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 通过不依赖于细胞质Ca浓度的途径调节收缩性的转导。Ca ~（2+）上游调控因子的结构域依赖性功能RhoA-Rho激酶途径、鸟嘌呤核苷酸交换因子（GEF）PDZ、RhoGEF和LARG的致敏作用将结合项目2中GEF结构域的结构鉴定和重组表达来确定。膜相关蛋白（p120连环蛋白）作为一个潜在的RhoA结合伴侣鸟嘌呤核苷酸解离抑制剂（GDI）的作用将被确定。将定量Rho激酶调节肌球蛋白磷酸酶的动力学及其对内源性磷酸酶抑制剂CP 1 -17表达水平的依赖性，以确定其相对生理相关性。基于两种异构体的比率的表达水平决定肌球蛋白磷酸酶活性的定位的假设，将评估肌球蛋白磷酸酶活性对在哺乳动物平滑肌中表达的调节亚基（MYPT 1）的特异性长和短剪接异构体的依赖性。 磷酸酶及其通过磷酸化位点和肌球蛋白结合特性差异进行调节的机制。该项目的一个主要的、新颖的目的是确定新发现的在平滑肌中选择性表达的蛋白质LPP对细胞表型的功能，包括形态、对基质的粘附、细胞骨架结构和运动性。将在项目3中进行信号蛋白的定位和刺激诱导的易位，蛋白质的共定位，如p120连环蛋白与RhoA或CP 1 -17与肌球蛋白磷酸酶或telokin，以及在双杂交筛选中鉴定的telokin和LPP伴侣。p120连环蛋白、LPP、重组RhoA、telokin、RhoA和其他蛋白突变体将由Core B提供。RhoA通路的阻断是 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.