G protein signal integration by multifunctional proteins

多功能蛋白的 G 蛋白信号整合

• 批准号: 6606450
• 负责人: T KENDALL HARDEN
• 金额: $ 23.9万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-10 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6606450
• 关键词: G protein; biological signal transduction; guanosinetriphosphatase activating protein; protein protein interaction

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 Ga-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 biologylbioinformatics (Siderovski), and structure (Sondek) of G protein signaling. A protein core will play a central role in the PPG by facilitating highthroughput 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-e 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-e in Ras-promoted cell transformation, and the domain(s) in PLC-e that interacts with Ga- subunits. Project III will focus on new signaling roles of the yeast Ga-subunit Gpa1, new effector roles of the yeast RGS protein Sst2, and the role of Scp160 in transmitting both downstream signals acting as a Gpa1 activated RNA binding protein and upstream signals as a modulator of Gpa1, potentially as a novel RGS protein. The delineation of novel signaling activities in yeast will guide complementary studies in mammalian systems. Project IV will establish the structural basis for several functional interactions mediating cross-talk in G protein signaling by solving the structures of the ¿5-R7 dimer and the GoLoco domain of R12-family RGS proteins, which has been shown to inhibit GDP release by Gai-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 Many cell stimulators act via cell surface receptors, G proteins, and effector enzymes. RGS proteins inactivate the signal by accelerating G protein GTPase activity. In yeast the beta/gamma subunits (Ste4/Ste18) have long been regarded as the signal-transmitting component of the G protein. We identified recently a positive signaling role for the G protein alpha subunit (Gpa1). Our goal is to identify effectors of the Gpa1-initiated signaling pathway(s). Our hypothesis is that the Gpa1 effectors include the RGS protein Sst2 and a novel RNA binding protein Scp160, since deletion of either blocks Gpa1 signaling. Our approach will be to determine how Sst2 and Scp160 transmit the downstream signal, and establish whether Scp160 accelerates Gpa1 GTPase activity, in the manner of Sst2. There are three specific aims of the proposal: Aim 1: Determine the mechanism of Sst2 effector activity. Two-hybrid screening revealed a number of proteins that bind selectively to Sst2 or Gpa1. We will confirm each interaction, identify additional binding partners; and through deletion and overexpression of each gene we will establish their role in Gpa1-initiated signaling in vivo Aim 2: Determine the mechanism of Scp160 effector activity. Scp160 was previously shown to bind mRNA. We will determine if Scp160 recruits selected mRNAs to the site of active signaling by sequencing RNAs bound to Scp160, and determine if these RNAs or their encoded protein translocate to the mating projection upon signal activation. We will determine if Scp160 transmits a signal through these RNA-encoded proteins, through deletion and overexpression of each gene. Aim 3: Determine the mechanism of Scp160 desensitization. Deletion of the SCP160 gene results in a pheromone-supersensitive phenotype, resembling that of the sst2 deletion mutant. We will map the Gpa1-binding domain of Scp160, and determine its ability to accelerate Gpa1 GTPase activity.

描述（由申请人提供）：G蛋白介导的信号传导是许多生长因子、激素和神经递质作用的基础。 虽然最初被认为是线性和单向的，但现在已知许多G蛋白相关的细胞信号通路在许多水平上会聚（和发散）。例如，RGS蛋白大家族的成员除了其促进Ga介导的GTP水解的特征能力之外还表现出多种生物化学活性。该计划项目（PPG）采用多学科方法，以获得G蛋白信号中多功能蛋白质的重要实例的机制和结构见解。我们聚集了具有癌症生物学（Der），酵母信号（Dohlman），分子药理学/生物化学（Harden），分子生物学/生物信息学（Siderovski）和G蛋白信号结构（Sondek）专业知识的研究人员。蛋白质核心将通过促进蛋白质的高通量克隆、表达、纯化和生物物理表征在PPG中发挥核心作用。R7家族RGS蛋白在受体/G蛋白相互作用和异源三聚体G蛋白之间的串扰的“上游”调节中的机制将在项目I中通过对哺乳动物和线虫蛋白的研究来确定。新鉴定的PLC-e同工酶的多功能性质将在项目II中通过鉴定N-末端RasGEF和C-末端Ras-缔合结构域的相互作用物、PLC-e在Ras促进的细胞转化中的作用以及PLC-e中与Ga-亚基相互作用的结构域的研究来描述。项目III将重点关注酵母Ga亚基Gpa 1的新信号作用，酵母RGS蛋白Sst 2的新效应子作用，以及Scp 160在传递下游信号（作为Gpa 1激活的RNA结合蛋白）和上游信号（作为Gpa 1的调节剂，可能作为一种新型RGS蛋白）中的作用。酵母中新的信号活性的描绘将指导哺乳动物系统中的补充研究。项目IV将通过解决R12家族RGS蛋白的<$5-R7二聚体和GoLoco结构域的结构来建立G蛋白信号传导中介导串扰的几种功能相互作用的结构基础，这些结构域已被证明可以抑制Gai亚基的GDP释放。这PPG将提供主要的新的机制的见解分子的复杂性，在G蛋白信号通路的功能，并应阐明新的药物靶点 许多细胞刺激因子通过细胞表面受体、G蛋白和效应酶起作用。RGS蛋白通过加速G蛋白GT3的活性来抑制信号。在酵母中，β/γ亚基（Ste 4/Ste 18）长期以来被认为是G蛋白的信号传递组分。我们最近确定了G蛋白α亚基（Gpa 1）的积极信号作用。我们的目标是确定Gpa 1启动的信号通路的效应子。我们的假设是，Gpa 1效应器包括RGS蛋白Sst 2和一种新的RNA结合蛋白Scp 160，因为缺失任何一种都会阻断Gpa 1信号传导。我们的方法将是确定Sst 2和Scp 160如何传输下游信号，并确定Scp 160是否以Sst 2的方式加速Gpa 1 GTdR活性。该提案有三个具体目标：目标1：确定Sst 2效应子活性的机制。双杂交筛选揭示了一些选择性结合Sst 2或Gpa 1的蛋白质。我们将确认每个相互作用，确定额外的结合伙伴，并通过删除和过表达的每个基因，我们将建立他们的作用，在体内Gpa 1启动信号目的2：确定Scp 160效应活性的机制。Scp 160先前显示结合mRNA。我们将通过对与Scp 160结合的RNA进行测序来确定Scp 160是否将选定的mRNA招募到活性信号传导的位点，并确定这些RNA或其编码的蛋白质是否在信号激活后易位到交配投射。我们将确定Scp 160是否通过这些RNA编码的蛋白质，通过每个基因的缺失和过表达来传递信号。目的3：探讨Scp 160的脱敏机制。SCP 160基因的缺失导致信息素超敏感表型，类似于Sst 2缺失突变体。我们将绘制Scp 160的Gpa 1结合结构域，并确定其加速Gpa 1 GTdR活性的能力。