Optical control of endogenous G protein Coupled Receptor and G Protein Signaling.

摘要 细胞主要通过G蛋白偶联受体(GPCRs)感知细胞外环境。他们 代表最大的细胞表面蛋白家族，在维持细胞生命方面起着关键的生理作用。 GPCRs利用异源三聚体G蛋白将信号传递到细胞内部。GPCR中也存在功能障碍 作为G蛋白信号，与一些最流行的人类疾病有关，因此，GPCRs已经成为 最大的毒品目标。在800多个成员中，有100多个GPCR由多肽或 小的蛋白质配体。甚至有一个这样的GPCRs家族，即蛋白酶激活受体(PAR)家族，也显示出一种 广泛的身体存在，从大脑到心脏，并调节许多已知和 可能还有更多未知的生理作用，从免疫到心脏。一个根本性的限制是使 人类生理学中PARS的进展是缺乏工具来控制内源性表达的受体 培养的细胞和体内。尽管Opsins可以通过空间和时间控制激活G蛋白信号，但它们 只是粗略地概括了内源性GPCRs的信号。同样，也不存在光遗传甚至化学物质 可用于控制内源性异源三聚体信号的工具。 因此，在目标1中，我们计划提供一个光配体文库来控制内源性par受体。 瞬间和可逆的。初步数据显示，野生型PAR1受体被一种 基因编码的光配体，并证明了所提出的可行性。尽管该提案的重点是 PAR家族GPCRs，在光配体设计中的更广泛的适应性将允许光学控制其他多肽或 小蛋白激活的GPCRs，扩展了AIM的未来生物医学意义1。我们的光配体将是 它们是第一个对亚细胞、细胞、组织甚至器官水平的gpr进行如此精确的调控的公司。 光学指令信号，满足未来生物医学研究的需求。 同样，尽管异三聚体G蛋白在所有GPCR的信号转导中起着核心作用， 除了少数几种可用的信号转导抑制剂外，没有直接的途径可以通过 可感知的空间或时间控制。尽管有光学控制，但可用的光遗传调节器仅旨在 G蛋白的下游效应器，并由于过表达的活性蛋白而引发更高的背景信号。 我们使用Aim 2直接进入内源性G蛋白异源三聚体来控制一个或两个G蛋白 亚单位光学信号。使用从G蛋白信号转导的本地控制器获得的多肽域，我们 显示光诱导的巨噬细胞通过G-βγ的局部生成而迁移。工程化的光遗传工具 在目标2中，不仅将提供绕过化学试剂限制的实验手段，而且还将 G蛋白亚单位功能的科学和促进未来的分子发现/筛选工作以控制 杂三聚体和或其选择亚基。

Abstract Cells sense the extracellular environment primarily using G protein-coupled receptors (GPCRs). They represent the largest family of cell surface proteins and play key physiological roles in maintaining cellular life. GPCRs employ heterotrimeric G protein to transduce signals to the cell interior. Dysfunctions in GPCR, as well as G protein signaling, contribute to some of the most prevalent human diseases and thus, GPCRs have become the largest drug target. Out of over 800 members, more than a hundred GPCRs are controlled by peptide or small protein ligands. Even one family of such GPCRs, the protease-activated receptor (PAR) family, shows an extensive physical presence throughout the body from the brain to the heart and regulates many known and possibly even more unknown physiological roles, from immune to cardiac. A fundamental limitation in making advances in PARs in human physiology is the lack of tools to control endogenously expressed receptors both in cultured cells and in vivo. Though opsins can activate G protein signaling with spatial and temporal control, they only loosely recapitulate signaling of endogenous GPCRs. Similarly, there are no optogenetic or even chemical tools available for controlling endogenous heterotrimer signaling. Therefore, in Aim 1, we plan to deliver a library of photoligands to control endogenous PAR receptors instantaneously and reversibly. The preliminary data shows optical activation of wild type PAR1 receptor by a genetically encoded photoligand and attests to the feasibility of the proposed. Though the proposal focuses on PAR family GPCRs, the broader adaptability in photoligand-design will allow optical control of other peptide or small protein activated GPCRs, expanding the future biomedical significance of Aim 1. Our photoligands will be the first of their kind to deliver such a precise regulation of subcellular, cellular, tissue, or even organ-level GPCR signaling on optical command, fulfilling the demands of future biomedical investigations. Similarly, despite the central roles of heterotrimeric G proteins in transducing signaling from all GPCRs, other than the few available inhibitors of their signaling, there are no direct routes to activate them with an appreciable spatial or temporal control. Despite the optical control, the available optogenetic regulators aim only downstream effectors of G proteins and elicit higher background signaling due to overexpressed active proteins. We use Aim 2 to gain direct access to endogenous G protein heterotrimers to control one or both G protein subunit signaling optically. Using a peptide domain derived from a native controller of G protein signaling, we show optically induced macrophage migration by the localized generation of Gβγ. Engineered optogenetic tools in Aim 2 will not only provide experimental means to bypass the limitations in chemical agents, but also inform the science on G protein subunit function and promote future molecule discovery/screening efforts to control heterotrimer and or its select-subunits.