Regulation of neutrophil receptor G protein interactions

G蛋白偶联趋化因子受体对人神经细胞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，我们确定p21激活 激酶（PAK）是Rho GT3信号传导的重要下游介质，并且调节 肌动蛋白和微管细胞骨架之间的串扰。我们确定了 PAK 1对细胞骨架的调节，包括GEF-H1、LIM激酶和肌球蛋白轻链激酶。在 根据目前的建议，我们将研究PAK 1的作用，以及它的目标GEF-H1， 利用生物化学、遗传学和分子方法进行细胞骨架调节。我们将继续 趋化过程中Rho GT3激活和功能的时空动态研究 使用独特的荧光成像技术。 我们将GEF-H1描述为一种微管调控的鸟嘌呤核苷酸交换因子， Rho GT3，并暗示GEF-H1在胞质分裂期间调节Rho活性， 定向细胞运动GEF-H1的生物学活性将使用遗传和 基于siRNA的方法。将研究PAK 1在调节GEF-H1功能中的作用。我们 已经确定了PAK 1在调节肌动蛋白逆行流（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） 结合遗传学和生物化学方法来确定PAK 1的下游介质 RAF的行动。最后，基于FRET的可视化Rac和Rho激活的方法将用于 分析这些GTP酶在趋化反应中的空间和时间动态， 人嗜中性粒细胞。Rac 2在尾足收缩中的作用将在生化水平上进行研究。 Rho激活的时空评价将与Rac 2活性以及特异性 a）化学引诱物受体信号传导，B）运动行为，和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.