Signaling through Rho GTP/GDP Exchange Factors

说明书(申请人提供)：Rho鸟嘌呤核苷酸交换因子(RhoGEF)包括一大类细胞内信号蛋白，它们将不同的输入耦合到小GTP酶Rho的激活，并最终耦合到Rho在修改细胞肌动蛋白细胞骨架中所起的作用所带来的动态细胞结构变化。Rho GTP酶调控的细胞过程包括细胞收缩、细胞黏附、细胞迁移、细胞增殖、突起伸展和收缩、基因表达和细胞分裂。一个由三个RhoGEF组成的亚家族被质膜上的异源三聚体G蛋白特异性激活，被称为G蛋白信号转导结构域调节因子(RGS-RhoGEF)。因此，RGS-RhoGEF介导了几个重要的G蛋白偶联受体(GPCR)对Rho的激活和细胞肌动蛋白细胞骨架的变化的信号。本申请中提出的研究将集中在一种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紧张素-II和内皮素对血管平滑肌细胞的收缩，以及LARG的基因缺失可以防止小鼠盐诱导的高血压。因此，LARG有可能成为治疗心血管疾病的靶点。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角色。在培养的细胞中，LARG定位于特定的有丝分裂结构，包括早期有丝分裂的中心体和有丝分裂纺锤体，以及晚期有丝分裂/胞质分裂的胞质分裂裂沟和中体。此外，LARG经历有丝分裂依赖性的磷酸化，而LARG的缺失导致强烈的晚期胞质分裂缺陷。本申请将侧重于定义和理解LARG的这一新角色。为了解决这个问题，这个建议的主要目标是：1)确定LARG在有丝分裂中的作用；2)表征有丝分裂依赖的LARG的磷酸化；以及3)研究LARG的有丝分裂定位以及LARG在招募关键蛋白质到中体中的作用。这些目标将通过各种实验方法来实现，包括培养细胞、时间推移显微镜、免疫荧光显微镜、药物抑制剂、突变分析和生化分析。 与公共健康相关：Rho鸟嘌呤核苷酸交换因子(RhoGEF)是关键的细胞内信号蛋白，将激活的细胞表面受体与细胞内部细胞骨架的动态变化联系起来。RhoGEF介导一系列涉及细胞形状变化的生理反应，包括平滑肌细胞收缩、细胞迁移和发育过程，并代表着高血压和癌症等疾病的潜在和新的治疗靶点。这一应用的研究将提供关于RhoGEF功能的新知识，从而更好地定义如何在不影响其关键正常功能的情况下，在疾病中对RhoGEF进行治疗抑制。

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.