节eNOS活性的机制尚不完全清楚。除了转录调控外，eNOS还受钙、磷酸化、蛋白-蛋白相互作用和亚细胞定位等翻译后机制的调控。在内皮细胞内，无论是培养的还是血管中的，eNOS都定位于质膜和核周高尔基复合体。eNOS在这两个位置的相对比例是可变的，最近发现的eNOS结合蛋白NOSIP调节这些区域内eNOS的数量。然而，eNOS在高尔基体膜或质膜上的单独功能尚不清楚。在初步结果中，我们已经表明，通过将eNOS仅靶向于高尔基体膜或质膜，细胞内位置可以直接影响NO的释放量和导致eNOS激活的机制。质膜eNOS具有较高的基础活性，对钙敏感，并在丝氨酸1179上构成磷酸化。相比之下，高尔基eNOS表现出较低的钙依赖性活性，并被Akt介导的磷酸化高度激活。基于这些发现，本提案的中心假设是利用Akt依赖通路的激动剂优先激活高尔基eNOS，钙依赖激动剂优先激活质膜eNOS。为了验证这一假设，提出了4个具体目标：第一个目标是确定利用Akt或钙依赖机制的激动剂是否优先激活高尔基体或质膜eNOS。第二个目标将确定高尔基体和质膜eNOS不同活性的机制（钙和磷酸化）。第三个目的是确定NOSIP蛋白是否在高尔基体中浓缩eNOS，从而调节特定激动剂的活性。第四个目标将确定eNOS在离体血管中脑膜下靶向对血管功能的重要性。

DESCRIPTION (provided by applicant): Cardiovascular disease is the leading cause of death for men and women regardless of ethnicity and up to 25% of the population live with some form of chronic cardiovascular disease. Reduced capacity of the endothelium to produce nitric oxide (NO), via the enzyme endothelial nitric oxide synthase (eNOS), is a hallmark of many cardiovascular diseases yet the mechanisms regulating eNOS activity are not fully understood. In addition to transcriptional control, eNOS is regulated by post-translational mechanisms including calcium, phosphorylation, protein-protein interactions and subcellular localization. Within endothelial cells, both cultured and in blood vessels, eNOS is localized to the plasma membrane and the perinuclear Golgi complex. The relative proportion of eNOS in both locations is variable and a recently discovered eNOS binding protein, NOSIP, regulates the amount of eNOS within these domains. However, the individual function of eNOS at the Golgi or plasma membrane is not yet known. In preliminary results we have shown that by targeting eNOS exclusively to the Golgi or plasma membrane, intracellular location can directly influence both the amount of NO released and the mechanisms leading to eNOS activation. Plasma membrane eNOS has elevated basal activity, is calcium-sensitive and is constitutively phosphorylated on serine 1179. In contrast, Golgi eNOS displays lower levels of calcium-dependent activity and is highly activated by Akt mediated phosphorylation. Based on these findings, the central hypothesis of this proposal is that agonists utilizing Akt- dependent pathways preferentially activate Golgi eNOS and that calcium-dependent agonists preferentially activate plasma membrane eNOS. To test this hypothesis, 4 specific aims are proposed: The first aim will determine whether agonists utilizing Akt or calcium-dependent mechanisms preferentially activate Golgi or plasma membrane eNOS. The second aim will determine the mechanisms (calcium and phosphorylation) underlying the divergent activities of Golgi and plasma membrane eNOS. The third aim will determine whether the protein NOSIP concentrates eNOS in the Golgi and thus modulates the activity of specific agonists. The fourth aim will determine the importance of eNOS subcetlutar targeting to vascular function in isolated blood vessels.