Spatial Regulation of RGS and G Protein Signaling

描述（由申请人提供）：在过去的5-7年中，我们鉴定了几种新的蛋白质，它们是新型G蛋白信号通路的组分。这些蛋白质的表征揭示了三聚体G蛋白在细胞增殖，细胞迁移和线粒体动力学中的新作用。我们在过去5年的研究成果包括：1）发现了G1在EGF受体（EGFR）下调和信号转导中的新的调节作用; 2）证明了GIPC在TrkA NGF信号转导和MAPK激活中的关键作用; 3）GIV，一种新的G1结合蛋白的鉴定，并证明G1 i3的激活及其与GIV的相互作用对于细胞迁移和细胞迁移期间Akt的激活是必不可少的; 4）hNOA 1的发现（以前称为MIG 78），一种新的动力蛋白样GT3，可能提供线粒体动力学、呼吸和凋亡之间的联系; 5）鉴定MIR 16（GDE 1），一种在磷酸肌醇代谢和G蛋白信号传导之间提供推定联系的新型酶;和6）发现网格蛋白包被的膜微区中G1 i信号传导的空间调节的新范例。提出的工作的总体目标是进一步确定我们发现的这些新途径中的两个，即G1 i3和GIV在细胞迁移和Akt信号传导中的作用以及G1在EGFR运输和信号传导调节中的作用。我们将专注于三个具体目标：具体目标#1：评估细胞迁移过程中G1 i3和GIV之间的功能相互作用。我们的工作模型是，GIV作为一个非受体GEF，是触发激活信号级联最终在有效的细胞运动。具体目标#2：目的探讨G1 i3和GIV相互作用及在迁移细胞中从高尔基体向PM移位的时空调控。我们将研究它们在哪里相互作用，以及在活细胞中使用荧光生物传感器FRET和FRAP激活G1 i3。具体目标#3：查明G1和RGS-PX 1影响EGF受体信号传导、运输和降解分选的机制。我们将研究它们对EGFR磷酸化、衔接子募集和下游信号传导以及通过内体系统的运输和下调分选的影响。这些研究有望深入了解正常细胞的功能以及与癌细胞转移相关的细胞增殖和迁移失调的分子基础。由于它们在调节细胞行为方面的关键功能，这些新分子成为开发药理学和抗肿瘤药物的有吸引力的靶点。 公共卫生相关性：在这项资助下，我们发现并表征了许多新的分子，这些分子是新型G蛋白信号网络的一部分，这些信号网络调节许多在癌症研究中重要的细胞过程，包括内吞作用，生长和增殖，细胞迁移和转移。该申请中计划的研究有望揭示正常细胞中新的G蛋白功能，并为癌症和其他疾病中发生的细胞增殖和迁移失调的分子基础提供关键见解。由于它们在细胞调节中的关键功能，这些新分子代表了开发药理学和抗肿瘤剂的有吸引力的靶点。

DESCRIPTION (provided by applicant): During the last 5-7 years, we identified several new proteins that are components of novel G protein signaling pathways. The characterization of these proteins has brought to light new roles for trimeric G proteins in cell proliferation, cell migration, and mitochondrial dynamics. Our accomplishments during the last 5 years of this grant include: 1) Discovery of a novel regulatory role for G1s in EGF receptor (EGFR) downregulation and signaling; 2) Demonstration of the crucial role played by GIPC in TrkA NGF signaling and MAPK activation; 3) Identification of GIV, a novel G1 binding protein, and demonstration that activation of G1i3 and its interaction with GIV is essential for cell migration and Akt activation during cell migration; 4) Discovery of hNOA1 (formerly called MIG78), a new dynamin-like GTPase which may provide a link between mitochondrial dynamics, respiration and apoptosis; 5) Identification of MIR16 (GDE1), a novel enzyme that provides a putative link between phosphoinositide metabolism and G protein signaling; and 6) Discovery of a new paradigm for spatial regulation of G1i signaling in clathrin-coated membrane microdomains. The overall goal of the work proposed is to further define two of these novel pathways we have discovered-namely, the role of G1i3 and GIV in cell migration and Akt signaling and the role of G1s in regulation of EGFR trafficking and signaling. We will focus on three specific aims: Specific Aim #1: To assess the functional interaction between G1i3 and GIV during cell migration. Our working model is that GIV serves as a non-receptor GEF and is the trigger that activates the signaling cascade culminating in efficient cell motility. Specific Aim #2: To investigate the spatial and temporal regulation of G1i3 and GIV's interaction and translocation from the Golgi to the PM in migrating cells. We will investigate where they interact and where G1i3 is activated using fluorescent biosensors, FRET and FRAP in living cells. Specific Aim #3: To pinpoint the mechanisms by which G1s and RGS-PX1 affect EGF receptor signaling, trafficking and sorting for degradation. We will investigate their effects on phosphorylation of EGFR, recruitment of adaptors, and downstream signaling as well as trafficking through the endosomal system, and sorting for down-regulation. These studies can be expected to provide insights into how normal cells function and the molecular basis for dysregulation of cell proliferation and migration associated with metastasis that occurs in cancer cells. Due to their crucial functions in regulating cell behavior these new molecules represent attractive targets for development of pharmacologic and anti-tumor agents. PUBLIC HEALTH RELEVANCE: Under this grant we have discovered and characterized a number of new molecules that are part of novel G protein signaling networks which regulate many cell processes important in cancer research including endocytosis, growth and proliferation, cell migration and metastasis. The studies planned in this application can be expected to shed light on new G protein functions in normal cells as well as to provide key insights into the molecular basis of the dysregulation of cell proliferation and migration that occur in cancer and other diseases. Due to their crucial functions in cell regulation these new molecules represent attractive targets for development of pharmacologic and anti-tumor agents.