Smooth Muscle Myosin Phosphatase Subunit Isoforms

描述(申请人提供)：肌球蛋白磷酸酶(MP)是平滑肌松弛的主要效应者，也是调节平滑肌张力的信号通路的关键靶点。我们和其他人提出了一个模型，在这个模型中，亮氨酸拉链(LZ)介导cGMP依赖的蛋白激酶(PKG1)和MP靶向亚基(MYPT1)的异源二聚化，激活MP，引起平滑肌松弛，以响应NO/cGMP信号。我们之前已经证明，鸡和大鼠的MYPT1亚型是由组织特异性的和发育调节的盒型外显子的选择性剪接产生的。我们推测，在正常和疾病状态下，MYPT异构体的调节表达决定了平滑肌对NO/cGMP信号的表型特异性反应。前一个资助阶段的目标是1)研究MYPT1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MYPT1 LZ+亚型的组织对cGMP有反应，1)PKG1与MYPT1的联系；2)通过cGMP(肌球蛋白去磷酸化)激活MP；3)在pCa4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亚型可抑制cGMP触发的肌球蛋白去磷酸化。我们使用MYPT1微型基因结构的突变/缺失、功能的获得和丢失以及RNA-蛋白质结合实验表明：1)TIA和SR蛋白与5‘剪接点附近的调控元件结合是强直性SM中ALT外显子剪接所必需的；2)在相性SM中剪接的抑制是由于TIA依赖的增强子活性的丧失以及可能的组织特异性顺式沉默。我们现在建议改进和扩展这些模型：1)MYPT：PKG联合：原始模型没有考虑MYPT的其他亚基。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是取代MYPT1的M21，还是与M21 LZ结合，以及PKG1/MYPT1的结合是否依赖于配体(CGMP)。2)功能意义：我们对模型进行了扩展，以显示MYPT 1 LZ+/-亚型在血管平滑肌组织中的特异性和发育调节的表达，以及以血管扩张为特征的疾病模型(门脉高压症)中的异构体转换。我们将测试MYPT亚型的表达与NO/cGMP信号在血管发育和疾病中的关系。作为进一步的测试，LZ的相互作用将在体内血管平滑肌中被特异性地破坏。3)MYPT1的调控剪接：我们建议a)检验PTB蛋白与TIA竞争并在SM期抑制TIA依赖剪接的新模型，以及b)更具体地定义推测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，了解肌球蛋白磷酸酶亚单位的调节表达、平滑肌表型多样性和血管功能在发育和疾病中的关系。

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.