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这种多样性产生的分子机制。我们选择了平滑肌肌球蛋白磷酸酶(SMMP)，作为平滑肌松弛的主要中介，作为了解平滑肌松弛特性的多样性的重点。SMMP的肌球蛋白靶向亚基(MYPT1)是调节平滑肌张力的信号通路的关键底物。通过cGMP依赖的蛋白激酶与MYPT1的C末端亮氨酸拉链的二聚化，松弛平滑肌的一氧化氮(NO)等信号分子增强了SMMP的活性。由于MYPT1亚型在C末端亮氨酸拉链上的存在存在差异，我们推测MYPT1亚型可能决定了不同的平滑肌表型对NO/cGMP介导的松弛的不同敏感性。为了验证这一点，我们描述了SMMP亚单位亚基的异构体，并将它们的表达与平滑肌组织的松弛特性相关联。鸡和大鼠的MYPT1亚型是由组织特异性和发育调节盒类型的中心和C末端外显子的选择性剪接产生的。初步实验表明，MYPT1亚型的表达与成熟的平滑肌组织的cGMP松弛反应有关，也与发育中的平滑肌的表型转换有关。表达含有C末端亮氨酸拉链的MYPT1亚型的组织在激活钙的存在下对cGMP的反应松弛，而那些表达含有C末端亮氨酸拉链的异构体的组织不松弛。鉴于MYPT1 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MYPT1亚型的组织特异性表达决定了平滑肌组织对NO/cGMP介导的松弛的不同敏感性。为了验证这一假说，我们建议：1)确定SMMP亚基亚基异构体表达与平滑肌松弛特性之间的关系。NO供体和cGMP类似物激活SMMP并导致平滑肌松弛的能力将在表达不同MYPT1亚型的发育和成熟的平滑肌组织中进行测量。2)通过在体外或体内强制表达SMMP亚基异构体，证明SMMP亚基异构体在决定cGMP介导的松弛敏感性中的作用。3)确定MYPT1亚型的组织特异性表达机制。使用体内基因递送系统的初步结果已经确定了MYPT1替代外显子剪接的假定内含子增强子和组织特异性抑制子。这些实验将有助于我们理解外显子选择性剪接导致SMMP亚基异构体和平滑肌表型多样性的分子机制，以及SMMP亚基异构体在决定平滑肌松弛特性多样性中的作用。

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.