Non-canonical activation of heterotrimeric G protein signaling in vivo

摘要 异三聚体G蛋白是细胞间通讯的典型介质。 确定这种广泛存在的机制的分子机制是至关重要的 重要性，因为它影响了广泛的生理过程和疾病。这是很好的例证 由于对G蛋白偶联受体（GPCR）的持续研究兴趣， 异源三聚体G蛋白和>30%的市售药物的靶标。一个研究较少的方面， 异源三聚体G蛋白调节，但具有深远的影响，是他们的“非典型”激活， 不是GPCR的蛋白质。 在这些非GPCR激活剂中，含有进化上保守的Gα-结合和- 激活基序（Activating motif，GBA），又称“GBA蛋白”（GBA proteins），在G蛋白的调控中扮演着重要的角色 健康和疾病的信号。GIV和DAPLE是这个G蛋白家族的原型成员 在人类神经发育障碍中，这两种调节因子都发生了突变， 缺陷DAPLE在非综合征型先天性脑积水（NSCH）中突变，而GIV在非综合征型先天性脑积水（NSCH）中突变。 进行性脑病伴水肿、心律失常和视神经萎缩（PEHO）样综合征。的 引起这些疾病的细胞和分子改变知之甚少。 背景和目标：迄今为止，我们的长期目标之一是确定和描述 具有激活G蛋白信号传导的GBA基序的细胞质蛋白家族。像GPCR一样， 体外鸟嘌呤-核苷酸交换因子（GEF）活性。在这种情况下，我们的目标是了解 这种体外GEF活性如何与G蛋白和G蛋白依赖性的G蛋白的活化有关， 活细胞中的信号传导，并建立这种机制在体内的重要性。 目的概要：我们将（1）剖析GIV和DAPLE，两个原型GBA，如何影响G蛋白 通过使用新的化学发生平台和G蛋白活性生物传感器在细胞中进行信号传导，（2）定义它们如何在细胞中表达。 通过使用细胞生物学测定和新颖的光遗传学工具，功能由外部线索调节，以及（3） 通过使用动物模型来描述它们对神经发育的影响。 创新和影响：总的来说，我们预计该项目将推动异源三聚体G 通过提供详细的机制洞察到一个知之甚少的方面，他们的调控蛋白质信号 影响人类健康，并通过生成一套化学遗传学，光遗传学和生物传感器工具， 以前所未有的准确度直接操纵和检测异源三聚体G蛋白活性。概念 和技术创新所产生的成功完成这一项目将有一个长期的持久 在信号转导领域的影响。

ABSTRACT SIGNIFICANCE: Heterotrimeric G proteins are quintessential mediators of intercellular communication. Defining the molecular mechanisms by which this widespread machinery is regulated is of paramount importance because it impacts a vast range of physiological processes and diseases. This is well exemplified by the ongoing research interest in G protein-coupled receptors (GPCRs), the canonical activators of heterotrimeric G proteins and the targets for >30% of marketed drugs. A much less studied aspect of heterotrimeric G protein regulation, but with far-reaching implications, is their “non-canonical” activation by proteins that are not GPCRs. Among these non-GPCR activators, proteins containing an evolutionarily conserved Gα-Binding-and- Activating (GBA) motif, namely “GBA proteins”, have emerged a key new players in the control of G protein signaling in health and disease. GIV and DAPLE are the prototype members of this family of G protein regulators and both of them are mutated in neurodevelopmental disorders in humans that result in brain birth defects. DAPLE is mutated in non-syndromic congenital hydrocephalus (NSCH) whereas GIV is mutated in Progressive Encephalopathy with edema, Hypsarrhythmia, and Optic atrophy (PEHO)-like syndrome. The cellular and molecular alterations that cause these diseases are poorly understood. BACKGROUND AND GOALS: One of our long-term goals to date has been to identify and characterize a family of cytoplasmic proteins with a GBA motif that activates G protein signaling. Like GPCRs, they have Guanine-nucleotide Exchange Factor (GEF) activity in vitro. Within this context, our goal here is to understand mechanistically how this in vitro GEF activity relates to activation of G proteins and G protein-dependent signaling in living cells, and to establish the importance of this mechanism in vivo. SYNOPSIS OF AIMS: We will (1) dissect how GIV and DAPLE, the two prototype GBAs, affect G protein signaling in cells by using novel chemogenetic platforms and G protein activity biosensors, (2) define how their function is regulated by extrinsic cues by using cell biological assays and novel optogenetic tools, and (3) characterize their impact on neurodevelopment by using animal models. INNOVATION AND IMPACT: Overall, we anticipate that this project will advance the field of heterotrimeric G protein signaling by providing detailed mechanistic insights into a poorly understood aspect of their regulation that impacts human health, and by generating a suite of chemogenetic, optogenetic and biosensor tools to directly manipulate and detect heterotrimeric G protein activity with unprecedented accuracy. The conceptual and technological innovations arising from the successful completion of this project would have a long lasting impact in the field of signal transduction.