Regulation of neutrophil receptor G protein interactions

G蛋白偶联趋化受体对人体功能反应的信号转导 白细胞涉及Rho小GTP酶。在以前的研究中，我们建立了p21-激活 蛋白激酶(PAK)是Rho GTP酶信号的重要下游介体，对Rho GTP酶信号传导具有重要的调节作用 肌动蛋白和微管细胞骨架之间的串扰。我们确定了分子靶标 PAK1对细胞骨架的调节，包括全球环境基金-H1、LIM激酶和肌球蛋白轻链激酶。在……里面 在目前的提案中，我们将调查PAK1的行动以及其在#年的目标全环基金-H1。 使用生化、遗传和分子方法的细胞骨架调节。我们将继续 趋化过程中Rho GTP酶活性和功能的时空动态研究 使用独特的基于荧光的成像技术。 我们已经鉴定了全球环境基金-H1是一个微管调节的鸟嘌呤核苷酸交换因子 Rho GTPase，并参与调控Rho在胞质分裂和细胞分裂中的活性 定向细胞运动。全球环境基金-H1的生物活性将用遗传和 基于siRNA的方法。我们将研究PAK1在调节全球环境基金-H1功能中的作用(S)。我们 已经确定了PAK1在调节肌动蛋白逆行流动(RAF)中的作用，RAF是一个动态过程 对细胞运动很重要。我们将使用定量荧光散斑显微镜(FSM) 结合遗传和生化方法确定PAK1的下游介体 在英国皇家空军的行动。最后，将使用基于FRET的可视化RAC和RHO激活的方法来 分析这些GTP酶在化学反应中的空间和时间动态 人类中性粒细胞。将从生化水平研究rac2在尾足类动物退缩中的作用。 Rho激活的时空评估将与rac2活性相关，以及特异性 A)趋化受体信号，b)运动行为，c)肌动蛋白-肌球蛋白 细胞骨架动力学。

Signaling from G protein-coupled chemoattractant receptors to the functional responses of human leukocytes involves Rho small GTPases. In previous studies, we established that p21-activated kinases (PAKs) are important downstream mediators of Rho GTPase signaling, and regulate crosstalk between the actin- and microtubule-cytoskeletons. We identified molecular targets for cytoskeletal regulation by PAK1, including GEF-H1, LIM kinase, and myosin light chain kinase. In the current proposal, we will investigate the action of PAK1, as well as its target GEF-H1 in cytoskeletal regulation using biochemical, genetic, and molecular approaches. We will continue studies of the spatio-temporal dynamics of Rho GTPase activation and function during chemotaxis using unique fluorescence-based imaging technologies. We have characterized GEF-H1 as a microtubule-regulated guanine nucleotide exchange factor for Rho GTPase, and implicated GEF-H1 in the regulation of Rho activity during cytokinesis and directional cell motility. The biological activities of GEF-H1 will be investigated using genetic and siRNA-based approaches. The role(s) of PAK1 in modulating GEF-H1 function will be studied. We have identified a role for PAK1 in regulating retrograde actin flow (RAF), a dynamic process important for cell motility. We will use quantitative fluorescence speckle microscopy (FSM) combined with genetic and biochemical approaches to define the downstream mediators of PAK1 action in RAF. Finally, FRET-based methods for visualizing Rac and Rho activation will be used to analyze the spatial and temporal dynamics of these GTPases in the chemotactically responding human neutrophil. The role of Rac2 in uropod retraction will be studied at the biochemical level. Spatio-temporal evaluation of Rho activation will be related to Rac2 activity, as well as specific aspects of a) chemoattractant receptor signaling, b) motile behavior, and c) actin-myosin cytoskeletal dynamics.