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

细胞相互交流和改变细胞反应的方式是所有生命的重要组成部分，并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。细胞之间的这种交流主要基于化学信使分子，其可以是小的（例如腺苷，肾上腺素）或大的（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）结合而起作用，这些蛋白质反过来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%的处方药物都针对这些受体。最近，我们已经发现了更多关于GPCR的物理结构，使用X射线晶体学。这使得人们更好地理解了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（正构）的激动剂分子刺激时如何变化。然而，在过去的十年中，药物也可以结合到一个额外的位点，称为变构位点，它位于GPCR蛋白的一个单独的位置。这些药物引起蛋白质结构的不同变化，可以改变激素或神经递质与正构结合位点的结合程度，并激活其受体。与小分子变构药物一样，邻近的细胞蛋白质（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的程度可能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（导致所谓的偏置信号）。传统的测量配体与受体结合的方式（它们的药理学）的方法需要大量的细胞来实现可测量的反应。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），以研究单个活细胞膜上非常小区域的GPCR药理学。我们已经集中在两个受体的激素腺苷-A1和A3受体。我们是英国唯一一个（也是世界上少数几个）应用FCS研究GPCR与药物和其他细胞信号蛋白相互作用的团队。此次更新的目的是将这项工作扩展到解决有关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。我们的重点将放在腺苷和肾上腺素的受体（β-肾上腺素受体）上，这对心血管系统很重要。我们将尝试回答的具体问题包括：（a）信号复合物中每种类型的受体有多少？(b)这些复合物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差异？(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？这些知识可以用来将药物靶向到具有特定组成的复合物吗？(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？

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?