Regulation of neutrophil receptor G protein interactions

中性粒细胞受体 G 蛋白相互作用的调节

• 批准号: 7901730
• 负责人: GARY M BOKOCH
• 金额: $ 7.96万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-31 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7901730
• 关键词: Actins; Behavior; Biochemical; Biochemical Genetics; Biological; Chemotaxis; Cytokinesis; Cytoskeleton; Evaluation; Fluorescence; Fluorescence Microscopy; Funding; GTP-Binding Proteins; Genetic; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate Phosphohydrolases; Human; Imaging technology; LIM Domain Kinase 1; Leukocytes; Mediator of activation protein; Methods; Microscopy; Microtubules; Molecular; Molecular Target; Monomeric GTP-Binding Proteins; Myosin ATPase; Myosin Light Chain Kinase; Normal Cell; Process; Proteins; Receptor Signaling; Regulation; Role; Signal Transduction; Small Interfering RNA; Tail; base; cell motility; directional cell; fMet-Leu-Phe receptor; neutrophil; p21 activated kinase; receptor; response; rho; rho GTP-Binding Proteins

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.

G 蛋白偶联趋化受体的信号传导至人类功能反应 白细胞涉及 Rho 小 GTP 酶。在之前的研究中，我们确定 p21 激活 激酶 (PAK) 是 Rho GTPase 信号传导的重要下游介质，并调节 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 的细胞骨架调节，包括 GEF-H1、LIM 激酶和肌球蛋白轻链激酶。在 在目前的提案中，我们将研究PAK1的作用，以及它的目标GEF-H1 使用生化、遗传和分子方法进行细胞骨架调节。我们将继续 趋化过程中 Rho GTPase 激活和功能的时空动态研究 使用独特的基于荧光的成像技术。 我们将 GEF-H1 描述为微管调节的鸟嘌呤核苷酸交换因子 Rho GTPase，并暗示 GEF-H1 参与胞质分裂和 Rho 活性的调节 定向细胞运动。 GEF-H1 的生物活性将通过遗传和 基于 siRNA 的方法。将研究 PAK1 在调节 GEF-H1 功能中的作用。我们 确定了 PAK1 在调节逆行肌动蛋白流 (RAF) 中的作用，这是一个动态过程 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来定义 PAK1 的下游介质 英国皇家空军的行动。最后，基于 FRET 的 Rac 和 Rho 激活可视化方法将用于 分析这些 GTPases 在趋化反应中的空间和时间动态 人类中性粒细胞。 Rac2 在尾足收缩中的作用将在生化水平上进行研究。 Rho 激活的时空评估将与 Rac2 活性以及特定的 a) 趋化受体信号传导、b) 运动行为和 c) 肌动蛋白-肌球蛋白的各个方面 细胞骨架动力学。

项目成果

Guanine nucleotide exchange factors regulate specificity of downstream signaling from Rac and Cdc42.

鸟嘌呤核苷酸交换因子调节 Rac 和 Cdc42 下游信号传导的特异性。

• DOI: 10.1074/jbc.273.27.16782
• 发表时间: 1998
• 期刊: The Journal of biological chemistry
• 作者: Zhou,K;Wang,Y;Gorski,JL;Nomura,N;Collard,J;Bokoch,GM
• 通讯作者: Bokoch,GM

rac, a novel ras-related family of proteins that are botulinum toxin substrates.

• 发表时间: 1989-10
• 期刊: The Journal of biological chemistry
• 作者: John DidsburyS;Richard F. Weber;Gary M. BokochQT;T. Evans;R. Snyderman

Human neutrophils coordinate chemotaxis by differential activation of Rac1 and Rac2.

• DOI: 10.4049/jimmunol.0900849
• 发表时间: 2009-08-15
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Zhang H;Sun C;Glogauer M;Bokoch GM
• 通讯作者: Bokoch GM

Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration.

• DOI: 10.1083/jcb.201010059
• 发表时间: 2011-06-27
• 期刊: The Journal of cell biology
• 作者: Delorme-Walker VD;Peterson JR;Chernoff J;Waterman CM;Danuser G;DerMardirossian C;Bokoch GM
• 通讯作者: Bokoch GM

Domains of the human neutrophil N-formyl peptide receptor involved in G protein coupling. Mapping with receptor-derived peptides.

人中性粒细胞 N-甲酰肽受体的结构域参与 G 蛋白偶联。

• 发表时间: 1994
• 期刊: The Journal of biological chemistry
• 作者: Schreiber,RE;Prossnitz,ER;Ye,RD;Cochrane,CG;Bokoch,GM
• 通讯作者: Bokoch,GM