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）包括细胞内信号传导蛋白的大家族，其将不同的输入偶联到小GTdR Rho的活化，并最终偶联到由Rho在修饰细胞的肌动蛋白细胞骨架中的作用所引起的动态细胞结构变化。在Rho GTP酶控制下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、神经突延伸和收缩、基因表达和细胞分裂。RhoGEFs是一个由三个RhoGEFs组成的亚家族，被称为G蛋白信号传导结构域调节因子RhoGEFs（RGS-RhoGEFs），它被质膜上的异源三聚体G蛋白特异性激活。因此，RGS-RhoGEFs介导从几个重要的G蛋白偶联受体（GPCR）到Rho激活和细胞肌动蛋白细胞骨架变化的信号传导。本申请中提出的研究将集中在一个RGS-RhoGEF上，称为白血病相关RhoGEF（LARG）。由LARG介导的关键生理途径包括血管平滑肌细胞响应于血管收缩剂血管紧张素II和内皮素的收缩，并且LARG的遗传缺失防止小鼠中盐诱导的高血压。因此，LARG具有作为治疗心血管疾病的治疗靶点的潜力。该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以及有丝分裂/胞质分裂晚期的胞质分裂沟和中间体。此外，LARG经历有丝分裂依赖性磷酸化，并且LARG的耗尽引起强烈的晚期胞质分裂缺陷。该应用程序将侧重于界定和理解LARG的这一新作用。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的有丝分裂依赖性磷酸化; 3）研究LARG的有丝分裂定位以及LARG在将关键蛋白质募集到中间体中的作用。这些目标将通过各种实验方法来实现，包括培养细胞，延时显微镜，免疫荧光显微镜，药理学抑制剂，突变分析和生化测定。 公共卫生相关性：Rho鸟嘌呤-核苷酸交换因子（RhoGEFs）是关键的细胞内信号传导蛋白，将活化的细胞表面受体与细胞内部细胞骨架的动态变化连接起来。RhoGEFs介导了许多生理反应，涉及细胞形状的变化，包括平滑肌细胞收缩性，细胞迁移和发育过程，并代表了高血压和癌症等疾病状态的潜在和新的治疗靶点。本申请中的研究将提供有关RhoGEFs功能的新知识，从而更好地定义RhoGEFs如何在疾病中被治疗性抑制而不影响其关键的正常功能。

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.