Optical control of endogeneous GPCR and G protein Signaling

内源 GPCR 和 G 蛋白信号转导的光学控制

• 批准号: 10542818
• 负责人: Welivitiya Kankanamlage Ajith Karunarathne
• 金额: $ 28.14万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10542818
• 关键词: Biological Availability; Brain; Bypass; Cardiac; Cardiovascular system; Cell Line; Cell Surface Proteins; Cell Surface Receptors; Cells; Chemical Agents; Chemicals; Color Visions; Cultured Cells; Data; Development; Disease; Drug Targeting; Engineering; Environment; Event; Eye; F2R gene; Family; Functional disorder; Future; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Generations; Genetic; Goals; Guanine Nucleotide Exchange Factors; Heart; Heterotrimeric G Protein Subunit; Heterotrimeric GTP-Binding Proteins; Human; Hydrolysis; Immune; Investigation; Libraries; Life; Ligands; Light; Macrophage; Metabolic Diseases; Methodology; Methods; Molecular; Neurologic; Opsin; Optics; Organ; PAR-1 Receptor; Pathologic; Pathway interactions; Peptide Hydrolases; Peptides; Phosphatidylinositol 4,5-Diphosphate; Physiological; Physiology; Play; Property; Protein Engineering; Protein Subunits; Proteinase-Activated Receptors; Proteins; RGS Domain; Receptor Cell; Recombinants; Regulation; Resolution; Role; Route; Science; Signal Transduction; Signaling Molecule; Signaling Protein; Solid; Tertiary Protein Structure; Testing; Therapeutic; Thrombin; Tissues; cell behavior; design; extracellular; genetic regulatory protein; human disease; in vivo; inhibitor; innovation; invention; light gated; member; method development; migration; optogenetics; overexpression; pharmacologic; protein function; receptor; response; screening; tool

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.

摘要 细胞主要使用G蛋白偶联受体（GPCR）感知细胞外环境。他们 代表细胞表面蛋白的最大家族，并在维持细胞生命中发挥关键的生理作用。 GPCR利用异源三聚体G蛋白将信号传递到细胞内部。气相色谱还原反应功能障碍，以及 作为G蛋白信号传导，导致一些最普遍的人类疾病，因此，GPCR已经成为 最大的毒品目标在超过800个成员中，超过100个GPCR由肽或 小蛋白配体。甚至一个这样的GPCR家族，蛋白酶激活受体（PAR）家族，也显示出与GPCR的相似性。 广泛的物理存在于整个身体从大脑到心脏，并调节许多已知的， 可能还有更多未知的生理作用，从免疫到心脏。一个基本的限制， PAR在人类生理学中的进展是缺乏控制内源性表达受体的工具， 培养的细胞和体内。虽然视蛋白可以通过空间和时间控制来激活G蛋白信号，但它们可以通过调节G蛋白信号来调节视蛋白的功能。 仅粗略地概括内源性GPCR的信号传导。同样，没有光遗传学甚至化学 可用于控制内源性异源三聚体信号传导的工具。 因此，在目标1中，我们计划提供光配体库来控制内源性PAR受体 瞬间和可逆地。初步数据显示野生型PAR 1受体通过一种 基因编码的光配体，并证明了所提出的可行性。虽然该提案的重点是 PAR家族GPCR，在光配体设计中更广泛的适应性将允许对其他肽或多肽进行光学控制。 小蛋白激活GPCR，扩展了Aim 1的未来生物医学意义。我们的光配体将是 这是第一个能够精确调控亚细胞、细胞、组织甚至器官水平GPCR的同类产品 光指令信号，满足未来生物医学研究的需求。 类似地，尽管异源三聚体G蛋白在从所有GPCR转导信号中起着中心作用， 除了少数可用的信号传导抑制剂外，没有直接的途径来激活它们， 空间或时间控制。尽管有光学控制，但可用的光遗传学调节剂仅针对 G蛋白的下游效应物，并由于过表达的活性蛋白引起更高的背景信号传导。 我们使用Aim 2来直接获得内源性G蛋白异源三聚体以控制一个或两个G蛋白 亚基光学信号传导。使用一个来自G蛋白信号传导的天然控制器的肽结构域，我们 显示通过Gβγ的局部产生光学诱导的巨噬细胞迁移。工程光遗传学工具 目标2中的技术不仅提供了绕过化学制剂限制的实验手段， G蛋白亚基功能科学，促进未来的分子发现/筛选工作， 异源三聚体和/或其选择亚基。

项目成果

Molecular regulation of PLCβ signaling.

PLCβ 信号传导的分子调节。

• DOI: 10.1016/bs.mie.2023.01.001
• 发表时间: 2023
• 期刊: Methods in enzymology
• 作者: Ubeysinghe,Sithurandi;Wijayaratna,Dhanushan;Kankanamge,Dinesh;Karunarathne,Ajith
• 通讯作者: Karunarathne,Ajith

A short C-terminal peptide in Gγ regulates Gβγ signaling efficacy.

• DOI: 10.1091/mbc.e20-11-0750
• 发表时间: 2021-08-01
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: Tennakoon M;Senarath K;Kankanamge D;Chadee DN;Karunarathne A
• 通讯作者: Karunarathne A

CaaX-motif-adjacent residues influence G protein gamma (Gγ) prenylation under suboptimal conditions.

• DOI: 10.1016/j.jbc.2023.105269
• 发表时间: 2023-11
• 期刊: The Journal of biological chemistry
• 作者: Tennakoon M;Thotamune W;Payton JL;Karunarathne A
• 通讯作者: Karunarathne A

Molecular basis of sequence-dependent differential prenylation inhibition and statin sensitivity of G proteins.

G 蛋白序列依赖性差异异戊二烯化抑制和他汀类药物敏感性的分子基础。

• 发表时间: 2022
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Tennakoon,MithilaI;Karunarathne,Ajith
• 通讯作者: Karunarathne,Ajith

The spatial distribution of GPCR and Gβγ activity across a cell dictates PIP3 dynamics.

• DOI: 10.1038/s41598-023-29639-0
• 发表时间: 2023-02-16
• 期刊: SCIENTIFIC REPORTS
• 影响因子: 4.6
• 作者: Wijayaratna, Dhanushan;Ratnayake, Kasun;Ubeysinghe, Sithurandi;Kankanamge, Dinesh;Tennakoon, Mithila;Karunarathne, Ajith
• 通讯作者: Karunarathne, Ajith