G protein signal integration by multifunctional proteins

描述（由申请人提供）：G蛋白介导的信号转导是 许多生长因子、激素和神经递质的作用。虽然 最初被认为是线性和单向，许多G蛋白相关细胞 现在已知信号通路在许多水平上会聚（和发散）。为 例如，RGS蛋白大家族的成员表现出多个 生物化学活动，除了他们的签名能力，以促进 G-α介导的GTP水解。本项目（PPG）适用于 多学科的方法，以获得机械和结构的见解， G蛋白信号传导中多功能蛋白的重要例子。我们有 聚集了具有癌症生物学（Der）专业知识的研究人员， 酵母（Dohlman），和分子药理学/生物化学（Harden）， 分子生物学/生物信息学（Siderovski）和G的结构（Sondek 蛋白质信号蛋白质核心将在PPG中发挥核心作用， 促进高通量克隆、表达、纯化和生物物理学 蛋白质的表征。R7-家族RGS蛋白在乳腺癌中的作用机制 受体/G蛋白相互作用的"上游"调节和 异源三聚体G蛋白将在项目I中通过以下研究确定： 哺乳动物和C. elegans蛋白。新技术的多功能性 项目II将通过研究确定一种已鉴定的PLC-β同工酶 识别N-末端RasGEF和C-末端的相互作用物 Ras相关结构域，PLC在Ras促进的细胞中的作用 转化，以及与转化相互作用的PLC-β中的结构域 G-α-亚单位项目III将重点研究酵母蛋白Sst 2， 第一个发现的RGS蛋白，包含N-末端DEP结构域 在研究的哺乳动物R7家族RGS蛋白中也发现了未知功能 项目一和项目四。酵母双杂交筛选已经鉴定出 结合Sst 2 N-末端结构域，并激活应激反应信号。 负责G蛋白效应子活性的Sst 2结构域将被 定义，正如压力反应信号的机制一样。项目四将 为几种功能性相互作用建立结构基础， 通过解决β 5-R7的结构来解决G蛋白信号传导中的串扰 二聚体和R12家族RGS蛋白的GoLoco结构域，其已被证明 抑制G-α-i-亚单位释放GDP。这一PPG将提供主要的新 对分子复杂性的机械见解， 蛋白质信号通路，并应阐明新的药物靶点， 癌症、心脏病和其他疾病的基础过程 疾病和精神障碍。

DESCRIPTION (provided by applicant): G protein-mediated signaling underlies the action of many growth factors, hormones, and neurotransmitters. Although initially conceived as linear and unidirectional, many G protein-related cell signaling pathways now are known to converge (and diverge) at many levels. For example, members of the large family of RGS proteins exhibit multiple biochemical activities in addition to their signature capacity to promote G-alpha-mediated GTP hydrolysis. This program project (PPG) applies a multidisciplinary approach to gain mechanistic and structural insight into important examples of multifunctional proteins in G protein signaling. We have assembled investigators with expertise in cancer biology (Der), in signaling in yeast (Dohlman), and in the molecular pharmacology/biochemistry (Harden), molecular biology/bioinformatics (Siderovski), and structure (Sondek) of G protein signaling. A Protein Core will play a central role in the PPG by facilitating high-throughput cloning, expression, purification, and biophysical characterization of proteins. The mechanism(s) of R7-family RGS proteins in "upstream" regulation of receptor/G protein interaction and cross-talk among heterotrimeric G proteins will be determined in Project I through studies with mammalian and C. elegans proteins. The multifunctional nature of the newly identified PLC-epsilon isozyme will be delineated in Project II through studies identifying interactors for the N-terminal RasGEF and C-terminal Ras-association domains, the role of PLC-epsilon in Ras-promoted cell transformation, and the domain(s) in PLC-epsilon that interacts with G-alpha-subunits. Project III will focus on the yeast protein Sst2, which was the first RGS protein discovered, and which contains an N-terminal DEP domain of unknown function also found in the mammalian R7-family RGS proteins studied in Projects I and IV. Yeast two-hybrid screens have identified proteins that bind the Sst2 N-terminal domain, and which activate the stress response signal. The domains of Sst2 responsible for G protein effector activity will be defined, as will the mechanism(s) of stress response signaling. Project IV will establish the structural basis for several functional interactions mediating cross-talk in G protein signaling by solving the structures of the beta5-R7 dimer and the GoLoco domain of R12-family RGS proteins, which has been shown to inhibit GDP release by G-alpha-i-subunits. This PPG will provide major new mechanistic insights into the molecular complexities that function across G protein signaling pathways and should illuminate new drug targets within fundamental processes that underlie diseases as diverse as cancer, heart disease, and mental disorders.