The organisational structure of class A GPCRs: Implications for pharmacology, function and therapeutic regulation

位于细胞表面的蛋白质是由大量激素、神经递质和各种其他细胞调节剂提供的信息的受体。在某些情况下，例如单个跨膜区受体-酪氨酸激酶，众所周知，为了将信使的结合传递到细胞内的反应中，受体必须二聚化，即受体的两个拷贝必须聚集在一起形成一个复合体并相互作用。G蛋白偶联受体(GPCRs)家族是关键荷尔蒙和神经递质的靶标，这些激素和神经递质控制从心率到情绪反应的一切。鉴于它们在生理学上的重要性，以及作为一系列非常广泛的缓解重大疾病的药物的靶标，这些蛋白质多年来一直被广泛研究。事实上，为了既了解通过与GPCRs结合而发挥作用的药物的分子作用基础，也为了改进下一代此类药物，近年来通过结晶某些GPCR取得了巨大进展，从而可以观察到它们结构的惊人细节。在最近的一些案例中，受体结晶为二聚体(两个拷贝)或四聚体(包含四个拷贝的受体)，每个拷贝的受体之间有明确的接触。尽管如此，而且有明确的证据表明，GPCRs可以而且确实以二聚体甚至多聚体的形式存在于活细胞中，但也有充分的证据表明，这种相互作用对于它们的主要功能并不是绝对必要的。因此，到目前为止还没有答案的关键悬而未决的问题包括，GPCRs复合体的大小范围是什么，在该家族的不同成员之间是否存在差异，在表达受体的不同细胞和组织中是否存在差异，如果是，这对健康和疾病的功能有何影响？然而，就目前而言，在这些话题上几乎没有达成一致。拟议研究的第一个实质性组成部分是处理和回答每一个问题。这些研究旨在回答关于这些受体的基本问题，但也是为了探索各种药物有效性答案的意义，以及如何利用这种差异来开发改进的药物。为了开发解决其中许多问题的方法，最近进行了一系列努力，以更好地将数学融入生物过程的分析，并认识到，用荧光标记标记的表达这些受体的细胞的摄影图像通常包含更多通常从这些细胞中提取的信息。因此，在为这一应用做准备的过程中，我们开发了一种方法，通过详细的数学分析，可以揭示迄今为止对单个细胞内受体复合体的大小和形状的认识。单独来看，这将是有趣的，但还不够。因此，这项工作的第二个主要内容将是使用晶体结构数据中的信息来尝试产生可拆解或防止形成此类复合体的修饰受体，然后使用这些修饰形式的受体来评估功能后果。这类研究将利用我和我的团队多年来建立和使用的药理学、生物化学和细胞信号分析方面的广泛方法。该提案的另一个主要组成部分反映了单个GPCRs不仅与同一受体的其他副本相互作用而形成复合体。它们还可以与GPCR超家族的其他成员相互作用，产生异构体。据报道，这些异构体与相应的同质异构体显示出非常不同的药理和功能，我们将解决与上面类似的问题，这些复合体包含神经递质多巴胺和谷氨酸的GPCR。

Proteins located at the surface of cells act as receptors for information provided by the presence of a bewildering number of hormones, neurotransmitters and various other cell modulators. In certain cases, such as the single transmembrane domain receptor-tyrosine kinases, it is well established that for binding of the messenger to be transmitted into a response inside the cell, the receptor has to dimerise, i.e. two copies of the receptor must come together into a complex and interact. The family of G protein-coupled receptors (GPCRs) are the targets for key hormones and neurotransmitters that control everything from heart rate to emotional responses. Given their importance in physiology and as the targets of a very broad group of medicines that alleviate major diseases, these proteins have been studied extensively for many years. Indeed, in a drive to both understand the molecular basis of action of medicines that function by binding to GPCRs and also to improve the next generation of such medicines, in recent years enormous progress has been made via crystallising certain GPCRs so that amazing detail of their structures can be observed. In a number of recent cases the receptor has crystallised as either a dimer (two copies) or a tetramer (containing four copies of the receptor) with well defined contacts between the individual copies of the receptor. Despite this, and clear evidence that GPCRs can and do exist as dimers or even, indeed, as multimers in living cells, it is also well established that such interactions are not absolutely required for their primary function. As such, key outstanding questions that so far lack answers include, what range of sizes of complexes of GPCRs exist, does this vary between different members of the family, does this vary in different cells and tissues in which the receptor is expressed and, if so, what are the consequences of this for function in both health and disease? However, as the moment there is little agreement on these topics. The first substantial component of the studies proposed is to address and answer each of these questions. These studies are designed to answer both fundamental questions about these receptors but also to explore the significiance of the answers for the effectiveness of various medicines and how such variation might be used to develop improved medicines.Integral to developing approaches to address many of these questions has been a series of recent efforts to better incorporate mathematics into analysis of biological processes and the appreciation that photographic images of cells expressing such receptors labelled with fluorescent markers often contain far more information that is usually abstracted from them. Thus, in preparation for this application we have developed ways in which detailed mathematical analysis can uncover hitherto unappreciated insights into the size and the shape of a receptor complex inside a single cell. In isolation, this would be interesting but insufficient. Therefore, the second major element of the work will be to use information from the crystal structure data to attempt to generate modified receptors that disassemble or that prevent the formation of such complexes and then use these modified forms of the receptor to assess the functional consequences. Such studies will take advantage of a wide array of approaches in pharmacology, biochemistry and cellular signalling assays that my team and I have built up and used over many years. The other major component of the proposal reflects that individual GPCRs do not only interact to form complexes with other copies of the same receptor. They can also interact with other members of the GPCR superfamily to generate heteromers. Such heteromers have been reported to display very distinct pharmacology and function than the corresponding homomers and we will address similar questions as above for such complexes incorporating GPCRs for the neurotransmitters dopamine and glutamate.