Regulation of Rho family GTPases by heterotrimeric G proteins

描述(由申请人提供)：G12家族的异三聚体G蛋白，由G12和G13组成，已被证明介导从GPCRs到RhoA激活的信号。单体GTP酶RhoA调控细胞过程，如肌动蛋白细胞骨架重排和基因表达。RGS-Rhogef是RhoA的特异性鸟苷核苷酸交换因子，具有氨基端RGS结构域，是G12/13和RhoA之间的直接纽带。研究表明，G12/13-RGS-Rhogef信号通路在多种生理功能中具有重要作用。这一信号通路在心脏肥大、免疫功能障碍或癌症转移等疾病中的作用也已得到证实。这一建议的长期目标是了解异源三聚体G蛋白介导的Rho家族GTP酶调节的分子机制和功能。这一建议的近期目标是阐明G12/13-Rhogef信号通路的分子机制和功能。在本资助期间，我们取得了以下进展：(1)我们开发了一种新的G12和G13的纯化方法，并测定了它们的晶体结构以及G13-p115RhoGEFRGS络合物。(2)我们成功地对GQ进行了类似的纯化策略，并确定了GQ-GRK2-Gbetagamma的晶体结构。此外，我们还发现了一种新的Rhogef，p63Rhogef作为GQ的直接效应因子。(3)利用表面等离子体共振(SPR)技术建立了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，LARG在与G13相互作用过程中发生了显著的构象变化。(4)我们发现G13与β3和β整合素的胞浆结构域直接相互作用，这种相互作用在介导整合素由外向内信号导致血小板扩散时是必需的。在这些进展的基础上，我们将调查这项提案的以下目标。(1)我们将测定G12或G13与p115Rhogef结构的络合物的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-p115Rhogef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重要的调节作用。结合特异性单克隆抗体和质谱仪，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途径和整合素途径的信号协调对癌细胞的侵袭和迁移是至关重要的。我们将使用X射线结晶学、重组分析和生物传感器来分析这一信号网络的机制，以监测RhoA的活性以及癌细胞的迁移和侵袭。

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.