Nitric Oxide Signaling Mechanisms in Vascular Cells

血管细胞中的一氧化氮信号传导机制

• 批准号: 7822184
• 负责人: MICHAEL E MENDELSOHN
• 金额: $ 1.59万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7822184
• 关键词: ATP phosphohydrolase; Actins; Age; Agonist; Animals; Binding; Biochemical; Biology; Blood Vessels; Calcium; Calmodulin; Cardiovascular Diseases; Cardiovascular Physiology; Cell physiology; Cells; Cellular Stress; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Data; Diagnosis; Dimerization; Family; Functional disorder; G alpha q Protein; Genes; Guanosine Triphosphate Phosphohydrolases; Human; Hypertension; Investigation; Ion Channel; Isoleucine; Knock-in Mouse; Leucine; Leucine Zippers; Life; Light; Mammals; Mediating; Molecular; Mus; Muscle Contraction; Muscle relaxation phase; Mutant Strains Mice; Myosin ATPase; Myosin Light Chain Kinase; Myosin Light Chains; N-terminal; Nitric Oxide; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Process; Protein Binding; Protein-Serine-Threonine Kinases; Proteins; RNA Splicing; Regulation; Relative (related person); Relaxation; Reporting; Research Personnel; Role; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Soluble Guanylate Cyclase; Stress Fibers; Testing; Thromboxane Receptor; Variant; Vasoconstrictor Agents; Vasodilator Agents; cell motility; gastrointestinal; human RGS2 protein; migration; mouse model; mutant; myosin phosphatase; programs; protein protein interaction; receptor; receptor coupling; response; rho; tool

DESCRIPTION (provided by applicant): This competitive renewal application explores molecular mechanisms regulating vascular smooth muscle cell (VSMC) relaxation by the nitric oxide/cGMP/cGMP-dependent protein kinase (PKG) pathway. VSMC contractile state is dynamically regulated by phosphorylation of the regulatory myosin light (MLC) chains controlling actinomyosin contraction. Increases in [Ca2+]in activate the Ca 2+/calmodulin-dependent MLC kinase (MLCK), which phosphorylates MLC, causing VSMC contraction. Conversely, increases in myosin phosphatase (PP1M) activity, such as occurs in response to the endogenous vasodilator nitric oxide (NO), dephosphorylate MLC, causing relaxation. Over the past decade, we have focused on understanding molecular mechanisms regulating vascular relaxation by identifying new PKGIalpha targets and testing their functional role. PKGI induces VSMC relaxation by multiple mechanisms involving phosphorylation of substrates that directly regulate (i) actinomyosin contractile stress fiber relaxation and (ii) inhibition of GPCR- mediated [Ca+2]in mobilization. We have characterized several PKGIa-target protein interactions important in VSMC relaxation, including interactions with (a) PP1M, (b) the regulator of G protein signaling 2 (RGS2), and (c) the formin homology domain-containing protein type 1, or FHOD1, a multi-functional protein that stimulates VSMC stress fiber formation. All 3 of these pathways are mediated by PKGIalpha binding/activation of these targets via its N-terminal leucine zipper (LZ) interaction domain, supporting the hypothesis that PKGIalpha has a central role in the regulation of normal VSMC tone and biology via PKGIalpha-target protein interactions mediated by the PKGIalpha LZ domain. To explore this hypothesis, we now have created mice expressing a LZ mutant (LZM) PKGIalpha defective for protein-protein interactions, but otherwise identical to wild-type PKGIa. LZM 'knock-in' mice have an exciting phenotype supporting the above hypothesis: LZM VSMC have excessive actinomyosin stress fibers; LZM mouse blood vessels relax abnormally; and intact PKGIalpha LZM mice are hypertensive, providing exciting tools with which to explore the central hypothesis of this application. We propose to investigate: the molecular mechanisms by which PKGIa inhibits VSMC actinomyosin stress fiber formation and contraction, with a focus on the FHOD1-PKG LZ domain interaction (SA1); PKGIa inhibition of GPCR-mediated [Ca+2]in mobilization, with a focus on LZ-mediated interactions regulating the (i) RGS2-GPCR-IP3 pathway and (ii) thromboxane receptor (SA2); and the functional role of the PKGIalpha LZ targeting domain in vascular regulation in intact blood vessels and animals, using WT and LZM mice (SA3). These studies are expected to increase our understanding of vascular tone regulation, with the potential to advance the diagnosis and therapy of human cardiovascular diseases.

描述(申请人提供)：这项竞争性更新申请探索了通过一氧化氮/cGMP/cGMP依赖的蛋白激酶(PKG)途径调节血管平滑肌细胞(VSMC)松弛的分子机制。VSMC收缩状态是通过控制放线肌球蛋白收缩的调节肌球蛋白轻链(MLC)的磷酸化来动态调节的。[Ca~(2+)]升高激活钙/钙调蛋白依赖的MLC激酶(MLCK)，使MLC磷酸化，引起VSMC收缩。相反，肌球蛋白磷酸酶(PP1M)活性的增加，如内源性血管扩张剂一氧化氮(NO)的反应，使MLC去磷酸化，导致松弛。在过去的十年里，我们一直致力于通过识别新的PKGIpha靶点并测试它们的功能作用来了解调节血管松弛的分子机制。PKGI通过底物的磷酸化直接调节(I)放线肌球蛋白收缩应激纤维的松弛和(Ii)抑制GPCR介导的[Ca+2]动员来诱导VSMC的松弛。我们已经确定了几个在VSMC松弛过程中重要的PKGIa-靶蛋白相互作用，包括与(A)PP1M，(B)G蛋白信号转导2(RGS2)的调节因子，以及(C)FHOD1，一种刺激VSMC应力纤维形成的多功能蛋白质。这3条通路都是由PKGIpha通过其N端亮氨酸拉链(LZ)相互作用结构域与靶蛋白的结合/激活而介导的，支持PKGIpha通过PKGIpha LZ结构域与靶蛋白相互作用在调节正常VSMC张力和生物学中起中心作用的假说。为了探索这一假设，我们现在创造了表达LZ突变体(LZM)PKGIpha的小鼠，该突变在蛋白质-蛋白质相互作用方面存在缺陷，但在其他方面与野生型PKGIa相同。LZM“敲入”小鼠有一个支持上述假说的令人兴奋的表型：LZM VSMC具有过量的放线肌球蛋白应激纤维；LZM小鼠血管异常松弛；完整的PKGIpha LZM小鼠患有高血压，为探索这一应用的中心假说提供了令人兴奋的工具。我们建议研究：PKGIa抑制VSMC放线肌球蛋白应激纤维形成和收缩的分子机制，重点是FHOD1-PKG LZ结构域相互作用(SA1)；PKGIa抑制GPCR介导的动员中的[Ca+2]，重点是LZ介导的相互作用，调节(I)RGS2-GPCRIP3途径和(Ii)血栓素受体(SA2)；以及PKGIalLZ靶向结构域在完整血管和动物血管调节中的功能作用，使用WT和LZM小鼠(SA3)。这些研究有望增加我们对血管张力调节的了解，并有可能促进人类心血管疾病的诊断和治疗。

项目成果

Steroid-sensitive gene 1 is a novel cyclic GMP-dependent protein kinase I substrate in vascular smooth muscle cells.

类固醇敏感基因 1 是血管平滑肌细胞中一种新型环 GMP 依赖性蛋白激酶 I 底物。

• DOI: 10.1074/jbc.m113.456244
• 发表时间: 2013
• 期刊: The Journal of biological chemistry
• 作者: Wang,Guang-rong;Surks,HowardK;Tang,KMary;Zhu,Yan;Mendelsohn,MichaelE;Blanton,RobertM
• 通讯作者: Blanton,RobertM