Regulation of Smooth Muscle Contractile Properties

一氧化氮（NO）介导的血管舒张是一种基本的血管反应，一氧化氮作为内皮源性舒张因子的鉴定是理解血管反应性的基本进展之一。NO介导的平滑肌细胞松弛的分子机制涉及鸟苷酸环化酶的激活，cGMP的增加以及随后的I型蛋白激酶G （PKGI）的激活。PKGI有多个靶点，导致平滑肌松弛，包括最大K+通道磷酸化，产生平滑肌的超极化，Ca2+通量减少，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20 kDa的未知功能亚基和一个110-130 kDa的肌球蛋白靶向亚基（MYPT1）组成。31 bp 3'外显子的选择性剪接负责亮氨酸拉链阳性（LZ+）或LZ MYPT1异构体的表达，最近已经证明MYPT1的c端LZ对介导血管对NO的反应很重要。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亚型的相对表达相关，并且在培养的平滑肌细胞中，LZ+或LZ MYPT1亚型的过表达可调节cGMP介导的MLC20去磷酸化。然而，我们也证明了LZ+和LZ MYPT1亚型都与PKGI结合。这些数据表明，尽管表达LZ的MYPT1是cGMP介导的MLC磷酸酶活性激活所必需的，但MLC磷酸酶活性的激活与PKGI与MYPT1的LZ-LZ相互作用无关。也有人认为卷曲结构域介导PKGI与MYPT1的关联。PKGI在nh2末端有一个螺旋结构域，MYPT1的螺旋结构域预测在aa 647-705和aa 888-928之间。因此，该应用所基于的假设是，在cGMP激活PKG之后，PKG和MYPT1通过盘绕结构域相互作用，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磷酸化的MYPT1位点（Specific Aim 1）；负责MYPT1和PKGI相互作用的位点（Specific Aim 2）；以及MYPT1结构变化对MLC磷酸酶活性调控的影响（Specific Aim 3）。这些特定目的旨在确定MYPT1在NO介导的平滑肌松弛中的结构-功能关系。初始实验将使用生化技术证明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的关键位点表达几个MYPT1截断突变体和MYPT1携带突变体，以确定这些变化对MLC磷酸酶活性调控的影响，以及cGMP介导的平滑肌松弛。因此，结果将确定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.