Smooth Muscle Myosin Phosphatase Subunit Isoforms

平滑肌肌球蛋白磷酸酶亚基同种型

基本信息

批准号：
7389670
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
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-02-01 至 2010-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7389670
关键词：
5&apos 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

项目摘要

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物，也是调节平滑肌张力的信号通路的关键靶标。我们和其他人提出了一种模型，其中亮氨酸拉链 (LZ) 介导 cGMP 依赖性蛋白激酶 (PKG1) 和 MP 靶向亚基 (MYPT1) 的异二聚化，激活 MP 并导致平滑肌松弛，以响应 NO/cGMP 信号传导。我们之前表明，鸡和大鼠的 MYPT1 亚型是通过组织特异性和发育调节的盒式外显子选择性剪接产生的。我们假设 MYPT 亚型的调节表达决定正常和疾病状态下平滑肌对 NO/cGMP 信号传导的表型特异性反应。上一资助期的目标是 1) 研究 MYPT1 同工型的功能意义，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 做出反应，证据如下：1) PKG1 与 MYPT1 的关联；2) cGMP 激活 MP（肌球蛋白去磷酸化）；3) 在 pCa4 处，平滑肌对 cGMP 完全松弛。在表达 MYPT1 LZ-亚型的组织中没有观察到这些反应。在培养的 SMC 中强制表达 MYPT1 LZ 同工型可抑制 cGMP 触发的肌球蛋白去磷酸化。我们使用 MYPT1 小基因构建体的突变/缺失、功能的获得和丧失以及 RNA 蛋白结合实验来表明 1) TIA 和 SR 蛋白与 5' 剪接位点附近的调节元件结合对于剪接强直 SM 中的 alt 外显子和 2) 阶段性 SM 中剪接的抑制是由于 TIA 依赖性增强子活性的丧失加上假定的组织特异性顺式沉默子所致。我们现在建议完善和扩展这些模型：1）MYPT：PKG关联：原始模型没有考虑其他MYPT亚基。 M21 是 MP 复合体的一部分，包含几乎相同的 LZ 基序。建议进行实验以确定 PKG1 是否从 MYPT1 中取代 M21，或与 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，以显示血管平滑肌组织特异性和发育调节的 MYPT 1 LZ+/- 同工型表达，以及以血管舒张为特征的疾病模型（门脉高压）中的同工型转换。我们将测试血管发育和疾病中 MYPT 同工型的表达与 NO/cGMP 信号传导之间的关系。作为进一步的测试，LZ 相互作用将在体内血管平滑肌中被特异性破坏。 3）MYPT1的剪接调节：我们建议a）测试PTB蛋白与TIA竞争并抑制阶段性SM中TIA依赖性剪接的新模型，以及b）更具体地定义推定的外显子组织特异性剪接抑制子。这些研究将推进我们的长期目标，即了解发育和疾病中肌球蛋白磷酸酶亚基的调节表达、平滑肌表型多样性和血管功能之间的关系。