The role of the small GTP binding protein, rho, in the vascular smooth muscle.

小G蛋白rho通过rho激酶磷酸化肌球蛋白轻链磷酸酶（MLCP）而抑制MLCP。因此，MLCP可能是rho介导的信号转导介导的血管平滑肌钙敏感性调节的关键蛋白。MLCP由催化亚基、调节小亚基和调节大亚基三个亚基组成。本课题主要研究MLCP的两个调节亚基在平滑肌收缩器中的作用。主要研究结果如下：1.在TritonX-100透化的猪肾动脉中研究了肌球蛋白轻链磷酸酶调节亚基（MLCPsr）对Ca^<2+>诱导的平滑肌收缩的影响。通过细菌表达系统获得的全长重组鸡MLCPsr在恒定的[Ca^<2+>] i下诱导额外的收缩，并使[Ca^<2+>] i-f ...更多信息 将关系曲线向左倾斜。与全长MLCPsr相比，含有MLCPsr的N-末端78个氨基酸的缺失突变体保留了全部作用，而该区域的缺失完全废除了其作用。含有N-末端78个氨基酸的片段也延迟了松弛过程。这些结果表明，MLCPsr增加了血管平滑肌收缩器对Ca^<2+>的敏感性，而N-末端78个氨基酸是导致这种作用的原因.在TritonX-100透化的猪肾动脉中研究了肌球蛋白轻链磷酸酶调节大亚基（MLCPlr）的氨基端片段对Ca^2+诱导的平滑肌收缩的影响。MLCPlr 1-374、MLCPlr 304-511和MLCPlr 297-374诱导了[Ca^2+] i-力关系的显著位移。从最有效的构建体MLCPlr 1-374中删除残基304-374，消除了Ca^2+-敏化作用。M1301-374在无Ca^<2+>培养基中减慢舒张速率。肌球蛋白轻链磷酸化水平（22.4%）和武力用300 nM Ca^<2+>获得的NH 2-末端MLCPlr片段（34.5%）分别被3 microMMLCPlr 1-374增加到35.7和92.2%。增加肌球蛋白轻链磷酸化和增加透化猪肾动脉收缩装置的Ca^2+敏感性.我们已经分离出心肌特异性肌球蛋白轻链磷酸酶（HS-MLCPsr）的小调节亚基的cDNA，该cDNA从MYPT 2基因的内含子13中的心肌特异性启动子转录。在透性化大鼠心肌细胞和猪肾动脉中的功能分析显示，HS-MLCPsr增强Ca^2+诱导的收缩，并且主要功能域定位于HS-MLCPsr的N-末端一半。此外，HS-MLCPsr与MYPT 1的C端三分之一结合，结合结构域也定位在HS-MLCPsr的N端一半。这些结果表明，HS-MLCPsr通过与MYPT 1结合在调节心肌收缩中起作用。少

The small G protein, rho, has been shown to inhibit myosin light chain phosphatase (MLCP) through the phosphorylation of this enzyme by rho kinase. It was thus obvious that MLCP may be the key protein for the rho-mediated signal transduction to induce modulation of calcium sensitivity of the vascular smooth muscle. MLCP is composed of three subunits, namely, catalytic subunit, small regulatory subunit and large regulatory subunit. We focused on the function of two regulatory subunits of MLCP on the smooth muscle contractile apparatus in the present research program. The major findings were as follows.1. The effects of the small regulatory subunit of myosin light chain phosphatase (MLCPsr) on the Ca^<2+>-induced contraction of smooth muscle were investigated in the Triton X-100-permeabilized porcine renal artery. The full-length recombinant chicken MLCPsr obtained by the bacterial expression system induced an additional contraction at a constant [Ca^<2+>] i and shifted the [Ca^<2+>] i-f … More orce relation curve to the left. A deletion mutant containing the N-terminal 78 amino acids of MLCPsr retained the full action, compared with the full-length MLCPsr, while the deletion of this region completely abolished its effect. The process of relaxation was also delayed by the fragment containing the N-terminal 78 amino acids. These results indicated that MLCPsr increases the Ca^<2+> sensitivity of the contractile apparatus while the N-terminal 78 amino acids are responsible for this effect in vascular smooth muscle.2. The effects of the NH2-terminal fragments of the large regulatory subunit of myosin light chain phosphatase (MLCPlr) on the Ca^<2+>-induced contraction of smooth muscle were investigated in the Triton X-100-permeabilized porcine renal artery. MLCPlr 1-374, MLCPlr 304-511 and MLCPlr 297-374 induced leftward shifts of the [Ca^<2+>] i-force relationship. Deletion of residues 304-374 from the most potent construct, MLCPlr 1-374, abolished the Ca^<2+>-sensitizing effect. M1301-374 slowed the rate of relaxation in Ca^<2+>-free medium. The levels of myosin light chain phosphorylation (22.4%) and force (34.5%) obtained with 300 nM Ca^<2+> were increased by 3 microMMLCPlr 1-374 to 35.7 and 92.2%, respectively.These results indicated that the NH2-terminal MLCPlr fragments containing residues 304-374 inhibited myosin phosphatase, increased myosin light chain phosphorylation and increased the Ca^<2+> sensitivity of the contractile apparatus in permeabilized porcine renal artery.3. We have isolated cDNAs for heart-specific small regulatory subunit of myosin light chain phosphatase (HS-MLCPsr) that were transcribed from a heart-specific promoter in intron 13 of the MYPT2 gene. Functional analysis in permeabilized rat cardiac myocytes and porcine renal artery revealed that HS-MLCPsr enhanced Ca^<2+>-induced contraction, and the main functional domain was mapped in the N-terminal half of HS-MLCPsr. In addition, HS-MLCPsr bound to C-terminal one-third of MYPT1, and the binding domain was also mapped in the N-terminal half of HS-MLCPsr. These results indicated that HS-MLCPsr plays a role in regulation of the cardiac muscle contraction via binding to MYPT1. Less