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

腺苷是细胞对一系列刺激物作出反应后释放的一种分子，它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特殊对接位点被称为腺苷受体。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。现在很清楚，有几种不同类型的腺苷受体（其中a3 -腺苷受体是一个例子），其次，这些受体定位在细胞膜上非常微小和高度特化的区域，称为微域。这些微域包含一系列不同的分子，这些分子参与告诉细胞如何对药物或激素作出反应。这项提议的目的是使用高度复杂的激光显微技术来研究药物与活细胞中这些小膜微域中的a3受体结合的方式。这是通过使用附着有荧光标记的药物分子来实现的。荧光药物可以在单分子水平上实时与腺苷A3受体结合。就其本身而言，这种小的荧光药物分子在激光束中快速移动并发出光（光子）。当药物与单一受体结合时，复合物会更大、更重，因此移动得更慢，发出不同的光模式。通过分析每个荧光分子在激光束内存在的时间，我们可以计算出存在的自由药物分子的数量和与受体结合的药物分子的数量。我们还可以监测单个受体-信号蛋白复合物的大小，从它们的扩散特性。这项工作的最终目的是将这些技术应用于患病的人类细胞。要做到这一点，我们需要开发非常特殊的荧光a3受体药物，它不会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在感染和炎症中起重要作用的专门的人类血细胞（中性粒细胞）中的a3受体。

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.