Regulation of Smooth Muscle Contractile Properties

一氧化氮(NO)介导的血管扩张是一种基本的血管反应，而确定NO为内皮源性松弛因子是了解血管反应性的基础进展之一。NO介导的平滑肌细胞松弛的分子机制包括激活鸟苷环化酶、增加cGMP和随后激活I型蛋白激酶G(PKGI)。PKGI有多个靶点可导致平滑肌松弛，包括最大钾通道的磷酸化，导致平滑肌超极化，钙流量减少，以及肌球蛋白轻链(MLC)磷酸酶的激活。MLC磷酸酶是一种全酶，由一个约38 kDa的PPIcDelta催化亚基、一个功能未知的20 kDa亚基和一个110-130 kDa的肌球蛋白靶向亚基(MYPT1)组成。31bp3‘外显子的选择性剪接导致亮氨酸拉链阳性(LZ)或LZ MYPT1亚型的表达，最近的研究表明MYPT1j的C端LZ在介导血管对NO的反应中起重要作用。我们已经证明，对cGMP介导的平滑肌松弛的敏感性与LZ和LZ MYPT1亚型的相对表达有关，培养的平滑肌细胞中LZ或LZ MYPT1亚型的过度表达调节了cGMP介导的MLC20去磷酸化。然而，我们也证明了LZ和LZ MYPT1亚型都能与PKGI结合。这些数据表明，尽管cGMP介导的MLC磷酸酶活性的激活需要表达LZ的MYPT1，但MLC磷酸酶活性的激活独立于PKGI与MYPT1的LZ-LZ相互作用。其他人则认为，盘绕结构域介导了PKGI与MYPT1的联系。PKGI在其NH2末端有一个螺旋结构域，在AA 647-705和AA 888-928之间预测了MYPT1的螺旋结构域。因此，这一应用所基于的假设是，在PKG的cGMP激活之后，PKG和MYPT1通过卷曲的结构域相互作用，而PKG通过MYPT1的磷酸化激活MLC磷酸酶活性。为了验证这一假设，我们将确定在cGMP刺激过程中PKGI磷酸化MYPT1的位点(特定目标1)；负责MYPT1和PKGI相互作用的位点(特定目标2)；以及MYPT1结构变化对MLC磷酸酶活性调节的后果(特定目标3)。这些特异性的目的是为了确定MYPT1在NO介导的平滑肌松弛中的结构与功能关系。初步实验将利用生化技术验证PKGI激活MLC磷酸酶活性的方法，然后我们将在培养的平滑肌细胞中表达几个MYPT1截断突变体和MYPT1突变，以确定这些变化对MLC磷酸酶活性的调节和cGMP介导的平滑肌松弛的影响。因此，结果将决定cGMP激活的分子机制！MLC磷酸酶活性产生平滑肌松弛。

Nitric oxide (NO) mediated vasodilatation is one of the basic vascular responses, and the identification of NO as the endothelial derived relaxing factor was one of the fundamental advances in understanding vascular reactivity. The molecular mechanism for NO mediated smooth muscle cells relaxation is known to involve the activation of guanylate cyclase, an increase in cGMP and the subsequent activation of type I protein kinase G (PKGI). PKGI has multiple targets that lead to relaxation of smooth muscle, including phosphorylation of the maxi K+ channel to produce a hyperpolarization of the smooth muscle, a decrease in Ca2+ flux, and an activation of myosin light chain (MLC) phosphatase. MLC phosphatase is a holoenzyme] consisting of an approximately 38 kDa PPIcdelta catalytic subunit, a 20 kDa subunit of unknown function, and a myosin targeting subunit (MYPT1) of 110-130 kDa. Alternative splicing of a 31 bp 3' exon is responsible for the expression of leucine zipper positive (LZ+) or LZ MYPT1 isoforms, and it has recently been demonstrated that the C-terminal LZ of MYPT1 jis important for mediating the vascular response to NO. We have demonstrated that the sensitivity to cGMP mediated smooth muscle relaxation correlates with the relative expression of LZ+ and LZ MYPT1 isoforms, and over expression of LZ+ or LZ MYPT1 isoforms in cultured smooth muscle cells modulates cGMP mediated MLC20 dephosphorylation. However, we also demonstrated that both LZ+ and LZ MYPT1 isoforms bind to PKGI. These data suggest that 'although a MYPT1 expressing a LZ is required for cGMP mediated activation of MLC phosphatase activity, the activation of MLC phosphatase activity is independent of a LZ-LZ interaction of PKGI with MYPT1. Others have suggested that coiled-coil domains mediate the association of PKGI with MYPT1. PKGI has a coiled-coil domain at its NH2-terminus, and there are predicted coiled-coil domains of MYPT1 between aa 647-705 and aa 888-928. Thus, the hypothesis on which this application is based is that following cGMP activation of PKG, PKG and MYPT1 interact via coiled-coil domains, and PKG activates MLC phosphatase activity by a phosphorylation of MYPT1. To test this hypothesis, we will determine the sites on MYPT1 phosphorylated by PKGI during cGMP stimulation (Specific Aim 1); the sites responsible for the interaction of MYPT1 and PKGI (Specific Aim 2); and the consequences of changes in MYPT1 structure on the regulation of MLC phosphatase activity (Specific Aim 3). These specific aims are designed to determine the structure-function relation of MYPT1 for NO mediated smooth muscle relaxation. Initial experiments will use biochemical techniques to demonstrate the method by which PKGI activates MLC phosphatase activity, and then we will express the several MYPT1 truncation mutants and MYPT1 bearing mutations at critical sites in cultured smooth muscle cells to determine the effect of these changes on the regulation of MLC phosphatase activity, and cGMP mediated smooth muscle relaxation. Thus the results will determine the molecular mechanism by which cGMP activates! MLC phosphatase activity to produce smooth muscle relaxation.