Smooth Muscle Myosin Phosphatase Subunit Isoforms

描述（由申请人提供）：肌球蛋白磷酸酶（Myosin phosphatase， MP）是平滑肌松弛的主要效应器，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，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并引起平滑肌松弛，以响应NO/cGMP信号。我们之前的研究表明，鸡和大鼠的MYPT1亚型是由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，在正常和疾病状态下，MYPT亚型的调控表达决定了平滑肌对NO/cGMP信号的表型特异性反应。上一个资助期的目标是：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+异构体的组织对cGMP有反应，证明如下：1)PKG1与MYPT1的关联2)cGMP激活MP（肌球蛋白的去磷酸化）3)pCa4处的cGMP完全平滑肌松弛。在表达MYPT1 LZ-异构体的组织中没有观察到这些反应。在培养的SMCs中强行表达MYPT1 LZ-异构体可抑制cgmp触发的肌球蛋白去磷酸化。我们使用MYPT1微小基因结构的突变/缺失、功能的获得和丧失以及rna -蛋白结合实验来证明：1)TIA和SR蛋白结合到5'剪接位点附近的调节元件是强直性SM中alt外显子剪接所必需的；2)剪接抑制是由于TIA依赖性增强子活性的丧失以及推测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：原始模型没有考虑到其他MYPT亚单位。M21是MP复合体的一部分，包含一个几乎相同的LZ基序。我们提出了实验来确定PKG1是否将M21从MYPT1中置换，或与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+/-亚型表达，以及以血管舒张为特征的疾病模型（门脉高压）中的亚型转换。我们将测试MYPT亚型的表达与血管发育和疾病中NO/cGMP信号之间的关系。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地破坏。3)调节MYPT1剪接：我们提出a)验证PTB蛋白与TIA竞争并抑制阶段性SM中TIA依赖剪接的新模型；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基的调控表达、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.