Smooth Muscle Myosin Phosphatase Subunit Isoforms

描述（由申请方提供）：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。我们和其他人已经提出了一个模型，其中亮氨酸拉链（LZ）介导的cGMP依赖性蛋白激酶（PKG 1）和MP靶向亚基（MYPT 1）的异源二聚体激活MP，并导致平滑肌松弛响应NO/cGMP信号。我们以前表明，鸡和大鼠MYPT 1亚型产生的组织特异性和发育调控盒型选择性剪接的外显子。我们假设，MYPT亚型的表达调控决定了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。上一个资助期的目标是1）研究MYPT 1亚型的功能意义，2）确定其组织特异性表达的分子机制。我们发现，表达MYPT 1 LZ+亚型的组织对cGMP有反应，如1）PKG 1与MYPT 1的结合，2）cGMP对MP的激活（肌球蛋白的去磷酸化）和3）在pCa 4时对cGMP的完全平滑肌松弛。在表达MYPT 1 LZ-同种型的组织中未观察到这些反应。在培养的SMC中强制表达MYPT 1 LZ-同种型抑制cGMP触发的肌球蛋白去磷酸化。我们使用MYPT 1微型基因构建体的突变/缺失、功能的获得和丧失以及RNA-蛋白结合实验来显示1）TIA和SR蛋白与5'剪接位点附近的调节元件的结合对于强直性SM中alt外显子的剪接是必需的，以及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推定的组织特异性顺式沉默子。我们现在建议改进和扩展这些模型：1）MYPT：PKG关联：原始模型没有考虑其他MYPT亚基。M21是MP复合物的一部分，包含几乎相同的LZ基序。建议进行实验以确定PKG 1是否从MYPT 1置换M21，或与M21 LZ结合，以及PKG 1/MYPT 1缔合是否是配体（cGMP）依赖性的。2)功能意义：我们扩展了该模型，以显示MYPT 1 LZ+/-亚型的血管平滑肌组织特异性和发育调节表达，以及以血管舒张为特征的疾病模型（门静脉高压症）中的亚型转换。我们将测试MYPT亚型的表达和NO/cGMP信号在血管发育和疾病之间的关系。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)MYPT 1的调节剪接：我们提出a）测试PT B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）更具体地定义假定的外显子组织特异性剪接抑制因子。这些研究将推进我们的长期目标，以了解肌球蛋白磷酸酶亚单位，平滑肌表型多样性和血管功能的发展和疾病的调节表达之间的关系。

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.