Use of fluorescence correlation spectroscopy to study GPCR oligomerisation and allosterism in membrane micro domains of single living cells.

使用荧光相关光谱研究单个活细胞膜微域中的 GPCR 寡聚和变构作用。

• 批准号: MR/N020081/1
• 负责人: Stephen Hill
• 金额: $ 244.28万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FN020081%2F1
• 关键词: Use; fluorescence; correlation; spectroscopy; study

The way in which cells communicate with each other and change cellular responses is an essential part of all life, and controls the inner workings of organs within the body allowing them to respond, adapt and survive. This communication between cells is largely based on chemical messenger molecules, which can be small (e.g. adenosine, adrenaline) or large (e.g. vascular endothelial growth factor, VEGF). These molecules work by binding to specific proteins (receptors) on the surface of their target cells that in turn activate signalling responses inside the cell. G Protein-Coupled Receptors (GPCRs) are the largest family of these cell surface proteins. They are major targets for drug discovery and over 30% of all prescribed drugs target these receptors. Recently we have discovered much more about the physical structure of GPCRs using x-ray crystallography. This has led to a better understanding of how the structure of these proteins changes when stimulated by agonist molecules that act at the same site (orthosteric) as the natural hormone or neurotransmitter. However, over the last decade it has become clear that drugs can also bind to an additional site, called the allosteric site, which is in a separate location on the GPCR protein. These drugs cause a different change in protein structure that can alter how well a hormone or neurotransmitter binds to the orthosteric binding site and activates its receptor. As well as small molecule allosteric drugs, neighbouring cellular proteins (including other GPCRs) can also bind to GPCRs and act as allosteric modulators to enhance or inhibit the binding and/or function of the natural ligand. This means that how well a drug binds or activates a GPCR can depend on where that receptor is in the cell and what other cellular proteins are present in that location. This can also change the signals stimulated by the receptor (leading to something called biased signalling). Traditional ways of measuring the way ligands bind to receptors (their pharmacology) require large numbers of cells to achieve a measurable response. During our current MRC programme grant we have developed new highly sensitive imaging approaches (based on a technique called fluorescence correlation spectroscopy or FCS) to study the pharmacology of GPCRs in very small areas of the membrane of single living cells. We have focused on two receptors for the hormone adenosine - the A1 and A3 receptors. We are the only group in the UK (and one of few worldwide) to have applied FCS to look at the interaction of GPCRs with both drugs and other cellular signaling proteins. The aim of this renewal is to extend this work to address key questions about the molecular pharmacology of GPCRs. This will use FCS to look at GPCRs in complex with other proteins (receptors and signaling proteins) in specific areas of living cell membranes. In particular, we will take advantage of the exquisite sensitivity of FCS to detect GPCRs at the low expression levels normally found in native cells. Our major emphasis will be on receptors for adenosine and adrenaline (beta-adrenoceptors) that are important for the cardiovascular system.Specific questions we will try and answer include: (a) How many receptors of each type do the signaling complexes contain? (b) What impact do changes in how these complexes are made up have on how each of the constituent receptors binds its ligand? (c) Does this vary between neighbouring cells and membrane locations? (d) To what extent does binding of a ligand to one receptor in the complex affect binding of ligands to the others? Can this knowledge be exploited to target drugs to complexes with a specific composition? (e) Can these complexes and their functional interactions be demonstrated in native cells from the cardiovascular system?

细胞相互沟通和改变细胞反应的方式是所有生命的重要组成部分，它控制着体内器官的内部运作，使它们能够做出反应、适应和生存。细胞之间的这种通讯很大程度上基于化学信使分子，这些分子可以很小（例如腺苷、肾上腺素），也可以很大（例如血管内皮生长因子、VEGF）。这些分子通过与靶细胞表面的特定蛋白质（受体）结合来发挥作用，从而激活细胞内的信号反应。 G 蛋白偶联受体 (GPCR) 是这些细胞表面蛋白中最大的家族。它们是药物发现的主要目标，超过 30% 的处方药物都以这些受体为目标。最近，我们利用 X 射线晶体学发现了更多有关 GPCR 物理结构的信息。这使得人们更好地了解这些蛋白质的结构在受到与天然激素或神经递质作用在同一位点（正位）的激动剂分子刺激时如何变化。然而，在过去十年中，人们已经清楚药物还可以与一个称为变构位点的附加位点结合，该位点位于 GPCR 蛋白上的一个单独位置。这些药物会引起蛋白质结构的不同变化，从而改变激素或神经递质与正位结合位点的结合程度并激活其受体。除了小分子变构药物外，邻近的细胞蛋白（包括其他 GPCR）也可以与 GPCR 结合并充当变构调节剂，以增强或抑制天然配体的结合和/或功能。这意味着药物结合或激活 GPCR 的效果取决于该受体在细胞中的位置以及该位置存在哪些其他细胞蛋白。这也可以改变受体刺激的信号（导致所谓的偏差信号传导）。测量配体与受体结合方式（其药理学）的传统方法需要大量细胞才能实现可测量的反应。在我们目前的 MRC 计划资助期间，我们开发了新的高灵敏度成像方法（基于称为荧光相关光谱或 FCS 的技术）来研究单个活细胞膜的非常小的区域中 GPCR 的药理学。我们重点关注激素腺苷的两种受体 - A1 和 A3 受体。我们是英国唯一一个（也是全球少数几个）应用 FCS 来研究 GPCR 与药物和其他细胞信号蛋白相互作用的团队之一。此次更新的目的是扩展这项工作以解决有关 GPCR 分子药理学的关键问题。这将使用 FCS 来观察活细胞膜特定区域中与其他蛋白质（受体和信号蛋白）复合的 GPCR。特别是，我们将利用 FCS 的精湛灵敏度来检测天然细胞中通常存在的低表达水平的 GPCR。我们的重点是对心血管系统很重要的腺苷和肾上腺素受体（β-肾上腺素受体）。我们将尝试回答的具体问题包括： (a) 信号复合物每种类型包含多少个受体？ (b) 这些复合物的组成方式的变化对每个组成受体如何结合其配体有什么影响？ (c) 这在相邻细胞和膜位置之间是否有所不同？ (d) 配体与复合物中一个受体的结合在多大程度上影响配体与其他受体的结合？是否可以利用这些知识将药物靶向具有特定成分的复合物？ (e) 这些复合物及其功能相互作用能否在心血管系统的天然细胞中得到证实？

项目成果

Correction to Synthesis and Evaluation of the First Fluorescent Antagonists of the Human P2Y2 Receptor Based on AR-C118925.

基于AR-C118925对人类P2Y2受体第一个荧光拮抗剂的合成和评价进行校正。

• DOI: http://dx.10.1021/acs.jmedchem.8b00606
• 发表时间: 2018
• 期刊: Journal of medicinal chemistry
• 影响因子: 7.3
• 作者: Conroy S

A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor

活细胞 NanoBRET 结合测定可以研究腺苷 A3 受体的配体结合动力学

• DOI: 10.1007/s11302-019-09650-9
• 发表时间: 2019-03-27
• 期刊: Purinergic Signalling
• 影响因子: 3.5
• 作者: Mónica Bouzo;Leigh A. Stoddart;Lizi Xia;A. IJzerman;L. Heitman;S. Briddon;S. Hill
• 通讯作者: S. Hill

The effect of two selective A1 -receptor agonists and the bitopic ligand VCP746 on heart rate and regional vascular conductance in conscious rats.

两种选择性 A1 受体激动剂和双位配体 VCP746 对清醒大鼠心率和局部血管电导的影响。

• DOI: http://dx.10.1111/bph.14870
• 发表时间: 2020
• 期刊: British journal of pharmacology
• 影响因子: 7.3
• 作者: Cooper SL

Fluorescently tagged nanobodies and NanoBRET to study ligand-binding and agonist-induced conformational changes of full-length EGFR expressed in living cells.

荧光标记的纳米抗体和 NanoBRET 用于研究活细胞中表达的全长 EGFR 的配体结合和激动剂诱导的构象变化。

• DOI: http://dx.10.3389/fimmu.2022.1006718
• 发表时间: 2022
• 期刊: Frontiers in immunology
• 作者: Comez D

Subtype-Selective Fluorescent Ligands as Pharmacological Research Tools for the Human Adenosine A2A Receptor.

亚型选择性荧光配体作为人腺苷 A2A 受体的药理学研究工具。

• DOI: http://dx.10.1021/acs.jmedchem.9b01856
• 发表时间: 2020
• 期刊: Journal of medicinal chemistry
• 影响因子: 7.3
• 作者: Comeo E