Spatial Regulation of RGS and G Protein Signaling

描述(申请人提供)：在这项资助下，我们发现了一些G蛋白结合蛋白，确定了它们的性质，并揭示了异源三聚体G蛋白在细胞中的新作用。在当前的资助期间，我们描述了GIV，一种激活GαI蛋白的非受体全环基金。GIV是一种多模块的信号转导分子，可组装蛋白质复合体，并将Gα亚单位与其他信号通路(生长因子和PI3K/Akt信号)以及几种重要的细胞功能联系起来，包括细胞迁移、自噬、分泌蛋白的运输和生长因子受体的内吞作用。GIV还与GαS结合，促进表皮生长因子受体下调，并阻断来自内小体的增殖信号。提出的工作的总体目标是利用我们开发的工具和见解来阐明G蛋白的一些众所周知但鲜为人知的功能，特别是在细胞内膜上。我们将集中于两个具体目标：具体目标1：明确GIV及其与GαI和GαS的相互作用调节EGFR转运和生长因子信号转导的机制。根据初步数据，我们的工作模型是：在表皮生长因子刺激后，GIV依次与GαI3和GαS结合，GIV的磷酸化调节与这两个G蛋白的相对结合，并决定细胞是迁移还是增殖。具体目标2：确定GαI3和GIV在高尔基体功能中的作用，并确定相关的机制。我们的数据表明，GIV或未能激活GαI3的GIV突变体的耗尽会减缓新合成的蛋白质通过高尔基体的运输。我们基于初步数据的工作假设是，GIV通过激活GαI3，下调Arf1活性，并促进囊泡脱壳来实现这一点。这些研究有望为GIV如何结合和调节两种不同的G蛋白并影响生长因子信号、高尔基体功能、细胞迁移和细胞增殖提供新的见解。基于GIV在调节细胞行为方面的关键功能，确定GIV的功能和相互作用是至关重要的。我们的研究不仅将深入了解G蛋白在细胞功能中的基本作用，还可能建立新的范式，有助于为开发药理和抗肿瘤药物寻找新的治疗靶点。通过利用我们手中的新工具、专业知识和信息，我们处于独特的地位，可以解决这些问题。

DESCRIPTION (provided by applicant): Under this grant we have discovered a number of G protein binding proteins, established their properties and revealed new roles for heterotrimeric G proteins in cells. During the current funding period we characterized GIV, a non-receptor GEF that activates GαI proteins. GIV serves as a multimodular signal transducer that assembles protein complexes and links Gα subunits to other signaling pathways (growth factor and PI3K/Akt signaling) and to several important cell functions, including cell migration, autophagy, transport of secretory proteins, and endocytosis of growth factor receptors. GIV also binds Gαs, promotes EGF receptor downregulation, and shuts down proliferative signaling from endosomes. The overall goal of the work proposed is to take advantage of the tools and insights we have developed to shed light on some well-known but poorly understood functions of G proteins especially on intracellular membranes. We will focus on two specific aims: Specific Aim #1: To pinpoint the mechanisms by which GIV and its interactions with GαI and Gαs regulate EGFR trafficking and growth factor signaling. Our working model based on preliminary data is that GIV sequentially binds Gαi3 and Gαs after EGF stimulation and that phosphomodification of GIV regulates the relative binding to the two G proteins and determines whether cells migrate or proliferate. Specific Aim #2: To determine the role of Gαi3 and GIV in Golgi functions and to define the mechanisms involved. Our data indicate that depletion of GIV or a GIV mutant that fails to activate Gαi3 slows transport of newly synthesized proteins through the Golgi. Our working hypothesis based on preliminary data is that GIV does so by activating Gαi3, down-regulating Arf1 activity, and facilitating vesicle uncoating. These studies can be expected to provide novel insights into how GIV binds and regulates two different G proteins and influences growth factor signaling, Golgi functions, cell migration and cell proliferation. Based on its crucil functions in regulating cell behavior, it is essential to define GIV's functions and interactions. Our studies will not only provide insights into fundamental roles of G proteins in cell functions, but also it may establish new paradigms that will help to identify new therapeutic targets for development of pharmacologic and anti-tumor agents. We are uniquely positioned to tackle these problems by taking advantage of the new tools, expertise, and information we have in hand.