Signaling through Rho GTP/GDP Exchange Factors

描述（由申请人提供）：Rho鸟嘌呤核苷酸交换因子（rhogef）包括一个大家族的细胞内信号蛋白，将不同的输入偶联到小GTPase Rho的激活，并最终导致Rho在修饰细胞肌动蛋白细胞骨架中的作用所带来的动态细胞结构变化。Rho gtpase控制的细胞过程包括平滑肌细胞收缩、细胞粘附、细胞迁移、细胞增殖、神经突伸展和收缩、基因表达和细胞分裂。一个由三个RhoGEFs组成的亚家族，被称为G蛋白信号结构域调控因子（RGS-RhoGEFs），在质膜上被异源三聚体G蛋白特异性激活。因此，rgs - rhogef介导几个重要的G蛋白偶联受体（GPCR）的信号传导到Rho的激活和细胞肌动蛋白细胞骨架的变化。本申请中提出的研究将集中于一种RGS-RhoGEF，称为白血病相关RhoGEF （LARG）。LARG介导的关键生理通路包括血管收缩剂血管紧张素II和内皮素对血管平滑肌细胞收缩的反应，以及LARG基因缺失可预防小鼠盐性高血压。因此，LARG有潜力作为治疗心血管疾病的治疗靶点。这个实验室的工作最近发现了LARG在细胞分裂中的一个新的和意想不到的作用。在培养的细胞中，LARG定位于特定的有丝分裂结构，包括有丝分裂早期的中心体和有丝分裂纺锤体，有丝分裂后期的分裂沟和中间体。此外，LARG经历有丝分裂依赖的磷酸化，LARG的耗尽导致强烈的晚期细胞分裂缺陷。这个应用程序将侧重于定义和理解LARG的这个新角色。为了解决这个问题，本提案的主要目标是：1)确定LARG在有丝分裂中的作用；2)表征LARG有丝分裂依赖性磷酸化；3)研究LARG在有丝分裂中的定位以及LARG在招募关键蛋白到中间体中的作用。这些目标将通过多种实验方法来实现，包括培养细胞、延时显微镜、免疫荧光显微镜、药理学抑制剂、突变分析和生化分析。

DESCRIPTION (provided by applicant): Rho guanine-nucleotide exchange factors (RhoGEFs) comprise a large family of intracellular signaling proteins that couple diverse inputs to the activation of the small GTPase Rho and ultimately to dynamic cell architecture changes brought about by Rho's role in modifying a cell's actin cytoskeleton. Cellular processes under the control of Rho GTPases include smooth muscle cell contractility, cell adhesion, cell migration, cell proliferation, neurite extension and retraction, gene expression and cell division. A sub-family of three RhoGEFs, termed regulator of G protein signaling domain-containing RhoGEFs (RGS-RhoGEFs), is specifically activated by heterotrimeric G proteins at the plasma membrane. The RGS-RhoGEFs thus mediate signaling from several important G protein-coupled receptors (GPCR) to activation of Rho and changes in a cell's actin cytoskeleton. The research proposed in this application will focus on one RGS-RhoGEF, termed leukemia-associated RhoGEF (LARG). Critical physiological pathways mediated by LARG include contraction of vascular smooth muscle cells in response to vasoconstrictors angiotensin II and endothelin, and genetic deletion of LARG prevents salt-induced hypertension in the mouse. Thus, LARG has potential as a therapeutic target in the treatment of cardiovascular disease. Work in this laboratory has recently uncovered a novel and unexpected role for LARG in cell division. In cultured cells, LARG is localized at specific mitotic structures, including centrosomes and mitotic spindles in early mitosis and the cytokinesis cleavage furrow and midbody in late mitosis/cytokinesis. Moreover, LARG undergoes mitotic-dependent phosphorylation, and depletion of LARG causes a strong late cytokinesis defect. This application will focus on defining and understanding this new role for LARG. To address this question, the major objectives of this proposal are 1) Define the role of LARG in mitosis; 2) Characterize mitotic-dependent phosphorylation of LARG; and 3) Investigate mitotic localization of LARG and the role LARG in recruiting critical proteins to the midbody. These objectives will be pursued by a variety of experimental approaches, including cultured cells, time-lapse microscopy, immunofluorescence microscopy, pharmacological inhibitors, mutational analysis, and biochemical assays. PUBLIC HEALTH RELEVANCE: Rho guanine-nucleotide exchange factors (RhoGEFs) are key intracellular signaling proteins, connecting activated cell-surface receptors to dynamic changes in a cell's interior cytoskeleton. The RhoGEFs mediate a number of physiological responses that involve changes in a cell's shape, including smooth muscle cell contractility, cell migration, and developmental processes, and represent potential and novel therapeutic targets in disease states such as hypertension and cancer. The research in this application will provide new knowledge about the functions of RhoGEFs and thus better define how the RhoGEFs can be therapeutically inhibited in disease without affecting their critical normal functions.