Use of fluorescence correlation spectroscopy to study the adenosine A3-receptor in microdomains of single living cells

使用荧光相关光谱研究单个活细胞微区中的腺苷 A3 受体

• 批准号: G0800006/1
• 负责人: Stephen Hill
• 金额: $ 161.4万
• 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: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=G0800006%2F1
• 关键词: Use; fluorescence; correlation; spectroscopy; study

Adenosine is a molecule that is released from cells in response to a range of stimulants and binds to specialised docking sites on the outside of neighbouring cells to pass on chemical signals in the form of changes in the level of intracellular messengers. The specialised docking sites on the surface of cells that recognise adenosine are called adenosine receptors. These are members of the G protein-coupled receptor (GPCR) family of cell surface receptors that mediate effects inside cells by binding to G proteins or other signalling proteins within the cell membrane and triggering changes in intracellular second messenger formation. It is now clear that there are several different types of adenosine receptors (of which the A3-adenosine receptor is one example) and secondly that these receptors are localised in very tiny and highly specialised regions of the cell membrane called microdomains. These microdomains contain a collection of different molecules that are involved in telling the cell how to respond to drugs or hormones. The aim of this proposal is to use highly sophisticated laser-based microscopy to study the way that drugs bind to A3-receptors in these small membrane microdomains in living cells. This is achieved by using a drug molecule that has a fluorescent label attached to it. The fluorescent drug can then be followed as it binds to the adenosine A3 receptor in real time at the single molecule level. On its own, the small fluorescent drug molecule moves quickly though a laser beam and gives off light (photons). When the drug binds to a single receptor, the complex is much bigger and heavier and so moves much more slowly and gives off a different pattern of light. By analysing the time that each fluorescent molecule is present within the laser beam, we can count the number of free drug molecules and the number of receptor-bound drug molecules that are present. We can also monitor the size of individual receptor-signalling protein complexes from their diffusional characterstics. The ultimate aim of this work is to use these techniques in human cells in disease. To do this we need to develop very specific fluorescent A3-receptor drugs that do not bind to other types of adenosine receptor. When we have designed and made these drugs we will use them to study A3-receptors in specialised human blood cells (neutrophils) that are have important roles during infection and inflammation.

腺苷是一种分子，细胞响应一系列刺激物而释放，并与邻近细胞外部的专门对接位点结合，以细胞内信使水平变化的形式传递化学信号。细胞表面识别腺苷的专门对接位点称为腺苷受体。这些是细胞表面受体 G 蛋白偶联受体 (GPCR) 家族的成员，通过与细胞膜内的 G 蛋白或其他信号蛋白结合并触发细胞内第二信使形成的变化来介导细胞内的作用。现在已经清楚，腺苷受体有几种不同类型（其中 A3-腺苷受体就是一个例子），其次这些受体位于细胞膜上非常微小且高度特化的区域，称为微域。这些微结构域包含一组不同的分子，这些分子参与告诉细胞如何对药物或激素做出反应。该提案的目的是使用高度复杂的激光显微镜来研究药物与活细胞中这些小膜微区中的 A3 受体结合的方式。这是通过使用附有荧光标记的药物分子来实现的。然后可以在单分子水平上实时跟踪荧光药物与腺苷 A3 受体的结合。就其本身而言，小的荧光药物分子通过激光束快速移动并发出光（光子）。当药物与单个受体结合时，复合物更大、更重，因此移动速度更慢，并发出不同模式的光。通过分析每个荧光分子在激光束中存在的时间，我们可以计算存在的游离药物分子的数量和受体结合的药物分子的数量。我们还可以根据单个受体信号蛋白复合物的扩散特征来监测其大小。这项工作的最终目标是将这些技术应用于疾病中的人类细胞。为此，我们需要开发非常特异的荧光 A3 受体药物，该药物不与其他类型的腺苷受体结合。当我们设计和制造这些药物时，我们将用它们来研究专门的人类血细胞（中性粒细胞）中的 A3 受体，这些受体在感染和炎症过程中发挥着重要作用。