Regulation of Rho family GTPases by heterotrimeric G proteins

描述（由申请人提供）：由G12和G13组成的异源三聚体G蛋白的G12家族已显示介导从GPCR到RhoA激活的信号。单体GTdR RhoA调节细胞过程，如肌动蛋白细胞骨架重排和基因表达。RGS-RhoGEF是RhoA的特异性GEF（guanine nucleotide exchange factor，鸟嘌呤核苷酸交换因子），具有氨基末端RGS结构域，直接连接G12/13和RhoA。研究表明G12/13-RGS-RhoGEF信号通路在多种生理功能中的重要性。这种信号通路参与疾病，如心脏肥大，免疫功能障碍，或癌症转移也已被证明。该提案的长期目标是了解异源三聚体G蛋白介导的Rho家族GTPases调节的分子机制和功能。本研究的近期目标是阐明G12/13-RhoGEF信号通路的分子机制和功能。在本项目资助期内，我们取得了以下进展：（1）开发了G12和G13的新的纯化方法，并测定了它们的晶体结构和G13-p115 RhoGEF RGS复合物。(2)我们成功地将类似的纯化策略应用于Gq，并确定了Gq-GRK 2-Gbetagamma复合物的晶体结构。此外，我们发现了一种新的RhoGEF，p63 RhoGEF作为Gq的直接效应子。(3)利用表面等离子体共振（SPR）技术，我们建立了一套G13-LARG相互作用的动力学和热力学测量系统。热力学参数表明，在与G13相互作用的过程中，LARG的构象发生了显着的变化。(4)我们发现G13直接与β 3和β整合素的胞质结构域相互作用，这种相互作用是介导整合素由外向内信号传导导致血小板扩散所必需的。基于这些进展，我们将在本提案中研究以下目标。(1)我们将测定G12或G13与p115 RhoGEF构建体的复合物的X射线晶体结构，包括RGS结构域和DH/PH结构域。我们还将进行SPR分析，以了解G12/13-p115 RhoGEF相互作用的动力学。(2)我们推测RGS-RhoGEF的磷酸化在G12/13介导的信号转导中起着至关重要的调节作用。结合特异性单克隆抗体和质谱技术，我们将确定内源性RGS-RhoGEF的磷酸化位点，并表征这些磷酸化修饰在G12/13介导的信号转导中的功能作用。(3)我们推测G12/13信号通路和整合素信号通路的相互协调对肿瘤细胞的侵袭和迁移起着关键作用。我们将使用X射线晶体学，重建分析和生物传感器来监测RhoA活性，以及癌细胞的迁移和侵袭来分析这个信号网络的机制。 公共卫生相关性：G蛋白介导的信号传导是控制人体多种生理功能的最广泛使用的分子机制。G蛋白偶联受体（GPCR）是人类基因组中最大的基因家族，也是最常见的药物靶点家族。目前市场上约有50%的药物靶向GPCR或其信号成分。因此，研究G蛋白介导的信号通路不仅对了解我们的生理机能至关重要，而且对未来开发新的药物也至关重要。在这个项目中，我将专注于G12/13介导的信号通路，这是一种特定的G蛋白信号通路，已知对各种重要的细胞过程非常重要，包括癌症进展，细胞转化，细胞形状变化或细胞生长和分化。实验拟阐明该途径的信号组分的结构以及这些组分之间相互作用的动态变化。最近，我们还发现了G12/13信号通路和细胞粘附信号通路之间的一个新的信号网络。这是一个新的发现，连接这两个重要的信号系统。从原子结构分析到细胞分析系统的实验都被提出来理解这个信号网络。该项目的结果将为了解癌症表型的进展做出重要贡献，并为未来的抗癌药物开发奠定坚实的基础。

DESCRIPTION (provided by applicant): The G12 family of heterotrimeric G proteins, which consists of G12 and G13, has been shown to mediate signals from GPCRs to RhoA activation. The monomeric GTPase RhoA modulates cellular processes, such as actin cytoskeletal rearrangement and gene expression. RGS-RhoGEF, specific GEFs (guanine nucleotide exchange factor) for RhoA with amino terminal RGS domain, acts as a direct link between G12/13 and RhoA. Studies have demonstrated the importance of G12/13-RGS-RhoGEF signaling pathway in a variety physiological functions. The involvement of this signaling pathway in disease conditions, such as cardiac hypertrophy, immunological dysfunction, or cancer metastasis has also been demonstrated. The long term goal of this proposal is to understand the molecular mechanism and function of heterotrimeric G protein-mediated regulation of Rho family GTPases. Immediate goal of this proposal is to elucidate the molecular mechanism and function of G12/13-RhoGEF signaling pathway. During the current funding period, we have made following progresses: (1) We developed a novel purification method for G12 and G13 and determined their crystal structures as well as G13-p115RhoGEF RGS complex. (2) We successfully applied similar purification strategy for Gq and determined the crystal structure of Gq-GRK2-Gbetagamma complex. Also, we discovered a novel RhoGEF, p63RhoGEF as a direct effector for Gq. (3) Using surface plasmon resonance (SPR) we established a system to measure kinetics and thermodynamics of G13-LARG interaction. The thermodynamic parameters suggested a significant conformational change of LARG during interaction with G13. (4) We discovered that G13 directly interacts with the cytoplasmic domain of the beta3andbeta integrin, and this interaction is required in mediating the integrin outside-in signaling leading to platelet spreading. Based on these progresses, we will investigate the following aims in this proposal. (1) We will determine X-ray crystal structures of the complex of G12 or G13 with p115RhoGEF construct including RGS domain and DH/PH domains. We will also perform SPR analysis to understand the dynamics of G12/13-p115RhoGEF interaction. (2) We hypothesize that phosphorylation of RGS-RhoGEF plays crucial regulatory role in G12/13-mediated signaling. With the combination of specific moloclonal antibodies and mass spectrometry, we will determine the phosphorylation sites of endogenous RGS-RhoGEF and characterize the functional role of these phosphorylation modifications in G12/13- mediated signal transduction. (3) We hypothesize that the signal coordination of G12/13-pathway and integrin-pathway is critical for cell invasion and migration of cancer cells. We will analyze the mechanism of this signal network using X-ray crystallography, reconstitution assays, and biosensor to monitor RhoA activity, and migration and invasion of cancer cells. PUBLIC HEALTH RELEVANCE: G protein-mediated signaling is the most widely used molecular mechanism to control a variety of physiological functions of our body. G protein-coupled receptors (GPCR) consist of the largest gene family of our genome and the most common drug target family. About 50% of current drugs on the market target GPCRs or their signaling components. Thus, the investigation of G protein- mediated signaling pathway is not only essential to understand our physiology but also critically important to develop novel drugs in future. In this project, I will focus on the G12/13-mediated signaling pathway, a specific G protein signaling pathway that is known to be important for various important cellular processes including cancer progression, cell transformation, cell shape changes, or cell growth and differentiation. Experiments were proposed to elucidate the structure of the signaling components of this pathway and also the dynamic changes of interaction between these components. Recently, we also found a novel signaling network between G12/13-signaling pathway and cell adhesion signaling pathway. This is a novel finding to connect these two important signaling systems. The experiments from atomic structure analysis to cellular assay system are proposed to understand this signal network. The results from this project will make crucial contribution to understand advancement of cancer phenotypes and form a solid basis for future anti-cancer drug development.