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、激素和神经递质作用的基础。虽然最初的设想是线性的和单向的，但现在已知许多与G蛋白相关的细胞信号通路在许多水平上汇聚(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水解的特征能力外，还表现出多种生化活性。该计划项目(PPG)应用多学科方法，以获得对G蛋白信号中多功能蛋白质的重要例子的机制和结构的洞察。我们汇集了在癌症生物学(Der)、酵母信号(Dohlman)以及G蛋白信号的分子药理学/生物化学(Harden)、分子生物生物信息学(Siderovski)和结构(Sondek)方面具有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、纯化和生物物理特性而在PPG中发挥核心作用。R7家族RGS蛋白“上游”调节受体/G蛋白相互作用和异源三聚体G蛋白之间串扰的机制(S)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结合结构域的相互作用，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(S)。项目III将侧重于酵母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结合蛋白传递下游信号和作为Gpa1的调节器的上游信号的作用，这可能是一种新的RGS蛋白。酵母中新的信号活性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，为G蛋白信号中介导串扰的几种功能相互作用建立结构基础，R12家族RGS蛋白已被证明抑制GAI-亚基释放GDP。这一PPG将为G蛋白信号通路中发挥作用的分子复杂性提供重要的新的机械学见解，并将照亮新的药物靶点 许多细胞刺激物通过细胞表面受体、G蛋白和效应酶发挥作用。RGS蛋白通过加速G蛋白GTP酶的活性而使信号失活。在酵母中，β/γ亚基(Ste4/Ste18)一直被认为是G蛋白的信号传递成分。我们最近发现了G蛋白α亚单位(Gpa1)的一个积极的信号作用。我们的目标是确定Gpa1启动的信号通路的效应者(S)。我们的假设是Gpa1效应器包括RGS蛋白Sst2和一种新的RNA结合蛋白Scp160，因为这两种蛋白中的任何一种的缺失都会阻止Gpa1信号转导。我们的方法是确定Sst2和Scp160如何传递下游信号，并确定Scp160是否以Sst2的方式加速Gpa1 GTP酶的活性。该提案有三个具体目标：目标1：确定Sst2效应器活性的机制。双杂交筛选发现许多蛋白质选择性地与Sst2或Gpa1结合。我们将确认每个相互作用，确定额外的结合伙伴；通过每个基因的缺失和过表达，我们将在体内确定它们在Gpa1启动的信号转导中的作用目标2：确定Scp160效应器活性的机制。先前的研究表明，Scp160可以与mRNA结合。我们将通过对与Scp160结合的RNA进行测序来确定Scp160是否将选定的mRNAs招募到激活信号的位置，并确定这些RNA或其编码的蛋白质是否在信号激活时移位到交配投影。我们将确定Scp160是否通过这些RNA编码的蛋白质，通过每个基因的缺失和过度表达来传递信号。目的3：探讨Scp160的脱敏机制。SCP160基因缺失导致信息素超敏表型，类似于Sst2缺失突变体。我们将定位Scp160的Gpa1结合域，并确定其促进Gpa1 GTPase活性的能力。

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.