G protein signal integration by multifunctional proteins

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收敛（和发散）。例如，RGS蛋白大家族的成员除了具有促进ga介导的GTP水解的标志性能力外，还表现出多种生化活性。本项目（PPG）应用多学科方法获得G蛋白信号中多功能蛋白的重要例子的机制和结构洞察力。我们汇集了具有癌症生物学（Der），酵母信号传导（Dohlman），分子药理学/生物化学（Harden），分子生物学/生物信息学（Siderovski）和G蛋白信号传导结构（Sondek）方面专业知识的研究人员。蛋白质核心通过促进蛋白质的高通量克隆、表达、纯化和生物物理表征，将在PPG中发挥核心作用。r7家族RGS蛋白“上游”调控受体/G蛋白相互作用和异源三聚体G蛋白间串扰的机制将在项目1中通过对哺乳动物和线虫蛋白的研究来确定。新发现的PLC-e同工酶的多功能性质将在项目II中通过研究确定n端RasGEF和c端ras -关联域的相互作用，PLC-e在ras -促进细胞转化中的作用，以及PLC-e中与Ga-亚基相互作用的域来描述。项目III将重点研究酵母ga亚基Gpa1的新信号作用，酵母RGS蛋白Sst2的新效应作用，以及Scp160作为Gpa1激活的RNA结合蛋白在传递下游信号和作为Gpa1调节剂的上游信号中的作用，并有可能作为一种新的RGS蛋白。酵母中新的信号活动的描述将指导哺乳动物系统的补充研究。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，建立介导G蛋白信号中串扰的几种功能相互作用的结构基础，这些功能相互作用已被证明可以抑制gai亚基释放GDP。该PPG将为G蛋白信号通路的分子复杂性提供重要的新机制见解，并应阐明新的药物靶点

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.