CALRETICULIN REGULATION OF LUNG ENDOTHELIAL CELL NOS

描述(申请人提供)：多功能钙(Ca~(2+))结合蛋白钙网蛋白(CRT)的合成增加已在以下文献中报道 不同组织中不同的生理和病理生理条件 包括血管内皮细胞。我们最近报道了血管紧张素 静脉刺激增加肺内皮细胞CRT的表达 CRT与血管内皮细胞亚型的蛋白质相互作用 一氧化氮合酶(ENOS)与持续性(12小时)有因果关系 ENOS在转录后水平的激活。其分子机制的研究进展 CRT介导的eNOS持续激活似乎是独特的，因为所有 参与eNOS激活的其他已知转录后机制 仅引起有限的或短暂的激活。目前CRT对此一无所知 内皮型一氧化氮合酶催化活性的分子相互作用及调控。 根据我们早先的报告和初步数据，我们假设 内皮型一氧化氮合酶的电子转移控制元件与CRT的相互作用 覆盖其假定的自抑制功能，导致电子增强 ENOS、NO/环鸟苷5‘-磷酸转移和持续激活 (CGMP)的产生和血管松弛反应。为了检验这一假设，我们将 1)确定内皮型一氧化氮合酶还原酶域中CRT是否与ETE相互作用 与电子转移和催化活性增加有因果关系 2)刻画了CRT不同分子间的相互作用 N-结构域，或P-结构域(高亲和力钙结合部位)，或C-结构域(低 亲和力、钙结合部位)和ETE对催化活性的增强起作用 以及3)验证CRT的表达增加还是其离散 分离的内皮细胞和肺动脉节段中的结构域增强 ENOS活性、NO/cGMP生成及血管松弛作用。身份的识别 内皮型一氧化氮合酶EtE与CRT的分子相互作用 ENOS的激活将促进我们对eNOS调控的理解 CRT增加时维持肺血管功能的研究 在不同的生理和病理生理条件下表达。

DESCRIPTION (provided by applicant): Increased synthesis of a multifunctional calcium (Ca2+) binding protein calreticulin (CRT) has been reported under diverse physiologic and pathophysiologic conditions in various tissues including vascular endothelium. We recently reported that angiotensin IV-stimulated increased expression of CRT in lung endothelial cells and the protein-protein interaction between CRT and the endothelial cell isoform of nitric oxide synthase (eNOS) are causally associated with sustained (> 12 hr) activation of eNOS at a post-transcriptional level. The molecular mechanisms of CRT-mediated sustained activation of eNOS appear distinctly unique since all other known post-transcriptional mechanisms involved in activation of eNOS cause only limited or transient activation. At present CRT knows nothing about the molecular interaction and regulation of the catalytic activity of eNOS. Based on our earlier report and preliminary data, we hypothesize that the interaction of CRT with the electron transfer control element (ETE) of eNOS overrides its putative autoinhibitory function resulting in enhanced electron transfer and sustained activation of eNOS, NO/cyclic guanosine 5'-monophosphate (cGMP) production and vasorelaxation response. To test this hypothesis, we will 1) determine whether CRT interaction with ETE in the reductase domain of eNOS is causally associated with increased electron transfer and catalytic activity of eNOS, 2) characterize the molecular interaction between CRT's distinct N-domain, or P-domain (high affinity Ca2+ binding site), or C-domain (low affinity Ca2+ binding site) and ETE responsible for enhanced catalytic activity of eNOS, and 3) verify whether increased expression of CRT or its discrete domains in isolated endothelial cells and in pulmonary artery segments enhances eNOS activity, NO/cGMP production, and vasorelaxation. Identification of a molecular interaction between ETE of eNOS and CRT that causes sustained activation of eNOS will advance our understanding of eNOS regulation for maintaining pulmonary vascular function in context with increased CRT expression under diverse physiologic and pathophysiologic conditions.