Signal Transduction Pathways of Smooth Muscle

本项目的总体目标是确定信号的生理相关机制 通过不依赖于胞浆钙浓度的途径调节收缩能力的转导。RhoA-Rho激酶途径钙敏化的上游调节因子，鸟嘌呤核苷酸交换因子(GEF)PDZ.RhoGf和LARG的结构鉴定和重组表达将结合项目2中的结构鉴定和重组表达来确定。膜相关蛋白(P120 Catenin)作为潜在的RhoA结合伙伴鸟嘌呤核苷酸解离抑制物(GDI)的作用将被确定。Rho-Kinase调节肌球蛋白磷酸酶的动力学及其与内源性磷酸酶抑制剂CP1-17表达水平的依赖关系将被定量，以确定它们之间的相对生理相关性。肌球蛋白磷酸酶活性与哺乳动物肌肉中表达的调节亚单位(MYPT1)的特定长短剪接亚基(MYPT1)亚基的相关性将被评估，其基础是这两种亚基的表达水平决定了肌球蛋白磷酸酶的定位。 磷酸酶及其通过磷酸化位点和肌球蛋白结合特性的不同而调节的机制。该项目的一个主要的、新颖的目标是确定一种新发现的在平滑肌中选择性表达的蛋白质LPP在细胞表型上的功能，包括形态、与底物的粘附性、细胞骨架结构和运动性。在项目3中，将执行双杂交筛选中确定的信号蛋白的定位和刺激诱导的转位、蛋白质的共定位，例如p120连环蛋白与RhoA或CP1-17与肌球蛋白磷酸酶或端粒蛋白以及端粒蛋白和LPP伙伴。p120连环蛋白、LPP、重组RhoA、端粒蛋白、RhoA-和其他蛋白质突变体将由Core B提供。RhoA途径的异常 被认为与哮喘和高血压有关。

The overall Aim of this Project is to determine the physiologically relevant mechanisms of signal transduction that regulate contractility by pathways not dependent on cytoplasmic Ca concentration. The domain dependent functions of the upstream regulators of Ca?+-sensitization by the RhoA-Rho kinase pathway, the guanine nucleotide exchange factors (GEFs) PDZ.RhoGEF and LARG, will be determined in conjunction with the structural identification and recombinant expression of GEF domains in Project 2. The role of a membrane associated protein (p120 catenin) as a potential RhoA-binding partner guanine nucleotide dissociations inhibitor (GDI) will be determined. The kinetics of myosin phosphatase regulation by Rho-kinase and its dependence on the expression level of the endogenous phosphatase inhibitor, CP1-17 will be quantitated in order to determine their relative physiological relevance. The dependence of myosin phosphatase activity on the specific long and short splice isoforms of the regulatory subunit (MYPT1) expressed in mammalian smooth muscle will be evaluated, based on the hypothesis that expression levels of the ratio of the two isoforms determine localization of the phosphatase and the mechanism of its regulation through differences in their phosphorylation sites and myosin binding properties. A major, novel, objective of this Project is to determine the functions of a protein, LPP, newly discovered to be selectively expressed in smooth muscle, on cellular phenotypes, including morphology, adhesion to substrate, cytoskeletal structure and motility. Localization and stimulus induced translocation of signaling proteins, co-localization of proteins, such as p120 catenin with RhoA or CP1-17 with myosin phosphatase or telokin and telokin and LPP partners identified in two-hybrid screens will be performed in Project 3. p120 catenin, LPP, recombinant RhoA, telokin, RhoA- and other protein-mutants will be supplied by Core B. Abnormalities of the RhoA pathway are thought to be involved in asthma and hypertension.