Smooth Muscle Myosin Phosphatase Subunit Isoforms

平滑肌肌球蛋白磷酸酶亚基同种型

• 批准号: 7169848
• 负责人: Steven A. Fisher
• 金额: $ 29.01万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7169848
• 关键词: 5' Splice Site; Accounting; Address; Age; Agonist; Alternative Splicing; Antibodies; Aorta; Arteries; Binding; Biological Assay; Blood Vessels; C-terminal; Calcium; Chickens; Complement; Complex; Cultured Cells; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Deletion Mutation; Development; Dimerization; Disease; Disease model; Dominant-Negative Mutation; Embryo; Enhancers; Exons; Funding; Gene Family; Genes; Gizzard; Goals; Hand; Leucine Zippers; Ligands; Light; Measures; Mediating; Mesentery; Modeling; Molecular; Muscle Tonus; Muscle relaxation phase; Myosin ATPase; Neonatal; Nitric Oxide; Phenotype; Phosphorylation; Polypyrimidine Tract-Binding Protein; Portal Hypertension; Protein Binding; Protein Isoforms; RNA Splicing; RNA-Binding Proteins; Range; Rattus; Regulation; Regulatory Element; Relaxation; Repression; Resistance; Signal Pathway; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Smooth Muscle Myosins; Testing; Tissues; Vascular Smooth Muscle; Vascular Smooth Muscle Tissue; Vasodilation; Vasodilation disorder; Vasodilator Agents; Veins; in vivo; loss of function; myosin phosphatase; novel; protein protein interaction; prototype; research study; response

DESCRIPTION (provided by applicant): Myosin phosphatase (MP) is the primary effector of smooth muscle relaxation and a key target of signaling pathways that regulate smooth muscle tone. We and others have proposed a model in which leucine zipper (LZ) mediated hetero-dimerization of the cGMP-dependent protein kinase (PKG1) and the MP targeting subunit (MYPT1) activates MP and causes smooth muscle relaxation in response to NO/cGMP signaling. We previously showed that chicken and rat MYPT1 isoforms are generated by tissue-specific and developmentally regulated cassette-type alternative splicing of exons. We hypothesized that regulated expression of MYPT isoforms determines smooth muscle phenotype-specific responses to NO/cGMP signaling in normal and disease states. The goals of the previous funding period were to 1) Investigate the functional significance of MYPT1 isoforms and 2) Define the molecular mechanisms for their tissue-specific expression. We showed that tissues that express the MYPT1 LZ+ isoform responded to cGMP as evidenced by 1) Association of PKG1 with MYPT1 2) Activation of MP by cGMP (de-phosphorylation of myosin) and 3) Complete smooth muscle relaxation to cGMP at pCa4. These responses were not observed in tissues that express MYPT1 LZ- isoform. Forced expression of MYPT1 LZ- isoform in cultured SMCs suppressed cGMP-triggered de-phosphorylation of myosin. We used mutation/deletion of MYPT1 mini-gene constructs, gain-and-loss of function, and RNA-protein binding experiments to show 1) TIA and SR protein binding to regulatory elements near the 5' splice site are necessary for splicing of the alt exon in tonic SM and 2) Supression of splicing in phasic SM is due to loss of TIA-dependent enhancer activity plus a putative tissue-specific cis-silencer. We now propose to refine and extend these models: 1) MYPT:PKG association: The original model did not account for other MYPT subunits. M21 is part of the MP complex and contains a nearly identical LZ motif. Experiments are proposed to determine if PKG1 displaces M21 from MYPT1, or binds to M21 LZ, and whether the PKG1/MYPT1 association is ligand (cGMP)-dependent. 2) Functional significance: We extended the model to show vascular smooth muscle tissue-specific and developmentally regulated expression of MYPT 1 LZ+/- isoforms, and isoform switching in a disease model (portal hypertension) characterized by vasodilatation. We will test the relationships between the expression of MYPT isoforms and NO/cGMP signaling in vascular development and disease. As a further test LZ interactions will be specifically disrupted in vascular smooth muscle in vivo. 3) Regulated splicing of MYPT1: We propose a) To test the novel model that PTB protein competes with TIA and suppresses TIA-dependent splicing in phasic SM and b) To more specifically define the putative exonic tissue-specific suppressor of splicing. These studies will advance our long-range goal to understand the relationship between regulated expression of myosin phosphatase subunits, smooth muscle phenotypic diversity and vascular function in development and disease.

描述（由申请人提供）：肌球蛋白磷酸酶（MP）是平滑肌松弛的主要效应因子，也是调节平滑肌张力的信号通路的关键目标。我们和其他人提出了一个模型，其中亮氨酸拉链（LZ）介导的CGMP依赖性蛋白激酶（PKG1）和MP靶向亚基（MYPT1）的异质差异化激活MP，并导致对NO/CGMP信号的响应，导致平滑肌弛豫。我们先前表明，鸡肉和大鼠MyPT1同工型是由组织特异性和发育调节的盒式盒式胶带的替代外显子产生的。我们假设调节MyPT同工型的表达决定了正常和疾病状态中对NO/CGMP信号的平滑肌表型特异性反应。上一个资金期的目标是1）研究MyPT1同工型的功能意义，以及2）定义其组织特异性表达的分子机制。我们表明，表达MYPT1 LZ+同工型的组织对CGMP做出了反应，这是1）PKG1与MyPT1 2）通过CGMP（肌球蛋白的去磷酸化）和3）在PCA4上通过CGMP激活MP的。这些反应在表达MYPT1 LZ-同工型的组织中没有观察到。在培养的SMC中，MyPT1 LZ-同工型的强制表达抑制了CGMP触发的肌球蛋白的去磷酸化。我们使用MyPT1微型基因构建体的突变/缺失，功能的增益和损害以及RNA - 蛋白结合实验显示1）1）TIA和SR蛋白与5'剪接位点附近的调节元素的结合是在张力SM和2）在Phasity Sm spliia sm的损失中剪接Alt外显子在Phasia sm中的剪接必不可少的，这是必不可少的。顺式传输者。现在，我们建议完善并扩展这些模型：1）MYPT：PKG关联：原始模型没有考虑其他Mypt子单位。 M21是MP复合物的一部分，包含一个几乎相同的LZ基序。提出了实验以确定PKG1是从MyPT1中取代M21还是与M21 LZ结合，以及PKG1/MYPT1关联是否是配体（CGMP） - 依赖性的。 2）功能意义：我们扩展了模型以显示MyPT 1 Lz +/-同工型的血管平滑肌组织特异性和发育调节的表达，并在疾病模型（Portal Hypertension）中进行的同工型切换，其特征在于血管舒张。我们将在血管发育和疾病中测试MyPT同工型表达与无/CGMP信号传导之间的关系。作为进一步测试的LZ相互作用将在体内的血管平滑肌中特异性破坏。 3）调节MYPT1的剪接：我们建议a）测试PTB蛋白与TIA竞争的新型模型，并抑制Phasic SM和B）中TIA依赖性剪接，以更具体地定义了推定的外观组织特异性固定抑制器。这些研究将促进我们的远程目标，以了解肌球蛋白磷酸酶亚基的调节表达，平滑肌表型多样性和在发育和疾病中的血管功能之间的关系。