SMOOTH MUSCLE MYOSIN PHOSPHATASE SUBUNIT ISOFORMS

平滑肌表现出显著的表型和功能多样性。这些研究的总体目标是了解产生这种多样性的分子机制。我们选择了平滑肌肌球蛋白磷酸酶（SMMP），平滑肌松弛的主要介质，作为了解平滑肌松弛特性的多样性的焦点。SMMP的肌球蛋白靶向亚基（MYPT 1）是调节平滑肌张力的信号传导途径的关键底物。放松平滑肌的信号分子如一氧化氮（NO）通过cGMP依赖性蛋白激酶与MYPT 1的C-末端亮氨酸拉链的二聚化增强SMMP活性。由于MYPT 1亚型已被描述为C-末端亮氨酸拉链的可变存在，我们假设MYPT 1亚型可能决定不同平滑肌表型对NO/cGMP介导的松弛的差异敏感性。为了检验这一点，我们的特点是SMMP亚基亚型和相关的表达与平滑肌组织的松弛特性。鸡和大鼠MYPT 1亚型是通过组织特异性和发育调节的盒型选择性剪接中心和C末端的外显子产生的。初步实验表明，MYPT 1亚型的表达与成熟平滑肌组织的cGMP舒张反应相关，以及在发育中的平滑肌的表型转换过程中。表达含有C-末端亮氨酸拉链的MYPT 1亚型的组织在活化钙存在下响应cGMP而松弛，而表达缺乏C-末端亮氨酸拉链的亚型的组织则不松弛。鉴于MYPT 1 C-末端亮氨酸拉链在cGMP松弛信号传递中的作用，以及该基序的存在与cGMP敏感性的相关性，我们假设MYPT 1亚型的组织特异性表达决定了平滑肌组织对NO/cGMP介导的松弛的差异敏感性。为了验证这一假设，我们提出：1）定义SMMP亚基亚型表达和平滑肌松弛特性之间的关系。NO供体和cGMP类似物激活SMMP并引起平滑肌松弛的能力将在表达不同MYPT 1同种型的发育和成熟平滑肌组织中测量。2)证明SMMP亚基亚型在体外或体内通过强制表达确定cGMP介导的舒张敏感性中的作用。3)确定MYPT 1亚型的组织特异性表达机制。使用体内基因传递系统的初步结果已经确定了一个假定的内含子增强子和MYPT 1选择性外显子剪接的组织特异性阻遏物。这些实验将推进我们的理解的分子机制，外显子的选择性剪接导致SMMP亚基亚型和平滑肌表型的多样性，以及SMMP亚基亚型在确定平滑肌松弛特性的多样性的作用。

Smooth muscle exhibits significant phenotypic and functional diversity. The overall goal of these studies is to understand the molecular mechanisms for the generation of this diversity. We have selected the smooth muscle myosin phosphatase (SMMP), the primary mediator of smooth muscle relaxation, as the focus for understanding the diversity in smooth muscle relaxant properties. The myosin targeting subunit (MYPT1) of SMMP is a critical substrate for signaling pathways that regulate smooth muscle tone. Signaling molecules such as nitric oxide (NO) that relax smooth muscle enhance SMMP activity via the dimerization of the cGMP-dependent protein kinase with the C-terminal leucine zipper of MYPT1. Since MYPT1 isoforms have been described with variable presence o the C-terminal leucine zipper, we postulated that MYPT1 isoforms might determine the differential sensitivity of distinct smooth muscle phenotypes to NO/cGMP-mediated relaxation. To examine this, we characterized the SMMP subunit isoforms and correlated their expression with relaxant properties of smooth muscle tissues. Chicken and rat MYPT1 isoforms are generated by tissue- specific and developmentally regulated cassette-type alternative splicing of exons at the center and C-terminus. Preliminary experiments suggest that the expression of the MYPT1 isoforms correlate with cGMP relaxant response of mature smooth muscle tissues, as well as during a phenotypic switch of developing smooth muscle. Tissues that express the MYPT1 isoform that contains the C-terminal leucine zipper relax in response to cGMP in the presence of activating calcium, while those that express the isoform lacking the C-terminal leucine zipper do not. Given the role of the MYPT1 C-terminal leucine zipper in the transmission of the cGMP relaxant signal, and the correlation of the presence of this motif with cGMP sensitivity, we hypothesize that tissue-specific expression of MYPT1 isoforms determines the differential sensitivity of smooth muscle tissues to NO/cGMP-mediated relaxation. To test this hypothesis we propose to: 1) Define the relationship between SMMP subunit isoform expression and smooth muscle relaxant properties. The ability of NO donors and cGMP analogues to activate SMMP and cause smooth muscle relaxation will be measured in developing and mature smooth muscle tissues that express distinct MYPT1 isoforms. 2) Demonstrate the role of the SMMP subunit isoforms in determining sensitivity to cGMP-mediated relaxation by forcing their expression in vitro or in vivo. 3) Identify the mechanisms for the tissue-specific expression of the MYPT1 isoforms. Preliminary results using an in vivo gene delivery system have identified a putative intronic enhancer and a tissue-specific repressor of splicing of a MYPT1 alternative exon. These experiments will advance our understanding of the molecular mechanisms by which alternative splicing of exons leads to SMMP subunit isoforms and smooth muscle phenotypic diversity, and the role of the SMMP subunit isoforms in determining the diversity in smooth muscle relaxant properties.