CALRETICULIN REGULATION OF LUNG ENDOTHELIAL CELL NOS

描述（由申请人提供）：多功能 钙（Ca 2+）结合蛋白钙网蛋白（CRT）已经在 各种组织中的不同生理和病理生理条件 包括血管内皮。我们最近报道血管紧张素 IV刺激的肺内皮细胞CRT表达增加， CRT和内皮细胞亚型之间的蛋白质-蛋白质相互作用 一氧化氮合酶（eNOS）与持续（> 12小时） 在转录后水平激活eNOS。的分子机制 CRT介导的eNOS持续激活似乎是独特的，因为所有 其它已知的参与eNOS激活的转录后机制 仅引起有限或短暂的激活。目前CRT对 eNOS催化活性的分子相互作用和调控。 根据我们先前的报告和初步数据，我们假设， CRT与eNOS的电子转移控制元件（埃特）的相互作用 推翻其假定的自身抑制功能，导致增强的电子 eNOS、NO/环鸟苷5 '-单磷酸的转移和持续激活 （cGMP）产生和血管舒张反应。为了验证这个假设，我们将 1)确定CRT是否与eNOS的还原酶结构域中的埃特相互作用 与增加的电子转移和催化活性有因果关系 2）表征CRT的不同的eNOS之间的分子相互作用， N-结构域或P-结构域（高亲和力Ca 2+结合位点）或C-结构域（低亲和力Ca 2+结合位点） 亲和力Ca 2+结合位点）和埃特负责增强催化活性 3）验证CRT表达增加或其离散性是否与内皮型一氧化氮合酶（eNOS）的表达有关。 在分离的内皮细胞和肺动脉段中， eNOS活性、NO/cGMP产生和血管舒张。识别 eNOS的埃特和CRT之间的分子相互作用， eNOS的激活将促进我们对eNOS调节的理解， 在CRT增加的背景下维持肺血管功能 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.