Regulation of Smooth Muscle Contractile Properties

一氧化氮（NO）介导的血管舒张是血管的基本反应之一，而NO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。已知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的增加和随后的I型蛋白激酶G（PKGI）的激活。PKGI具有导致平滑肌松弛的多个靶点，包括最大K+通道的磷酸化以产生平滑肌的超极化、Ca 2+通量的减少和肌球蛋白轻链（MLC）磷酸酶的活化。MLC磷酸酶是一种全酶，由一个约38 kDa的PPIc δ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（MYPT 1）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 MYPT 1亚型的表达，并且最近已经证明MYPT 1的C末端LZ对于介导血管对NO的反应是重要的。我们已经证明对cGMP介导的平滑肌松弛的敏感性与LZ+和LZ MYPT 1亚型的相对表达相关，并且在培养的平滑肌细胞中LZ+或LZ MYPT 1同种型的过表达调节cGMP介导的MLC 20去磷酸化。然而，我们也证明了LZ+和LZ MYPT 1亚型都与PKGI结合。这些数据表明，虽然表达LZ的MYPT 1是cGMP介导的MLC磷酸酶活性活化所必需的，但MLC磷酸酶活性的活化不依赖于PKGI与MYPT 1的LZ-LZ相互作用。其他人认为卷曲螺旋结构域介导PKGI与MYPT 1的关联。PKGI在其NH 2-末端具有卷曲螺旋结构域，并且在aa 647-705和aa 888-928之间存在MYPT 1的预测卷曲螺旋结构域。因此，本申请所基于的假设是，在PKG的cGMP活化后，PKG和MYPT 1通过卷曲螺旋结构域相互作用，并且PKG通过MYPT 1的磷酸化活化MLC磷酸酶活性。为了检验这一假设，我们将确定cGMP刺激期间PKGI磷酸化MYPT 1的位点（具体目标1）;负责MYPT 1和PKGI相互作用的位点（具体目标2）;以及MYPT 1结构变化对MLC磷酸酶活性调节的影响（具体目标3）。这些特定的目的是为了确定MYPT 1的NO介导的平滑肌松弛的结构-功能关系。最初的实验将使用生物化学技术来证明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中表达几种MYPT 1截短突变体和MYPT 1携带突变的关键位点，以确定这些变化对MLC磷酸酶活性调节和cGMP介导的平滑肌松弛的影响。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.