Non-canonical activation of heterotrimeric G protein signaling in vivo

摘要 意义：异三聚体G蛋白是细胞间通讯的典型媒介。 确定调节这一广泛存在的机制的分子机制至关重要。 这很重要，因为它影响到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(GPCRs)的持续研究兴趣，G蛋白偶联受体(GPCRs)的典型激活物 异三聚体G蛋白和30%上市药物的靶标。一个很少被研究的方面 异源三聚体G蛋白的调节，但具有深远的影响，是它们通过 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(GBA)基序，即“GBA蛋白”，已成为控制G蛋白的关键新分子 健康和疾病方面的信号。GIV和DAPLE是这个G蛋白家族的原型成员 在导致脑出生的人类神经发育障碍中，调节因子及其两者都发生了突变 缺陷。非综合征性先天性脑积水(NSCH)患者DAPLE基因突变，而GIV基因突变 进行性脑病伴有水肿、低心率失常和视神经萎缩(PEHO)样综合征。这个 导致这些疾病的细胞和分子变化还知之甚少。 背景和目标：到目前为止，我们的长期目标之一是识别和描述一种 胞质蛋白家族，具有激活G蛋白信号的GBA基序。与GPCR一样，它们也有 鸟嘌呤核苷酸交换因子的体外活性。在这种情况下，我们的目标是了解 这种体外全球环境基金活性如何与G蛋白的激活和G蛋白依赖有关 在活细胞中的信号，并建立这一机制的重要性在体内。 AIMS简介：我们将(1)剖析GIV和DAPLE这两个原型GBA是如何影响G蛋白的 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传递信号，(2)定义它们的 功能是通过使用细胞生物学检测和新型光遗传工具的外在提示来调节的，以及(3) 通过动物模型描述它们对神经发育的影响。 创新和影响：总体而言，我们预计该项目将推动异三聚体G领域的发展 通过提供对其调节的一个鲜为人知的方面的详细的机械性见解来传递蛋白质信号 影响人类健康，并通过产生一套化学遗传、光遗传和生物传感器工具来 直接操纵和检测异源三聚体G蛋白活性，具有前所未有的准确性。概念性 而这一项目的成功完成所带来的技术创新将会产生持久的影响 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.